Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
ASSAYS FOR NUCLEAR RECEPTOR LIGANDS USING FRET
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional
Application No. 60/061,385, filed 10/7/97, the contents of which are
incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
This invention relates to methods of identifying novel
agonists and antagonists of nuclear receptors utilizing the agonist-
dependent interaction of such receptors with CREB-binding protein
(CBP) or other nuclear receptor co-activators in which this interaction is
detected by fluorescence resonance energy transfer.
BACKGROUND OF THE INVENTION
Nuclear receptors are a superfamily of ligand-activated
transcription factors that bind as homodimers or heterodimers to their
cognate DNA elements in gene promoters. The superfamily, with more
than 150 members, can be divided into subfamilies (e.g. the steroid,
retinoid, thyroid hormone, and peroxisome proliferator-activated
[PPAR] subfamilies). Each subfamily may consist of several members
which are encoded by individual genes (e.g. PPARa, PPAR~y, and
PPARB). In addition, alternative mRNA splicing can result in more
than one isoform of these genes as in the case of specific PPARs (e.g.
PPAR~y1 and PPAR~y2). The nuclear receptor superfamily is involved in
a wide variety of physiological functions in mammalian cells: e.g.,
differentiation, proliferation, and metabolic homeostasis. Dysfunction
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or altered expression of specific nuclear receptors has been found to be
involved in disease pathogenesis.
The PPAR subfamily of nuclear receptors consists of three
members: PPARa, PPARy, and PPARB. PPARa is highly expressed in
S liver and kidney. Activation of PPARa by peroxisome proliferators
(including hypolipidimic reagents such as fibrates) or medium and
long-chain fatty acids is responsible for the induction of acyl-CoA
oxidase and hydratase-dehydrogenase (enzymes required for
peroxisomal ~i-oxidation), as well as cytochrome P450 4A6 (an enzyme
required for fatty acid w-hydroxylase). Thus, PPARa has an important
role in the regulation of lipid metabolism and is part of the mechanism
through which hypolipidimic compounds such as fibrates exert their
effects. PPARY is predominantly expressed in adipose tissue. Recently,
a prostaglandin J2 metabolite, 15-Deoxy-D12,14-prostaglandin J2, has
been identified as a potential physiological ligand of PPAR~y. Both 15-
Deoxy-D12,14-prostaglandin J2 treatment of preadipocytes or retroviral
expression of PPAR~y2 in fibroblasts induced adipocyte differentiation,
demonstrating the role of PPAR~y in adipocyte differentiation and lipid
storage. The demonstration that anti-diabetic and lipid-lowering
insulin sensitizing compounds known as thiazolidinediones are high
affinity ligands for PPARy suggests a broad therapeutic role for PPAR~y
ligands in the treatment of diabetes and disorders associated with
insulin resistance (e.g. obesity and cardiovascular disease).
Nuclear receptor proteins contain a central DNA binding
domain (DBD) and a COOH-terminal ligand binding domain (LBD). The
DBD is composed of two highly conserved zinc fingers .that target the
receptor to specific promoter/enhancer DNA sequences known as
hormone response elements (HREs). The LBD is about 200-300 amino
acids in length and is less well conserved than the DBD. There are at
least three functions for the LBD: dimerization, ligand binding, and
transactivation. The transactivation function can be viewed as a
molecular switch between a transcriptionally inactive and a
transcriptionally active state of the receptor. Binding of a ligand which
is an agonist flips the switch from the inactive state to the active state.
The COON-terminal portion of the LBD contains an activation function
domain (AF2)~ that is required for the switch.
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The ligand-induced nuclear receptor molecular switch is
mediated through interactions with members of a family of nuclear
receptor co-activators (e.g., CBP/p300, SRC-1/NcoA-1, TIF2/GRIP-
1/NcoA-2, and p/CIP). Upon binding of agonist to its cognate receptor
LBD, a conformational change in the receptor protein creates a co-
activator binding surface and results in recruitment of co-activator(s) to
the receptor and subsequent transcriptional activation. The binding of
antagonist ligands to nuclear receptors will not induce the required
conformational change and prevents recruitment of co-activator and
subsequent induction of transcription. The co-activators CREB-binding
protein (CBP) and p300 are two closely related proteins that were
originally discovered by virtue of their ability to interact with the
transcription factor CREB. These two proteins share extensive amino
acid sequence homology. CBP can form a bridge between nuclear
receptors and the basic transcriptional machinery (Kamei et al., 1996,
Cell 85:403-414; Chakravarti et al., 1996, Nature 383:99-103; Hanstein et
al., 1996, Proc. Natl. Acad. Sci. USA 93:11540-11545; Heery et al., 1997,
Nature 387:733-736). CBP also contains intrinsic histone
acetyltransferase activity which could result in local chromatin
rearrangement and further activation of transcription. Ligand- and
AF2-dependent interaction between certain nuclear receptors and CBP
has been demonstrated in in vitro pull down assays and far-western
assays. This interaction is both necessary and sufficient for the
transcriptional activation that is mediated by these nuclear receptors.
Thus, an AF2 mutant of the estrogen receptor (ER) which abolishes the
transcriptonal function of the receptor is incapable of interacting with
CBP.
The N-terrmini of CBP and p300 have been shown to interact
with the ligand-binding domains of some nuclear receptors (Kamei et
al., 1996, Cell 85:403-414, hereinafter "Kamei"). Kamei was able to
demonstrate direct interaction of CBP and p300 with nuclear receptors
by several different methods:
(1) Kamei produced GST fusion proteins of the first 100
amino acids of the N-terminus of CBP. These fusion proteins were run
out on a polyacrylamide gel, transferred to a membrane, and the
membrane was exposed to 32P-labeled ligand-binding domains of
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nuclear receptors. In the presence of ligand, a specific binding
interaction between the CBP and nuclear receptor fragments was
detected in that the 32P-labeled ligand-binding domains were observed to
bind to the bands on the membrane containing the GST-CBP fusion
proteins.
(2) Kamei also utilized the yeast two-hybrid system. The
ligand-binding domain of the nuclear receptor fused to the DNA-binding
domain of the LexA protein was used as bait. The amino terminal
domain of CBP fused to the gal4 transactivation domain was used as
prey. In the presence of ligand, a specific binding interaction (occurring
in viuo, i.e., within the yeast) was observed between the CBP and nuclear
receptor fragments.
(3) Kamei observed ligand-induced binding between CBP
and nuclear receptors via a gel-shift assay. This assay is based on the
observation that, in the presence of ligand, nuclear receptors will bind to
oligonucleotides containing their target recognition sequence. Such
binding results in the formation of a nuclear receptor-ligand-
oligonucleotide complex having a higher molecular weight than the
oligonucleotide alone. This difference in molecular weight is detected
via a shift in position of the 32P-labeled oligonucleotide when it is run out
on a polyacrylamide gel. Kamei found that a fragment of CBP (the N-
terminal 100 amino acids) was capable of binding to the nuclear
receptor-ligand-oligonucleotide complex and shifting the complex's
position on the gel to an even higher molecular weight.
(4) Kamei was able to co-immunoprecipitate CBP using
antibodies to nuclear receptors in extracts from a variety of cells in the
presence of ligand.
(5) By the use of transcriptional activation assays, Kamei
was able to demonstrate that nuclear receptors and CBP interact in a
functional manner. Such transcriptional activation assays can indicate
that two proteins are involved in a pathway that results in
transcriptional activation but these assays do not prove that the
interaction between the proteins is one of direct binding.
By the above-described methods, Kamei was able to
demonstrate specific binding interactions between CBP and the retinoic
acid receptor (RAR), glucocorticoid receptor (GR), thyroid hormone
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receptor (T3R), and retinoid X receptor (RXR). Kamei also demonstrated
specific binding between the N-terminus of p300 and RAR. However,
Kamei did not demonstrate specific binding between CBP, p300, or any
other nuclear receptor co-activators and PPARs.
What is striking about the methods used by Kamei is their
extremely laborious and time consuming nature. Such methods
involve, among other things, the construction of fusion proteins, the
preparation of 32P-labeled proteins, the construction of specialized
expression vectors for the yeast two-hybrid assay and the transcriptional
activation assays, the running of many gels, and the raising of
antibodies. Most of these assays take days to carry out and preparing the
reagents needed to carry them out may take weeks. Because of the
complicated reagents that are involved in these assays and the time
needed to prepare and run the assays, these assays tend to be costly.
Investigators other than Kamei who have studied the interaction
between nuclear receptors and CBP have also been forced to rely on such
cumbersome methods (see, e.g., Chakravarti et al., 1996, Nature 383:99-
103; Hanstein et al., 1996, Proc. Natl. Acad. Sci. USA 93:11540-11545;
Heery et al., 1997, Nature 387:733-736).
Kamei did not use the above-described methods to identify
novel agonists or antagonists of nuclear receptors. The focus of Kamei
was not on agonists or antagonists, but rather on the interaction
between nuclear receptors and CBP. Although modifying the methods
of Kamei to identify agonists or antagonists might be possible, such
methods would suffer from serious disadvantages. This is because, as
discussed above, all of the assays employed by Kamei to study the
interaction of CBP and p300 with nuclear receptors are very laborious,
slow, and costly. Given the therapeutic importance of steroid hormones
such as estrogen, cortisol, progesterone, and other nuclear receptor
agonists such as thyroid hormone and antidiabetic thiazolidinedione
compounds, the need for improved high-throughput screening assays to
identify potential pharmaceutical compounds affecting nuclear
receptors is clear. Historically, therapeutically useful nuclear receptor
ligand compounds were identified by screening animal models, an
approach which is even more labor intensive and time consuming than
the methods used by Kamei. Also, approaches such as those used by
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Kamei are ill-suited for the identification of antagonists of nuclear
receptors. It is now widely appreciated that antagonists of nuclear
receptors can be valuable therapeutic agents. Examples of such
therapeutically useful antagonists are tamoxifene, raloxifene, and RU-
486.
What is needed is a high throughput, time and labor-
saving, non-radioactive, inexpensive, and very reliable assay for the
identification and characterization of both agonists and antagonists of
nuclear receptors. Such an assay is provided by the present invention.
