Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
PROTEIN-PROTEIN INTERACTIONS
BACKGROUND OF THE INVENTION
The present invention relates to the discovery of novel protein-protein
interactions that are
involved in mammalian physiological pathways, including physiological
disorders or diseases.
Examples of physiological disorders and diseases include non-insulin dependent
diabetes mellitus
(NIDDM), neurodegenerative disorders, such as Alzheimer's Disease (AD), and
the like. Thus, the
present invention is directed to complexes of these proteins and/or their
fragments, antibodies to the
complexes, diagnosis of physiological generative disorders (including
diagnosis of a predisposition
to and diagnosis of the existence of the disorder), drug screening for agents
which modulate the
interaction of proteins described herein, and identification of additional
proteins in the pathway
common to the proteins described herein.
The publications and other materials used herein to illuminate the background
of the
invention, and in particular, cases to provide additional details respecting
the practice, are
incorporated herein by reference, and for convenience, are referenced by
author and date in the
following text and respectively grouped in the appended List of References.
Many processes in biology, including transcription, translation and metabolic
or signal
transduction pathways, are mediated by non-covalently associated protein
complexes. The
formation of protein-protein complexes or protein-DNA complexes produce the
most efficient
chemical machinery. Much of modern biological research is concerned with
identifying proteins
involved in cellular processes, determining their functions, and how, when and
where they interact
with other proteins involved in specific pathways. Further, with rapid
advances in genome
sequencing, there is a need to define protein linkage maps, i.e., detailed
inventories of protein
interactions that make up functional assemblies of proteins or protein
complexes or that make up
physiological pathways.
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Recent advances in human genomics research has led to rapid progress in the
identification
of novel genes. In applications to biological and pharmaceutical research,
there is a need to
determine functions of gene products. A first step in defining the function of
a novel gene is to
determine its interactions with other gene products in appropriate context.
That is, since proteins
make specific interactions with other proteins or other biopolymers as part of
functional assemblies
or physiological pathways, an appropriate way to examine function of a gene is
to determine its
physical relationship with other genes. Several systems exist for identifying
protein interactions and
hence relationships between genes.
There continues to be a need in the art for the discovery of additional
protein-protein
interactions involved in mammalian physiological pathways. There continues to
be a need in the
art also to identify the protein-protein interactions that are involved in
mammalian physiological
disorders and diseases, and to thus identify drug targets.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of protein-protein interactions
that are
involved in mammalian physiological pathways, including physiological
disorders or diseases, and
to the use of this discovery. The identification of the interacting proteins
described herein provide
new targets for the identification of useful pharmaceuticals, new targets for
diagnostic tools in the
identification of individuals at risk, sequences for production of transformed
cell lines, cellular
models and animal models, and new bases for therapeutic intervention in such
physiological
pathways
Thus, one aspect of the present invention is protein complexes. The protein
complexes are
a complex of (a) two interacting proteins, (b) a first interacting protein and
a fragment of a second
interacting protein, (c) a fragment of a first interacting protein and a
second interacting protein, or
(d) a fragment of a first interacting protein and a fragment of a second
interacting protein. The
fragments of the interacting proteins include those parts of the proteins,
which interact to form a
complex. This aspect of the invention includes the detection of protein
interactions and the
production of proteins by recombinant techniques. The latter embodiment also
includes cloned
sequences, vectors, transfected or transformed host cells and transgenic
animals.
A second aspect of the present invention is an antibody that is immunoreactive
with the
above complex. The antibody may be a polyclonal antibody or a monoclonal
antibody. While the
antibody is immunoreactive with the complex, it is not immunoreactive with the
component parts
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of the complex. That is, the antibody is not immunoreactive with a first
interactive protein, a
fragment of a first interacting protein, a second interacting protein or a
fragment of a second
interacting protein. Such antibodies can be used to detect the presence or
absence of the protein
complexes.
A third aspect of the present invention is a method for diagnosing a
predisposition for
physiological disorders or diseases in a human or other animal. The diagnosis
of such disorders
includes a diagnosis of a predisposition to the disorders and a diagnosis for
the existence of the
disorders. In accordance with this method, the ability of a first interacting
protein or fragment
thereof to form a complex with a second interacting protein or a fragment
thereof is assayed, or the
genes encoding interacting proteins are screened for mutations in interacting
portions of the protein
molecules. The inability of a first interacting protein or fragment thereof to
form a complex, or the
presence of mutations in a gene within the interacting domain, is indicative
of a predisposition to,
or existence of a disorder. In accordance with one embodiment of the
invention, the ability to form
a complex is assayed in a two-hybrid assay. In a first aspect of this
embodiment, the ability to form
a complex is assayed by a yeast two-hybrid assay. In a second aspect, the
ability to form a complex
is assayed by a mammalian two-hybrid assay. In a second embodiment, the
ability to form a
complex is assayed by measuring in vitro a complex formed by combining said
first protein and said .
second protein. In one aspect the proteins are isolated from a human or other
animal. In a third
embodiment, the ability to form a complex is assayed by measuring the binding
of an antibody,
which is specific for the complex. In a fourth embodiment, the ability to form
a complex is assayed
by measuring the binding of an antibody that is specific for the complex with
a tissue extract from
a human or other animal. In a fifth embodiment, coding sequences of the
interacting proteins
described herein are screened for mutations.
A fourth aspect of the present invention is a method for screening for drug
candidates which
are capable of modulating the interaction of a first interacting protein and a
second interacting
protein. In this method, the amount of the complex formed in the presence of a
drug is compared
with the amount of the complex formed in the absence of the drug. If the
amount of complex
formed in the presence of the drug is greater than or less than the amount of
complex formed in the
absence of the drug, the drug is a candidate for modulating the interaction of
the first and second
interacting proteins. T'he drug promotes the interaction if the complex formed
in the presence of the
drug is greater and inhibits (or disrupts) the interaction if the complex
formed in the presence of the
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drug is less. The drug may affect the interaction directly, i.e., by
modulating the binding of the two
proteins, or indirectly, e.g., by modulating the expression of one or both of
the proteins.
A fifth aspect of the present invention is a model for such physiological
pathways, disorders
or diseases. The model may be a cellular model or an animal model, as further
described herein.
In accordance with one embodiment of the invention, an animal model is
prepared by creating
transgenic or "knock-out" animals. The knock-out may be a total knock-out,
i.e., the desired gene
is deleted, or a conditional knock-out, i.e., the gene is active until it is
knocked out at a determined
time. In a second embodiment, a cell line is derived from such animals for use
as a model. In a
third embodiment, an animal model is prepared in which the biological activity
of a protein complex
of the present invention has been altered. In one aspect, the biological
activity is altered by
disrupting the formation of the protein complex, such as by the binding of an
antibody or small
molecule to one of the proteins which prevents the formation of the protein
complex. In a second
aspect, the biological activity of a protein complex is altered by disrupting
the action of the
complex, such as by the binding of an antibody or small molecule to the
protein complex which
interferes with the action of the protein complex as described herein. In a
fourth embodiment, a cell
model is prepared by altering the genome of the cells in a cell line. In one
aspect, the genome of
the cells is modified to produce at least one protein complex described
herein. In a second aspect,
the genome of the cells is modified to eliminate at least one protein of the
protein complexes
described herein.
A sixth aspect of the present invention are nucleic acids coding for novel
proteins discovered
in accordance with the present invention.
A seventh aspect of the present invention is a method of screening for drug
candidates useful
for treating a physiological disorder. In this embodiment, drugs are screened
on the basis of the
association of a protein with a particular physiological disorder. This
association is established in
accordance with the present invention by identifying a relationship of the
protein with a particular
physiological disorder. The drugs are screened by comparing the activity of
the protein in the
presence and absence of the drug. If a difference in activity is found, then
the drug is a drug
candidate for the physiological disorder. The activity of the protein can be
assayed in vitro or in
vivo using conventional techniques, including transgenic animals and cell
lines of the present
invention.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention is the discovery of novel interactions between proteins
described
herein. The genes coding for some of these proteins may have been cloned
previously, but their
potential interaction in a physiological pathway or with a particular protein
was unknown.
5 Alternatively, the genes coding for some of these proteins have not been
cloned previously and
represent novel genes. These proteins are identified using the yeast two-
hybrid method and
searching a human total brain library, as more fully described below.
According to the present invention, new protein-protein interactions have been
discovered.
The discovery of these interactions has identified several protein complexes
for each protein-protein
interaction. The protein complexes for these interactions are set forth below
in Tables 1-73, which
also identify the new protein-protein interactions of the present invention.
TABLE 1
Protein Complexes of Glut4/CARP Interaction
Glucose Transporter 4 (Glut4) and Clone C-193 (CARP)
A fragment of Glut4 and CARP
Glut4 and a fragment of CARP
A fragment of Glut4 and a fragment of CARP
TABLE 2
Protein Complexes of Glutl/DRAL(FHL2) Interaction
Glucose Transporter 1 (Glutl) and DRAL(FHL2)
A fragment of Glutl and DRAL(FHL2)
Glutl and a fragment of DRAL(FHL2)
A fragment of Glutl and a fragment of DRAL(FHL2)
TABLE 3
Protein Complexes of Glutl/Myosin Heaw Chain Interaction
Glucose Transporter 1 (Glutl) and myosin heavy chain
A fragment of Glutl and myosin heavy chain
Glutl and a fragment of myosin heavy chain
A fragment of Glutl and a fragment of myosin heavy chain
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TABLE 4
Protein Complexes of Glutl/HSS Interaction
Glucose Transporter 1 (Glutl) and human sperm surface protein (HSS)
A fragment of Glutl and HSS
Glutl and a fragment of HSS
A fragment of Glutl and a fragment of HSS
TABLE 5
Protein Complexes of OGTase/Mvosin Heavy Chain Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and myosin heavy chain
A fragment of OGTase and myosin heavy chain
OGTase and a fragment of myosin heavy chain
A fragment of OGTase and a fragment of myosin heavy chain
TABLE 6
Protein Complexes of IRAP/14-3-3 Beta Interaction
Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and 14-3-3 beta
A fragment of IRAP and 14-3-3 beta
IRAP and a fragment of 14-3-3 beta
A fragment of IRAP and a fragment of 14-3-3 beta
TABLE 7
Protein Complexes of IRAP/HSS Interaction
Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and human sperm
surface
protein (HSS)
A fragment of IRAP and HSS
IRAP and a fragment of HSS
A fragment of IRAP and a fragment of HSS
TABLE 8
Protein Complexes of PI-3K110/Complement Protein C4 Interaction
PI-3 Kinase pl 10 subunit (PI-3K110) and Complement Protein C4
A fragment of PI-3K110 and Complement Protein C4
PI-3K110 and a fragment of Complement Protein C4
A fragment of PI-3K110 and a fragment of Complement Protein C4
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TABLE 9
Protein Complexes of PI-3K110/Tenascin XB Interaction
PI-3 Kinase pl 10 subunit (PI-3K110) and Tenascin XB
A fragment of PI-3K110 and Tenascin XB
PI-3K110 and a fragment of Tenascin XB
A fragment of PI-3K110 and a fragment of Tenascin XB
TABLE 10
Protein Complexes of PI-3K110/GAA Interaction
PI-3 Kinase p110 subunit (PI-3K110) and Alpha Acid Glucosidase (GAA)
A fragment of PI-3K110 and GAA
PI-3K110 and a fragment of GAA
A fragment of PI-3K110 and a fragment of GAA
TABLE 11
Protein Complexes of MM-1/C-Napl Interaction
C-myc Binding Protein (MM-1) and C-Napl
A fragment of MM-1 and C-Napl
MM-1 and a fragment of C-Napl
A fragment of MM-1 and a fragment of C-Napl
TABLE 12
Protein Complexes of MM-lBeta Spectrin Interaction
C-myc Binding Protein (MM-1) and Beta Spectrin
A fragment of MM-1 and Beta Spectrin
MM-1 and a fragment of Beta Spectrin
A fragment of MM-1 and a fragment of Beta Spectrin
TABLE 13
Protein Complexes of MM-1/KIAA0477 Interaction
C-myc Binding Protein (MM-1) and KIAA0477
A fragment of MM-1 and KIAA0477
MM-1 and a fragment of KIAA0477
A fragment of MM-l and a fragment of KIAA0477
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TABLE 14
Protein Complexes of D~~namin/CALM Interaction
Dynamin and Calthrin Assembly Protein (CALM)
A fragment of Dynamin and CALM
Dynamin and a fragment of CALM
A fragment of Dynamin and a fragment of CALM
TABLE 15
Protein Complexes of Dvnamin/Psme3 Interaction
Dynamin and Proteosome Activator Subunit Psme3 (Psme3)
A fragment of Dynamin and Psme3
Dynamin and a fragment of Psme3
A fragment of Dynamin and a fragment of Psme3
TABLE 16
Protein Complexes ofNaflb/I-TRAF Interaction
Nef Associated Factor 1 beta (Naflb) and I-TRAF
A fragment of Nafl b and I-TRAF
Naflb and a fragment of I-TRAF
A fragment of Naflb and a fragment of I-TRAF
TABLE 17
Protein Complexes of Aktl/NuMAI Interaction
Akt kinase 1 (Akt 1 ) and NuMA 1
A fragment of Akt 1 and NuMA 1
Aktl and a fragment of NuMAl
A fragment of Akt 1 and a fragment of NuMA 1
TABLE 18
Protein Complexes of Akt2/NuMAI Interaction
Akt kinase 2 (Akt2) and NuMAl
A fragment of Akt2 and NuMAl
Akt2 and a fragment of NuMAI
A fragment of Akt2 and a fragment of NuMAI
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TABLE 19
Protein Complexes of Akt2/BAP31 Interaction
Akt kinase 2 (Akt2) and BAP31
A fragment of Akt2 and BAP31
Akt2 and a fragment of BAP31
A fragment of Akt2 and a fragment of BAP31
TABLE 20
Protein Complexes of Akt2/Beta Adaptin Interaction
Akt kinase 2 (Akt2) and beta adaptin
A fragment of Akt2 and beta adaptin
Akt2 and a fragment of beta adaptin
A fragment of Akt2 and a fragment of beta adaptin
TABLE 21
Protein Complexes of OGTase/Desmin Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and desmin
A fragment of OGTase and desmin
OGTase and a fragment of desmin
A fragment of OGTase and a fragment of desmin
TABLE 22
Protein Complexes of OGTase/Alpha-karyopherin Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and alpha-karyopherin
A fragment of OGTase and alpha-karyopherin
OGTase and a fragment of alpha-karyopherin
A fragment of OGTase and a fragment of alpha-karyopherin
TABLE 23
Protein Complexes of OGTase/Glutaminyl tRNA Synthetase Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and glutaminyl tRNA
synthetase
A fragment of OGTase and glutaminyl tRNA synthetase
OGTase and a fragment of glutaminyl tRNA synthetase
A fragment of OGTase and a fragment of glutaminyl tRNA synthetase
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TABLE 24
Protein Complexes of OGTase/Clone 25100 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and clone 25100
A fragment of OGTase and clone 25100
OGTase and a fragment of clone 25100
A fragment of OGTase and a fragment of clone 25100
TABLE 25
Protein Complexes of PTPlb/VAP-A Interaction
10 PTP 1 b and VAMP-associated protein A (VAP-A)
A fragment of PTP 1 b and VAP-A
PTP 1 b and a fragment of VAP-A
A fragment of PTP 1 b and a fragment of VAP-A
TABLE 26
Protein Complexes of Rab4/Alpha-catenin-like Protein Interaction
Rab4 and alpha-cantein-like protein
A fragment of Rab4 and alpha-cantein-like protein
Rab4 and a fragment of alpha-cantein-like protein
A fragment of Rab4 and a fragment of alpha-cantein-like protein
TABLE 27
Protein Complexes of Rab4/Rab2 Interaction
Rab4 and Rab2
