Note: Descriptions are shown in the official language in which they were submitted.
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STRUCTURAL MODELS FOR CYTOPLASMIC
DOMAINS OF TRANSMEMBRANE RECEPTORS
Introduction
This invention was made in the course of research
sponsored by the National Institutes of Health. The U.S.
Government may have certain rights in this invention.
Background of the Invention
In eukaryotic cells, many proteins extend through the
cell membrane and therefore contain a cytoplasmic domain, a
transmembrane domain and an extracellular domain. Many of
these proteins are involved in signal transduction, cell
adhesion and cell-cell interactions.
Among the proteins that fall into this category are
the integrins. Integrins are involved in a number of
pathological and physiological processes, including
thrombosis, inflammation, and cancer. Other physiological
and pathological conditions involving changes in cell
adhesiveness are also mediated through integrins.
Many transmembrane proteins are oligomeric, being
noncovalent associations of two or more different types of
polypeptide subunits. In particular, integrins are
heterodimers of two different protein subunits, designated
a and ~. The a subunits vary in size between 120 and 180
kDa and are each noncovalently associated with a ~3 subunit.
The extracellular domain of the integrin molecule forms a
ligand binding site; both the a and (3 subunits are involved
in forming the ligand binding site. A number of different
ligands for integrins are known, including collagens,
laminin, fibronectin, vitronectin, complement components,
thrombospondin, and integral membrane proteins of the
immunoglobulin superfamily such as ICAM-1, ICAM-2, and
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VCAM-1. The integrins recognize various short peptide
sequences in their ligands. Examples of these are Arg-Gly-
Asp (RGD), Lys-Gln-Ala-Gly-Asp-Val (KQAGDV; SEQ ID NO: 1),
Asp-Gly-Glu-Ala (DGEA; SEQ ID NO: 2), and Glu-Ile-Leu-Asp-
Val (EILDV; SEQ ID NO: 3). Variations in integrin function
are often caused by changes in the ligand binding affinity
of the extracellular domain of the integrins (J. S. Bennett
& G. Vilaire J. Clin. Invest. 64:1393-1401 (1979); Altieri
et al. J. Cell Biol. 107:1893-1900 (1988); Faull et al. J.
Cell Biol. 121:155-162 (1993); Lollo et al. J. Biol. Chem.
268:21693-21700 (1993)).
Integrin allb(33 (platelet GPIIb-IIIa), a heterodimer of
two type I transmembrane protein subunits, manifests highly
regulated changes in ligand binding affinity. Affinity
state-specific antibodies, e.g., PACl (Shattil et al. J.
Biol. Chem. 260:1107-1114 (1985)), are useful for analysis
of recombinant allb(33 in heterologous cells (O'Toole et al.
Cell Regulation 1:883-893 (1990)). Platelet agonists
increase the affinity of allb(33 (activation) probably by
causing changes in the conformation of the extracellular
domain (O'Toole et al. Cell Regulation 1:883-893 (1990);
Sims et al. J. Biol. Chem. 266:7345-7352 (1991)).
Cytoplasmic signaling pathways involving heterotrimeric GTP
binding proteins, phospholipid metabolism, and serine-
threonine kinases initiate these conformational changes in
the extracellular domain; these changes may also involve
calcium fluxes, tyrosine kinases, and low molecular weight
GTP binding proteins (Sims et al. J. Biol. Chem. 266:7345-
7352 (1991); Shattil et al. J. Biol. Chem. 267: 18424-18431
(1992); S.J. Shattil & J.S. Brugge Curr. Opin. Cell Biol.
3:869-879 (1991); Ginsberg et al. Cold Spring Harbor
Symposium of Quantitative Biology: The Cell Surface
57:221-231 (1992); Ginsberg et al. Curr. Opin. Cell Biol.