SUMMARY OF THE INVENTION
The present invention provides novel methods of identifying
agonists and antagonists of nuclear receptors. The methods take
advantage of the agonist-dependent binding of nuclear receptors and
CBP, p300, or other nuclear receptor co-activators. In the absence of
agonist, binding between the nuclear receptor and CBP, p300, or other
nuclear receptor co-activators does not occur. If agonist is present,
however, such binding occurs and can be detected by fluorescence
resonance energy transfer (FRET) between a fluorescently-labeled
nuclear receptor and fluorescently-labeled CBP, p300, or other nuclear
receptor co-activator. Antagonists can be identified by virtue of their
ability to prevent or disrupt the agonist-induced interaction of nuclear
receptors and CBP, p300, or other nuclear receptor co-activators. In
contrast to prior art methods of identifying agonists and antagonists of
nuclear receptors, the methods of the present invention, are simple,
rapid, and less costly.
The present invention provides a nuclear receptor or ligand
binding domain thereof labeled with a fluorescent reagent for use in the
above-described methods of identifying agonists and antagonists of
nuclear receptors. The present invention also provides CBP, p300, or
other nuclear receptor co-activator, or a binding portion thereof, labeled
with a fluorescent reagent.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates a method of fluorescently labelling a
protein or polypeptide with Europium cryptate (Eu3+K).
S Figure 2 illustrates the format for experiments 1 and 2 of
Table 1.
Figure 3 illustrates the format for experiment 3 of
Table 1.
Figure 4 illustrates the format for experiment 4 of
Table 1.
Figure 5 shows the results of studies using the methods of
the present invention with four known PPARY agonists. --o-- = AD5075;
--O-- = Fioglitazone; --X -- = Troglitazone; --0-- = BRL49653.
Figure 6 shows a measurement of the binding constant for
the interaction between hCBP and PPAR~yILBD.
Figure 7A shows the amino acid sequence of human CBP
(SEQ.ID.NO.:1).
Figure 7B shows the nucleotide sequence of a cDNA
encoding human CBP (SEQ.ID.N0.:2). The open reading frame is at
positions 76-1290.
Figure 8A shows the amino acid sequence of human
PPARa (SEQ.ID.N0.:3).
Figure 8B shows the nucleotide sequence of a cDNA
encoding human PPARa (SEG.~.ID.N0.:4). The open reading frame is at
positions 217-1623.
Figure 9A shows the amino acid sequence of human
PPAR~yl (SEQ.ID.N0.:5).
Figure 9B shows the nucleotide sequence of a cDNA
encoding human PPAR~y1 (SEfa.ID.N0.:6). The open reading frame is at
positions I73-1609.
Figure l0A shows the amino acid sequence of human
PPARB (SEQ.ID.N0.:7).
Figure lOB-C shows the nucleotide sequence of a cDNA
encoding human PPARB (SEQ.ID.N0.:8). The open reading frame is at
positions 338-1663.
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DETAILED DESCRIPTION OF THE INVENTION
For the purposes of this invention:
- an "agonist" is a substance that binds to nuclear receptors
in such a way that a specific binding interaction between the nuclear
receptor and CBP or other nuclear receptor co-activator can occur.
- an "antagonist" is a substance that is capable of preventing
or disrupting the agonist-induced specific binding interaction between a
nuclear receptor and CBP, p300, or another nuclear receptor co-
activator.
- a "ligand" of a nuclear receptor is an agonist or an
antagonist of the nuclear receptor.
- a "specific binding interaction," "specific binding," and
the like, refers to binding between a nuclear receptor and CBP, p300, or
other nuclear receptor co-activator which results in the occurrence of
fluorescence resonance energy transfer between a fluorescent reagent
bound to the nuclear receptor and a fluorescent reagent bound to CBP,
p300, or other nuclear receptor co-activator.
With respect to CBP, p300, or other nuclear receptor co-
activators, a "binding portion" is that portion of CBP, p300, or other
nuclear receptor co-activators that is sufficient for specific binding
interactions with nuclear receptors.
With respect to nuclear receptors, a "ligand binding
domain" is that portion of a nuclear receptor that is sufficient to bind an
agonist or antagonist of the nuclear receptor.
The present invention provides a high throughput, time
and labor-saving, non-radioactive, inexpensive, and very reliable assay
for the identification and characterization of both agonists and
antagonists of nuclear receptors. In a general embodiment, the present
invention provides methods of identifying agonists and antagonists for
any nuclear receptor for which CBP, p300, or another nuclear receptor
binding protein is a co-activator. Such agonists and antagonists are
identified by virtue of their ability to induce or prevent binding between
the ligand binding domain of a nuclear receptor and CBP, p300, or other
nuclear receptor co-activator. The interaction between the nuclear
receptor and CBP, p300, or other nuclear receptor co-activator is
monitored by observing the occurrence of fluorescence resonance energy
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transfer (FRET) between two fluorescent reagents. One fluorescent
reagent is bound to the nuclear receptor; the other fluorescent reagent is
bound to CBP, p300, or other nuclear receptor co-activator. The binding
of fluorescent reagent to nuclear receptor, CBP, p300, or other nuclear
receptor co-activator can be by a covalent linkage or a non-covalent
linkage.
The present invention makes use of fluorescence resonance
energy transfer (FRET). FRET is a process in which energy is
transferred from an excited donor fluorescent reagent to an acceptor
fluorescent reagent by means of intermolecular long-range dipole-dipole
coupling. FRET typically occurs over distances of about lOb to 100 and
requires that the emission spectrum of the donor reagent and the
absorbance spectrum of the acceptor reagent overlap adequately and that
the quantum yield of the donor and the absorption coe~cient of the
acceptor be su~ciently high. In addition, the transition dipoles of the
donor and acceptor fluorescent reagents must be properly oriented
relative to one another. For a review of FRET and its applications to
biological systems, see Clegg, 1995, Current Opinions in Biotechnology
6:103-110.
The present invention makes use of a nuclear receptor or
ligand binding domain thereof labeled with a first fluorescent reagent
and CBP, p300, or other nuclear receptor co-activator, or a binding
portion thereof, labeled with a second fluorescent reagent. The second
fluorescent reagent comprises a fluorophore capable of undergoing
energy transfer by either (a) donating excited state energy to the first
fluorescent reagent, or (b) accepting excited state energy from the first
fluorescent reagent. In other words, according to the present invention,
either the first or the second fluorescent reagents can be the donor or the
acceptor during FRET.
The first and second fluorescent reagents are
spectropscopically complementary to each other. This means that their
spectral characteristics are such that excited state energy transfer can
occur between them. FRET is highly sensitive to the distance between
the first and second fluorescent reagents. For example, FRET varies
inversely with the sixth power of the distance between the first and
second fluorescent reagents. In the absence of agonist, the first
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fluorescent reagent, bound to the nuclear receptor or ligand binding
domain thereof, will not be near the second fluorescent reagent, bound to
CBP, p300, or other nuclear receptor co-activator, or binding portion
thereof. Thus, no FRET, or very little FRET, will be observed. In the
presence of agonist, however, interaction between the nuclear receptor
and CBP, p300, or other nuclear receptor co-activator will occur, thus
bringing close together the first and the second fluorescent reagents,
allowing FRET to occur and be observed.
Accordingly, the present invention provides a method of
identifying an agonist of a nuclear receptor that comprises providing:
(a) a nuclear receptor or ligand binding domain thereof
labeled with a first fluorescent reagent;
(b) CBP, p300, or other nuclear receptor co-activator, or a
binding portion thereof, labeled with a second fluorescent reagent; and
(c) a substance suspected of being an agonist of the
nuclear receptor;
under conditions such that, if the substance is an agonist of
the nuclear receptor, binding between the nuclear receptor or ligand
binding domain thereof and CBP, p300, or other nuclear receptor co-
activator, or a binding portion thereof, will occur; and
(d) measuring fluorescence resonance energy transfer
(FRET) between the first and second fluorescent reagents;
where the occurrence of FRET indicates that the substance
is an agonist of the nuclear receptor.
In particular embodiments, the nuclear receptor is selected
from the group consisting of steroid receptors, thyroid hormone
receptors, retinoic acid receptors, peroxisome proliferator-activated
receptors, retinoid X receptors, glucocorticoid receptors, vitamin D
receptors, and "orphan nuclear receptors" such as LXR, FXR, etc.
In a particular embodiment, the nuclear receptor or ligand
binding domain thereof is a full-length nuclear receptor. In another
embodiment, the nuclear receptor or ligand binding domain thereof is a
ligand binding domain of a nuclear receptor. In another embodiment,
the nuclear receptor or ligand binding domain thereof comprises an AF-
2 site of a nuclear receptor.
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In a particular embodiment, the nuclear receptor or ligand
binding domain thereof is a full-length PPAR. In another embodiment,
the nuclear receptor or ligand binding domain thereof is the ligand
binding domain of a PPAR. In a further embodiment, the PPAR is
selected from the group consisting of PPARa, PPARyl, PPARy2, and
PPARB. In a further embodiment, the ligand binding domain of the
PPAR contains amino acid residues 176-478 of human PPARyl.
In a particular embodiment, the nuclear receptor or ligand
binding domain thereof contains amino acids 143-462 of human RARa.
In another embodiment, the nuclear receptor or ligand binding domain
thereof contains amino acids 122-410 of rat T3Ral. In another
embodiment, the nuclear receptor or ligand binding domain thereof
contains amino acids 227-463 of mouse RXRy. In another embodiment,
the nuclear receptor or ligand binding domain thereof contains amino
acids 251-595 of human ER.
In a particluar embodiment, the above-described methods
utilize full-length CBP, either mouse or human. In other embodiments,
the methods utilize amino acid residues 1-113 of human CBP. In
another embodiment, the methods utilize amino acid residues 1-453 of
human CBP.
The conditions under which the methods described above
are carried out are conditions that are typically used in the art for the
study of protein-protein interactions: e.g., physiological pH; salt
conditions such as those represented by such commonly used buffers as
PBS; a temperature of about 4°C to about 55°C. The presence
of
commonly used non-ionic detergents, e.g., NP-40~, sarcosyl, Triton X-
100~, is optional. When europium cryptates are used as fluorescent
reagents, reactions should contain KF at a concentration of at least 200
mM.