A fragment of Rab4 and Rab2
Rab4 and a fragment of Rab2
A fragment of Rab4 and a fragment of Rab2
TABLE 28
Protein Complexes of Glut4/PN7065 Interaction
Glucose Transporter 4 (Glut4) and Novel Protein Fragment PN7065 (PN7065)
A fragment of Glut4 and PN7065
Glut4 and a fragment of PN7065
A fragment of Glut4 and a fragment of PN7065
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TABLE 29
Protein Complexes of Glut4/PN7386 Interaction
Glucose Transporter 4 (Glut4) and Novel Protein Fragment PN7386 (PN7386)
A fragment of Glut4 and PN7386
Glut4 and a fragment of PN7386
A fragment of Glut4 and a fragment of PN7386
TABLE 30
Protein Complexes of OGTase/PN6931 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and Novel Protein Fragment
PN6931
(PN6931 )
A fragment of OGTase and PN6931
OGTase and a fragment of PN6931
A fragment of OGTase and a fragment of PN6931
TABLE 31
Protein Complexes of Nafl b/PN7582 Interaction
Nef Associated Factor 1 beta (Naflb) and Novel Protein Fragment PN7582
(PN7582)
A fragment of Naflb and PN7582
Naflb and a fragment of PN7582
A fragment of Naflb and a fragment of PN7582
TABLE 32
Protein Complexes of OGTase/Talin Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and Talin
A fragment of OGTase and Talin
OGTase and a fragment of Talin
A fragment of OGTase and a fragment of Talin
TABLE 33
Protein Complexes of OGTase/MOP2 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and MOP2
A fragment of OGTase and MOP2
OGTase and a fragment of MOP2
A fragment of OGTase and a fragment of MOP2
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TABLE 34
Protein Complexes of OGTase/Clone 25100 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and Clone 25100
A fragment of OGTase and Clone 25100
OGTase and a fragment of Clone 25100
A fragment of OGTase and a fragment of Clone 25100
TABLE 35
Protein Complexes of OGTase/KIAA0443 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and KIAA0443
A fragment of OGTase and KIAA0443
OGTase and a fragment of KIAA0443
A fragment of OGTase and a fragment of KIAA0443
TABLE 36
Protein Complexes of OGTase/EGR1 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and EGR1
A fragment of OGTase and EGR1
OGTase and a fragment of EGRl
A fragment of OGTase and a fragment of EGR1
TABLE 37
Protein Complexes of OGTase/Dynamin II Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and Dynamin II
A fragment of OGTase and Dynamin II
OGTase and a fragment of Dynamin II
A fragment of OGTase and a fragment of Dynamin II
TABLE 38
Protein Complexes of OGTase/INT-6 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and INT-6
A fragment of OGTase and INT-6
OGTase and a fragment of INT-6
A fragment of OGTase and a fragment of INT-6
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TABLE 39
Protein Complexes of OGTase/HSPC028 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and HSPC028
A fragment of OGTase and HSPC028
OGTase and a fragment of HSPC028
A fragment of OGTase and a fragment of HSPC028
TABLE 40
Protein Complexes of OGTase/BAP31 Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and BAP31
A fragment of OGTase and BAP31
OGTase and a fragment of BAP31
A fragment of OGTase and a fragment of BAP3 I
TABLE 41
Protein Complexes of OGTase/Interferon-Ind Prot Interaction
O-linked N-acetylglucosaminyltransferase (OGTase) and Interferon-Ind Protein
A fragment of OGTase and Interferon-Ind Protein
OGTase and a fragment of Interferon-Ind Protein
A fragment of OGTase and a fragment of Interferon-Ind Protein
TABLE 42
Protein Complexes of Glut4/Beta-Catenin Interaction
Glucose Transporter 4 (Glut4) and Beta-catenin
A fragment of Glut4 and Beta-catenin
Glut4 and a fragment of Beta-catenin
A fragment of Glut4 and a fragment of Beta-catenin
TABLE 43
Protein Complexes of GIut4/Alpha-SNAP Interaction
Glucose Transporter 4 (Glut4) and Alpha-SNAP
A fragment of GIut4 and Alpha-SNAP
GIut4 and a fragment of Alpha-SNAP
A fragment of Glut4 and a fragment of Alpha-SNAP
3~
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TABLE 44
Protein Complexes of Glut4/MAPKKK6 Interaction
Glucose Transporter 4 (Glut4) and MAPKKK6
A fragment of Glut4 and MAPKKK6
GIut4 and a fragment of MAPKKK6
A fragment of Glut4 and a fragment of MAPKKK6
TABLE 45
Protein Complexes of Glut4/Tropomyosin 3 Interaction
Glucose Transporter 4 (Glut4) and Tropomyosin 3
A fragment of Glut4 and Tropomyosin 3
GIut4 and a fragment of Tropomyosin 3
A fragment of Glut4 and a fragment of Tropomyosin 3
TABLE 46
Protein Complexes of Glutl/DRAL/FHL2 Interaction
Glucose Transporter 1 (Glutl) and DRAL/FHL2
A fragment of Glutl and DRAL/FHL2
Glutl and a fragment of DRAL/FHL2
A fragment of Glutl and a fragment of DRAL/FHL2
TABLE 47
Protein Complexes of Glutl/MYSA Interaction
Glucose Transporter 1 (Glutl) and cardiac muscle myosin heavy chain (MYSA)
A fragment of Glutl and MYSA
Glutl and a fragment of MYSA
A fragment of Glutl and a fragment of MYSA
TABLE 48
Protein Complexes of IRAP/SLAP-2 Interaction
Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and SLAP-2
A fragment of IRAP and SLAP-2
IRAP and a fragment of SLAP-2
A fragment of IRAP and a fragment of SLAP-2
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TABLE 49
Protein Complexes of IRAP/SG2NA Interaction
Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and SG2NA
A fragment of IRAP and SG2NA
5 IRAP and a fragment of SG2NA
A fragment of IRAP and a fragment of SG2NA
TABLE 50
Protein Complexes of OGTase/14-3-3-epsilon
10 O-linked N-acetylglucosaminyltransferase (OGTase) and 14-3-3-epsilon
A fragment of OGTase and 14-3-3-epsilon
OGTase and a fragment of 14-3-3-epsilon
A fragment of OGTase and a fragment of 14-3-3-epsilon
15 TABLE 51
Protein Complexes of PI-3K85/Chromo~ranin Interaction
PI-3 Kinase p85 subunit (PI-3K85) and Chromogranin
A fragment of PI-3K85 and Chromogranin
PI-3K85 and a fragment of Chromogranin
A fragment of PI-3K85 and a fragment of Chromogranin
TABLE 52
Protein Complexes of PI-3K85/SLP-76 Interaction
PI-3 Kinase p85 subunit (PI-3K85) and SLP-76
A fragment of PI-3K85 and SLP-76
PI-3K85 and a fragment of SLP-76
A fragment of PI-3K85 and a fragment of SLP-76
TABLE 53
Protein Complexes of PI-3K85/14-3-3-zeta Interaction
PI-3 Kinase p85 subunit (PI-3K85) and 14-3-3-zeta
A fragment of PI-3K85 and 14-3-3-zeta
PI-3K85 and a fragment of 14-3-3-zeta
A fragment of PI-3K85 and a fragment of 14-3-3-zeta
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TABLE 54
Protein Complexes of PI-3K85/14-3-3-eta Interaction
PI-3 Kinase p85 subunit (PI-3K85) and 14-3-3-eta
A fragment of PI-3K85 and 14-3-3-eta
PI-3K85 and a fragment of 14-3-3-eta
A fragment of PI-3K85 and a fragment of 14-3-3-eta
TABLE 55
Protein Complexes of PI-3K85/TACC2 Interaction
PI-3 Kinase p85 subunit (PI-3K85) and TACC2
A fragment of PI-3K85 and TACC2
PI-3K85 and a fragment of TACC2
A fragment of PI-3K85 and a fragment of TACC2
TABLE 56
Protein Complexes of Glut4/MM-1 Interaction
Glucose Transporter 4 (Glut4) and C-Myc Binding Protein (MM-1)
A fragment of Glut4 and MM-1
Glut4 and a fragment of MM-1
A fragment of Glut4 and a fragment of MM-1
TABLE 57
Protein Complexes of Glutl/KIAA0144 Interaction
Glucose Transporter 1 (Glutl) and KIAA0144 (KIAA)
A fragment of Glutl and KIAA
Glutl and a fragment of KIAA
A fragment of Glutl and a fragment of KIAA
TABLE 58
Protein Complexes of Glutl/Dynamin Interaction
Glucose Transporter 1 (Glutl) and Dynamin
A fragment of Glutl and Dynamin
Glutl and a fragment of Dynamin
A fragment of Glutl and a fragment of Dynamin
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TABLE 59
Protein Complexes of Glutl/Clone 25204 Interaction
Glucose Transporter 1 (Glutl) and Clone 25204
A fragment of Glutl and Clone 25204
Glutl and a fragment of Clone 25204
A fragment of Glutl and a fragment of Clone 25204
TABLE 60
Protein Complexes of IRAP/VAP-A Interaction
Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP; oxytocinase) and
VAMP-
Associated Protein A (VAP-A)
A fragment of IRAP and VAP-A
IRAP and a fragment of VAP-A
A fragment of IRAP and a fragment of VAP-A
TABLE 61
Protein Complexes of OGTase/Nafla Interaction
O-Linked-N-AcetylglucosaminylTransferase (OGTase) and NEF-Associated Factor 1
Alpha
(Nafl a)
A fragment of OGTase and Nafl a
OGTase and a fragment of Nafla
A fragment of OGTase and a fragment of Nafl a
TABLE 62
Protein Complexes of OGTase/Alpha-2-Catenin Interaction
O-Linked-N-AcetylglucosaminylTransferase (OGTase) and Alpha-2-Catenin
A fragment of OGTase and Alpha-2-Catenin
OGTase and a fragment of Alpha-2-Catenin
A fragment of OGTase and a fragment of Alpha-2-Catenin
TABLE 63
Protein Complexes of PI-3K110/TRIP15 Interaction
PI-3 Kinase p110 subunit (PI-3K110) and TRIP15
A fragment of PI-3K110 and TRIP15
PI-3K110 and a fragment of TRIP15
A fragment of PI-3K110 and a fragment of TRIP15
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TABLE 64
Protein Complexes of Glut4/14-3-3 Zeta Interaction
Glucose Transporter 4 (Glut4) and 14-3-3 Zeta
A fragment of Glut4 and 14-3-3 Zeta
Glut4 and a fragment of 14-3-3 Zeta
A fragment of Glut4 and a fragment of 14-3-3 Zeta
TABLE 65
Protein Complexes of Glut4/KIAA0282 Interaction
Glucose Transporter 4 (Glut4) and KIAA0282 (an efp-like protein)
A fragment of Glut4 and KIAA0282
Glut4 and a fragment of KIAA0282
A fragment of Glut4 and a fragment of KIAA0282
TABLE 66
Protein Complexes of Glut4/Tankyrase Interaction
Glucose Transporter 4 (Glut4) and Tankyrase
A fragment of GIut4 and Tankyrase
GIut4 and a fragment of Tankyrase
A fragment of Glut4 and a fragment of Tankyrase
TABLE 67
Protein Complexes of IRAP/PTPZ Interaction
Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and protein tyrosine
phosphatase zeta (PTPZ)
A fragment of IRAP and PTPZ
IRAP and a fragment of PTPZ
A fragment of IRAP and a fragment of PTPZ
TABLE 68
Protein Complexes of IRAP/~iSpectrin Interaction
Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and (3-spectrin
A fragment of IRAP and (3-spectrin
IRAP and a fragment of (3-spectrin
A fragment of IRAP and a fragment of (3-spectrin
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TABLE 69
Protein Complexes of IRAP/PI-3K85 Interaction
Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and PI-3 Kinase p85
subunit (PI-3K85)
A fragment of IRAP and PI-3K85
IRAP and a fragment of PI-3K85
A fragment of IRAP and a fragment of PI-3K85
TABLE 70
Protein Complexes of PPS/HSP89 Interaction
Protein Phosphatase 5 (PPS) and Heat Shock Protein 89 (HSP89)
A fragment of PPS and HSP89
PPS and a fragment of HSP89
A fragment of PPS and a fragment of HSP89
TABLE 71
Protein Complexes of PPS/Tankyrase Interaction
Protein Phosphatase 5 (PPS) and Tankyrase
A fragment of PPS and Tankyrase
PPS and a fragment of Tankyrase
A fragment of PPS and a fragment of Tankyrase
TABLE 72
Protein Complexes of PI-3K85/Tankyrase Interaction
PI-3 Kinase p85 subunit (PI-3K85) and Tankyrase
A fragment of PI-3K85 and Tankyrase
PI-3K85 and a fragment of Tankyrase
A fragment of PI-3K85 and a fragment of Tankyrase
TABLE 73
Protein Complexes of PI-3K110/APP Interaction
PI-3 Kinase pl 10 subunit (PI-3K110) and Amyloid Precursor Protein (APP)
A fragment of PI-3K110 and APP
PI-3K110 and a fragment of APP
A fragment of PI-3K110 and a fragment of APP
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The proteins newly associated with a particular physiological pathway
described herein are
used to screen for drug candidates useful for treating a physiological
disorder. These proteins are
set forth in Table 74.
5 TABLE 74
Proteins for Drua Screening and Physiological Disorder
PI-3K110 Alzheimer's Disease
PI-3K110 Diabetes
TRIP15 Alzheimer's Disease
10 TRIP 15 Diabetes
All Others Diabetes
The involvement of above interactions in particular pathways is as follows.
One of the key questions which must be answered in order to understand and
treat non-
15 insulin dependent diabetes mellitus (NIDDM) is how glucose uptake is
regulated in the cell. It is
known that in adipose and muscle tissues there exists an insulin-regulated
membrane-spanning
glucose transporter called Glut4 that shuttles between the interior of the
cell to the plasma
membrane by means of vesicle-mediated endocytosis and exocytosis (Garvey et
al., 1998; Haruta
et a1.,1995; Pessin et al., 1999). Fat and muscle cells from diabetic patients
appear to be defective
20 in this type of regulated glucose transport (Zierath et al., 1998). The
mechanisms by which this
transport to the plasma membrane and return back to the interior of the cell
are regulated are not
well understood. Toward this end, there has been much interest in analyzing
the Glut4 protein and
the additional factors that participate in glucose uptake in fat and muscle
cells. As described below,
the yeast two-hybrid assay has been employed as a means of identifying
proteins that interact with
Glut4 as well as other molecules that have been implicated in the insulin-
dependent transport of
glucose. Thus, by understanding the regulation of glucose transport in normal
cells, medical
interventions can be discovered that would serve to increase sugar uptake in
the cells of diabetic
patients.
One approach to understanding how glucose transport is achieved has been to
use the Glut4
molecule as a molecular bait in the search for other molecules which can
interact with it. In this
way, it might be possible to find ways to influence the function of Glut4 and
perhaps find ways to
cause it to go to the plasma membrane and subsequently remove glucose from the
outside of the cell
and bring it into the interior. Using the yeast two-hybrid assay, we have been
able to demonstrate
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21
that clone C-193 (known as CARP in other systems) can bind to Glut4. CARP is a
cytokine-
inducible gene that reportedly acts as a negative regulator of cardiac-
specific genes (Jeyaseelan et
al., 1997). This interaction may serve as a tie between heart disease and
diabetes.
Using the yeast two-hybrid assay, we have also been able to detect the
interaction of Glut4
with two novel proteins that have been named PN7065 and PN7386. PN7065 bears a
striking
similarity to a rat salt-induced protein kinase (GenBank accession AB020480).
Experiments in rats
have shown that this salt-induced kinase may play an important role in the
regulation of
adrenocortical functions in response to high plasma salt and ACTH stimulation
(Wang et al., 1999).
It is possible that Glut4 may act as a substrate for the kinase. PN7386 is
identical to a human
chromosome 20 clone called 850H21 (GenBank accession AL031680) that is
uncharacterized in the
literature. It is possible that the protein product of this clone may
participate in protein trafficking
or in the signal transduction mechanism that regulates this process.
Using the yeast two-hybrid assay, we have been able to detect four more
proteins, beta
catenin, alpha-SNAP, tropomyosin 3 and MAPKKK6, which can bind to Glut4. Beta-
catenin is a
protein containing so-called Armadillo repeats that is involved in two
important cellular processes:
signal transduction via the Wingless pathway and cell adhesion (Ben-Ze'ev et
al., 1998). This
interaction between Glut4 and beta-catenin may shed light on the regulation of
insulin-responsive
glucose transport since it links the transporter to an important signaling
pathway. The alpha-SNAP
is an important mediator in the cellular process of intracellular transport
(St-Denis, et al., 1998).
The finding that alpha-SNAP and Glut4 interact provides a link between glucose
transportation and
the machinery required to perform movement of the glucose transporter between
the outside and the
interior of the cell. Glut4 has been demonstrated to interact with tropomyosin
3, a protein involved
in muscle contraction (Squire et al., 1998). This interaction may represent a
link between glucose
uptake and muscle function. Finally, the Glut4 glucose transporter has been
shown to interact with
a putative protein kinase, MAPKKK6. This enzyme has not been well-
characterized, but it was
identified by virtue of its ability to bind to another protein kinase (Wang et
al., 1998). Once again,
this may provide a clue to the mechanism of regulation of glucose transport
since Glut4 can interact
with another potential signal tranduction mediator.
Using the yeast two-hybrid assay, we have been able to detect three proteins
which can bind
to Glut4, 14-3-3 zeta, the efp-like protein (KIAA0282) and tankyrase. The 14-3-
3 zeta is a signal
transduction protein which has been shown to interact specifically with
phospherine residues
(Thorson et al., 1998). 14-3-3 zeta is part of a pathway that links the
insulin receptor molecule at
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22
the cell surface to the Glut4 protein located either in the interior of the
cell or also at the cell surface.
Interestingly, the same small region of Glut4 that interacts with 14-3-3 zeta
has been shown to be
phosphorylated on a critical serine residue by the kinase Akt-2 that has also
been implicated in
glucose uptake (Kupriyanova et al., 1999). The efp-like protein is a putative
transcription factor
(Orimo et al., 1995). Its function is not well described but the Glut4 protein
could influence the
transcriptional activation of various genes, some of which might be involved
in cellular metabolism.
Tankyrase is a known telomere-asscoiated protein (Smith et al., 1998).
Using the yeast two-hybrid assay, we have been able to detect an additional
protein, which
can bind to Glut4. This protein is called MM-1 and was identified by virtue of
its ability to interact
with the proto-oncogene c-myc (Mori et al., 1998). Other than this original
characterization, there
is not much else known about MM-1 but on the basis of the interaction found
here, MM-1 may play
critical roles in both cancer and diabetes.