4:766-771 (1992); Nemoto et al. J. Biol. Chem. 267:20916-
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20920 (1992)). How cytoplasmic signals result in changes
in the conformation and ligand binding affinity of the
extracellular domain ("inside-out signal transduction") of
the integrin remains unknown. Studies with chimeras
containing the cytoplasmic domains of various a and
subunits joined to the transmembrane and extracellular
domain of aIIbG3 indicate that integrin cytoplasmic domains
transduce cell type-specific signals that modulate ligand
binding affinity. These signals require active cellular
processes in both a and (3 cytoplasmic tails of the
integrin, suggesting that they reflect physiologically
relevant signals. In addition, deletion of a highly
conserved motif, Gly-Phe-Phe-Lys-Arg (GFFKR; SEQ ID NO: 4),
at the amino-terminus of the a subunit cytoplasmic domain,
also resulted in high affinity binding of ligands to
integrin aii~3. In contrast to the chimeras, high affinity
ligand binding to GFFKR deletion mutants was independent of
cellular metabolism, cell type, and the bulk of the
subunit cytoplasmic domain. Thus, integrin cytoplasmic
tails are targets for the modulation of integrin affinity.
However, technical difficulties have greatly limited
the application of high resolution techniques for
determination of the structures of these proteins. In
fact, molecular structures are available for only two
intact transmembrane proteins, a bacterial photoreaction
center (Deisenhofer et al. Nature 318:618-624 (1985)), and
a porin (Weiss et al. FEBS Lett. 267:268-272 (1990)).
Structures of receptor extracellular domains have been
determined using soluble truncated extracellular domains as
models (DeVos et al. Science 255:306-312 (1992); Milburn et
al. Science 254:1342-1347 (1991)). These structures have
contributed to the understanding of the basis of ligand
recognition, but have provided less insight into the
mechanism of signal transduction. Many membrane proteins
that transduce signals are members of the Type I
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transmembrane protein family, the defining feature of which
is a single membrane spanning region. These include the T
cell receptor (A. Weiss Cell 73:209-212 (1993)); growth
factor receptors (L. Patthy Cell 61:13-14 (1990)), and
cytokine receptors (Miyajima et al. TIBS 17:378-382
(1992)). In general, the cytoplasmic domain of these
proteins is critical for signaling. Thus, to understand
signal transduction through such receptors, it is essential
to understand the structure and function of the cytoplasmic
domain. This is especially difficult for multisubunit
Type I proteins.
A strategy for the chemical synthesis of structural
models of the cytoplasmic domain of multisubunit
transmembrane receptors has been previously proposed (Muir
et al. Biochemistry 33:7701 (1994)). The cytoplasmic
domains of integrin aiib(33 were covalently linked via a
helical coiled-coil made up of a series of identical heptad
repeats. Coiled-coil tertiary structure was utilized to
mimic the presumed helical membrane spanning domain and as
a topological constraint, fixing the two integrin tails in
a parallel orientation with the appropriate vertical
stagger (Muir et al. Biochemistry 33:7701 (1994)).
However, this synthetic approach poses limitations upon
the polypeptide length and has a relatively modest yield.
Accordingly, there is a need for improved methods of
producing structural models of the cytoplasmic domain of
multisubunit transmembrane receptors. These models are
useful in evaluating agents which control and modulate the
activity of integrins and other transmembrane proteins,
detecting their activity, and modulating their activity to
detect and control physiological conditions.
Summary of the Invention
In the present invention, a method is provided for
preparation of proteins for use in structural models or
mimics of the cytoplasmic face of multimeric transmembrane
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proteins such as integrins. Proteins of the present
invention may be prepared recombinantly or synthetically.
However, by using recombinant proteins, limitations of
polypeptide length and modest yield encountered in the
initial synthetic approaches of the prior art are avoided.
Accordingly, it is preferred that at least a portion of the
structural model of the present invention be prepared
recombinantly. In the model of the present invention, the
heterodimeric nature of the (3 cytoplasmic domain is
mimicked by use of covalent heterodimers of these domains.