Heery et al., 1997, Nature 387:733-736 showed that
interactions between nuclear receptors and a variety of nuclear receptor
co-activators are mediated by a short amino acid sequence in the nuclear
receptor co-activators having the amino acid sequence LXXLL, where L
is leucine and X represents any amino acid. Accordingly, the present
invention can be practiced with a binding portion of a nuclear receptor
co-activator, provided that the binding portion contains the amino acid
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sequence LXXLL. Therefore, the present invention includes a method of
identifying an agonist of a nuclear receptor that comprises providing:
(a) a nuclear receptor or ligand binding domain thereof
labeled with a first fluorescent reagent;
(b) a binding portion of a nuclear receptor co-activator,
where the binding portion contains the amino acid sequence LXXLL,
and where the binding portion is labeled with a second fluorescent
reagent; and
(c) a substance suspected of being an agonist of the
nuclear receptor;
under conditions such that, if the substance is an agonist of
the nuclear receptor, binding between the nuclear receptor or ligand
binding domain thereof and the binding portion of the nuclear receptor
co-activator will take place; and
(d) measuring fluorescence resonance energy transfer
(FRET) between the first and second fluorescent reagents;
where the occurrence of FRET indicates that the substance
is an agonist of the nuclear receptor.
In a particular embodiment, the nuclear receptor co-
activator is selected from the group consisting of human RIP-140,
human SRC-1, mouse TIF-2, human or mouse CBP, human or mouse
p300, mouse TIF-1, and human TRIP proteins.
In a particular embodiment, the nuclear receptor co-
activator is human RIP-140 and the binding portion includes a
contiguous stretch of amino acids of human RIP-140 selected from the
group consisting of positions 20-29,132-139,184-192, 266-273, 379-387,
496-506, 712-719, 818-825, 935-944, and 935-942.
In another embodiment, the nuclear receptor co-activator is
human SRC-1 and the binding portion includes a contiguous stretch of
amino acids of human SRC-1 selected from the group consisting of
positions 45-53, 632-640, 689-696, ?48-755, and 1434-1441.
In another embodiment, the nuclear receptor co-activator is
mouse TIF-2 and the binding portion includes a contiguous stretch of
amino acids of mouse TIF-2 selected from the group consisting of:
positions 640-650, 689-699, and 744-754.
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In another embodiment, the nuclear receptor co-activator is
human or mouse CBP and the binding portion includes a contiguous
stretch of amino acids of human or mouse CBP selected from the group
consisting of positions 68-78 and 356-366.
In another embodiment, the nuclear receptor co-activator is
human or mouse p300 and the binding portion includes a contiguous
stretch of amino acids of human or mouse p300 selected from the group
consisting of positions 80-90 and 341-351.
In another embodiment, the nuclear receptor co-activator is
mouse TIF-1 and the binding portion includes a contiguous stretch of
amino acids of mouse TIF-1 containing positions ?22-732.
In another embodiment, the nuclear receptor co-activator is
human TRIP2 and the binding portion includes a contiguous stretch of
amino acids of human TRIP2 containing positions 23-33.
In another embodiment, the nuclear receptor co-activator is
human TRIPS and the binding portion includes a contiguous stretch of
amino acids of human TRIPS containing positions 97-107.
In another embodiment, the nuclear receptor co-activator is
human TRIP4 and the binding portion includes a contiguous stretch of
amino acids of human TRIP4 containing positions 36-46.
In another embodiment, the nuclear receptor co-activator is
human TRIPS and the binding portion includes a contiguous stretch of
amino acids of human TRIPS containing positions 26-36.
In another embodiment, the nuclear receptor co-activator is
human TRIP8 and the binding portion includes a contiguous stretch of
amino acids of human TRIP8 containing positions 36-46.
In another embodiment, the nuclear receptor co-activator is
human TRIPS and the binding portion includes a contiguous stretch of
amino acids of human TRIPS selected from the group consisting of
positions 73-83, 256-266 and 288-298.
For amino acid sequences of nuclear receptor co-activators,
see Yao et al., 1996, Proc. Natl. Acad. Sci. USA 93:10626-10631 (SRC-1);
O~ate et al., 1995, Science 270:1354-1357 (SRC-1); Cavaill~s et al., 1995,
EMBO J. 14:3741-3751 (RIP-140); Voegel et al., 1996, EMBO J. 15:101-108
(TIF-2); Kwok et al., 1994, Nature 370:223-226 (CBP); Arias et al., 1994,
Nature 370:226-229 (CBP); Eckner et al., 1994, Genes Dev. 8:869-884
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(p300); Le Douarin et al., 1995, EMBO J. 14:2020-2033 (TIF-1); Lee et al.,
1995, Nature 3?4:91-94 (TRIP proteins).
The particular embodiments of the present invention
described above are all particular embodiments of a more general
S method that is also part of the present invention. That general method
is a method of identifying an agonist of a nuclear receptor that
comprises providing:
(a) a nuclear receptor or ligand binding domain thereof
labeled with a first fluorescent reagent;
(b) a polypeptide containing the amino acid sequence
LXXLL where the polypeptide is labeled with a second fluorescent
reagent; and
(c) a substance suspected of being an agonist of the
nuclear receptor;
under conditions such that, if the substance is an agonist of
the nuclear receptor, binding between the nuclear receptor or ligand
binding domain thereof and the polypeptide will take place; and
(d) measuring fluorescence resonance energy transfer
(FRET) between the first and second fluorescent reagents;
where the occurrence of FRET indicates that the substance
is an agonist of the nuclear receptor.
In a particular embodiment, the amino acid sequence
LXXLL is present in an a helical portion of the polypeptide. In another
embodiment, the amino acid sequence LXXLL is present in an a helical
portion of the polypeptide and the leucines form a hydrophobic face.
The present invention provides methods for identifying
antagonists of a nuclear receptor. Such methods are based on the ability
of the antagonist to prevent the occurrence of agonist-induced binding
between a nuclear receptor and CBP, p300, or other nuclear receptor co-
activator, or to disrupt such binding after it has occurred. Thus, the
present invention provides a method for identifying antagonists of
nuclear receptors that comprises providing:
(a) a nuclear receptor or ligand binding domain thereof
labeled with a first fluorescent reagent;
(b) CBP, p300, or other nuclear receptor co-activator, or a
binding portion thereof, labeled with a second fluorescent reagent;
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(c) an agonist of the nuclear receptor; and
(d) a substance suspected of being an antagonist of the
nuclear receptor;
under conditions such that, in the absence of the substance,
binding between the nuclear receptor or ligand binding domain thereof
and CBP, p300, or other nuclear receptor co-activator, or a binding
portion thereof will occur; and
(e) measuring fluorescence resonance energy transfer
(FRET) between the first and second fluorescent reagents when the
substance is present and measuring FRET between the first and second
fluorescent reagents when the substance is absent;
where the a decrease in FRET when the substance is
present indicates that the substance is an antagonist of the nuclear
receptor.
In particular embodiments, the nuclear receptor is selected
from the group consisting of steroid receptors, thyroid hormone
receptors, retinoic acid receptors, peroxisome proliferator-activated
receptors, retinoid X receptors, glucocorticoid receptors, vitamin D
receptors, and "orphan nuclear receptors" such as LXR, FXR, etc.
In a particular embodiment, the nuclear receptor or ligand
binding domain thereof is a full-length nuclear receptor. In another
embodiment, the nuclear receptor or ligand binding domain thereof is a
ligand binding domain of a nuclear receptor. In another embodiment,
the nuclear receptor or ligand binding domain thereof is an AF-2 site of
a nuclear receptor.
In a particular embodiment, the nuclear receptor or ligand
binding domain thereof is a full-length PPAR. In another embodiment,
the nuclear receptor or ligand binding domain thereof is the ligand
binding domain of a PPAR. In a further embodiment, the PPAR is
selected from the group consisting of PPAR,a, PPAR,~y, and PPAR8. In a
further embodiment, the ligand binding domain of the PPAR contains
amino acid residues 176-47$ of human PPAR,Yl.
In a particular embodiment, the nuclear receptor or ligand
binding domain thereof contains amino acids 143-462 of human RAR,a.
In another embodiment, the nuclear receptor or ligand binding domain
thereof contains amino acids 122-410 of rat T3Ral. In another
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WO 99/18124 PCT/US98/21049
embodiment, the nuclear receptor or ligand binding domain thereof
contains amino acids 227-463 of mouse RxR,~. In another embodiment,
the nuclear receptor or ligand binding domain thereof contains amino
acids 251-595 of human ER.
In a particular embodiment, the above-described methods
utilize full-length CBP, either mouse or human. In other embodiments,
the methods utilize amino acid residues 1-113 of human CBP. In
another embodiment, the methods utilize amino acid residues 1-453 of
human CBP.:
The conditions under which the methods described above
are carried out are conditions that are typically used in the art for the
study of protein-protein interactions: e.g., physiological pH; salt
conditions such as those represented by such commonly used buffers as
PBS; a temperature of about 4°C to about 55°C. The presence
of
commonly used non-ionic detergents, e.g., NP-400, sarcosyl, Triton X
1000, is optional. When europium cryptates are used as fluorescent
reagents, reactions should contain KF at a concentration of at least 200
mM.
In principle, one could measure FRET by monitoring either
(a) a decrease in the emission of the donor fluorescent reagent following
stimulation at the donor's absorption wavelength and/or (b) an increase
in the emission of the acceptor reagent following stimulation at the
donor's absorption wavelength. In practice, FRET is most effectively
measured by emission ratioing. Emission ratioing monitors the change
in the ratio of emission by the acceptor over emission by the donor. An
increase in this ratio signifies that energy is being transferred from
donor to acceptor and thus that FRET is occurring. Emission ratioing
can be measured by employing a laser-scanning confocal microscope.
Emission ratioing is preferably done by splitting the emitted light from a
sample with a dichroic mirror and measuring two wavelength bands
(corresponding to the donor and the acceptor emission wavelengths)
simultaneously with two detectors. Alternatively, the emitted light can
be sampled consecutively at each wavelength (by using appropriate
filters) with a single detector. In any case, these and other methods of
3 S measuring FRET are well known in the art.
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Although a variety of donor and acceptor fluorescent
reagents can be used in the practice of the present invention, preferred
embodiments of the present invention make use of cryptates of
fluorescent reagents as donor reagents. Inclusion of a substrate into the
intramolecular cavity of a macropolycyclic ligand results in the
formation of a cryptate. The macropolycyclic ligand shields the
substrate from interaction with solvent and other solute molecules. If
the substrate is a fluororescent reagent, formation of a cryptate may
result in markedly different spectroscopic characteristics for the reagent
as compared to the spectroscopic characteristics of the free reagent.