Other than this original characterization, there was not much else known about
MM-1,
however because of its association with Glut4 and its tie to Diabetes, we have
used MM-1 in two
hybrid assays. Using the yeast two-hybrid assay, we have identified the large
centrosomal protein
C-Napl as an interactor of MM-1. C-Napl was originally identified as a protein
that could interact
with the Nek2 cell cycle-regulated protein kinase (Fry et al., 1998). The
finding that MM-1 can
interact with C-Napl serves to tie Glut4 and glucose transport in general to
the control of the cell
cycle. The second protein shown to interact with MM-1 is beta spectrin.
Spectrins give flexibility
to the cell and also act as a scaffold for other cellular proteins (Grum et
al., 1999). Interestingly, we
have linked beta spectrin to glucose transport and Diabetes in a previous
finding where it was shown
that beta spectrin could interact with the vesicle-associated protein IRAP.
The finding that MM-1
can bind to beta spectrin further strengthens the argument that beta spectrin
plays a role in glucose
transport. MM-1 was shown to bind to a third protein, KIAA0477, which has no
known function.
KIAA0477 was originally isolated from brain but its tissue distribution is not
known. The finding
that KIAA0477 interacts with MM-1 suggests that KIAA0477 plays a role in
glucose transport or
in some cellular function associated with vesicular transport.
Another approach to understanding the mechanism of glucose transport has been
to use the
Glutl molecule (Hresko et al., 1994), a gene highly related to Glut4 and
possessing similar
biological function, as a molecular bait in the search for other molecules
that can interact with it.
Using the yeast two-hybrid assay, we have identified three more proteins which
can bind to Glutl
DRAL, HSS and myosin heavy chain. DRAL is a LIM domain-containing protein that
is also
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23
known as FHL-2 or SLIM3. DRAL was identified as a protein that was expressed
in normal muscle
tissue culture cells but was down-regulated in cancerous rhabdomyosarcoma
cells (Genini et al.,
1997). It is possible that DRAL plays a critical role in the terminal
differentiation of muscle cells
such as cardiac muscle, and that its misregulation could result in an
undifferetiated cancerous
phenotype. Glutl was also shown to interact with HSS or human sperm surface
protein. HSS is a
testis-specific protein that has no known function (Shankar et al., 1998). It
does contain a putative
transmembrane domain and a leucine-zipper dimerization domain. Since it
interacts with Glutl and
it is presumably membrane-bound, HSS could potentially act with Glutl or Glut4
to affect glucose
transport in the testis. Glutl has also been demonstrated to interact with a
form of the myosin heavy
chain. Myosin heavy chain plays a key role in muscle structure and contraction
(Eddinger et al.,
1998). The interaction between Glutl and myosin suggests that glucose uptake
and muscle function
may be interrelated via the association of these two proteins.
Using the yeast two-hybrid assay, we identified dynamin as an interactor of
Glutl. Dynamin
has been implicated in the movement of glucose transporters via vesicular
trafficking and likely
plays a critical role in endocytosis (or movement from the cell surface to the
interior of the cell)
{Kao et al., 1998). Because of dynamin's link to Diabetes and glucose
transport, we used it in two-
hybrid assays and found two proteins that could interact with it. The first
protein is called CALM,
and it is a clathrin assembly protein similar to the AP-3 family of adaptor
proteins. CALM was
originally found in a lymphoid myeloid leukemia cell line containing a
chromosome translocation
resulting in the fusion of the AF 10 gene with CALM (Dreyling et al., 1996).
Clathrin and its
associated proteins have a long history of involvement in the transport of
vesicles from the cell
surface to the interior of the cell. The association of dynamin and CALM
further supports this role
and ties CALM to glucose transport. Dynamin also binds to a proteosome
activator subunit termed
Psme3. The human Psme3 gene maps to the region of the BRCAI gene, and its
function was
deduced by its similarity to the mouse gene that is also referred to as the Ki
antigen (Kohda et al.,
1998). Since the proteosome is required for the post-translational processing
and the specific
degradation of certain proteins, the fording that Psme3 can bind to dynamin
implies that this type
of protease activity may play a key role in glucose transport.
Using the yeast two-hybrid assay, we have identified two more proteins which
can bind to
Glutl, DRAL/FHL2 and cardiac muscle myosin heavy chain. The first protein,
DRAL/FHL2, is
a protein shown to be down-regulated in rhabdomyosarcoma (Genini et al.,
1997). It is entirely
composed of LIM domains, polypeptide motifs that form double zinc fingers and
may function by
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24
facilitating binding to nucleic acid or other proteins. The same region of
Glutl has been shown to
bind to a cardiac muscle myosin heavy chain (MYSA)(Metzger et al., 1999). The
significance of
this interaction is unknown with regard to glucose transport, however myosin
is known to function
in muscle contraction and cell structure.
Using the yeast two-hybrid assay, we have identified three proteins which can
bind to Glutl,
dynamin, and two proteins of unknown function, KIAA0144 and clone 25204.
Dynamin has been
implicated in the movement of glucose transporters via vesicular trafficking
and likely plays a
critical role in endocytosis (or movement from the cell surface to the
interior of the cell) (Kao et al.,
1998). Although the KIAA0144 gene is uncharacterized to date, the region of it
that contacts Glutl
is highly enriched for serine, threonine and proline residues, possibly
providing a clue to its
function. Other proteins with similar "STP" domains include the extracellular
portions of cell
surface receptors. Finally, a potential translation product of clone 25204
bears a striking
resemblance to a previously identified mouse gene called SEZ-6. This gene was
found by virtue
of its increased transcript levels in brain tissue following exposure to a
seizure producing drug
(Shimizu-Nishikawa et al., 1995).
The insulin-regulated membranse-spanning aminopeptidase or IRAP (also known as
vp165,
gp160 and oxytocinase) co-localizes with the Glut4 transporter in specified
endocytic vesicles
(Keller et al., 1995; Malide et al., 1997). Since expression of the N-terminal
fragment of IRAP has
been shown to result in the translocation of Glut4 to the plasma membrane,
IRAP is thought to play
a key role in glucose transport (Waters et al., 1997). Using the two-hybrid
system, we have detected
the interaction of IRAP with two proteins, 14-3-3 beta and HSS. The 14-3-3
family of proteins are
critical signal transduction proteins that bind to phosphoserine residues (Jin
et al., 1996). The
interaction of IRAP with 14-3-3 beta strongly suggests that the function of
IRAP could be regulated
by phosphorylation and by the subsequent binding by 14-3-3 family members.
Since IRAP and the
Glut4 glucose transporter co-localize in the same intracellular vesicles, it
is possible that Glut4 may
participate in signal transduction mechanisms mediated by the 14-3-3 proteins
such as 14-3-3 beta.
IRAP has also been shown to bind to the HSS protein described above. Like
Glutl and Glut4, IRAP
is membrane-bound and could potentially bind to HSS in the membrane. This
finding points to HSS
as playing a role in glucose transport or in other important functions
performed in intracellular
vesicles.
Using the two-hybrid system, we have detected the interaction of IRAP with two
more
proteins. The N-terminal portion of IR.AP has been shown to interact with SLAP-
2. The rabbit
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homolog of SLAP-2 has been demonstrated to localize to the sarcolemma or the
membrane of
muscle cells although its function has not been elucidated (Wigle et al.,
1997). SLAP-2 may play
a role in vesicular transport or may at least participate in it since it has
been shown to be membrane-
associated and localizes to both the cell membrane as well as to intracellular
stores in the
5 endoplasmic reticulum. The C-terminal extracellular portion of IRAP has been
demonstrated to
interact with SG2NA. SG2NA is a cell cycle nuclear autoantigen that contains
so-called WD-40
repeats that are present in a variety of signal transduction proteins (Muro et
al., 1995). Once again,
the significance of this interaction is unclear however it is possible that
SG2NA binding to IRAP
is part of a more complex regulatory mechanism.
10 Using the two-hybrid system, we have detected the interaction of IRAP with
one more
protein. The N-terminal portion of IRAP has been shown to interact VAMP-
associated protein A
(VAP-A or VAP-33). This protein has been implicated in intracellular transport
(specifically
exocytosis or movement to the cell surface) in A. californica and likely plays
a similar role in
humans (Skehel et al., 1995; Weir et al., 1998).
15 Using the two-hybrid system, we have detected the interaction of IRAP with
three proteins.
The N-terminal portion of IRAP has been shown to interact with another signal
transduction
protein, the zeta polypeptide of protein tyrosine phosphatase. This protein is
not well characterized
but could play a role in regulating glucose transport by dephosphorylating
critical proteins that cause
or prevent glucose transport (Nishiwaki et al., 1998). The C-terminal portion
of IR.AP has been
20 shown to interact with non-erythrocytic beta-spectrin. This protein is
thought to be involve in
secretion and could play a role in the movement of Glut 4 vesicles through
IRAP (Hu et al., 1992).
The C-terminus of IRAP has also been shown in the two-hybrid assay to interact
with the p85
regulatory subunit of phosphatidylinositol-3 (PI-3) kinase. This protein is a
central player in cellular
signal transduction and participates in transmitting signals from the outside
of the cell into the
25 interior. Interestingly, one of the functions of PI-3 kinase p85 involves
the insulin receptor (Martin
et al., 1996). Further, it is well known that the movement of Glut 4 between
the plasma membrane
and the interior of the cell depends on the action of the PI-3 kinase signal
transduction pathway
since inhibitors of this kinase prevent the cycling of GIut4. We also
discovered that PI-3 kinase p85
interacts with tankyrase. Two-hybrid interactions have been detected using the
p l 10 catalytic
subunit of PI-3 kinase, and these include interactions with the p85 and p55
subunits of the same
enzyme complex as well as non-subunit interactions.
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Protein phosphatase 5 (PPS) is a TPR domain containing protein that seems to
be part of
larger multiprotein complexes that possess several cellular functions
(Silverstein et al., 1997). Our
two-hybrid studies have confirmed the biochemical interaction between protein
phosphatase 5 and
Hsp90, and the association between these proteins has been previously
demonstrated using
biochemical methods. PPS has also been demonstrated using the two-hybrid assay
herein to interact
with another related heat shock protein, Hsp89, and also with tankyrase.
Phosphatidyl inositol-3 kinase is a very- important signal transduction
protein and likely
plays a critical role in Glut4-mediated glucose uptake (Shankar et al., 1998).
This protein
participates in transmitting signals from the outside of the cell into the
interior. It is composed of
two subunits, the p85 regulatory subunit and the p110 catalytic subunit, and
functions by facilitating
the transmission of signals from the outside of the cell into the interior. PI-
3 kinase has been
implicated in insulin-regulation and glucose uptake since one of its functions
involves the insulin
receptor (Martin et al., 1996). Further, the movement of Glut4 between the
plasma membrane and
the interior of the cell depends on the action of the PI-3 kinase signal
transduction pathway since
inhibitors of this kinase prevent the cycling of Glut4.
The p85 regulatory subunit of PI-3 kinase has been shown to interact with
several proteins
in the two-hybrid assay. Here we report the identification of five more
proteins that can interact
with p85. SLP-76 is a tyrosine phosphoprotein that participates in T cell
signaling (Clements et
al., 1998; Jackman et al., 1995). It is thought that SLP-76 acts as a so-
called adaptor protein since
it plays a role in intermediate steps of signal transduction. This is achieved
by bridging factors that
act at the plasma membrane with other molecules that perform functions within
the interior of the
cell. Our results indicate that SLP-76 may play a critical role in the PI-3
kinase signal transduction
pathway by virtue of its ability to bind the p85 regulatory subunit. The p85
subunit of PI-3 kinase
has also been demonstrated to bind to two more important signal transduction
proteins: 14-3-3 zeta
and 14-3-3 eta. These proteins bind specifically to phosphoserine residues in
a number of proteins
(Oghira et al., 1997; Thorson et al., 1998; Yaffe et al., 1997).
Interestingly, our studies have shown
that the Glut4 glucose transporter can also interact with 14-3-3 zeta. Thus,
PI-3 kinase can also be
connected to glucose uptake mechanisms by its interaction with the 14-3-3
signal transduction
proteins. PI-3 kinase p85 was shown to interact with chromogranin C, a
neuroendocrine secretory
granule protein in the granin family (Ozawa et al., 1995). Members of the
granin family localize
to specialized secretory vesicles and are thought to serve an important
function in protein sorting
and secretion (Leitner et al., 1999). Finally, TACC2 has been shown to
interact with PI-3 kinase
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p85 in the yeast two-hybrid assay. TACC2 is a member of a family of
"transforming coiled coil"
proteins that have been implicated in cellular growth control and cancer
(Still et al., 1999).
Although the function of TACC2 remains unknown, its interaction with p85
demonstrates that it
may also be a part of an important signal transduction pathway.
The pl 10 subunit of this protein has been shown to interact with complement
protein C4,
tenascin XB and alpha acid glucosidase (GAA). The complement C4 protein plays
a key role in
acitvating the classical complement pathway, and it is involved in evoking
histamine release from
basophils and mast cells. Naturally occurnng deficiencies of C4 have been
correlated with a number
of immune-related human diseases such as Systemic Lupus Erythmatosus, kidney
disease, hepatitis,
dementia and the propensity for recurrent infectiions (Mascart-Lemone et al.,
1983; Vergani et al.,
1985; Waters et al., 1997; Nerl et al., 1984; Lhotta et al., 1990). The
fording that C4 interacts with
the p110 subunit of PI-3 kinase suggests that the function of C4 is somehow
regulated by this signal
transduction, perhaps in response to some immunologic stimulus. The p110
catalytic subunit of PI-
3 kinase has been demonstrated to interact with tenascin XB. Tenascin XB is an
extracellular
structural protein. Deficiency of tenascin XB is linked to the connective
tissue disorder Ehler-Danlos
syndrome (Burch et al., 1997). This finding that p110 can bind to tenascin XB
suggest that the
function of tenascin XB could be modified or regulated by the PI-3 kinase
signal trasduction
pathway. PI-3 kinase p110 has also been shown to bind to GAA or alph acid
glucosidase. GAA
is a lysosomal enzyme that catalyzes the release of glucose from glycosylated
substrates, and defects
in GAA result in a glycogen storage disease (Raben et al., 1995). The fording
that PI-3 kinase can
bind to GAA suggests that GAA activity may be influenced by the PI-3 kinase
signaling
mechanism. Our previous two-hybrid results have linked the PI-3 kinase to
human diseases such
as Diabetes and Alzheimer's disease. These new findings therefore link
complement protein C4,
tenascin XB and alpha acid glucosidase to these diseases as well as to the
other diseases already
described.
The p110 subunit of this protein has been shown to interact with the thyroid
hormone
interacting protein TRIP 1 S in the two-hybrid assay. TRIP 15 is part of a
larger multiprotein complex
termed the signalsome that is likely to be involved in cell signaling (Seeger
et al., 1998). Our two
hybrid results have linked the PI-3 kinase to human diseases such as Diabetes
and Alzheimer's
disease.
We have detected the interaction of PI-3 kinase pl 10 subunit with the ~3-
amyloid precursor
protein (APP). In brief, there is now growing evidence that APP metabolism and
A(3 generation are
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28
central events to AD pathogenesis. For a more extensive review of APP and AD
pathogenesis, see
U.S. patent application Serial No. 09/466,139. filed 21 December 1999 and
international patent
application No. PCT/LJS99/30396, filed 21 December 1999, each incorporated
herein by reference.
Although several candidates (cathepsin G. cathepsin D, chymotrypsin, and
others) have been
suggested, the (3-secretase enzyme has not yet been identified. Even less is
known about oc- and y-
secretases. The biochemical link between the presenilins and APP processing
has not been
established. The proteins that mediate the neurotrophic and neuroprotective
effects of sAPP are
unknown. This last point is of utmost importance because an alteration of APP
metabolism could
result in both the generation of a toxic product (A/3) and the impairment of
sAPP trophic activity
(Saitoh et a1.1994; Roch et a1.1993; Saitoh and Roch, 1995). In this respect,
it is interesting that one
APP mutation associated with Alzheimer's results in a defective neurite
extension activity of sAPP
(Li et al., 1997). Moreover, the balance of phosphorylation cascades is deeply
altered in Alzheimer
brains (Saitoh and Roch, 1995; Jin and Saitoh, 1995; Mook-Jung and Saitoh,
1997; Saitoh et al.,
1991; Shapiro et al., 1991). Since APP is thought to play a major role in the
pathology of
Alzheimer's disease, the elucidation of a tie betwreen PI-3 kinase and APP
provides a new target for
discovering the treatment for this neurological disorder (Russo et al., 1998).
O-linked N-acetylglucosaminyltransferase or OGTase is an enzyme implicated in
intracellular signal transduction (Kreppel et al., 1997). It has been
speculated that OGTase may play
a key role in glucose uptake and may therefore participate in the Diabetes
related pathways
(Cooksey et al., 1999). OGTase has been shown to also interact with myosin
heavy chain. The
binding of myosin heavy chain to OGTase suggests that the function of myosin
in muscle structure
or function could be influenced by OGTase in its signal transductive capacity.
This could
potentially affect many cellular processes in the muscle cell, including
glucose transport. OGTase
has been shown to bind to a novel protein termed PN6931. PN6931 is very
similar to the mouse
kinesin light chain gene (GenBank accession AF055666). Kinesin is a molecular
motor involved
in cellular transport and chromosome movement (Kirchner et al., 1999). Perhaps
by post-
translationally modifying this novel kinesin-like protein, OGTase can
influence its function.