Helical coiled-coil architecture provides the desired
parallel topology and vertical stagger of the tails. The
model is useful in studying protein interactions with
transmembrane proteins such as integrin and screening
agents for integrin inhibitory activity and in obtaining
structures of integrin cytoplasmic domains. For example,
using a model comprising an a4 cytoplasmic tail, it has now
been found that paxillin and paxillin related molecules
such as leupaxin and Hic-5 have high affinity interactions
with a4 integrin. Accordingly, agents which inhibit the
interaction of paxillin and paxillin related molecules with
a4 integrin are believed to be useful in inhibiting
biological responses associated with a4 integrins. Thus,
these agents may be useful in inhibiting normal a4 integrin
activity such as that occurring in wound healing which can
lead to scarring. These agents can also be used in
inhibiting pathological responses of a4 integrin such as in
atherosclerosis and immune responses associated with
conditions including, but not limited to inflammatory bowel
disease, arthritis, multiple sclerosis and asthma.
Brief Description of the Drawincrs
Figure 1 exemplifies amino acid sequences of
recombinant model proteins of integrin cytoplasmic domains.
Figure lA shows the N-terminal (SEQ ID NO: 5) and heptad-
repeat (SEQ ID N0: 6) structures common to all constructs.
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In the example shown, these are connected to the G1-(31A
cytoplasmic domain (SEQ ID NO: 7). Arrows indicate the
positions of hydrophobic residues corresponding to
positions a and d of the heptad repeats. Positions of the
additional Gly insertions in the
G2-, G3- and G4-constructs are also indicated. Figure 1B
shows the integrin-specific sequences of the constructs
used in experiments described herein including B1A (SEQ ID
NO: 8), B1A (U788A) (SEQ ID NO: 9), B1B (SEQ ID NO: 10),
B1C (SEQ ID NO: 11), B1D (SEQ ID NO: 12) and B7 (SEQ ID NO:
13). All integrin peptides correspond to the reported
human integrin sequences.
Detailed Description of the Invention
The present invention relates to the production of
mimics of the cytoplasmic face of occupied and clustered
transmembrane proteins such as integrins consisting of
polypeptides comprising a series of a-helical heptad
repeats, preferably 2 to 20, more preferably 3 to 6, most
preferably 4, that mimic a transmembrane domain connected
to a cytoplasmic domain of a selected multisubunit
transmembrane receptors such as integrins. By "mimic" it is
meant that the series of heptad repeats, imitates or
replaces the structural features of the transmembrane
domain. In one embodiment, an immobilizing epitope such as
a His-Tag sequence or glutathione-S-transferase, is linked
to the N-terminus for immobilization of the polypeptide in
affinity chromatography. In this embodiment, it is
preferred that the immobilizing epitope be linked to the
polypeptide via a Cys-Gly linker. For convenience, a
prokaryotic or chemical cleavage site such as a thrombin
cleavage site can also be incorporated into the polypeptide
at this linkage site.
For the purposes of the present invention, by "a-
helical heptad-repeat" it is meant a sequence consisting of
substantially helical amphiphilic amino acids having
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hydrophobic residues at selected positions in the repeat,
preferably positions a and d as depicted in Figure 1. In
such an embodiment, each repeat is seven amino acids with
hydrophobic residues at the first and fourth positions.
For example, in a preferred embodiment, the heptad repeat
comprises the amino acid sequence G-X1-L-XZ-X3-L-X4-G, (SEQ
ID NO: 14) wherein X1 is a lysine, arginine or ornithine, X2
and X~ are glutamic acid or aspartic acid, and X3 is
alanine, serine or threonine. The heptad repeats of the
polypeptide are preferably identical. However, in some
embodiments, each heptad repeat may differ in amino acid
sequence.
In a preferred embodiment, the cytoplasmic tail of a
transmembrane receptor such as an integrin is linked to the
heptad repeat via a glycine residue at the C-terminus of
the heptad repeat. In this embodiment the polypeptide is
predicted to form parallel coiled-coil dimers under
physiological conditions. However, trimers and tetramers
can also be designed based upon current methods for coiled
coil protein design. These coiled-coil structures are
likely to better mimic the proximity of transmembrane
helices in the natural system and also ensure that a
defined topology is maintained between the a and ~3
cytoplasmic tails. In other words, the coiled-coil of the
a-helical heptad repeat can act as a structural template
onto which the cytoplasmic domain of the integrin or other
transmembrane protein is attached. This ensures that the
two cytoplasmic tails are staggered with respect to one
another in a manner that approximates the intact protein.