The present invention includes the use of europium (Eunl)
or terbium (Tbul) cryptates as donor fluorescent reagents. Such Eu~ or
Tbnl cryptates, as well as methods for their formation, are well known
in the art. For example, see Alpha et al., 1987, Angew. Chem. Int. Ed.
Engl. 26:266-267; Mathis, 1995, Clin. Chem. 41:1391-1397. A europium
cryptate is formed by the inclusion of a europium ion into the
intramolecular cavity of a macropolycyclic ligand which contains
bipyridine groups as light absorbers. When europium cryptates are
present in solution together with fluoride ions, a total shielding of the
europium cryptate fluorescence is occurs. The molecular structure of a
europium cryptate is shown below.
NH2 NH2
C2H4 C2H4
NH NH
O=C, .C=O
- 17-
~=N N=C
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WO 99/18124 PCT/US98/21049
Europium cryptates can be conjugated to proteins by the use
of well-known heterobifunctional reagents (see, e.g., International
Patent Application WO 89/05813; Prat et al., 1991, Anal. Biochem.
195:283-289; Lopez et al., 1993, Clin. Chem. 39:196-201 ).
The present invention includes the use of XL665 as the
acceptor fluorescent reagent. XL665 is a crosslinked derivative of
allophycocyanin (APC). APC is a porphyrin containing protein which is
derived from the light harvesting system of algae (Kronick, 1986, M.
Immunol. Meth. 92:1-13). XL665 has an absorption maximum at =620
nm and an emission maximum at 665 nm. In some embodiments of the
invention, XL665 is labeled with streptavidin in order to eil'ect the
binding of the streptavidin-labled XL665 to a biotin-labeled substance,
e.g. , CBP or the ligand binding domain of a nuclear receptor.
Streptavidin labeling of XL655 and biotin labeling of CBP, or the ligand
binding domain of a nuclear receptor, can be performed by well known
methods.
In a preferred embodiment of the invention, XL665 as the
acceptor fluorescent reagent is combined with Europium cryptate
(Eu3+K) as the donor fluorescent reagent. Europium cryptate (Eu3+K)
has a large Stokes shift, absorbing light at 337 nm and emitting at 620
nm. Thus, the emission maximum of Europium cryptate (Eu3+K)
overlaps the absorption maximum of XL665. Europium cryptate
(Eu3+K) has a large temporal shift; the time between absorption and
emission of a photon is about 1 millisecond. This is advantageous
because most background fluorescence signals in biological samples are
short-lived. Thus the use of a fluorescent reagent such as europium
cryptate, with a long fluorescent lifetime, permits time-resolved
detection resulting in the reduction of background interference.
The spectral and temporal properties of europium cryptate
(Eu3+K) result in essentially no fluorescence background and thus
assays using this fluorescent reagent can be carried out in a "mix and
read" mode, greatly facilitating its use as a high throughput screening
tool. For the embodiment using Europium cryptate (Eu3+K) and XL665,
the measuring instrument irradiates the sample at 337 nm and
measures the fluorescence output at two wavelengths, 620 nm (B counts,
europium fluorescence) and 665 nm (A counts, XL665 fluorescence).
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The extent of flurorescent resonance energy transfer is measured as the
ratio between these two values. Typically this ratio is multiplied by
10,000 to give whole numbers.
Other FRET donor-acceptor pairs are suitable for the
practice of the present invention. For example, the following donor-
acceptor pairs can be used: dansyl/fluorescein; fluorescein/rhodamine;
tryptophan/aminocoumarin.
The present invention provides a nuclear receptor or ligand
binding domain thereof labeled with a fluorescent reagent for use in the
above-described methods of identifying agonists and antagonists of
nuclear receptors. The present invention also provides CBP, p300, or
other nuclear receptor co-activator, or a binding portion thereof, labeled
with a fluorescent reagent.
In a particular embodiment, the nuclear receptor or ligand
binding domain thereof is selected from the group consisting of PPARa,
PPARy, PPAR8, a ligand binding domain of PPARa, PPAR~y, or PPARB,
and amino acid residues 176-478 of human PPAR71 and the fluorescent
reagent is selected from the group consisting of XL665 and Europium
cryptate (Eu3+K).
In a particular embodiment, CBP, p300, or other nuclear
receptor co-activator is labeled with a fluorescent reagent selected from
the group consisting of XL665 and Europium cryptate (Eu3+K).
The following non-limiting examples are presented to better
illustrate the invention.
EXAMPLE 1
To test whether human CBP can interact with PPARs in an
agonist-dependent manner, we cloned the human cDNA fragments
encoding the NH2-terminal 1-113 amino acids (hCBPl-113) and 1-453
amino acids (hCBPl-453) of human CBP by the polymerase chain
reaction (PCR). The DNA and amino acid sequences of human CBP are
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WO 99/18124 PCT/US98/21049
disclosed in Borrow et al., 1996, Nature Genet. 14:33-41 and in GenBank,
accession no. U47741.
The primers used for hCBPl-113 were:
5'-ACTCGGATCCAAGCCATGGCTGAGAACTTGCTGGACGG-3'
(SEQ.ID.N0.:9) and
5'-CACAAAGCTTAGGCCATGTTAGCACTGTTCGG-3' (SEQ.ID.NO.:
10).
These primers were expected to amplify a 0.9 kb DNA fragment.
The primers for hCBPl-453 were:
5'-ACTCGGATCCAAGCCATGGCTGAGAACTTGCTGGACGG-3'
(SEQ.ID.N0.:9) and
5'CTCAGTCGACTTATTGAATTCCACTAGCTGGAGATCC-3'
(SEQ.ID.NO.:11).
These primers were expected to amplify a 1.5 kb DNA fragment..
The template for the PCR reaction was a human fetal brain
cDNA library (Stratagene, Catalogue #IS 937227). Of course, any
human cDNA library from a tissue expressing CBP could have been
used. The PCR amplified 0.9 kb and 1.5 kp DNA fragments which were
digested with restriction endonucleases and ligated into pBluescript II
vector. DNA sequencing analysis confirmed that the amplified
fragments were identical to the corresponding published nucleic acid
sequences of human CBP.
Based on the publicly available sequences for human CBP
cited above, other primers could be readily identified and prepared by
those skilled in the art in order to amplify and clone other portions of
cDNA encoding human CBP from appropriate cDNA libraries. Once
such portions of human CBP are produced, they could be used in the
methods of the present invention in a manner similar to that described
herein for hCBPl-113 and hCBPl-453. The amino acid sequence of
human CBP is shown in Figure 7A; the nucleic acid sequence of the
cDNA encoding human CBP is shown in Figure 7B.
To express the polypeptides encoded by the PCR fragments,
vectors encoding fusion proteins of the polypeptides and glutathione S-
transferase (GST) were constructed and expressed in E. coli. The PCR
fragments were subcloned into the expression vector pGEX (Pharmacia
Biotech) to generate pGEXhCBPl-113 and pGEXhCBPl-453.
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pGEXhCBPl-113 and pGEXhCBPI-453 were transfected into the DHSa
strain of E. coli (GIBCO BRL) and the bacteria hosting either
pGEXhCBPl-113 or pGEXhCBPl-453 were cultured in LB medium
(GIBCO BRL) to a density of OD6pp = 0.7-1.0 and induced for
overexpression of the GST-CBP fusion proteins by addition of IPTG
(isopropylthio-~3-galactoside) to a final concentration of 0.2 mM. The
IPTG induced cultures were further grown at room temperature for 2-5
hrs. The cells were harvested by centrifugation for 10 min at 5000g. The
cell pellet was used for GST-CBP fusion protein purification by following
the procedure from Pharmacia Biotech using Glutathione Sepharose
beads. hCBPI-113 and hCBPl-453 proteins were generated by cleaving
the corresponding GST fusion proteins with thrombin. SDS-
polyacrylamide gel electrophoresis analysis showed that the preparation
from pGEXhCBPl-113 gave two polypeptide bands, with apparent
molecular weight of 12 kd and 10 kd. The 12 kd band is the expected size
of hCBPl-113 and the 10 kd band is most likely a premature translational
termination product. The preparation from pGEXhCBPI-450 gave a
single band with the expected size, 50 kd.
cDNAs encoding full-length PPARa and PPAR~yl were
subcloned into pGEX vectors for the production of GST-PPARa and GST-
PPARyl fusion proteins in E.coli. PPAR~yI was cloned from a human fat
cell cDNA library (see Elbrecht et al., 1996, Biochem. Biophys. Res.
Comm. 224:431-437). A cDNA encoding the human PPARYl ligand
binding domain (PPARyILBD; amino acids 176-478 of PPAR~yl) was
subcloned from a modified pSGS vector as a Xho I (site located in the N-
terminus of the LBD)/ Xba I (site located in the pSG5 vector) fragment.
The Xba I site was blunt-ended with T4 DNA polymerase. The 1.1 kb
fragment containing the LBD was purified from an agarose gel and
ligated into pGEX-KG (see Guan & Dixon, 1991, Anal. Biochem. 192:262-
267) that had been digested with Xho I and Hind III (the Hind III site
had been blunt-ended with T4 DNA polymerase). This construct was
used for the production of GST-hPPARyILBD and hPPARyILBD (the
ligand binding domain cleaved free of GST). The overexpression and
purification of PPARa, PPAR~yl, and PPAR~yILBD were as described
above for CBP.
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WO 99/18124 PCT/US98/21049
The DNA and amino acid sequences of human PPARa are
disclosed in Schmidt et al., 1992, Mol. Endocrinol. 6:1634-1641 and in
GenBank, accession no. L07592. See Fieure 8A and 8B.
The DNA and amino acid sequences of human PPARyl are
disclosed in Greene et al., 1995, Gene Expr. 4:281-299; Qi et al., 1995, Mol.
Cell. Biol. 15:1817-1825; Elbrecht et al.,1996, Biochem. Biophys. Res.
Comm. 224:431-437; and in GenBank, accession no. L40904. See Figure
9A and 9B. Human PPARy2 contains the same amino acid sequence as
human PPARyl except for an amino terminal addition of 24 amino acids
(see Elbrecht et al., 1996, Biochem. Biophys. Res. Comm. 224:431-437).
Thus, the amino acid sequence of the ligand binding domain of human
PPARy2 is the same as the amino acid sequence of the ligand binding
domain of human PPARyl, although the numbering of the amino acids
differs (176-478 for human PPARyl and 200-502 for human PPARy2).