Amino acids 250 to 450 of OGTase has been shown to interact with a member of
the 14-3-3
protein family, 14-3-3 epsilon. The 14-3-3 proteins function in intracellular
signal transduction
pathways by specifically binding to phosphoserine residues on other critical
signaling molecules
(Ogihara et al., 1997; Yaffe et al., 1997). The interaction between OGTase and
14-3-3 epsilon may
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29
serve to link two different signal transduction pathways, one which involves
phosphorylation and
a second which involves another type of protein modification.
Amino acids 250 to 450 of OGTase has been shown to interact with two proteins,
alpha-2
catenin and Nafl a. Alpha-catenin is a protein related to vinculins that
functions in cell-cell contact
by binding to cadherins (Rudiger et al., 1998). The same region of OGTase can
interact with Naflb,
a protein identified by virtue of its ability to bind the Nef gene product of
HIV in the two-hybrid
assay (Fukushi et al., 1999). Over-expression of Nafla was observed to cause
an increase in cell
surface expression of the CD4 antigen, therefore this protein may also
function in intracellular
trafficking and could potentially participate in a number of diseases related
to this general process
such as Diabetes and Alzheimer's.
Using the yeast two-hybrid assay, we identified Nafl b as an interactor of
OGTase. Nafl b
is a protein which was identified by virtue of its ability to bind the Nef
gene product of HIV in the
two-hybrid assay (Fukushi et al., 1999). Over-expression of Naflb was observed
to cause an
increase in cell surface expression of the CD4 antigen, therefore this protein
may also function in
intracellular trafficking and could potentially participate in a number of
diseases related to this
general process such as Diabetes and Alzheimer's. In a two-hybrid search using
Naflb as a bait, the
TRAF-interacting protein I-TRAF was found to be an interactor. I-TRAF appears
to act as a
regulator of the TRAF signal transduction pathway that transmits signals from
TNF (tumor necrosis
factor) receptor family members (Rothe et al., 1996). The observation that
Naflb interacts with I-
TRAF serves to link an extracellular stimulated signal transduction mechanism
with the OGTase
pathway involved in glucose transport. A two-hybrid search using Naflb as a
bait has also
identified a novel protein fragment called PN7582. This small protein fragment
does not appear to
have any strong similarity to known proteins therefore its function is not
readily apparent. It is very
possible that it may participate in protein trafficking or signal transduction
by its association with
Naflb.
In a two-hybrid search using OGTase as a bait, four new interactors were
found. The first
interactor, desmin, is a cytoplasmic intermediate filament protein found in
muscle (Capetanaki et
al., 1998). It functions in striated muscle by connecting myofibrils with
themselves and with the
plasma membrane. Desmin is broken down into three functional domains, the
head, rod and tail,
and OGTase can bind to the rod structure. The second protein shown to interact
with OGTase is
called alpha-karyopherin. Alpha-karyopherin (also known as importin and SRP1)
is a ubiquitously
expressed protein that plays a role in trafficking nuclear localization signal-
containing proteins into
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the nucleus through the nuclear pore (Moroianu, 1997). The third protein that
OGTase has been
shown to bind is glutanimyl-tRNA synthetase. The aminoacyl-tRNA synthetases
not only play a
key role in protein synthesis, but recent studies have shown that they also
impact a number of other
cellular processes such as tRNA processing. RNA splicing, RNA trafficking,
apoptosis, and
5 transcriptional and translational regulation (Martinis et al., 1999). The
fourth protein shown to bind
to OGTase in the yeast two-hybrid assay is called clone 25100 and it has no
known function. The
gene for clone 25100 was isolated from human infant brain and appears to
encode a small protein
with no structural characteristics that shed light on its function. All four
of these OGTase-
interacting proteins may act as substrates for OGTase or may affect its
function in some way. The
10 finding that they interact with OGTase suggests that they may play a role
in glucose transport or in
the pathogenesis of Diabetes.
Our yeast two-hybrid studies have demonstrated that OGTase can interact with a
variety of
proteins that can fall into a number of functional categories. Two proteins
that have been implicated
in vesicular transport bind to OGTase. The first is called BAP31, and it
likely plays a critical role
15 in sorting and transporting membrane proteins between intracellular
compartments (Annaert et al.,
1997). The second protein is called dynamin Ih and it is implicated in the
movement of transport
vesicles from the plasma membrane to sites within the interior of the cell
(Sontag et al., 1994).
OGTase has been demonstrated to interact with two proteins that function in
transcription, as well.
The first is called EGRI for early growth response protein 1, and it has been
shown to be highly
20 induced in cells following mitogenic stimulation (Sukhatme et al., 1988).
It is a zinc-finger
containing protein, and following its own stimulation, goes on to activate the
transcription of genes
involved in mitogenesis and differentiation. Additionally, OGTase can interact
with MOP2, a basic
helix-loop-helix containing transcription factor that is involved in the
induction of oxygen regulated
genes (Hogenesch et al., 1997). OGTase binds to a structural protein called
talin. Talin is a
25 cytoplasmic protein that serves to link integrins with the actin
cytoskeleton (Calderwood etal.,
1999). Finally, OGTase has been shown in the yeast two-hybrid assay to bind to
five proteins of
unknown or poorly characterized function. OGTase binds to Int-6, a protein
that has also been
demonstrated to bind to the HTLV-I Tax transactivator and it is a component of
promyelocytic
leukemia nuclear bodies (Desbois et al., 1996). OGTase binds to interferon-
induced protein 54, an
30 uncharacterized protein that shows a large increase in its transcript
following treatment with alpha-
and beta-interferons (Levy et al., 1986). Lastly, HSPC028, KIAA0443, and clone
25100 interact
with OGTase.
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31
Akt kinase is a serine/threonine protein kinase that has been implicated in
insulin-regulated
glucose transport and the development of non-insulin dependent diabetes
mellitus (Krook et al.,
1998). Because of this link, Akt kinase has been used in two-hybrid assays to
determine what
proteins interact with it either because they are substrates of Akt kinase or
because they are
regulators of the kinase. Two closely related Akt proteins were used: Aktl and
its closely related
family-member Akt2. Aktl and Akt2 were both shown to interact with the nuclear
mitotic apparatus
protein NuMAl. NuMAI displays a distinct pattern of immunofluorescent staining
that varies
throughout the cell cycle. It is nuclear throughout interphase but re-
localizes to the spindle
apparatus during mitosis (Lydersen et al., 1980). Phosphorylation is thought
to play a critical role
in NuMA function. It appears to become phosphorylated just prior to mitosis
and becomes
dephosphorylated after mitosis has occurred in the G1 phase of the cell cycle
mitosis (Sparks et al.,
1995). Aktl and Akt2 may be capable of phosphorylating NuMA, especially since
the region of
NuMA that interacts with Akt2 (amino acids 98 to 365) contains 23 serines and
9 threonines. Akt2
was also shown to interact with two proteins involved in vesicular transport.
The first protein,
BAP31, likely plays a role in the movement of membrane-bound proteins from the
Golgi appartus
to the plasma membrane (Annaert et al., 1997). Since BAP31 was also shown to
interact with
OGTase in our previous experiments, two signal transduction proteins
implicated in glucose
transport have been tied to BAP31. Akt2 was also shown to bind to another
vesicular transport
protein termed beta-adaptin or AP2-beta. Beta-adaptin is a part of the AP2
coat assembly complex
that links clathrin and to transmembrane receptors resident in coated vesicles
(Pearse, 1989). This
finding suggests a role for Akt2 in the regulation of endocytosis, while the
finding that Akt2 binds
to BAP31 provides for a tie between Akt2 and exocytosis. All of the proteins
that can interact with
the Akt kinase may play a role in glucose transport by virtue of their
association with Akt.
PTPIb is a protein tyrosine phosphatase that plays a critical role in signal
transduction. It
has been implicated as a negative regulator of insulin-responsive glucose
transport (Chen et al.,
1997), and therefore it has been used in yeast two-hybrid assays in an attempt
to find more proteins
involved in this function, perhaps by acting as substrates. PTP 1 b was shown
to interact with
VAMP-associated protein A (VAP-A or VAP-33). This protein has been implicated
in intracellular
transport (specifically exocytosis or movement to the cell surface) in A.
californica and likely plays
a similar role in humans (Skehel et al., 1995; weir et al., 1998). The
interaction between PTPlb
and VAP-A serves as a potential tie between PTP 1 b and IR.AP since IRAP was
also shown to bind
to VAP-A in two-hybrid experiments: IRAP, insulin-regulated membrane-spanning
aminopeptidase
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32
(also known as vp165, gp160 and oxytocinase) co-localizes with the Glut4
transporter in specified
endocytic vesicles (Keller et al., 1995; Malide et al., 1997). Since
expression of the N-terminal
fragment of IRAP has been shown to result in the translocation of Glut4 to the
plasma membrane,
IRAP is thought to play a key role in glucose transport (Waters et al., 1997).
Thus, the result that
PTP 1 b interacts with VAP-A serves to strengthen the tie between PTP 1 b and
VAP-A to glucose
transport and Diabetes.
The small GTP-binding protein Rab4 is another signal transduction factor that
has been
implicated in the regulation of insulin-stimulated glucose uptake
(Vollenweider et al., 1997;
Cormont et al., 1996). Rab4, therefore, has also been used in the yeast two-
hybrid assay to find
additional proteins involved in glucose transport and in Diabetes. Two
proteins have been shown
to bind to Rab4, an alpha-catenin-like protein (sometimes called alpha-
catulin) and another small
GTP-binding protein called Rab2. The alpha-catenin-like protein resembles
alpha-catenin and
vinculin however its function has not yet been well-characterized (Janssens et
al., 1999). Alpha-
catenin itself is a protein related to vinculins that functions in cell-cell
contact by binding to
cadherins (Rudinger et al., 1998). Interestingly, alpha-catenin was found in
our previous studies to
bind to OGTase and, as a consequence, has been implicated in glucose
transport. The second
protein shown to bind to Rab4 is Rab2. These two small GTP-binding proteins
are highly related
with a 50% amino acid identity. Unlike Rab4, Rab2 has not been thought to play
a role in insulin-
stimulated glucose transport (Uphues et al., 1994) although it does play an
important role in
vesicular transport in the cell (Tisdale et al., 1998). The finding that Rab4
and Rab2 interact
suggests that they may be capable of influencing each others cellular
functions, thus Rab2 could
potentially affect Rab4's role in glucose transport.
The proteins disclosed in the present invention were found to interact with
their
corresponding proteins in the yeast two-hybrid system. Because of the
involvement of the
corresponding proteins in the physiological pathways disclosed herein, the
proteins disclosed herein
also participate in the same physiological pathways. Therefore, the present
invention provides a list
of uses of these proteins and DNA encoding these proteins for the development
of diagnostic and
therapeutic tools useful in the physiological pathways. This list includes,
but is not limited to, the
following examples.
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JJ
Two-Hybrid System
The principles and methods of the yeast two-hybrid system have been described
in detail
elsewhere (e.g., Bartel and Fields, 1997; Bartel et al., 1993; Fields and
Song, 1989; Chevray and
Nathans, 1992). The following is a description of the use of this system to
identify proteins that
interact with a protein of interest.
The target protein is expressed in yeast as a fusion to the DNA-binding domain
of the yeast
Gal4p. DNA encoding the target protein or a fragment of this protein is
amplified from cDNA by
PCR or prepared from an available clone. The resulting DNA fragment is cloned
by ligation or
recombination into a DNA-binding domain vector (e.g., pGBT9, pGBT.C, pAS2-1)
such that an in-
frame fusion between the Gal4p and target protein sequences is created.
The target gene construct is introduced. by transformation, into a haploid
yeast strain. A
library of activation domain fusions (i.e., adult brain cDNA cloned into an
activation domain vector)
is introduced by transformation into a haploid yeast strain of the opposite
mating type. The yeast
strain that carries the activation domain constructs contains one or more
Gal4p-responsive reporter
gene(s), whose expression can be monitored. Examples of some yeast reporter
strains include Y190,
PJ69, and CBYl4a. An aliquot of yeast carrying the target gene construct is
combined with an
aliquot of yeast carrying the activation domain library. The two yeast strains
mate to form diploid
yeast and are plated on media that selects for expression of one or more Gal4p-
responsive reporter
genes. Colonies that arise after incubation are selected for further
characterization.
The activation domain plasmid is isolated from each colony obtained in the two-
hybrid
search. The sequence of the insert in this construct is obtained by the
dideoxy nucleotide chain
termination method. Sequence information is used to identify the gene/protein
encoded by the
activation domain insert via analysis of the public nucleotide and protein
databases. Interaction of
the activation domain fusion with the target protein is confirmed by testing
for the specificity of the
interaction. The activation domain construct is co-transformed into a yeast
reporter strain with either
the original target protein construct or a variety of other DNA-binding domain
constructs.
Expression of the reporter genes in the presence of the target protein but not
with other test proteins
indicates that the interaction is genuine.
In addition to the yeast two-hybrid system, other genetic methodologies are
available for the
discovery or detection of protein-protein interactions. For example, a
mammalian two-hybrid system
is available commercially (Clontech, Inc.) that operates on the same principle
as the yeast two
hybrid system. Instead of transforming a yeast reporter strain, plasmids
encoding DNA-binding and
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34
activation domain fusions are transfected along with an appropriate reporter
gene (e.g., lacZ) into
a mammalian tissue culture cell line. Because transcription factors such as
the Saccharomyces
cerevisiae Gal4p are functional in a variety of different eukaryotic cell
types, it would be expected
that a two-hybrid assay could be performed in virtually any cell line of
eukaryotic origin (e.g., insect
cells (SF9), fungal cells, worm cells, etc.). Other genetic systems for the
detection of protein-protein
interactions include the so-called SOS recruitment system (Aronheim et al.,
1997).
Protein-protein interactions
Protein interactions are detected in various systems including the yeast two-
hybrid system,
affinity chromatography, co-immunoprecipitation, subcellular fractionation and
isolation of large
molecular complexes. Each of these method is well characterized and can be
readily performed by
one skilled in the art. See, e.g., U.S. Patents No. 5,622,852 and 5,773,218,
PCT published
application No. WO 97/27296 and PCT published application No. WO 99/65939,
each of which are
incorporated herein by reference.
The protein of interest (or an interacting domain thereof) can be produced in
eukaryotic or
prokaryotic systems. A cDNA encoding the desired protein is introduced in an
appropriate
expression vector and transfected in a host cell (which could be bacteria,
yeast cells, insect cells, or
mammalian cells). Purification of the expressed protein is achieved by
conventional biochemical
and immunochemical methods well known to those skilled in the art. The
purified protein is then
used for affinity chromatography studies: it is immobilized on a matrix and
loaded on a column.
Extracts from cultured cells or homogenized tissue samples are then loaded on
the column in
appropriate buffer, and non-binding proteins are eluted. After extensive
washing, binding proteins
or protein complexes are eluted using various methods such as a gradient of pH
or a gradient of salt
concentration. Eluted proteins can then be separated by two-dimensional gel
electrophoresis, eluted
from the gel, and identified by micro-sequencing. The purified proteins can
also be used for affinity
chromatography to purify interacting proteins disclosed herein. All of these
methods are well
known to those skilled in the art.
Similarly, both proteins of the complex of interest (or interacting domains
thereof) can be
produced in eukaryotic or prokaryotic systems. The proteins (or interacting
domains) can be under
control of separate promoters or can be produced as a fusion protein. The
fusion protein may
include a peptide linker between the proteins (or interacting domains) which,
in one embodiment,
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WO 00/65340 PCT/US00/10651
serves to promote the interaction of the proteins (or interacting domains).
All of these methods are
also well known to those skilled in the art.
Purified proteins of interest, individually or a complex, can also be used to
generate
antibodies in rabbit, mouse, rat, chicken, goat, sheep, pig, guinea pig,
bovine, and horse. The
5 methods used for antibody generation and characterization are well known to
those skilled in the
art. Monoclonal antibodies are also generated by conventional techniques.
Single chain antibodies
are further produced by conventional techniques.°
DNA molecules encoding proteins of interest can be inserted in the appropriate
expression
vector and used for transfection of eukaryotic cells such as bacteria, yeast,
insect cells, or
10 mammalian cells, following methods well known to those skilled in the art.
Transfected cells
expressing both proteins of interest are then lysed in appropriate conditions,
one of the two proteins
is immunoprecipitated using a specific antibody, and analyzed by
polyacrylamide gel
electrophoresis. The presence of the binding protein (co-immunoprecipitated)
is detected by
immunoblotting using an antibody directed against the other protein. Co-
immunoprecipitation is
15 a method well known to those skilled in the art.
Transfected eukaryotic cells or biological tissue samples can be homogenized
and
fractionated in appropriate conditions that will separate the different
cellular components. Typically,
cell lysates are run on sucrose gradients, or other materials that will
separate cellular components
based on size and density. Subcellular fractions are analyzed for the presence
of proteins of interest
20 with appropriate antibodies, using immunoblotting or immunoprecipitation
methods. These methods
are all well known to those skilled in the art.