A cystine bridge ensures a parallel orientation and a
correct stagger of the coiled-coil sequences within this
dimer configuration. Examples of cytoplasmic tails of
integrins which can be used include, but are not limited to
which, integrin ~ subunits such as (31A (SEQ ID NO: 8),
~31A(Y788A) (SEQ ID NO: 9), (31B (SEQ ID NO: 10), (31C (SEQ ID
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NO: 11), ~1B (SEQ ID NO: 12), (37 (SEQ ID NO: 13), and (33
and a integrin subunits such as aIIb, a4, a3A, a5 or a6A.
It is preferred that at least a portion of the
polypeptides used in the mimics of the present invention be
prepared recombinantly. Recombinant preparation of
polypeptides overcomes limitations of polypeptide length
and modest yield encountered in the initial synthetic
approaches of the prior art. Methods for recombinant
preparation of at least a portion of a polypeptide are well
known in the art. Polypeptides of the mimics or portions
thereof may also be prepared synthetically. Methods for
synthetic preparation of polypeptides are well known in the
art. Further, methods for combining portions of
synthetically and recombinantly prepared peptides into a
single polypeptide are known. In the present invention,
if both polypeptides of the mimic are prepared
synthetically, at least one heptad repeat in the series of
heptad repeats forming the coiled-coil sequences must
differ in amino acid sequence from the other heptad repeats
in the series.
Polypeptides of the model of the present invention are
preferably >90o homogenous as determined by reverse phase
C18 high pressure liquid chromatography and have a monomer
mass that varies by less than O.lo from that of the desired
monomer sequence as determined by electrospray mass
spectrometry. In this embodiment, formation of covalent
dimers in aqueous solution can be observed by mass
spectrometry and by SDS-PAGE, thus confirming the parallel
orientation of the helices.
In this embodiment, the beginning of the integrin
cytoplasmic domain sequence provides the hydrophobic
residues of a fifth heptad repeat (Figure 1).
Consequently, direct linkage of the coiled-coil sequence of
the a-helical heptad repeat could induce helical structure
in the tail. To address this possibility, embodiments of
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the protein model containing additional glycines between
the a-helical heptad repeats and the cytoplasmic domain
sequence were synthesized (Figure 1). Comparison was made
of the CD-spectra of (31 integrin constructs containing
either only one glycine (G1-(31A) or three additional
glycines (G4-~31A) between the heptad repeats and the
cytoplasmic domain. Insertion of glycines sharply reduced
the minima at 208 and 222 nm. Consequently, predicted a-
helical content in the protein model was reduced from 650
to 36%. The four heptad repeats constitute 27o of the mass
of the construct; therefore, 36o helical content is
consistent with the helical structure being limited to
these repeats. Thus, the Gly insertion appears to
eliminate a-helical structure induced in. the cytoplasmic
domain coiled-coil sequence.
To study possible influences of the structural changes
induced by the Gly insertions, protein models were produced
having the (~lA cytoplasmic domain with one, two and three
additional Gly residues inserted after the heptad-repeat
motif (G2-, G3-, G4-(31A) and compared with the G1-(31A
construct. As an additional control, a variant of the G4-
(31A peptide was produced with a Tyr to Ala substitution in
the membrane-proximal NPXY-motif (G4-~31A-Y788A) (Figure 1).