The DNA and amino acid sequences of human PPARB are
disclosed in Sher et al., 1993, Biochemistry 32:5598-5604 and in GenBank,
accession no. L02932. See Figure l0A-C.
EXAMPLE 2
Interaction between PPARs and hCBP fra n s
Experiments were first conducted using hCBPl-113 and
hPPARyILBD. Purified hPPARyILBD was biotinylated with Sulfo-NHS-
LC-Biotin (PIERCE) to a biotin:hPPARyILBD ratio of 3:1 according to the
procedure provided by PIERCE. Purified hCBPl-113 was directly labeled
with europium cryptate (Eu3+K) by the method illustrated in Figure 1.
Biotin-labeled hPPARyILBD, Eu3+K-labeled hCBPl-113, and
streptavidin-labeled XL665 (SA-XL665; from PACKARD) were incubated
together in the presence or absence of 1 ~,M of known PPARy agonist
(BRL49653 or AD5075).
Thus, this experimental format made use of the fluorescent
reagent pair europium cryptate (Eu3+K), which acted as donor, and
XL665, which acted as acceptor. hCBPl-113 was directly labeled with
europium cryptate (Eu3+K); hPPARyILBD was indirectly labeled with
XL665 by means of a biotin-streptavidin link. The emission maximum
CA 02305711 2000-04-06
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of europium cryptate (Eu3+K) overlaps with the absorption maximum of
XL665. Therefore, when europium cryptate (Eu3+K) and XL665 are in
close proximity, and the sample is illuminated with light at 337 nm (the
absorption maximum of europium cryptate (Eu3+K)), FRET can occur
between europium cryptate (Eu3+K) and XL665. This FRET manifests
itself as increased emission at 665 nm by XL665. Figure 2 shows a
schematic of the format used in this experiment (experiment 1 of Table
1). When agoniat is bound to hPPAR~yILBD, a specific interaction occurs
between hPPAR~yILBD and hCBPl-113, thus bringing europium cryptate
(Eu3+K) and XL665 into close enough proximity for FRET to occur. In
the absence of agonist, no interaction occurs between hPPAR~yILBD and
hCBPl-113 and thus europium cryptate (Eu3+K) and XL665 are not
brought into close proximity and no FRET occurs. When FRET occurs,
the amount of light given off by the sample at the emission maximum of
XL665 (665 nm) is increased relative to the amount of light given off by
the sample at the emission maximum of europium cryptate {Eu3+K)
(620 nm). Therefore, measuring the ratio of emission at 665 nm to 620
nm in the presence and the absence of a substance suspected of being an
agonist allows for the determination of whether that substance actually
is an agonist. If the substance is an agoniat, an increase in the ratio of
emission at 665 nm to 620 nm in the presence of the substance will be
ob served.
Reactions were carried out in microtiter plates. Reaction
conditions were: appropriate volume (total 250 ~1) of the reaction buffer
(either PBS or HEPES, see below, containing 500 mM KF, 0.1% bovine
serum albumin, BSA) was added to each well, followed by addition of
ligands (BRL49653 or AD5075 at a final concentration of 1 ~,M and 0.1%
dimethylaulfoxide (DMSO) or vehicle control (0.1% DMSO), Eu3+K
labeled hCBP (100 nM), biotin-hPPAR~yILBD (100 nM), and streptavidin-
labeled XL665 (100 nM) to appropriate wells. After mixing, 200 ~.1 of
reaction mixture was transferred to a new well. The plate was either
directly measured for fluorescence resonance energy transfer (FRET) or
covered with sealing tape (PACKARD) to avoid evaporation and
incubated at room temperature for up to 24 hrs before measuring FRET.
The results of this experiment and others described below
yielded ratio values as follows:
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Table 1
Experiment Buffer Emission ratioEmission ratio
with AD5075 with vehicle
1 PBS 1134 1074
2 HEPES + 0.05% 967 617
NP40
3 HEPES + 0.05% 1078 536
NP40
4 HEPES + 0.05% 1883 487
CHAPS
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Experiment 1 of Table 1 was carried out using PBS ( 13? mM
NaCl, 2.7 mM KCl, 4.3 mM Na2HP04, 1.4 mM KH~04, pH 7.4). The
greater emission ratio observed in the presence of AD5075 demonstrated
that a specific interaction between hCBPl-113 and hPPARyILBD
occurred in the presence of the agonist AD5075. Although it was clear
that FRET was occurring, the signal-noise ratio was small. In
experiment 2 of Table 1, HEPES buffer (N-2-hydroxyethylpiperazine-N'-
2-ethane sulfonic acid, 100 mM, pH 7.0) containing 0.05% NP40 (Nonidet
P-40) was used instead of PBS and an improved signal-noise ratio was
obtained.
In order to get an even better signal-noise ratio, the above-
described format was modified slightly for experiment 3. In experiment
3, SA-XL665 {500 nM), biotin-labeled hPPARyILBD (100 nM), GST-
hCBPl-113, and Eu3+K labeled anti-GST antibody (2.5 ~.l) were incubated
in the presence or absence of AD5075 (1 ~t.M) in HEPES buffer containing
0.05% NP40. A two-fold signal- noise ratio was obtained. Figure 3 shows
a schematic of the format used in experiment 3.
The anti-GST antibody was a goat antibody to GST from
Pharmacia (catalogue number 27-4577-O1) that was labeled with Eu3+K
according to the procedure summarized below.
- Make up @ 10
mg/mL in H20.
Need 42.2 ug (4.2
pL, 96.6 nmol) for
49.0 ~tg Eu3+
Reagent
O
O
N.O N
Na03S ~ O
Eus* NH2 O
FW = 436.4
-Resuspended Qa -FW = 1465 2.9 Equiv SULFO-SMCC,
2.5 mg/mL in 10% Use 49.0 pg 20 mM Pi buffer, 10% DMF
DMF/PBS (19.6 pL, 33.4 nmol) RT, 30 minutes
_~r_
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O
O
Eu~* H \
N
O
equiv. Eu3+
complex
1 ) Add Eu reagent
O 2) [Pr] 4.2 mg/mL.
Onight at 4°C.
~ti-csT Ant~t~ay. N ~ S. S N~
f cat # 27-as77-of ~ H I Lower pH: Add 12 pL of
1 M NaPi, pH 7Ø
pH drops to 7.18.
350 pM TCEP
(35 mM stock is
10.0 mg/mL- PBS, pH
7.0), 2.4 pL,
min rt then 15 min
4°C
O
S O
N S' ~ ~N
~t~-csT ~t~i~ay, N H
Cat # 27-4577-01 2 (>
O
From Pharmacia, 5.0 mg/mL, 5.0 Equiv SPDP, FW =312, Dissolve
FW = 150 kD Use 200 pL (1 mg, RT, 5 hours @ 1.00 mg/mL in EtOH.
6.66 nmol) exchange into 10 mM Add 10.4 pL (5 equiv.,
Borate, 350 mM NaCI, 10% Gly, 10.4
pH 8.5 with BioSpin-30 rotei~n.~ 33.4 nmol) to
P
To further improve the signal to noise ratio, a series of
experiments were conducted. Experiment 4 of Table 1 exemplifies
5 results obtained from those efforts. cDNA encoding a longer fragment of
hCBP was cloned and expressed to get hCBPl-453. hCBPl-453 was
biotinylated. Biotin-labeled hCBPl-453 (25 nM), SA-XL665 (100 nM),
GST-hPPAR~yILBD (1 nM), and Eu3+K-labeled anti-GST antibody (2 nM)
were mixed together in the presence or absence of 1 ~,M AD5075. The
10 detergent was changed from 0.05% NP40 to 0.5% CHAPS (3-~[3-
cholamidopropyl]dimethyl-ammoniol}-1-propanesulfonate). A three- to
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four-fold signal-noise ratio was obtained. Figure 4 shows the strategy
used for experiment 4 and similar experiments.
The correlation between results from the above-described
assays and previously reported results from in aitro binding and
transcriptional activation assays of selected antidiabetic insulin
sensitizers that are known to be PPARy agonists (Elbrecht et al., 1996,
Biochem Biophys Res Comm 224:431-437} was analyzed by titrating those
known PPARy agonists in the assays described above and comparing
ECSOs so obtained with previously described values for potency in
binding or transcriptional activation assays for the known agoniats. The
results are shown in Figure 5. From Figure 5, the following ECSps can
be derived:
AD5075 = 8 nM
BRL49653 = 53 nM
Troglitazone = 646 nM
Pioglitazone = 890 nM.
These ECSOs generated in the above-described assays are in close
agreement with those generated by in vitro binding and transcriptional
activation studies (Elbrecht et al., 1996, Biochem Biophys Res Comm
224:431-437).
The above-described assay can also be used to characterize
the interaction between nuclear receptors with co-activators as, e.g., by
determining the binding constant for that interaction. Figure 6 shows
an example of such an application. Saturating amounts of PPARy
agonist (10 ~.M BRL49653) were used. Increasing concentrations of non-
biotinylated hCBPl-453 were used to titrate away biotin-hCBP-
PPAR~yILBD complex and decrease the fluorescence energy transfer. A
Kd of 300 nM for the interaction between hCBPl-453 and PPAR~yILBD
can be derived from the results illustrated in Figure 6 and this Kd (300
nM) is a measurement ofthe affinity between CBP and PPAR~y.
The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various modifications
of the invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description. Such
modifications are intended to fall within the scope of the appended
claims.
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Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Merck & Co., Inc.
(ii) TITLE OF INVENTION: ASSAYS FOR NUCLEAR RECEPTOR
AGONISTS AND ANTAGONISTS USING FLUORESCENCE RESONANCE
ENERGY TRANSFER
(iii) NUMBER OF SEQUENCES: 11
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Merck & Co., Inc.
(B) STREET: P.O. Box 2000, 126 E. Lincoln Ave.