Disruption of protein-protein interactions
It is conceivable that agents that disrupt protein-protein interactions can be
beneficial in
25 many physiological disorders, including, but not-limited to NIDDM, AD and
others disclosed
herein. Each of the methods described above for the detection of a positive
protein-protein
interaction can also be used to identify drugs that will disrupt said
interaction. As an example, cells
transfected with DNAs coding for proteins of interest can be treated with
various drugs, and co
immunoprecipitations can be performed. Alternatively, a derivative of the
yeast two-hybrid system,
30 called the reverse yeast two-hybrid system (Leanna and Hannink, 1996), can
be used, provided that
the two proteins interact in the straight yeast two-hybrid system.
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36
Modulation of protein-protein interactions
Since the interaction described herein is involved in a physiological pathway,
the
identification of agents which are capable of modulating the interaction will
provide agents which
can be used to track the physiological disorder or to use as lead compounds
for development of
therapeutic agents. An agent may modulate expression of the genes of
interacting proteins, thus
affecting interaction of the proteins. Alternatively, the agent may modulate
the interaction of the
proteins. The agent may modulate the interaction of wild-type with wild-type
proteins, wild-type
with mutant proteins, or mutant with mutant proteins. Agents can be tested
using transfected host
cells, cell lines, cell models or animals, such as described herein, by
techniques well known to those
of ordinary skill in the art, such as disclosed in U.S. Patents No. 5,622,852
and 5,773,218, PCT
published application No. WO 97/27296 and PCT published application No. WO
99/65939, each
of which are incorporated herein by reference. The modulating effect of the
agent can be screened
in vivo or in vitro. Exemplary of a method to screen agents is to measure the
effect that the agent
has on the formation of the protein complex.
Mutation screening
The proteins disclosed in the present invention interact with one or more
proteins known to
be involved in a physiological pathway, such as in NIDDM or AD. Mutations in
interacting
proteins could also be involved in the development of the physiological
disorder, such as NIDDM
or AD, for example, through a modification of protein-protein interaction, or
a modification of
enzymatic activity, modification of receptor activity, or through an unknown
mechanism.
Therefore, mutations can be found by sequencing the genes for the proteins of
interest in patients
having the physiological disorder, such as insulin, and non-affected controls.
A mutation in these
genes, especially in that portion of the gene involved in protein interactions
in the physiological
pathway, can be used as a diagnostic tool, and the mechanistic understanding
the mutation provides
can help develop a therapeutic tool.
Screening for at-risk individuals
Individuals can be screened to identify those at risk by screening for
mutations in the protein
disclosed herein and identified as described above. Alternatively, individuals
can be screened by
analyzing the ability of the proteins of said individual disclosed herein to
form natural complexes.
Further, individuals can be screened by analyzing the levels of the complexes
or individual proteins
CA 02371006 2001-10-22
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37
of the complexes or the mRNA encoding the protein members of the complexes.
Techniques to
detect the formation of complexes, including those described above, are known
to those skilled in
the art. Techniques and methods to detect mutations are well known to those
skilled in the art.
Techniques to detect the level of the complexes. proteins or mRNA are well
known to those skilled
in the art.
Cellular models of Physiological Disorders
A number of cellular models of many physiological disorders or diseases have
been
generated. The presence and the use of these models are familiar to those
skilled in the art. As an
example, primary cell cultures or established cell lines can be transfected
with expression vectors
encoding the proteins of interest, either wild-t<~pe proteins or mutant
proteins. The effect of the
proteins disclosed herein on parameters relevant to their particular
physiological disorder or disease
can be readily measured. Furthermore, these cellular systems can be used to
screen drugs that will
influence those parameters, and thus be potential therapeutic tools for the
particular physiological
disorder or disease. Alternatively, instead of transfecting the DNA encoding
the protein of interest,
the purified protein of interest can be added to the culture medium of the
cells under examination,
and the relevant parameters measured.
Animal models
The DNA encoding the protein of interest can be used to create animals that
overexpress
said protein, with wild-type or mutant sequences (such animals are referred to
as "transgenic"), or
animals which do not express the native gene but express the gene of a second
animal (referred to
as "transplacement"), or animals that do not express said protein (referred to
as "knock-out"). The
knock-out animal may be an animal in which the gene is knocked out at a
determined time. The
generation of transgenic, transplacement and knock-out animals (normal and
conditioned) uses
methods well known to those skilled in the art.
In these animals, parameters relevant to the particular physiological disorder
can be
measured. These parametes may include receptor function, protein secretion in
vivo or in vitro,
survival rate of cultured cells, concentration of particular protein in tissue
homogenates, signal
transduction, behavioral analysis, protein synthesis, cell cycle regulation,
transport of compounds
across cell or nuclear membranes, enzyme activity, oxidative stress,
production of pathological
products, and the like. The measurements of biochemical and pathological
parameters, and of
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38
behavioral parameters, where appropriate, are performed using methods well
known to those skilled
in the art. These transgenic, transplacement and knock-out animals can also be
used to screen drugs
that may influence the biochemical, pathological, and behavioral parameters
relevant to the
particular physiological disorder being studied. Cell lines can also be
derived from these animals
for use as cellular models of the physiological disorder, or in drug
screening.
Rational drug design
The goal of rational drug design is to produce structural analogs of
biologically active
polypeptides of interest or of small molecules with which they interact (e.g.,
agonists, antagonists,
inhibitors) in order to fashion drugs which are, for example, more active or
stable forms of the
polypeptide, or which, e.g., enhance or interfere v~~ith the function of a
polypeptide in vivo. Several
approaches for use in rational drug design include analysis of three-
dimensional structure, alanine
scans, molecular modeling and use of anti-id antibodies. These techniques are
well known to those
skilled in the art.
1 S Following identification of a substance which modulates or affects
polypeptide activity, the
substance may be further investigated. Furthermore, it may be manufactured
and/or used in preparation,
i.e., manufacture or formulation, or a composition such as a medicament,
pharmaceutical composition
or drug. These may be administered to individuals.
A substance identified as a modulator of polypeptide function may be peptide
or non-peptide
in nature. Non-peptide "small molecules" are often preferred for many in vivo
pharmaceutical uses.
Accordingly, a mimetic or mimic of the substance (particularly if a peptide)
may be designed for
pharmaceutical use.
The designing of mimetics to a known pharmaceutically active compound is a
known
approach to the development of pharmaceuticals based on a "lead" compound.
This approach might
be desirable where the active compound is difficult or expensive to synthesize
or where it is
unsuitable for a particular method of administration, e.g., pure peptides are
unsuitable active agents
for oral compositions as they tend to be quickly degraded by proteases in the
alimentary canal.
Mimetic design, synthesis and testing is generally used to avoid randomly
screening large numbers
of molecules for a target property.
Once the pharmacophore has been found, its structure is modeled according to
its physical
properties, e.g., stereochemistry, bonding, size and/or charge, using data
from a range of sources,
e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational
analysis, similarity
CA 02371006 2001-10-22
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39
mapping (which models the charge and/or volume of a pharmacophore, rather than
the bonding
between atoms) and other techniques can be used in this modeling process.
A template molecule is then selected, onto which chemical groups that mimic
the
pharmacophore can be grafted. The template molecule and the chemical groups
grafted thereon can
be conveniently selected so that the mimetic is easy to synthesize, is likely
to be pharmacologically
acceptable, and does not degrade in vivo, while retaining the biological
activity of the lead
compound. Alternatively, where the mimetic is peptide-based, further stability
can be achieved by
cyclizing the peptide, increasing its rigidity. The mimetic or mimetics found
by this approach can
then be screened to see whether they have the target property, or to what
extent it is exhibited.
Further optimization or modification can then be carried out to arrive at one
or more final mimetics
for in vivo or clinical testing.
Diagnostic Assays
The identification of the interactions disclosed herein enables the
development of diagnostic
assays and kits, which can be used to determine a predisposition to or the
existence of a
physiological disorder. In one aspect, one of the proteins of the interaction
is used to detect the
presence of a "normal" second protein (i.e., normal with respect to its
ability to interact with the first
protein) in a cell extract or a biological fluid, and further, if desired, to
detect the quantitative level
of the second protein in the extract or biological fluid. The absence of the
"normal" second protein
would be indicative of a predisposition or existence of the physiological
disorder. In a second
aspect, an antibody against the protein complex is used to detect the presence
and/or quantitative
level of the protein complex. The absence of the protein complex would be
indicative of a
predisposition or existence of the physiological disorder.
EXAMPLES
The present invention is further detailed in the following Examples, which are
offered by
way of illustration and are not intended to limit the invention in any manner.
Standard techniques
well known in the art or the techniques specifically described below are
utilized.
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EXAMPLE 1
Yeast Two-Hvbrid System
The principles and methods of the yeast t<vo-hybrid systems have been
described in detail
(Bartel and Fields, 1997). The following is thus a description of the
particular procedure that we
5 used, which was applied to all proteins.
The cDNA encoding the bait protein was generated by PCR from brain cDNA. Gene-
specific primers were synthesized with appropriate tails added at their 5'
ends to allow
recombination into the vector pGBTQ. The tail for the forward primer was 5'-
GCAGGAAACAGCTATGACCATACAGTCAGCGGCCGCCACC-3' (SEQ ID NO:1 ) and the tail for
the reverse
10 primer was 5'-ACGGCCAGTCGCGTGGAGTGTTATGTCATGCGGCCGCTA-3' (SEQ ID N0:2). The
tailed
PCR product was then introduced by recombination into the yeast expression
vector pGBTQ, which
is a close derivative of pGBTC (Bartel et al., 1996) in which the polylinker
site has been modified
to include M 13 sequencing sites. The new construct was selected directly in
the yeast J693 for its
ability to drive tryptophane synthesis (genotype of this strain: Mat a, ade2,
his3, leu2, trill,
15 URA3::GAL1-lacZ LYS2::GAL1-HIS3 ga14de1 ga180de1 cyhR2). In these yeast
cells, the bait is
produced as a C-terminal fusion protein with the DNA binding domain of the
transcription factor
Gal4 (amino acids 1 to 147). A total human brain (37 year-old male Caucasian)
cDNA library
cloned into the yeast expression vector pACT2 was purchased from Clontech
(human brain
MATCHMAKER cDNA, cat. # HL4004AH), transformed into the yeast strain J692
(genotype of
20 this strain: Mat a, ade2, his3, leu2, trill, URA3::GAL1-lacZ LYS2::GAL1-
HIS3 gal4del ga180de1
cyhR2), and selected for the ability to drive leucine synthesis. In these
yeast cells, each cDNA is
expressed as a fusion protein with the transcription activation domain of the
transcription factor
Gal4 (amino acids 768 to 881) and a 9 amino acid hemagglutinin epitope tag.
J693 cells (Mat a
type) expressing the bait were then mated with J692 cells (Mat a type)
expressing proteins from the
25 brain library. The resulting diploid yeast cells expressing proteins
interacting with the bait protein
were selected for the ability to synthesize tryptophane, leucine, histidine,
and (3-galactosidase. DNA
was prepared from each clone, transformed by electroporation into E. coli
strain KC8 (Clontech
KC8 electrocompetent cells, cat # C2023-1), and the cells were selected on
ampicillin-containing
plates in the absence of either tryptophane (selection for the bait plasmid)
or leucine (selection for
30 the brain library plasmid). DNA for both plasmids was prepared and
sequenced by di-
deoxynucleotide chain termination method. The identity of the bait cDNA insert
was confirmed and
the cDNA insert from the brain library plasmid was identified using BLAST
program against public
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nucleotides and protein databases. Plasmids from the brain library (preys)
were then individually
transformed into yeast cells together with a plasmid driving the synthesis of
lamin fused to the Gal4
DNA binding domain. Clones that gave a positive signal after (3-galactosidase
assay were considered
false-positives and discarded. Plasmids for the remaining clones were
transformed into yeast cells
together with plasmid for the original bait. Clones that gave a positive
signal after (3-galactosidase
assay were considered true positives.
EXAMPLE 2
Identification of Glut4/CARP Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(Swiss Protein (SP) accession no. P 14672) as bait was performed. One clone
that was identified by
this procedure included the amino acids encoded by nucleotides 312-1155 of
CARP (GenBank (GB)
accession no. X83703).
EXAMPLE 3
Identification of GLUTl/DRAL~FHL2) Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 448-492
of Glutl
(SP accession no. P11166) as bait was performed. One clone that was identified
by this procedure
included amino acids 1-279 of DRAL(FHL2) (SP accession no. Q13229).
EXAMPLE 4
Identification of GLUT1/Myosin Heavy Chain Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 448-492
of Glutl
(SP accession no. P11166) as bait was performed. One clone that was identified
by this procedure
included amino acids 1589-1909 of myosin heavy chain (SP accession no.
P13533).
EXAMPLE 5
Identification of GLUTl/HSS Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 448-492
of Glutl
(SP accession no. P11166) as bait was performed. One clone that was identified
by this procedure
included amino acids encoded by nucleotides 1-? of HSS (GB accession no.
X91879).
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EXAMPLE 6
Identification of OGTase/Mvosin Heavy Chain Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was
performed. One clone
that was identified by this procedure included amino acids encoded by
nucleotides 1773-2283 of
myosin heavy chain (GB accession no. AF 111785).
EXAMPLE 7
Identification of IRAP/14-3-3 beta Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 449-880 of IRAP (GB accession no. U62768) as bait was performed.
One clone that
was identified by this procedure included amino acids 97-236 of 14-3-3 beta
(SP accession no.
P31946).
EXAMPLE 8
Identification of IRAP/HSS Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 62-880 of IRAP (GB accession no. U62768) as bait was performed.
One clone that was
identified by this procedure included amino acids encoded by nucleotides 726-
1446 of HSS (GB
accession no. X91879).
EXAMPLE 9
Identification of PI-3K110/Complement Protein C4 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of
PI-3
kinase p110 subunit (SP accession no. P42338) as bait was performed. One clone
that was
identified by this procedure included amino acids 1056-1277 of complement]
protein C4 (SP
accession no. P01028).
EXAMPLE 10
Identification of PI-3K110/Tenascin XB Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of
PI-3
kinase p110 subunit (SP accession no. P42338) as bait was performed. One clone
that was
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identified by this procedure included amino acids 3573-3787 of tenascin XB (SP
accession no.
P78530).
EXAMPLE 11
Identification of PI-3K110/GAA Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of
PI-3
kinase p110 subunit (SP accession no. P42338) as bait was performed. One clone
that was
identified by this procedure included amino acids 513-? of GAA (SP accession
no. P10253).
EXAMPLE 12
Identification of MM-1/C-Napl Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 27-175
of MM-1
(SP accession no. Q99471) as bait was performed. One clone that was identified
by this procedure
included amino acids encoded by nucleotides 1508-1949 of C-Napl (GB accession
no. AF049105).
EXAMPLE 13
Identification of MM-lBeta Spectrin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-175 of
MM-1 (SP
accession no. Q99471 ) as bait was performed. One clone that was identified by
this procedure
included amino acids 1545-1789 of beta spectrin (SP accession no. Q01082).
EXAMPLE 14
Identification of MM-1/KIAA0477 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 27-175
of MM-1
(SP accession no. Q99471) as bait was performed. One clone that was identified
by this procedure
included amino acids encoded by nucleotides 2448-3207 of KIAA0477 (GB
accession no.
AB007946).
EXAMPLE 15
Identification of Dynamin/CALM Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 250-700
of
dynamin (SP accession no. Q05193) as bait was performed. One clone that was
identified by this
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procedure included amino acids encoded by nucleotides 948-1599 of CALM (GB
accession no.
U45976).
EXAMPLE 16
Identification of Dvnamin/Psme3 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 250-700
of
dynamin (SP accession no. Q05193) as bait was performed. One clone that was
identified by this
procedure included amino acids encoded by nucleotides 378-966 of Psme3 (GB
accession no.
U11292).
EXAMPLE 17
Identification ofNaflb/I-TRAF Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 590-1610 ofNaflb (GB accession no. Q05193) as bait was performed.
One clone that
was identified by this procedure included amino acids encoded by nucleotides
209-1420 of I-TRAF
(GB accession no. U59863).
EXAMPLE 18
Identification of Aktl/NuMAl Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-118 of
Aktl (SP
accession no. P31749) as bait was performed. One clone that was identified by
this procedure
included amino acids encoded by nucleotides 813-1341 of NuMAI (GB accession
no. Z11584).
EXAMPLE 19
Identification of Akt2/NuMA 1 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-152 of
Akt2 (SP
accession no. P31751) as bait was performed. One clone that was identified by
this procedure
included amino acids encoded by nucleotides 552-1353 of NuMAI (GB accession
no. Z11584).
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EXAMPLE 20
Identification of Akt2/BAP31 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 915-1311 of Akt2 (GB accession no. M95936) as bait was performed.
One clone that
was identified by this procedure included amino acids encoded by nucleotides
469-877 of BAP31
(GB accession no. NM00574).
EXAMPLE 21
Identification of Akt2Beta Adaptin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 915-1311 of Akt2 (GB accession no. M95936) as bait was performed.
One clone that
was identified by this procedure included amino acids 214-594 of beta adaptin
(SP accession no.
P21851).
EXAMPLE 22
Identification of OGTase/Desmin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was
performed. One clone
that was identified by this procedure included amino acids 219-449 of desmin
(SP accession no.
P17661).
EXAMPLE 23
Identification of OGTase/Alpha-karyopherin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was
performed. One clone
that was identified by this procedure included amino acids 152-444 of alpha-
karyopherin (SP
accession no. P52294).
EXAMPLE 24
Identification of OGTase/Glutaminyl tRNA Synthetase Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was
performed. One clone
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that was identified by this procedure included amino acids 36-161 of
glutaminyl tRNA synthetase
(SP accession no. P47897).
EXAMPLE 25
Identification of OGTase/Clone 25100 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was
performed. One clone
that was identified by this procedure included amino acids 11-338 of clone
25100 (GB accession
no. AF 131780).
EXAMPLE 26
Identification of PTP 1 b/VAP-A Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 280-
436of PTPlb
(SP accession no. P 18031 ) as bait was performed. One clone that was
identified by this procedure
included amino acids encoded by nucleotides 9-? of VAP-A (GB accession no.
AF086627).
EXAMPLE 27
Identification of Rab4/Alpha-catenin-like Protein Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-214 of
Rab4 (SP
accession no. P20338) as bait was performed. One clone that was identified by
this procedure
included amino acids encoded by nucleotides 1573-1987 of alpha-catenin-like
protein (GB
accession no. U97067).
EXAMPLE 28
Identification of Rab4/Rab2 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-214 of
Rab4 (SP
accession no. P20338) as bait was performed. One clone that was identified by
this procedure
included amino acids 27-177 of Rab2 (SP accession no. P08886).
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EXAMPLE 29
Identification of Glut4/PN7065 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(Swiss Protein (SP) accession no. P14672) as bait was performed. One clone
that was identified by
this procedure included novel protein fragment PN7065. The DNA sequence and
the predicted
protein sequence for PN7065 are set forth in 7~ and 76, respetively.
TABLE 75
Nucleotide Sequence of PN7065 (SEO ID N0:3)
CGCCGGTGGATGCGGGCTGAGCCCTGCTTGCCGGGACCCGCCTGCCCCGCCTTCTCCGCA
CACAGCTACACCTCCAACCTGGGCGACTACGATGAGCAGGCGCTGGGTATCATGCAGACC
CTGGGCGTGGACCGGCAGAGGACGGTGGAGTCACTGCAAAACAGCAGCTATAACCACTTT
GCTGCCATTTATTACCTCCTCCTTGAGCGGCTCAAGGAGTATCGGAATGCCCAGTGCGCC
CGCCCCGGGCCTGCCAGGCAGCCGCGGCCTCGGAGCTCGGACCTCAGTGGTTTGGAGGTG
CCTCAGGAAGGTCTTTCCACCGACCCTTTCCGACCTGCCTTGCTGTGCCCGCAGCCGCAG
ACCTTGGTGCAGTCCGTCCTCCAGGCCGAGATGGACTGTGGGCTCCAGAGCTCGCTGCAG
TGGCCCTTGTTCTTCCCGGTGGATGCCAGCTGCAGCGGAGTGTTCCGGCCCCGGCCCGTG
TCCCCAAGCAGCCTGCTGGACACAGCCATCAGTGAGGAGGCCAGGCAGGGGCCGGGCCTA
GAGGAGGAGCAGGACACGCAGGAGTCCCTGCCCAGCAGCACGGGCCGGGGGCACACCCTG
GCCGAGGTCTCCACCCGCCTCTCCCCACTCACCGCGCCAG
TABLE 76
Predicted Protein Sequence of PN7065 (SEQ ID N0:4)
RRWMRAEPCLPGPACPAFSAHSYTSNLGDYDEQALGIMQTLGVDRQRTVESLQNSSYNHF
AAIYYLLLERLKEYRNAQCARPGPARQPRPRSSDLSGLEVPQEGLSTDPFRPALLCPQPQ
TLVQSVLQAEMDCGLQSSLQWPLFFPVDASCSGVFRPRPVSPSSLLDTAISEEARQGPGL
EEEQDTQESLPSSTGRGHTLAEVSTRLSPLTAP
EXAMPLE 30
Identification of Glut4/PN7386 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 478-509
of Glut4
(SP accession no. P 14672) as bait was performed. One clone that was
identified by this procedure
included novel protein fragment PN7386. The DNA sequence and the predicted
protein sequence
for PN7386 are set forth in Tables 77 and 78, respectively.
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TABLE 77
Nucleotide Sequence of PN7386 (SEQ ID NO:S)
GGCCAATGTTGACGTTTTGGTAGGCTATGCAGACATCCATGGAGACTTACTACCTATAAA
TAATGATGATAATTATCACAAAGCTGTTTCAACGGCCAATCCACTGCTTAGGATATTTAT
ACAAAAGAAGGAAGAAGCAGACTACAGTGCCTTTGGTACAGACACGCTAATAAAGAAGAA
GAATGTTTTAACCAACGTATTGCGTCCTGACAACCATAGAAAAAAGCCACATATAGTCAT
TAGTATGCCCCAAGACTTTAGACCTGTGTCTTCTATTATAGACGTGGATATTCTCCCAGA
AACGCATCGTAGGGTACGTCTTTACAAATACGGCACGGAGAAACCCCTAGGATTCTACAT
CCGGGATGGCTCCAGTGTCAGGGTAACACCACATGGCTTAGAAAAGGTTCCAGGGATCTT
TATATCCAGGCTTGTCCCAGGAGGTCTGGCTCAAAGTACAGGACTATTAGCTGTTAATGA
TGAAGTTTTAGAAGTTAATGGCATAGAAGTTTCAGGGAAGAGCCTTGATCAAGTAACAGA
CATGATGATTGCAAATAGCCGTAACCTCATCATAACAGTGAGACCGGCAAACCAGAGGAA
TAATGTTGTGAGGAACAGTCGGACTTCTGGCAGTTCCGGTCAGTCTACTGATAACAGCCT
TCTTGGCTACCCACAGCAGATTGAACCAAGCTTTGAGCCAGAGGATGAAGACAGCGAAGA
AGATGACATTATCATTGAAGACAATGGAGTGCCACAGCAGATTCCAAAAGCTGTTCCTAA
TACTGAGAGCCTGGAGTCATTAACACAGATAGAGCTAAGCTTTGAGTCTGGACAGAATGG
CTTTATTCCCTCTAATGAAGTGAGCTTAGCAGCCATAGCAAGCAGCTCAAACACGGAATT
TGAAACACATGCTCCAGATCAAAAACTCTTAGAAGAAGATGGAACAATCATAACATTATG
AAACCGTGGTTTGAATGTTTTCAGAGTGAGGATGCCATGAGGACTTGTACATTTGGCTAG
TTTAGGCCAATGTTGACGTTTTGGTAGGCTATGCAGACATCCATG
TABLE 78
Predicted Amino Acid Sequence of PN7386 (SEQ ID N0:6)
ANVDVLVGYADIHGDLLPINNDDNYHKAVSTANPLLRIFIQKKEEADYSAFGTDTLIKKK
NVLTNVLRPDNHRKKPHIVISMPQDFRPVSSIIDVDILPETHRRVRLYKYGTEKPLGFYI
RDGSSVRVTPHGLEKVPGIFISRLVPGGLAQSTGLLAVNDEVLEVNGIEVSGKSLDQVTD
MMIANSRNLIITVRPANQRNNVVRNSRTSGSSGQSTDNSLLGYPQQIEPSFEPEDEDSEE
DDIIIEDNGVPQQIPKAVPNTESLESLTQIELSFESGQNGFIPSNEVSLAAIASSSNTEF
ETHAPDQKLLEEDGTIITL
EXAMPLE 31
Identification of OGTase/PN6931 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1618 of OGTase (GenBank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included novel protein
fragment PN6931. The DNA
sequence and the predicted protein sequence for PN6931 are set forth in Tables
79 and 80,
respectively.
TABLE 79
Nucleotide Sequence of PN6931 (SEQ ID N0:7)
AGGGAGGAGAAGCTGAGCCAGGATGAGATCGTGCTGGGCACCAAGGCTGTCATCCAGGGA
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CTGGAGACTCTGCGTGGGGAGCATCGTGCCCTGCTGGCTCCTCTGGTTGCACCTGAGGCC
GGCGAAGCCGAGCCTGGCTCGCAGGAGCGCTGCATCCTCCTGCGTCGCTCCCTGGAAGCC
ATTGAGCTTGGGCTGGGGGAGGCCCAGGTGATCTTGGCATTGTCGAGCCACCTGGGGGCT
GTAGAATCAGAGAAGCAGAAGCTGCGGGCGCAGGTGCGGCGTCTGGTGCAGGAGAACCAG
TGGCTGCGTGAGGAGCTGCCGGGGACACAGCAKAAGCTGCAGCGCAGTGAGCAGGCCGTG
GCCCAGCTCGAGGAGGAGAAGCAGCACTTGCTGTTCATGARCCAGATCCGCAGTTGGATG
AAGACGCCTYCCCTAACGAGGAGAAGGGGGACGTCCCCAAAGACACACTGGATGACCTGT
TCCCCAATGAGGATGAGCAGAGCCCAGCCCCTAGCCCAGGAGGAGGGGATGTGTCTGGTC
AGCATGGGGGATACGAGATCCCGGCCCGGCTCCGCACCCTGCACAACTGGTGATCCAATA
CGCCTCACAGGGCCGCTACGAGGTAGCTGTGCCACTCTGCAAGCAGGCACTCGAAGACTG
GAGAAGACGTCAGGCCACGACCACCCTGACGTTGCCACCATGCTGAACATCCTGGCACTG
GTCTATCGGGATCAGAACAAGTACAAGGAGGCTGCCCACCTGCTCAATGATGCTCTGGCC
ATCCGGGAGAAAACACTGGGCAAGGACCACCCAGCCGTGGCTGCGACACTAAACAACCTG
GCAGTCCTGTATAGCGCAGAG
TABLE 80
Predicted Protein Sequence of PN6931 (SEQ ID N0:8)
REEKLSQDEIVLGTKAVIQGLETLRGEHRALLAPLVAPEAGEAEPGSQERCILLRRSLEA
IELGLGEAQVILALSSHLGAVESEKQKLRAQVRRLVQENQWLREELPGTQXKLQRSEQAV
AQLEEEKQHLLFMXQIRSWMKTPXLTRRRGTSPKTHWMTCSPMRMSRAQPLAQEEGMCLV
SMGDTRSRPGSAPCTTGDPIRLTGPLRGSCATLQAGTRRLEKTSGHDHPDVATMLNILAL
VYRDQNKYKEAAHLLNDALAIREKTLGKDHPAVAATLNNLAVLYSAE
EXAMPLE 32
Identification ofNaflb/PN7582 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 233-1568 ofNaflb (GB accession no: AJOl 1896) as bait was
performed. One clone that
was identified by this procedure included novel protein fragment PN7582. The
DNA sequence and
the predicted protein sequence for PN7582 are set forth in Tables 81 and 82,
respectively.
TABLE 81
Nucleotide Sequence for PN7582 (SEQ ID N0:9~
ATTTCAAGGGGGCTGCTGTACCCCCAGGCATGTGTCTGTATATCGCACAGGAAGAAGGAA
AGTAAGGACATTGCCAGCAAATATCTTACATCTCATCAGCCTATACTGTGTCTCCTGACC
ACTCCTAACTGCAAAGGATGCTGGGAAAAAAAGAGCATTGTAGCTTTTCCAGCCTCTGTG
GTAGGCGCAGATAAGGGATTAGAGTTGGGTGTTACTGAATCAATGTATCAGACACTTCTC
AGTCAGGCTAGAGCCAGATTTAACTAGATTTAGCAGGAAAAGTATGTTTCTTTCACCTGC
ATGTAATGAAGGAAATCTATGTCCTTCATACACTTAATAAACCTGTAAGTCTCTACTATG
GGCAGGTACTGTGCTAGCTAGACATTACAATGTGTGGGGGCAGACACAAAGATGGGAACA
GTAGACACTGGGGAACCCTAGAGGGGGGAGGTTGGGAGTAGGGGAAGGGTTGAAAAATGA
TTGGGTACTATGCTCACTACCTGGGTGATGGGATCATTTGTACACCAAATGCCAGCAACA
CACAATTTACCCGTGTAACACACCGGCACGTGTACCCCCTGAACCTAAGATGAAAGCCGA
A
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TABLE 82
Predicted Amino Acid Sequence for PN7582 ~SEO ID NO:10)
ISRGLLYPQACVCISHRKKESKDIASKYLTSHQPILCLLTTPNCKGCWEKKSIVAFPASV
VGADKGLELGVTESMYQTLLSQARARFN
EXAMPLE 33
Identification of OGTase/Talin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids 812 et
seq. of talin (GB
accession no. AF078828).
EXAMPLE 34
Identification of OGTase/MOP2 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids 397-546
of MOP2 (Swiss
Protein (SP) accession no. Q99814).
EXAMPLE 3 5
Identification of OGTase/Clone 25100 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids encoded
by nucleotides 31-
1014 of clone 25100 (GB accession no. AF 121780).
EXAMPLE 36
Identification of OGTase/KIAA0443 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
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One clone that was identified by this procedure included amino acids encoded
by nucleotides 3468-
4106 of KIAA0443 (GB accession no. AB007903).
EXAMPLE 37
Identification of OGTase/EGR1 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids 415-544
of EGR1 (SP
accession no. P 18146).
EXAMPLE 38
Identification of OGTase/Dynamin II Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids 515-823
of dynamin II (SP
accession no. P50570).
EXAMPLE 39
Identification of OGTase/INT-6 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids encoded
by nucleotides 824-
1191 of Int-6 (GB accession no. U62962).
EXAMPLE 40
Identification of OGTase/HSPC028 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids encoded
by nucleotides 58-443
of HSPC028 (GB accession no. AF083246).
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EXAMPLE 41
Identification of OGTase/BAP31 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids 116-215
of BAP31 (SP
accession no. P51572).
EXAMPLE 42
Identification of OGTase/Interferon-Ind Protein Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait
was performed.
One clone that was identified by this procedure included amino acids 1 et seq.
of interferon-ind
protein (SP accession no. P09913).
EXAMPLE 43
Identification of Glut4/Beta-catenin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(Swiss Protein (SP) accession no. P 14672) as bait was performed. One clone
that was identified by
this procedure included amino acids 579-782 of Beta-catenin (SP accession no.
P35222).
EXAMPLE 44
Identification of GIut4/Alpha-SNAP Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(SP accession no. P14672) as bait was performed. One clone that was identified
by this procedure
included amino acids 36-241 of Alpha-SNAP (SP accession no. P54920).
EXAMPLE 45
Identification of Glut4/MAPKKK6 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(SP accession no. P14672) as bait was performed. One clone that was identified
by this procedure
incl~a~ed amino acids 824-1012 of MAPKKK6 (GenBank (GB) accession no.
AF100318).
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EXAMPLE 46
Identification of GLUT4/Tropomyosin 3 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 434-509
of Glut4
(SP accession no. P 14672) as bait was performed. One clone that was
identified by this procedure
included amino acids 171-286 of tropomyosin 3 (SP accession no. P06753).
EXAMPLE 47
Identification of GLUT1/DRAL/FHL2 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 448-492
of Glutl
(SP accession no. P11166) as bait was performed. One clone that was identified
by this procedure
included amino acids 1-280 of DR.AL/FHL2 (SP accession no. Q13229).
EXAMPLE 48
Identification of GLUT1/MYSA Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 448-492
of Glutl
(SP accession no. P11166) as bait was performed. One clone that was identified
by this procedure
included amino acids 1589-1909 of MYSA (SP accession no. P13533).
EXAMPLE 49
Identification of IRAP/SG2NA Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 2311-3136 of IRAP (GB accession no. U62768) as bait was performed.
One clone that
was identified by this procedure included amino acids 623-713 of SG2NA (SP
accession no.
P70483).
EXAMPLE 50
Identification of IR.AP/SLAP-2 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 62-880 of IRAP (GB accession no. U62768) as bait was performed.
One clone that was
identified by this procedure included amino acids 236-453 of SLAP-2 (GB
accession no.
AF 100750).
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EXAMPLE 51
Identification of OGTase/14-3-3 Epsilon Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1618 of OGTase (GB accession no. U77413) as bait was
performed. One clone
that was identified by this procedure included amino acids 21-232 of 14-3-3
epsilon (SP accession
no. P29360).
EXAMPLE 52
Identification of PI-3K8~/Chromo~ranin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-250 of
PI-3
kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone
that was identified
by this procedure included amino acids 2-322 of chromogranin (SP accession no.
P 13521 ).
EXAMPLE 53
Identification of PI-3K85/SLP-76 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 320-440
of PI-3
kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone
that was identified
by this procedure included amino acids 291-486 of SLP-76 (GB accession no.
U20158).
EXAMPLE 54
Identification of PI-3K85/SLP-76 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 600-724
of PI-3
kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone
that was identified
by this procedure included amino acids 291-486 of SLP-76 (GB accession no.
U20158).
EXAMPLE 55
Identification of PI-3K85/14-3-3-zeta Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 425-630
of PI-3
kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone
that was identified
by this procedure included amino acids 79-246 of 14-3-3-zeta (AP accession no.
P29213).
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EXAMPLE 56
Identification of PI-3K85/14-3-3-eta Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 425-630
of PI-3
kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone
that was identified
5 by this procedure included amino acids beginning with residue 91 of 14-3-3-
eta (SP accession no.