(C) CITY: Rahway
(D) STATE: NJ
(E) COUNTRY: USA
(F) ZIP: 07065-0900
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: Windows
(D) SOFTWARE: FastSEQ for Windows Version 2.Ob
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Coppola, Joseph A
(B) REGISTRATION NUMBER: 38,413
(C) REFERENCE/DOCKET NUMBER: 20017PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 732-594-6734
(B) TELEFAX: 732-594-4720
(C) TELEX:
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 405 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-1_
CA 02305711 2000-04-06
WO 99/18124 PCTNS98/21049
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Met Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys Arg Ala Lys
1 5 10 15
Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Ser Thr Asp Phe Gly Ser
20 25 30
Leu Phe Asp Leu Glu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly
35 40 45
Gly Glu Leu Gly Leu Leu Asn Ser Gly Asn Leu Val Pro Asp Ala Ala
50 55 60
Ser Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser Gly Ser
65 70 75 g0
Ser Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln
85 90 95
Gln Gly Leu Gly Gly Gln Ala Gln Gly Gln Pro Asn Ser Ala Asn Met
100 105 110
Ala Ser Leu Ser Ala Met Gly Lys Ser Pro Leu Ser Gln Gly Asp Ser
115 120 125
Ser Ala Pro Ser Leu Pro Lys Gln Ala Ala Ser Thr Ser Gly Pro Thr
130 135 140
Pro Ala Ala Ser Gln Ala Leu Asn Pro Gln Ala Gln Lys Gln Val Gly
145 150 155 160
Leu Ala Thr Ser Ser Pro Ala Thr Ser Gln Thr Gly Pro Gly Ile Cys
165 170 275
Met Asn Ala Asn Phe Asn Gln Thr His Pro Gly Leu Leu Asn Ser Asn
180 185 190
Ser Gly His Ser Leu Ile Asn Gln Ala Ser Gln Gly Gln Ala Gln VaI
195 200 205
Met Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met
210 215 220
Pro Tyr Pro Thr Pro Ala Met Gln Gly Ala Ser Ser Ser Val Leu Ala
225 230 235 240
Glu Thr Leu Thr Gln Val Ser Pro Gln Met Thr Gly His Ala Gly Leu
245 250 255
Asn Thr Ala Gln Ala Gly Gly Met Ala Lys Met Gly Ile Thr Gly Asn
260 265 270
Thr Ser Pro Phe Gly Gln Pro Phe Ser Gln Ala Gly Gly Gln Pro Met
275 280 285
Gly Ala Thr Gly Val Asn Pro Gln Leu Ala Ser Lys Gln Ser Met Val
290 295 300
Asn Ser Leu Pro Thr Phe Pro Thr Asp Ile Lys Asn Thr Ser Val Thr
305 310 315 320
Asn Val Pro Asn Met Ser Gln Met Gln Thr Ser Val Gly Ile Val Pro
325 330 335
Thr Gln Ala Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys
340 345 350
Leu Ile Gln Gln Gln Leu Val Leu Leu Leu His Ala His Lys Cys Gln
355 360 365
Arg Arg Glu Gln Ala Asn Gly Glu Val Arg Ala Cys Ser Leu Pro His
370 375 380
Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gln Ala
385 390 395 400
Gly Lys Ala Cys Gln
405
-2-
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WO 99!18124 PCT/US98/21049
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1290 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
CGAGCCCCGACCCCCGTCCG GGCCGCGCCGCCCGTGCCCG GGGCTGTTTT60
GGCCCTCGCC
CCCGAGCAGGTGAAAATGGCTGAGAACTTGCTGGACGGACCGCCCAACCC CAAAAGAGCC120
AAACTCAGCTCGCCCGGTTTCTCGGCGAATGACAGCACAGATTTTGGATC ATTGTTTGAC180
TTGGAAAATGATCTTCCTGATGAGCTGATACCCAATGGAGGAGAATTAGG CCTTTTAAAC240
AGTGGGAACCTTGTTCCAGATGCTGCTTCCAAACATAAACAACTGTCGGA GCTTCTACGA300
GGAGGCAGCGGCTCTAGTATCAACCCAGGAATAGGAAATGTGAGCGCCAG CAGCCCCGTG360
CAGCAGGGCCTGGGTGGCCAGGCTCAAGGGCAGCCGAACAGTGCTAACAT GGCCAGCCTC420
AGTGCCATGGGCAAGAGCCCTCTGAGCCAGGGAGATTCTTCAGCCCCCAG CCTGCCTAAA480
CAGGCAGCCAGCACCTCTGGGCCCACCCCCGCTGCCTCCCAAGCACTGAA TCCGCAAGCA540
CAAAAGCAAGTGGGGCTGGCGACTAGCAGCCCTGCCACGTCACAGACTGG ACCTGGTATC600
TGCATGAATGCTAACTTTAACCAGACCCACCCAGGCCTCCTCAATAGTAA CTCTGGCCAT660
AGCTTAATTAATCAGGCTTCACAAGGGCAGGCGCAAGTCATGAATGGATC TCTTGGGGCT720
GCTGGCAGAGGAAGGGGAGCTGGAATGCCGTACCCTACTCCAGCCATGCA GGGCGCCTCG780
AGCAGCGTGCTGGCTGAGACCCTAACGCAGGTTTCCCCGCAAATGACTGG TCACGCGGGA840
CTGAACACCGCACAGGCAGGAGGCATGGCCAAGATGGGAATAACTGGGAA CACAAGTCCA900
TTTGGACAGCCCTTTAGTCAAGCTGGAGGGCAGCCAATGGGAGCCACTGG AGTGAACCCC960
CAGTTAGCCAGCAAACAGAGCATGGTCAACAGTTTGCCCACCTTCCCTAC AGATATCAAG1020
AATACTTCAGTCACCAACGTGCCAAATATGTCTCAGATGCAAACATCAGT GGGAATTGTA1080
CCCACACAAGCAATTGCAACAGGCCCCACTGCAGATCCTGAAAAACGCAA ACTGATACAG1140
CAGCAGCTGGTTCTACTGCTTCATGCTCATAAGTGTCAGAGACGAGAGCA AGCAAACGGA1200
GAGGTTCGGGCCTGCTCGCTCCCGCATTGTCGAACCATGAAAAACGTTTT GAATCACATG1260
ACGCATTGTCAGGCTGGGAAAGCCTGCCAA 1290
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 468 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Val Asp Thr Glu Ser Pro Leu Cys Pro Leu Ser Pro Leu Glu Ala
1 5 10 15
Gly Asp Leu Glu Ser Pro Leu Ser Glu Glu Phe Leu Gln Glu Met Gly
20 25 30
Asn Ile Gln Glu Ile Ser Gln Ser Ile Gly Glu Asp Ser Ser Gly Ser
35 40 45
Phe Gly Phe Thr Glu Tyr Gln Tyr Leu Gly Ser Cys Pro Gly Ser Asp
50 55 60
-3-
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Gly Ser Val Ile Thr Asp Thr Leu Ser Pro Ala Ser Ser Pro Ser Ser
65 70 75 80
Val Thr Tyr Pro Val Val Pro Gly Ser Val Asp Glu Ser Pro Ser Gly
85 90 g5
Ala Leu Asn Ile Glu Cys Arg Ile Cys Gly Asp Lys Ala Ser Gly Tyr
100 105 110
His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg
115 120 125
Thr Ile Arg Leu Lys Leu Val Tyr Asp Lys Cys Asp Arg Ser Cys Lys
130 135 140
Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys Arg Phe His Lys
145 _ 150 155 160
Cys Leu Ser Val Gly Met Ser His Asn Ala Ile Arg Phe Gly Arg Met
165 170 175
Pro Arg Ser Glu Lys Ala Lys Leu Lys Ala Glu Ile Leu Thr Cys Glu
180 185 190
His Asp Ile Glu Asp Ser Glu Thr Ala Asp Leu Lys Ser Leu Ala Lys
195 200 205
Arg Ile Tyr Glu Ala Tyr Leu Lys Asn Phe Asn Met Asn Lys Val Lys
210 215 220
Ala Arg Val Ile Leu Ser Gly Lys Ala Ser Asn Asn Pro Pro Phe Val
225 230 235 240
Ile His Asp Met Glu Thr Leu Cys Met Ala Glu Lys Thr Leu Val Ala
245 250 255
Lys Leu Val Ala Asn Gly Ile Gln Asn Lys Glu Val Glu Val Arg Ile
260 265 270
Phe His Cys Cys Gln Cys Thr Ser Val Glu Thr Val Thr Glu Leu Thr
275 280 285
Glu Phe Ala Lys Ala Ile Pro Ala Phe Ala Asn Leu Asp Leu Asn Asp
290 295 300
Gln Val Thr Leu Leu Lys Tyr Gly Val Tyr Glu Ala Ile Phe Ala Met
305 310 315 320
Leu Ser Ser Val Met Asn Lys Asp Gly Met Leu Val Ala Tyr Gly Asn
325 330 335
Gly Phe Ile Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Cys
340 345 350
Asp Ile Met Glu Pro Lys Phe Asp Phe Ala Met Lys Phe Asn Ala Leu
355 360 365
Glu Leu Asp Asp Ser Asp Ile Ser Leu Phe Val Ala Ala Ile Ile Cys
370 375 380
Cys Gly Asp Arg Pro Gly Leu Leu Asn Val Gly His Ile Glu Lys Met
385 390 395 400
Gln Glu Gly Ile Val His Val Leu Arg Leu His Leu Gln Ser Asn His
405 410 415
Pro Asp Asp Ile Phe Leu Phe Pro Lys Leu Leu Gln Lys Met Ala Asp
420 425 430
Leu Arg Gln Leu Val Thr Glu His Ala Gln Leu Val Gln Ile Ile Lys
435 440 445
Lys Thr Glu Ser Asp Ala Ala Leu His Pro Leu Leu Gln Glu Ile Tyr
450 455 460
Arg Asp Met Tyr
465
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
-4-