Q04917).
EXAMPLE 57
Identification of PI-3K85/TACC2 Interaction
10 A yeast two-hybrid system as described in Example 1 using amino acids 600-
724 of PI-3
kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone
that was identified
by this procedure included amino acids 350-600 of TACC2 (GB accession no.
AF095791).
EXAMPLE 58
15 Identification of Glut4/MM-1 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(Swiss Protein (SP) accession no. P14672) as bait was performed. One clone
that was identified by
this procedure included amino acids 27-168 of MM-1 (GenBank (GB) accession no.
D89667).
20 EXAMPLE 59
Identification of Glutl/KIAA0144 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-492
of Glutl
(SP accession no. P 11166) as bait was performed. One clone that was
identified by this procedure
included amino acids beginning with residue 691 of KIAA0144 (GB accession no.
D63478).
EXAMPLE 60
Identification of Glutl/Dvnamin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-492
of Glutl
(Swiss. Protein (SP) accession no. P 11166) as bait was performed. One clone
that was identified by
3fl this procedure included amino acids 104-365 of dynamin (GB accession no.
L07807).
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EXAMPLE 61
Identification of Glut 1 /Clone 25204 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-492
of Glutl
(Swiss Protein (SP) accession no. Pl 1166) as bait was performed. One clone
that was identified by
this procedure included undetermined amino acids residues of Clone 25204 (GB
accession no.
AF 131749).
EXAMPLE 62
Identification of IRAP/VAP-A Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 62-880 of IRAP (GB accession no. U62768) as bait was performed.
One clone that was
identified by this procedure included amino acids 3-243 of VAP-A (GB accession
no. AF086627).
EXAMPLE 63
Identification of OGTase/Nafl a Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 1016-1618 of OGTase (GB accession no. U77413) as bait was
performed. One clone
that was identified by this procedure included amino acids 41-486 of Nafla (GB
accession no.
AJ011895).
EXAMPLE 64
Identification of OGTasePPS/Alnha-2-Catenin
A yeast two-hybrid system as described in Example 1 using acids encoded by
nucleotides
1016-1618 of OGTase (GB accession no. U77413) as bait was performed. One clone
that was
identified by this procedure included amino acids 366-469 of Alpha-2-Catenin
(GB accession no.
M94151 ).
EXAMPLE 65
Identification of PI-3K110/TRIP15 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of
PI-3K110
(SP accession no. P42338) as bait was performed. One clone that was identified
by this procedure
included amino acids 24-233 of TRIP15 (GB accession no. L40388).
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EXAMPLE 66
Identification of Glut4/14-3-3 Zeta Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(Swiss Protein (SP) accession no. P14672) as bait was performed. Three clones
that were identified
by this procedure included amino acids 1-121. 1-200 and 1-209 of 14-3-3 zeta
(SP accession no.
P29213).
EXAMPLE 67
Identification of Glut4/KIAA0282 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(Swiss Protein (SP) accession no. P14672) as bait was performed. One clone
that was identified by
this procedure included amino acids 229-379 of KIAA0282 (GenBank (GB)
accession no. D87458),
an efp-like protein.
EXAMPLE 68
Identification of Glut4/Tankvrase Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 463-509
of Glut4
(Swiss Protein (SP) accession no. P14672) as bait was performed. One clone
that was identified by
this procedure included amino acids encoded by nucleotides 1110-1750 of
tankyrase (GB accession
no. AF082557).
EXAMPLE 69
Identification of IRAP/PTPZ Interaction
2~ A yeast two-hybrid system as described in Example 1 using amino acids
encoded by
nuci~tides 62-880 of IRAP (GB accession no. U62768) as bait was performed. One
clone that was
identified by this procedure included amino acids 1420-1780 of PTPZ (SP
accession no. P23471).
EXAMPLE 70
3fl Identification of IRAP/~3Spectrin Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 2311-3136 of IRAP (GB accession no. U62768) as bait was performed.
One clone that
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58
was identified by this procedure included amino acids 1487-1993 of (3-spectrin
(SP accession no.
Q01082).
EXAMPLE 71
Identification of IRAP/PI-3K85 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids encoded
by
nucleotides 2311-3136 of IRAP (GB accession no. U62768) as bait was performed.
One clone that
was identified by this procedure included amino acids 498-544 of PI-3 kinase
p85 subunit (SP
accession no. P27986).
EXAMPLE 72
Identification of PP~/HSP89 Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-150 of
PPS (SP
accession no. P53041) as bait was performed. One clone that was identified by
this procedure
included amino acids 517-681 of Hsp89 (SP accession no. P07900).
EXAMPLE 73
Identification of PPS/Tankvrase Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-150 of
PPS (SP
accession no. P53041 ) as bait was performed. One clone that was identified by
this procedure
included amino acids encoded by nucleotides 1110-1750 and nucleotides 1809-
2257 of tankyrase
(GB accession no. AF082557).
EXAMPLE 74
Identification of PI-3K85/Tankyrase Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 320-440
of PI-3
kinase p85 subunit (SP accession no. P27986) as bait was performed. Two clones
that were
identified by this procedure included amino acids encoded by nucleotides 1110-
1750 of tankyrase
(GB accession no. AF082557).
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EXAMPLE 75
Identification of PI-3K110/APP Interaction
A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of
PI-3
kinase p110 subunit (SP accession no. P42338) as bait was performed. One clone
that was
identified by this procedure included amino acids 374-546 of APP (SP accession
no. P05067).
EXAMPLE 76
Generation of Polyclonal Antibody Against Protein Complexes
As shown above, Glut4 interacts with CARP to form a complex. A complex of the
two
proteins is prepared, e.g., by mixing purified preparations of each of the two
proteins. If desired,
the protein complex can be stabilized by cross-linking the proteins in the
complex, by methods
known to those of skill in the art. The protein complex is used to immunize
rabbits and mice using
a procedure similar to that described by Harlow et al. (1988). This procedure
has been shown to
generate Abs against various other proteins (for example, see Kraemer et al.,
1993).
Briefly, purified protein complex is used as immunogen in rabbits. Rabbits are
immunized
with 100 ~.g of the protein in complete Freund's adjuvant and boosted twice in
three-week intervals,
first with 100 ~g of immunogen in incomplete Freund's adjuvant, and followed
by 100 p,g of
immunogen in PBS. Antibody-containing serum is collected two weeks thereafter.
The antisera
is preadsorbed with Glut4 and CARP, such that the remaining antisera comprises
antibodies which
bind conformational epitopes, i.e., complex-specific epitopes, present on the
Glut4-CARP complex
but not on the monomers.
Polyclonal antibodies against each of the complexes set forth in Tables 1-73
are prepared
in a similar manner by mixing the specified proteins together, immunizing an
animal and isolating
antibodies specific for the protein complex, but not for the individual
proteins.
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EXAMPLE 77
Generation of Monoclonal Antibodies Specific for Protein Com In exec
Monoclonal antibodies are generated according to the following protocol. Mice
are
immunized with immunogen comprising Glut4/CARP complexes conjugated to keyhole
limpet
5 hemocyanin using glutaraldehyde or EDC as is well known in the art. The
complexes can be
prepared as described in Example 76, and may also be stabilized by cross-
linking. The immunogen
is mixed with an adjuvant. Each mouse receives four injections of 10 to 100 pg
of immunogen, and
after the fourth injection blood samples are taken from the mice to determine
if the serum contains
antibody to the immunogen. Serum titer is determined by ELISA or RIA. Mice
with sera indicating
10 the presence of antibody to the immunogen are selected for hybridoma
production.
Spleens are removed from immune mice and a single-cell suspension is prepared
(Harlow
et al., 1988). Cell fusions are performed essentially as described by Kohler
et al. (1975). Briefly,
P3.65.3 myeloma cells (American Type Culture Collection, Rockville, MD) or NS-
1 myeloma cells
are fused with immune spleen cells using polyethylene glycol as described by
Harlow et al. (1988).
15 Cells are plated at a density of 2x105 cells/well in 96-well tissue culture
plates. Individual wells are
examined for growth, and the supernatants of wells with growth are tested for
the presence of
Glut4/CARP complex-specific antibodies by ELISA or RIA using Glut4/CARP
complex as target
protein. Cells in positive wells are expanded and subcloned to establish and
confirm monoclonality.
Clones with the desired specificities are expanded and grown as ascites in
mice or in a
20 hollow fiber system to produce sufficient quantities of antibodies for
characterization and assay
development. Antibodies are tested for binding to Glut4 alone or to CARP
alone, to determine
which are specific for the GIut4/CARP complex as opposed to those that bind to
the individual
proteins.
Monoclonal antibodies against each of the complexes set forth in Tables 1-73
are prepared
25 in a similar manner by mixing the specified proteins together, immunizing
an animal, fusing spleen
cells with myeloma cells and isolating clones which produce antibodies
specific for the protein
complex, but not for the individual proteins.
EXAMPLE 78
30 In vitro Identification of Modulators for Protein-Protein Interactions
The present invention is useful in screening for agents that modulate the
interaction of Glut4
and CARP. The knowledge that GIut4 and CARP form a complex is useful in
designing such
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61
assays. Candidate agents are screened by mixing Glut4 and CARP (a) in the
presence of a candidate
agent, and (b) in the absence of the candidate agent. The amount of complex
formed is measured
for each sample. An agent modulates the interaction of GIut4 and CARP if the
amount of complex
formed in the presence of the agent is greater than (promoting the
interaction), or less than
(inhibiting the interaction) the amount of complex formed in the absence of
the agent. The amount
of complex is measured by a binding assay, which shows the formation of the
complex, or by using
antibodies immunoreactive to the complex.
Briefly, a binding assay is performed in which immobilized Glut4 is used to
bind labeled
CARP. The labeled CARP is contacted with the immobilized Glut4 under aqueous
conditions that
permit specific binding of the two proteins to form an Glut4/CARP complex in
the absence of an
added test agent. Particular aqueous conditions may be selected according to
conventional methods.
Any reaction condition can be used as long as specific binding of Glut4/CARP
occurs in the control
reaction. A parallel binding assay is performed in which the test agent is
added to the reaction
mixture. The amount of labeled CARP bound to the immobilized Glut4 is
determined for the
1 S reactions in the absence or presence of the test agent. If the amount of
bound, labeled CARP in the
presence of the test agent is different than the amount of bound labeled CARP
in the absence of the
test agent, the test agent is a modulator of the interaction of Glut4 and
CARP.
Candidate agents for modulating the interaction of each of the protein
complexes set forth
in Tables 1-73 are screened in vitro in a similar manner.
EXAMPLE 79
In vivo Identification of Modulators for Protein-Protein Interactions
In addition to the in vitro method described in Example 78, an in vivo assay
can also be used
to screen for agents which modulate the interaction of Glut4 and CARP.
Briefly, a yeast two-hybrid
system is used in which the yeast cells express ( 1 ) a first fusion protein
comprising Glut4 or a
fragment thereof and a first transcriptional regulatory protein sequence,
e.g., GAL4 activation
domain, (2) a second fusion protein comprising CARP or a fragment thereof and
a second
transcriptionai regulatory protein sequence, e.g., GAL4 DNA-binding domain,
and (3) a reporter
gene, e.g., (3-galactosidase, which is transcribed when an intermolecular
complex comprising the
first fusion protein and the second fusion protein is formed. Parallel
reactions are performed in the
absence of a test agent as the control and in the presence of the test agent.
A functional Glut4/CARP
complex is detected by detecting the amount of reporter gene expressed. If the
amount of reporter
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62
gene expression in the presence of the test agent is different than the amount
of reporter gene
expression in the absence of the test agent, the test agent is a modulator of
the interaction of Glut4
and CARP.
Candidate agents for modulating the interaction of each of the protein
complexes set forth
in Tables 1-73 are screened in vivo in a similar manner.
While the invention has been disclosed in this patent application by reference
to the details
of preferred embodiments of the invention, it is to be understood that the
disclosure is intended in
an illustrative rather than in a limiting sense, as it is contemplated that
modifications will readily
occur to those skilled in the art, within the spirit of the invention and the
scope of the appended
claims.
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1 S PCT Published Application No. WO 97/27296
PCT Published Application No. WO 99/65939
U.S. Patent No. 5,622,852
U.S. Patent No. 5,773,218
CA 02371006 2001-10-22
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SEQUENCE LISTING
<110> Heichman, Karen
Bartel, Paul L.
Myriad Genetics, Inc.