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(A) LENGTH: 1854 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GGCCCAGGCTGAAGCTCAGG GCCCTGTCTG CTCTGTGGACTCAACAGTTT 60
GTGGCAAGAC
AAGCTCAGAACTGAGAAGCT GTCACCACAG TTCTGGAGGCTGGGAAGTTCAAGATCAAAG120
TGCCAGCAGATTCAGTGTCA TGTGAGGACG TGCTTCCTGCTTCATAGATAAGAGTAGCTT180
GGAGCTCGGCGGCACAACCA GCACCATCTG GTCGCGATGGTGGACACGGAAAGCCCACTC240
TGCCCCCTCTCCCCACTCGA GGCCGGCGAT CTAGAGAGCCCGTTATCTGAAGAGTTCCTG300
CAAGAAATGGGAAACATCCA AGAGATTTCG CAATCCATCGGCGAGGATAGTTCTGGAAGC360
TTTGGCTTTACGGAATACCA GTATTTAGGA AGCTGTCCTGGCTCAGATGGCTCGGTCATC420
ACGGACACGCTTTCACCAGC TTCGAGCCCC TCCTCGGTGACTTATCCTGTGGTCCCCGGC480
AGCGTGGACGAGTCTCCCAG TGGAGCATTG AACATCGAATGTAGAATCTGCGGGGACAAG540
GCCTCAGGCTATCATTACGG AGTCCACGCG TGTGAAGGCTGCAAGGGCTTCTTTCGGCGA600
ACGATTCGACTCAAGCTGGT GTATGACAAG TGCGACCGCAGCTGCAAGATCCAGAAAAAG660
AACAGAAACAAATGCCAGTA TTGTCGATTT CACAAGTGCCTTTCTGTCGGGATGTCACAC720
AACGCGATTCGTTTTGGACG AATGCCAAGA TCTGAGAAAGCAAAACTGAAAGCAGAAATT780
CTTACCTGTGAACATGACAT AGAAGATTCT GAAACTGCAGATCTCAAATCTCTGGCCAAG840
AGAATCTACGAGGCCTACTT GAAGAACTTC AACATGAACAAGGTCAAAGCCCGGGTCATC900
CTCTCAGGAAAGGCCAGTAA CAATCCACCT TTTGTCATACATGATATGGAGACACTGTGT960
ATGGCTGAGAAGACGCTGGT GGCCAAGCTG GTGGCCAATGGCATCCAGAACAAGGAGGTG1020
GAGGTCCGCATCTTTCACTG CTGCCAGTGC ACGTCAGTGGAGACCGTCACGGAGCTCACG1080
GAATTCGCCAAGGCCATCCC AGCGTTCGCA AACTTGGACCTGAACGATCAAGTGACATTG1140
CTAAAATACGGAGTTTATGA GGCCATATTC GCCATGCTGTCTTCTGTGATGAACAAAGAC1200
GGGATGCTGGTAGCGTATGG AAATGGGTTT ATAACTCGTGAATTCCTAAAAAGCCTAAGG1260
AAACCGTTCTGTGATATCAT GGAACCCAAG TTTGATTTTGCCATGAAGTTCAATGCACTG1320
GAACTGGATGACAGTGATAT CTCCCTTTTT GTGGCTGCTATCATTTGCTGTGGAGATCGT1380
CCTGGCCTTCTAAACGTAGG ACACATTGAA AAAATGCAGGAGGGTATTGTACATGTGCTC1440
AGACTCCACCTGCAGAGCAA CCACCCGGAC GATATCTTTCTCTTCCCAAAACTTCTTCAA1500
AAAATGGCAGACCTCCGGCA GCTGGTGACG GAGCATGCGCAGCTGGTGCAGATCATCAAG1560
AAGACGGAGTCGGATGCTGC GCTGCACCCG CTACTGCAGGAGATCTACAGGGACATGTAC1620
TGAGTTCCTTCAGATCAGCC ACACCTTTTC CAGGAGTTCTGAAGCTGACAGCACTACAAA1680
GGAGACGGGGGAGCAGCACG ATTTTGCACA AATATCCACCACTTTAACCTTAGAGCTTGG1740
ACAGTCTGAGCTGTAGGTAA CCGGCATATT ATTCCATATCTTTGTTTTAACCAGTACTTC1800
TAAGAGCATAGAACTCAAAT GCTGGGGGAG GTGGCTAATCTCAGGACTGGGAAG 1854
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 478 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Thr Met Val Asp Thr Glu Ile Ala Phe Trp Pro Thr Asn Phe Gly
1 5 10 15
-5-
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Ile Ser Ser Val Asp Leu Ser Val Met Glu Asp His Ser His Ser Phe
20 25 30
Asp Ile Lys Pro Phe Thr Thr Val Asp Phe Ser Ser Ile Ser Thr Pro
35 40 45
His Tyr Glu Asp Ile Pro Phe Thr Arg Thr Asp Pro Val Val Ala Asp
50 55 60
Tyr Lys Tyr Asp Leu Lys Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val
65 70 75 g0
Glu Pro Ala Ser Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn
85 90 95
Lys Pro His Glu Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg
100 105 110
Val Cys Gly Asp Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys
115 120 125
Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Ile
130 135 140
Tyr Asp Arg Cys Asp Leu Asn Cys Arg Ile His Lys Lys Ser Arg Asn
145 150 155 160
Lys Cys Gln Tyr Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser
165 170 175
His Asn Ala Ile Arg Phe Gly Arg Ile Ala Gln Ala Glu Lys Glu Lys
180 185 190
Leu Leu Ala Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser
195 200 205
Ala Asp Leu Arg Gln Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile
210 215 220
Lys Ser Phe Pro Leu Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly
225 230 235 240
Lys Thr Thr Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu
245 250 255
Met Met Gly Glu Asp Lys ile Lys Phe Lys His Ile Thr Pro Leu Gln
260 265 270
Glu Gln Ser Lys Glu Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe
275 280 285
Arg Ser Val Glu Ala Val Gln Glu Ile Thr Glu Tyr Ala Lys Ser Ile
290 295 300
Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys
305 310 315 320
Tyr Gly Val His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn
325 330 335
Lys Asp Gly Val Leu Ile Ser Glu Gly Gln Gly Phe Met Thr Arg Glu
340 345 350
Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys
355 360 365
Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp
370 375 380
Leu Ala Ile Phe Ile Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly
385 390 395 400
Leu Leu Asn Val Lys Pro Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln
405 410 415
Ala Leu Glu Leu Gln Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu
420 425 430
Phe Ala Lys Leu Leu Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr
435 440 445
Glu His Val Gln Leu Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met
450 455 460
-s-
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Ser Leu His Pro Leu Leu Gln Glu Ile Tyr Lys Asp Leu Tyr
465 470 475
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1811 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CCGACCTTACCCCAGGCGGCCTTGACGTTG CAGGAGACAGCACCATGGTG60
GTCTTGTCGG
GGTTCTCTCTGAGTCTGGGAATTCCCGAGCCCGAGCCGCAGCCGCCGCCTGGGGGGCTTG120
GGTCGGCCTCGAGGACACCGGAGAGGGGCGCCACGCCGCCGTGGCCGCAGAAATGACCAT180
GGTTGACACAGAGATCGCATTCTGGCCCACCAACTTTGGGATCAGCTCCGTGGATCTCTC240
CGTAATGGAAGACCACTCCCACTCCTTTGATATCAAGCCCTTCACTACTGTTGACTTCTC300
CAGCATTTCTACTCCACATTACGAAGACATTCCATTCACAAGAACAGATCCAGTGGTTGC360
AGATTACAAGTATGACCTGAAACTTCAAGAGTACCAAAGTGCAATCAAAGTGGAGCCTGC420
ATCTCCACCTTATTATTCTGAGAAGACTCAGCTCTACAATAAGCCTCATGAAGAGCCTTC480
CAACTCCCTCATGGCAATTGAATGTCGTGTCTGTGGAGATAAAGCTTCTGGATTTCACTA540
TGGAGTTCATGCTTGTGAAGGATGCAAGGGTTTCTTCCGGAGAACAATCAGATTGAAGCT600
TATCTATGACAGATGTGATCTTAACTGTCGGATCCACAAAAAAAGTAGAAATAAATGTCA660
GTACTGTCGGTTTCAGAAATGCCTTGCAGTGGGGATGTCTCATAATGCCATCAGGTTTGG720
GCGGATCGCACAGGCCGAGAAGGAGAAGCTGTTGGCGGAGATCTCCAGTGATATCGACCA780
GCTGAATCCAGAGTCCGCTGACCTCCGTCAGGCCCTGGCAAAACATTTGTATGACTCATA840
CATAAAGTCCTTCCCGCTGACCAAAGCAAAGGCGAGGGCGATCTTGACAGGAAAGACAAC900
AGACAAATCACCATTCGTTATCTATGACATGAATTCCTTAATGATGGGAGAAGATAAAAT960
CAAGTTCAAACACATCACCCCCCTGCAGGAGCAGAGCAAAGAGGTGGCCATCCGCATCTT1020
TCAGGGCTGCCAGTTTCGCTCCGTGGAGGCTGTGCAGGAGATCACAGAGTATGCCAAAAG1080
CATTCCTGGTTTTGTAAATCTTGACTTGAACGACCAAGTAACTCTCCTCAAATATGGAGT1140
CCACGAGATCATTTACACAATGCTGGCCTCCTTGATGAATAAAGATGGGGTTCTCATATC1200
CGAGGGCCAAGGCTTCATGACAAGGGAGTTTCTAAAGAGCCTGCGAAAGCCTTTTGGTGA1260
CTTTATGGAGCCCAAGTTTGAGTTTGCTGTGAAGTTCAATGCACTGGAATTAGATGACAG1320
CGACTTGGCAATATTTATTGCTGTCATTATTCTCAGTGGAGACCGCCCAGGTTTGCTGAA1380
TGTGAAGCCCATTGAAGACATTCAAGACAACCTGCTACAAGCCCTGGAGCTCCAGCTGAA1440
GCTGAACCACCCTGAGTCCTCACAGCTGTTTGCCAAGCTGCTCCAGAAAATGACAGACCT1500
CAGACAGATTGTCACGGAACACGTGCAGCTACTGCAGGTGATCAAGAAGACGGAGACAGA1560
CATGAGTCTTCACCCGCTCCTGCAGGAGATCTACAAGGACTTGTACTAGCAGAGAGTCCT1620