<120> Protein-Protein Interactions
<130> Protein Interactions II
<140>
<141>
<150> US 60/130,389
<151> 1999-04-22
<150> US 60/140,693
<151> 1999-06-24
<150> US 60/156,947
<151> 1999-09-30
<150> US 60/163,073
<151> 1999-11-02
<150> US 60/168,376
<151> 1999-12-02
<150> US 60/168,378
<151> 1999-12-02
<160> 10
<170> PatentIn Ver. 2.0
<210> 1
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:tail for PCR
primers
<400> 1
gcaggaaaca gctatgacca tacagtcagc ggccgccacc 40
<210> 2
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:tail for PCR
primers
<400> 2
acggccagtc gcgtggagtg ttatgtcatg cggccgcta 39
<210> 3
<211> 640
<212> DNA
<213> Homo sapiens
CA 02371006 2001-10-22
WO 00/65340 PCT/LJS00/10651
2
<220>
<221>
CDS
<222> )..(639)
(1
<400>
3
cgccggtgg atgcggget gagccctgc ttgccggga cccgcctgc ccc 48
ArgArgTrp MetArgAla GluProCys LeuProGly ProAlaCys Pro
1 5 10 15
gccttctcc gcacacagc tacacctcc aacctgggc gactacgat gag 96
AlaPheSer AlaHisSer TyrThrSe= AsnLeuGly AspTyrAsp Glu
20 25 30
caggcgctg ggtatcatg cagaccctg ggcgtggac cggcagagg acg 144
GlnAlaLeu GlyIleMet GlnThrLeu GlyValAsp ArgGlnArg Thr
35 40 45
gtggagtca ctgcaaaac agcagctat aaccacttt getgccatt tat 192
ValGluSer LeuGlnAsn SerSerTyr AsnHisPhe AlaAlaIle Tyr
50 55 60
tacctcctc cttgagcgg ctcaaggag tatcggaat gcccagtgc gcc 240
TyrLeuLeu LeuGluArg LeuLysGlu TyrArgAsn AlaGlnCys Ala
65 70 75 80
cgccccggg cctgccagg cagccgcgg cctcggagc tcggacctc agt 288
ArgProGly ProAlaArg GlnProArg ProArgSer SerAspLeu Ser
85 90 95
ggtttggag gtgcctcag gaaggtctt tccaccgac cctttccga cct 336
GlyLeuGlu ValProGln GluGlyLeu SerThrAsp ProPheArg Pro
100 105 110
gccttgctg tgcccgcag ccgcagacc ttggtgcag tccgtcctc cag 384
AlaLeuLeu CysProGln ProGlnThr LeuValGln SerValLeu Gln
115 120 125
gccgagatg gactgtggg ctccagagc tcgctgcag tggcccttg ttc 432
AlaGluMet AspCysGly LeuGlnSer SerLeuGln TrpProLeu Phe
130 135 140
ttcccggtg gatgccagc tgcagcgga gtgttccgg ccccggccc gtg 480
PheProVal AspAlaSer CysSerGly ValPheArg ProArgPro Val
145 150 155 160
tccccaagc agcctgctg gacacagcc atcagtgag gaggccagg cag 528
SerProSer SerLeuLeu AspThrAla IleSerGlu GluAlaArg Gln
165 170 175
gggccgggc ctagaggag gagcaggac acgcaggag tccctgccc agc 576
GlyProGly LeuGluGlu GluGlnAsp ThrGlnGlu SerLeuPro Ser
180 185 190
agcacgggc cgggggcac accctggcc gaggtctcc acccgcctc tcc 624
SerThrGly ArgGlyHis ThrLeuAla GluValSer ThrArgLeu Ser
195 200 205
ccactcacc gcgccag 640
ProLeuThr AlaPro
210
<210>
4
<211> 13
2
<212>
PRT
CA 02371006 2001-10-22
WO 00/65340 PCT/US00/10651
3
<213> Homo Sapiens
<400> 4
Arg Arg Trp Met Arg Ala Glu Pro Cys Leu Pro Gly Pro Ala Cys Pro
1 5 10 15
Ala Phe Ser Ala His Ser Tyr Thr Ser Asn Leu Gly Asp Tyr Asp Glu
20 25 30
Gln Ala Leu Gly Ile Met Gln Thr Leu Gly Val Asp Arg Gln Arg Thr
35 40 45
Val Glu Ser Leu Gln Asn Ser Ser Tyr Asn His Phe Ala Ala Ile Tyr
50 55 60
Tyr Leu Leu Leu Glu Arg Leu Lys Glu Tyr Arg Asn Ala Gln Cys Ala
65 70 75 80
Arg Pro Gly Pro Ala Arg Gln Pro Arg Pro Arg Ser Ser Asp Leu Ser
85 90 95
Gly Leu Glu Val Pro Gln Glu Gly Leu Ser Thr Asp Pro Phe Arg Pro
100 105 110
Ala Leu Leu Cys Pro Gln Pro Gln Thr Leu Val Gln Ser Val Leu Gln
115 120 125
Ala Glu Met Asp Cys Gly Leu Gln Ser Ser Leu Gln Trp Pro Leu Phe
130 135 140
Phe Pro Val Asp Ala Ser Cys Ser Gly Val Phe Arg Pro Arg Pro Val
145 150 155 160
Ser Pro Ser Ser Leu Leu Asp Thr Ala Ile Ser Glu Glu Ala Arg Gln
165 170 175
Gly Pro Gly Leu Glu Glu Glu Gln Asp Thr Gln Glu Ser Leu Pro Ser
180 185 190
Ser Thr Gly Arg Gly His Thr Leu Ala Glu Val Ser Thr Arg Leu Ser
195 200 205
Pro Leu Thr Ala Pro
210
<210> 5
<211> 1065
<212> DNA
<213> Homa Sapiens
<220>
<221> CDS
<222> (2)..(958)
<400> 5
g gcc aat gtt gac gtt ttg gta ggc tat gca gac atc cat gga gac tta 49
Ala Asn Val Asp Val Leu Val Gly Tyr Ala Asp Ile His Gly Asp Leu
1 5 10 15
cta cet ata aat aat gat gat aat tat cac aaa get gtt tca acg gec 97
Leu Pro Ile Asn Asn Asp Asp Asn Tyr His Lys Ala Val Ser Thr Ala
20 25 30
CA 02371006 2001-10-22
WO 00/65340 PCT/US00/10651
4
aat ccactgctt aggata tttatacaa aagaaggaa gaagcagac tac 145
Asn ProLeuLeu ArgIle PheIleGln LysLysGlu GluAlaAsp Tyr
35 40 45
agt gcctttggt acagac acgctaata aagaagaag aatgtttta acc 193
Ser AlaPheGly ThrAsp ThrLeuIle LysLysLys AsnValLeu Thr
50 55 60
aac gtattgcgt cctgac aaccataga aaaaagcca catatagtc att 291
Asn ValLeuArg ProAsp AsnHisArg LysLysPro HisIleVa1 Ile
65 70 75 80
agt atgccccaa gacttt agacctgtg tcttctatt atagacgtg gat 289
Ser MetProGln AspPhe ArgProVal SerSerIle IleAspVal Asp
85 90 95
att ctcccagaa acgcat cgtagggta cgtctttac aaatacggc acg 337
Ile LeuProGlu ThrHis ArgArgVal ArgLeuTyr LysTyrGly Thr
100 105 110
gag aaaccccta ggattc tacatccgg gatggctcc agtgtcagg gta 385
Glu LysProLeu GlyPhe TyrIleArg AspGlySer SerValArg Val
115 120 125
aca ccacatggc ttagaa aaggttcca gggatcttt atatccagg ctt 433
Thr ProHisGly LeuGlu LysValPro GlyIlePhe IleSerArg Leu
130 135 140
gtc ccaggaggt ctgget caaagtaca ggactatta getgttaat gat 481
Val ProGlyGly LeuAla GlnSerThr GlyLeuLeu AlaValAsn Asp
145 150 155 160
gaa gttttagaa gttaat ggcatagaa gtttcaggg aagagcctt gat 529
Glu ValLeuGlu ValAsn GlyIleGlu ValSerGly LysSerLeu Asp
165 170 175
caa gtaacagac atgatg attgcaaat agccgtaac ctcatcata aca 577
Gln ValThrAsp MetMet IleAlaAsn SerArgAsn LeuIleIle Thr
180 185 190
gtg agaccggca aaccag aggaataat gttgtgagg aacagtcgg act 625
Val ArgProAla AsnGln ArgAsnAsn ValValArg AsnSerArg Thr
195 200 205
tct ggcagttcc ggtcag tctactgat aacagcctt cttggctac cca 673
Ser GlySerSer GlyGln SerThrAsp AsnSerLeu LeuGlyTyr Pro
210 215 220
cag cagattgaa ccaagc tttgagcca gaggatgaa gacagcgaa gaa 721
Gln GlnIleGlu ProSer PheGluPro GluAspGlu AspSerGlu Glu
225 230 235 240
gat gacattatc attgaa gacaatgga gtgccacag cagattcca aaa 769
Asp AspIleIle IleGlu AspAsnGly ValProGln GlnIlePro Lys
245 250 255
get gttcctaat actgag agcctggag tcattaaca cagatagag cta 817
Ala ValProAsn ThrGlu SerLeuGlu SerLeuThr GlnIleGlu Leu
260 265 270
agc tttgagtct ggacag aatggcttt attccctct aatgaagtg agc 865
Ser PheGluSer GlyGln AsnGlyPhe I1eProSer AsnGluVal Ser
275 280 285
CA 02371006 2001-10-22
WO 00/65340 PCT/US00/10651
tta gca gcc ata gca agc agc tca a~~ acg gaa ttt gaa aca cat get 913
Leu Ala Ala Ile Ala Ser Ser Ser As:~ Thr Glu Phe Glu Thr His Ala
290 295 300
cca gat caa aaa ctc tta gaa gaa gay gga aca atc ata aca tta 958
Pro Asp Gln Lys Leu Leu Glu Glu Asc_ Gly Thr Ile Ile Thr Leu
305 310 315
tgaaaccgtg gtttgaatgt tttcagagtg agga~gccat gaggacttgt acatttggct 1018
agtttaggcc aatgttgacg ttttggtagg c~atgcagac atccatg 1065
<210> 6
<211> 319
<212> PRT
<213> Homo Sapiens
<400> 6
Ala Asn Val Asp Val Leu Val Gly Tyr Ala Asp Ile His Gly Asp Leu
1 5 10 15
Leu Pro Ile Asn Asn Asp Asp Asn Tyr His Lys Ala Val Ser Thr Ala
20 25 30
Asn Pro Leu Leu Arg Ile Phe Ile Gin Lys Lys Glu Glu Ala Asp Tyr
35 40 45
Ser Ala Phe Gly Thr Asp Thr Leu Ile Lys Lys Lys Asn Val Leu Thr
50 55 60
Asn Val Leu Arg Pro Asp Asn His Arg Lys Lys Pro His Ile Val Ile
65 70 75 80
Ser Met Pro Gln Asp Phe Arg Pro Val Ser Ser Ile Ile Asp Val Asp
85 90 95
Ile Leu Pro Glu Thr His Arg Arg Val Arg Leu Tyr Lys Tyr Gly Thr
100 105 110
Glu Lys Pro Leu Gly Phe Tyr Ile Arg Asp Gly Ser Ser Val Arg Val
115 120 125
Thr Pro His Gly Leu Glu Lys Val Pro Gly Ile Phe Ile Ser Arg Leu
130 135 140
Val Pro Gly Gly Leu Ala Gln Ser Thr Gly Leu Leu Ala Val Asn Asp
145 150 155 160
Glu Val Leu Glu Val Asn Gly Ile Glu Val Ser Gly Lys Ser Leu Asp
165 170 175
Gln Val Thr Asp Met Met Ile Ala Asn Ser Arg Asn Leu Ile Ile Thr
180 185 190
Val Arg Pro Ala Asn Gln Arg Asn Asn Val Val Arg Asn Ser Arg Thr
195 200 205
Ser Gly Ser Ser Gly Gln Ser Thr Asp Asn Ser Leu Leu Gly Tyr Pro
210 215 220
Gln Gln Ile Glu Pro Ser Phe Glu Pro Glu Asp Glu Asp Ser Glu Glu
225 230 235 240
CA 02371006 2001-10-22
WO 00/65340 PCT/US00/10651
6
Asp Asp Ile Ile Ile Glu Asp Asn Gly Val Pro Gln Gln Ile Pro Lys
245 250 255
Ala Val Pro Asn Thr Glu Ser Leu Glu Ser Leu Thr Gln Ile Glu Leu
260 265 270
Ser Phe Glu Ser Gly Gln Asn Gly Phe Ile Pro Ser Asn Glu Val Ser
275 280 285
Leu Ala Ala Ile Ala Ser Ser Ser As.~. T~:r Glu Phe Glu Thr His Ala
290 295 300
Pro Asp Gln Lys Leu Leu Glu Glu Asp Gly Thr Ile Ile Thr Leu
305 310 315
<210>
7
<211> 1
86
<212> A
DN
<213> mo apiens
Ho S
<220>
<221>
CDS
<222> )..(861)
(1
<400>
7
agg gaggagaag ctgagc caggatgag atcgtgctg ggcaccaag get 48
Arg GluGluLys LeuSer GlnAspGlu I1eValLeu GlyThrLys Ala
1 5 10 15
gtc atccaggga ctggag actctgcgt ggggagcat cgtgccctg ctg 96
Val IleGlnGly LeuGlu ThrLeuArg G1yGluHis ArgAlaLeu Leu
20 25 30
get cctctggtt gcacct gaggccggc gaagccgag cctggctcg cag 144
Ala ProLeuVal AlaPro GluAlaGly GluAlaGlu ProGlySer Gln
35 40 45
gag cgctgcatc ctcctg cgtcgctcc ctggaagcc attgagctt ggg 192
Glu ArgCysIle LeuLeu ArgArgSer LeuGluAla IleGluLeu Gly
50 55 60
ctg ggggaggcc caggtg atcttggca ttgtcgagc cacctgggg get 240
Leu GlyGluAla GlnVal IleLeuAla LeuSerSer HisLeuGly Ala
65 70 75 80
gta gaatcagag aagcag aagctgcgg gcgcaggtg cggcgtctg gtg 288
Val GluSerGlu LysGln LysLeuArg AlaGlnVal ArgArgLeu Val
85 90 95
cag gagaaccag tggctg cgtgaggag ctgccgggg acacagcak aag 336
Gln GluAsnGln TrpLeu ArgGluGlu LeuProGly ThrGlnXaa Lys
100 105 110
ctg cagcgcagt gagcag gccgtggcc cagctcgag gaggagaag cag 384
Leu GlnArgSer GluGln AlaValAla GlnLeuGlu GluGluLys Gln
115 120 125
cac ttgctgttc atgarc cagatccgc agttggatg aagacgcct ycc 432
His LeuLeuPhe MetXaa GlnIleArg SerTrpMet LysThrPro Xaa
130 135 140
cta acgaggaga aggggg acgtcccca aagacacac tggatgacc tgt 480
Leu ThrArgArg ArgGly ThrSerPro LysThrHis TrpMetThr Cys
145 150 155 160
CA 02371006 2001-10-22
WO 00/65340 PCT/US00/10651
7
tccccaatg aggatgagc agagcc cagccccta gcccaggag gagggg 528
SerProMet ArgMetSer ArgAla GlnProLeu AlaGlnGlu GluGly
165 170 175
atgtgtctg gtcagcatg ggggat acgagatcc cggcccggc tccgca 576
MetCysLeu ValSerMet GlyAsp ThrArgSer ArgProGly SerAla
180 185 190
ccctgcaca actggtgat ccaata cgcc~caca gggccgcta cgaggt 624
ProCysThr ThrGlyAsp ProIle ArgLeuThr GlyProLeu ArgGly
195 200 205
agctgtgcc actctgcaa gcaggc actcgaaga ctggagaag acgtca 672
SerCysAla ThrLeuGln AlaGly ThrArgArg LeuGluLys ThrSer
210 215 220
ggccacgac caccctgac gttgcc accatgctg aacatcctg gcactg 720
GlyHisAsp HisProAsp ValAla ThrMetLeu AsnIleLeu AlaLeu
225 230 235 240
gtctatcgg gatcagaac aagtac aaggagget gcccacctg ctcaat 768
ValTyrArg AspGlnAsn LysTyr LysGluAla AlaHisLeu LeuAsn
245 250 255
gatgetctg gccatccgg gagaaa acactgggc aaggaccac ccagcc 816
AspAlaLeu AlaIleArg GluLys ThrLeuGly LysAspHis ProAla
260 265 270
gtggetgcg acactaaac aacctg gcagtcctg tatagcgca gag 861
ValAlaAla ThrLeuAsn AsnLeu AlaValLeu TyrSerAla Glu
275 280 285
<210> 8
<211> 287
<212> PRT
<213> Homo Sapiens
<400> 8
Arg Glu Glu Lys Leu Ser Gln Asp Glu Ile Val Leu Gly Thr Lys Ala
1 5 10 15
Val Ile Gln Gly Leu Glu Thr Leu Arg Gly Glu His Arg Ala Leu Leu
20 25 30
Ala Pro Leu Val Ala Pro Glu Ala Gly Glu Ala Glu Pro Gly Ser Gln
35 40 45
Glu Arg Cys Ile Leu Leu Arg Arg Ser Leu Glu Ala Ile Glu Leu Gly
50 55 60
Leu Gly Glu Ala Gln Val Ile Leu Ala Leu Ser Ser His Leu Gly Ala
65 70 75 80
Val Glu Ser Glu Lys Gln Lys Leu Arg Ala Gln Val Arg Arg Leu Val
85 90 95
Gln Glu Asn Gln Trp Leu Arg Glu Glu Leu Pro Gly Thr Gln Xaa Lys
100 105 110
Leu Gln Arg Ser Glu Gln Ala Val Ala Gln Leu Glu Glu Glu Lys Gln
115 120 125
CA 02371006 2001-10-22
WO 00/65340 PCT/US00/10651
8
His Leu Leu Phe Met Xaa Gln Ile Arg Ser Trp Met Lys Thr Pro Xaa
130 135 140
Leu Thr Arg Arg Arg Gly Thr Ser Pro Lys Thr His Trp Met Thr Cys
145 150 155 160
Ser Pro Met Arg Met Ser Arg Ala Gln Pro Leu Ala Gln Glu Glu Gly
165 170 175
Met Cys Leu Val Ser Met Gly Asp Thr Arg Ser Arg Pro Gly Ser Ala
180 185 190
Pro Cys Thr Thr Gly Asp Pro Ile Arg Leu Thr Gly Pro Leu Arg Gly
195 200 205
Ser Cys Ala Thr Leu Gln Ala Gly Thr Arg Arg Leu Glu Lys Thr Ser
210 215 220
Gly His Asp His Pro Asp Val Ala Thr Met Leu Asn Ile Leu Ala Leu
225 230 235 240
Val Tyr Arg Asp Gln Asn Lys Tyr Lys Giu Ala Ala His Leu Leu Asn
245 250 255
Asp Ala Leu Ala Ile Arg Glu Lys Thr Leu Gly Lys Asp His Pro Ala
260 265 270
Val Ala Ala Thr Leu Asn Asn Leu Ala Val Leu Tyr Ser Ala Glu
275 280 285
<210> 9
<211> 601
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(264)
<400> 9
att tca agg ggg ctg ctg tac ccc cag gca tgt gtc tgt ata tcg cac 48
Ile Ser Arg Gly Leu Leu Tyr Pro Gln Ala Cys Val Cys Ile Ser His
1 5 10 15
agg aag aag gaa agt aag gac att gcc agc aaa tat ctt aca tct cat 96
Arg Lys Lys Glu Ser Lys Asp Ile Ala Ser Lys Tyr Leu Thr Ser His
20 25 30
cag cct ata ctg tgt ctc ctg acc act cct aac tgc aaa gga tgc tgg 144
Gln Pro Ile Leu Cys Leu Leu Thr Thr Pro Asn Cys Lys Gly Cys Trp
35 40 45
gaa aaa aag agc att gta get ttt cca gcc tct gtg gta ggc gca gat 192
Glu Lys Lys Ser Ile Val Ala Phe'Pro Ala Ser Val Val Gly Ala Asp
50 55 60
aag gga tta gag ttg ggt gtt act gaa tca atg tat cag aca ctt ctc 240
Lys Gly Leu Glu Leu Gly Val Thr Glu Ser Met Tyr Gln Thr Leu Leu
65 70 75 80
agt cag get aga gcc aga ttt aac tagatttagc aggaaaagta tgtttctttc 294
Ser Gln Ala Arg Ala Arg Phe Asn
CA 02371006 2001-10-22
WO 00/65340 PCT/US00/10651
9
acctgcatgt aatgaaggaa atctatgtcc ttcatacact taataaacct gtaagtctct 354
actatgggca ggtactgtgc tagctagaca ttacaatgtg tgggggcaga cacaaagatg 414
ggaacagtag acactgggga accctagagg ggggaggttg ggagtagggg aagggttgaa 474
aaatgattgg gtactatgct cactacctgg gtgatgggat catttgtaca ccaaatgcca 534
gcaacacaca atttacccgt gtaacacacc ggcacgtgta ccccctgaac ctaagatgaa 594
agccgaa 601
<210> 10
<211> 88
<212> PRT
<213> Homo Sapiens
<400> 10
Ile Ser Arg Gly Leu Leu Tyr Pro Gln Ala Cys Val Cys Ile Ser His
1 5 10 15
Arg Lys Lys G1u Ser Lys Asp Ile Ala Ser Lys Tyr Leu Thr Ser His
20 25 30
Gln Pro Ile Leu Cys Leu Leu Thr Thr Pro Asn Cys Lys Gly Cys Trp
35 40 45
Glu Lys Lys Ser Ile Val Ala Phe Pro Ala Ser Val Val Gly Ala Asp
50 55 60
Lys Gly Leu Glu Leu Gly Val Thr Glu Ser Met Tyr Gln Thr Leu Leu
65 70 75 80
Ser Gln Ala Arg Ala Arg Phe Asn