GAGCCACTGCCAACATTTCCCTTCTTCCAGTTGCACTATTCTGAGGGAAAATCTGACCAT1680
AAGAAATTTACTGTGAAAAAGCGTTTTAAAAAGAAAAGGGTTTAGAATATGATCTATTTT1740
ATGCATATTGTTTATAAAGACACATTTACAATTTACTTTTAATATTAAAAATTACCATAT1800
TATGAAATTGC 1811
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
-7-
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Met Glu Gln Pro Gln Glu Glu Ala Pro Glu Val Arg Glu Glu Glu Glu
1 5 10 15
Lys Glu Glu Val Ala Glu Ala Glu Gly Ala Pro Glu Leu Asn Gly Gly
20 25 30
Pro Gln His Ala Leu Pro Ser Ser Ser Tyr Thr Asp Leu Ser Arg Ser
35 40 45
Ser Ser Pro Pro Ser Leu Leu Asp Gln Leu Gln Met Gly Cys Asp Gly
50 55 60
Ala Ser Cys Gly Ser Leu Asn Met Glu Cys Arg Val Cys Gly Asp Lys
65 70 75 80
Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly
85 90 95
Phe Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Glu
100 105 110
Arg Ser Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys
115 120 125
Arg Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile Arg
130 135 140
Phe Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly Leu
145 150 155 160
Thr Ala Asn Glu Gly Ser Gln Tyr Asn Pro Gln Val Ala Asp Leu Lys
165 170 175
Ala Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met
180 185 190
Thr Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ala Ser His Thr
195 200 205
Ala Pro Phe Val Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys
210 215 220
Gly Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Lys Glu
225 230 235 240
Ile Ser Val His Val Phe Tyr Arg Cys Gln Cys Thr Thr Val Glu Thr
245 250 255
Val Arg Glu Leu Thr Glu Phe Ala Lys Ser Ile Pro Ser Phe Ser Ser
260 265 270
Leu Phe Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu
275 280 285
Ala Ile Phe Ala Met Leu Ala Ser Ile Val Asn Lys Asp Gly Leu Leu
290 295 300
Val Ala Asn Gly Ser Gly Phe Val Thr Arg Glu Phe Leu Arg Ser Leu
305 310 315 320
Arg Lys Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val
325 330 335
Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile
340 345 350
Ala Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro
355 360 365
Arg Val Glu Ala Ile Gln Asp Thr Ile Leu Arg Ala Leu Glu Phe His
370 375 380
Leu Gln Ala Asn His Pro Asp Ala Gln Tyr Leu Phe Pro Lys Leu Leu
385 390 395 400
Gln Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala Gln Met
405 410 415
_8_
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Met Gln Arg Ile Lys Lys Thr Glu Thr Glu Thr Ser Leu His Pro Leu
420 425 430
Leu Gln Glu Ile Tyr Lys Asp Met Tyr
435 440
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3301 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GAATTCTGCGGAGCCTGCGG TAGGCAGCCG 60
GACGGCGGCG GGACAGTGTT
GGTTGGCCCG
GTACAGTGTTTTGGGCATGCACGTGATACTCACACAGTGGCTTCTGCTCACCAACAGATG 120
AAGACAGATGCACCAACGAGGGTCTGGAATGGTCTGGAGTGGTCTGGAAAGCAGGGTCAG 180
ATACCCCTGGAAAACTGAAGCCCGTGGAGCAATGATCTCTACAGGACTGCTTCAAGGCTG 240
ATGGGAACCACCCTGTAGAGGTCCATCTGCGfiTCAGACCCAGACGATGCCAGAGCTATGA 300
CTGGGCCTGCAGGTGTGGCGCCGAGGGGAGATCAGCCATGGAGCAGCCACAGGAGGAAGC 360
CCCTGAGGTCCGGGAAGAGGAGGAGAAAGAGGAAGTGGCAGAGGCAGAAGGAGCCCCAGA 420
GCTCAATGGGGGACCACAGCATGCACTTCCTTCCAGCAGCTACACAGACCTCTCCCGGAG 480
CTCCTCGCCACCCTCACTGCTGGACCAACTGCAGATGGGCTGTGACGGGGCCTCATGCGG 540
CAGCCTCAACATGGAGTGCCGGGTGTGCGGGGACAAGGCATCGGGCTTCCACTACGGTGT 600
TCATGCATGTGAGGGGTGCAAGGGCTTCTTCCGTCGTACGATCCGCATGAAGCTGGAGTA 660
CGAGAAGTGTGAGCGCAGCTGCAAGATTCAGAAGAAGAACCGCAACAAGTGCCAGTACTG 720
CCGCTTCCAGAAGTGCCTGGCACTGGGCATGTCACACAACGCTATCCGTTTTGGTCGGAT 780
GCCGGAGGCTGAGAAGAGGAAGCTGGTGGCAGGGCTGACTGCAAACGAGGGGAGCCAGTA 840
CAACCCACAGGTGGCCGACCTGAAGGCCTTCTCCAAGCACATCTACAATGCCTACCTGAA 900
AAACTTCAACATGACCAAAAAGAAGGCCCGCAGCATCCTCACCGGCAAAG.CCAGCCACAC 960
GGCGCCCTTTGTGATCCACGACATCGAGACATTGTGGCAGGCAGAGAAGGGGCTGGTGTG 1020
GAAGCAGTTGGTGAATGGCCTGCCTCCCTACAAGGAGATCAGCGTGCACGTCTTCTACCG 1080
CTGCCAGTGCACCACAGTGGAGACCGTGCGGGAGCTCACTGAGTTCGCCAAGAGCATCCC 1140
CAGCTTCAGCAGCCTCTTCCTCAACGACCAGGTTACCCTTCTCAAGTATGGCGTGCACGA 1200
GGCCATCTTCGCCATGCTGGCCTCTATCGTCAACAAGGACGGGCTGCTGGTAGCCAACGG 1260
CAGTGGCTTTGTCACCCGTGAGTTCCTGCGCAGCCTCCGCAAACCCTTCAGTGATATCAT 1320
TGAGCCTAAGTTTGAATTTGCTGTCAAGTTCAACGCCCTGGAACTTGATGACAGTGACCT 1380
GGCCCTATTCATTGCGGCCATCATTCTGTGTGGAGACCGGCCAGGCCTCATGAACGTTCC 1440
ACGGGTGGAGGCTATCCAGGACACCATCCTGCGTGCCCTCGAATTCCACCTGCAGGCCAA 1500
CCACCCTGATGCCCAGTACCTCTTCCCCAAGCTGCTGCAGAAGATGGCTGACCTGCGGCA 1560
ACTGGTCACCGAGCACGCCCAGATGATGCAGCGGATCAAGAAGACCGAAACCGAGACCTC 1620
GCTGCACCCTCTGCTCCAGGAGATCTACAAGGACATGTACTAACGGCGGCACCCAGGCCT 1680
CCCTGCAGACTCCAATGGGGCCAGCACTGGAGGGGCCCACCCACATGACTTTTCCATTGA 1740
CCAGCTCTCTTCCTGTCTTTGTTGTCTCCCTCTTTCTCAGTTCCTCTTTCTTTTCTAATT 1800
CCTGTTGCTCTGTTTCTTCCTTTCTGTAGGTTTCTCTCTTCCCTTCTCCCTTCTCCCTTG 1860
CCCTCCCTTTCTCTCTCCTATCCCCACGTCTGTCCTCCTTTCTTATTCTGTGAGATGTTT 1920
TGTATTATTTCACCAGCAGCATAGAACAGGACCTCTGCTTTTGCACACCTTTTCCCCAGG 1980
AGCAGAAGAGAGTGGGCCTGCCCTCTGCCCCATCATTGCACCTGCAGGCTTAGGTCCTCA 2040
CTTCTGTCTCCTGTCTTCAGAGCAAAAGACTTGAGCCATCCAAAGAAACACTAAGCTCTC 2100
TGGGCCTGGGTTCCAGGGAAGGCTAAGCATGGCCTGGACTGACTGCAGCCCCCTATAGTC 2160
ATGGGGTCCCTGCTGCAAAGGACAGTGGCAGACCCCGGCAGTAGAGCCGAGATGCCTCCC 2220
CAAGACTGTCATTGCCCCTCCGATCGTGAGGCCACCCACTGACCCAATGATCCTCTCCAG 2280
CAGCACACCTCAGCCCCACTGACACCCAGTGTCCTTCCATCTTCACACTGGTTTGCCAGG 2340
-9-
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CCAATGTTGC TGATGGCCCC TCCAGCACAC ACACATAAGC ACTGAAATCA 2400
CTTTACCTGC
AGGCACCATG CACCTCCCTT CCCTCCCTGA GGCAGGTGAG AACCCAGAGA 2460
GAGGGGCCTG
CAGGTGAGCA GGCAGGGCTG GGCCAGGTCT CCGGGGAGGC AGGGGTCCTG 2520
CAGGTCCTGG
TGGGTCAGCC CAGCACCTCG CCCAGTGGGA GCTTCCCGGG ATAAACTGAG 2580
CCTGTTCATT
CTGATGTCCA TTTGTCCCAA TAGCTCTACT GCCCTCCCCT TCCCCTTTAC 2640
TCAGCCCAGC
TGGCCACCTA GAAGTCTCCC TGCACAGCCT CTAGTGTCCG GGGACCTTGT 2700
GGGACCAGTC
CCACACCGCT GGTCCCTGCC CTCCCCTGCT CCCAGGTTGA GGTGCGCTCA 2760
CCTCAGAGCA
GGGCCAAAGC ACAGCTGGGC ATGCCATGTC TGAGCGGCGC AGAGCCCTCC 2820
AGGCCTGCAG
GGGCAAGGGG CTGGCTGGAG TCTCAGAGCA CAGAGGTAGG AGAACTGGGG 2880
TTCAAGCCCA
GGCTTCCTGG GTCCTGCCTG GTCCTCCCTC CCAAGGAGCC ATTCTATGTG 2940
ACTCTGGGTG
GAAGTGCCCA GCCCCTGCCT GACGGNNNNN NNGATCACTC TCTGCTGGCA 3000
GGATTCTTCC
CGCTCCCCAC CTACCCAGCT GATGGGGGTT GGGGTGCTTC TTTCAGCCAA 3060
GGCTATGAAG
GGACAGCTGC TGGGACCCAC CTCCCCCCTT CCCCGGCCAC ATGCCGCGTC 3120
CCTGCCCCCA
CCCGGGTCTG GTGCTGAGGA TACAGCTCTT CTCAGTGTCT GAACAATCTC 3180
CAAAATTGAA
ATGTATATTT TTGCTAGGAG CCCCAGCTTC CTGTGTTTTT AATATAAATA 3240
GTGTACACAG
ACTGACGAAA CTTTAAATAA ATGGGAATTA AATATTTAAA AAAAAAAGCG 3300
GCCGCGAATT
C
3301
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ACTCGGATCC AAGCCATGGC TGAGAACTTG CTGGACGG 3g
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
CACAAAGCTT AGGCCATGTT AGCACTGTTC GG 32
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
-1~-
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
CTCAGTCGAC TTATTGAATT CCACTAGCTG GAGATCC
-11-