This mutation interferes with focal adhesion targeting and
activation of integrins. The purified proteins were bound
via their N=terminal His-Tag to a Ni2'-resin and used in
affinity chromatography experiments with lysates of NHS-
biotin-labeled human platelets. Marked changes in the
pattern of protein binding were observed as a consequence
of the Gly insertions. Polypeptides migrating at 45, 56,
58, 140 and 240 kDa bound only to the mimics with Gly
insertions. The Y788A mutation in the G4-~1A construct
(YA) suppressed the interaction with the 240 kDa, but not
with the other components. Using monoclonal antibodies,
the 240 kDa and 45 kDa proteins were identified as filamin
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and actin, respectively. The enriched 56, 58 and 140 kDa
polypeptides have not been identified but have failed to
react with antibodies specific for pp60sr~, paxillin,
pp125fa'', a_actinin, vinculin and pp725Y'' in Western blotting
experiments. Talin bound to the G1- and G4-(31A construct
but not to the Y788A-G4(31A construct. Thus, the structural
changes in the model induced by the insertion of glycines
into the coiled-coil motif and the integrin cytoplasmic
domain sequence alter interactions of these proteins with
cellular components. Alterations of the (31A tail that
block cytoskeletal interactions, such as the Y788 mutation
and (31B- and ~1C-splice variants also abrogate binding to
talin and filamin. Consequently, the observed in vitro
interactions are likely to be biologically relevant.
Models of the present invention were also constructed
with G1- and G4- polypeptides of the muscle-specific splice
variant (31D and the (37 integrin subunits (Figure 1) to
study binding interactions of various integrin binding
proteins. When used with NHS-biotinylated platelet
lysates, the ~31D constructs bound more talin and (37
constructs bound more filamin, compared to (31A. In
addition, these differences in binding were consistently
observed when lysates of a human T-cell leukemia cell line
(Jurkat), a human fibrosarcoma cell line (HT 1080), and a
differentiated myotubes derived from a mouse myoblast cell
line (C2C12), were used for affinity-chromatography.
Moreover, stronger binding of the ~1D constructs to talin
and of the (37 constructs to filamin was independently
observed, both with the G1- as well as the G4-variants of
the model proteins, indicating that the structural changes
induced by Gly insertions do not strongly influence these
differential interactions.
Purified preparations of these proteins were then used
to demonstrate that the observed interactions with talin
and filamin in the cell extracts are direct. The relative
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amounts of purified filamin and talin bound to the model
proteins were similar to those observed with cell lysates.
Specifically, ~31D constructs bound more talin and (37
constructs bound more filamin than (31A protein models. In
addition, binding of both cytoskeletal proteins to the G4-
Y788A-(31A construct and to the G4-(31B and G4-(31C variants
was functionally reduced compared to G4-~lA. Moreover, G4-
constructs of (31A, (31D and ~7 integrin cytoplasmic domains
bound more purified filamin than the corresponding 61-
constructs. However, the G1-~7 model protein still bound
more filamin than G4-(31A or G4-(31D. A densitometric
evaluation of the Coomassie blue-stained gels indicated
that the (31D construct bound about nine times more talin,
and the (37 construct bound 8.4 times more filamin than the
~31A model protein. In these experiments, there was a >10
fold molar excess of model proteins relative to the
quantity of talin and filamin. Thus, the affinity of (31A
for filamin is at least eight fold less than that of (37,
and its affinity for talin is at least nine fold less than
that of (31D.
Cytoplasmic domain mimics of the a4 integrin have also
been prepared in accordance with the present invention.
The a4 integrin subunit is indispensible for embryogenesis,
hematopoiesis and the immune response (Stewart et al. Curr.
Opin. Cell Biol. 7, 690-696 (1995); Shimizu et al. Adv.
Immunol. 72, 325-380 (1999)). Because of their central
role in the immune response a4 integrins are strongly
implicated as potential therapeutic targets for
inflammatory bowel disease, arthritis, multiple sclerosis
and asthma. It has been suggested that a4 may regulate
cell migration, cytoskeletal organization and gene
expression differently from other integrin a subunits
(Hemler et al. Cold Spring Harbor Symposia on Quantitative
Biology: The Cell Surface 57, 213-220 (1992)). These
biological properties are dependent on the a4 cytoplasmic
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domain (Stewart et al. Curr. Opin. Cell Biol. 7, 690-696
(1995); Hemler et al. Cold Spring Harbor Symposia on
Quantitative Biology: The Cell Surface 57, 213-220 (1992);
Newton et al. J. Leukocyte Biol. 61, 422-426 (1997)).
Structural mimics of the present invention comprising the
a4 cytoplasmic tails were prepared and used to identify
molecules involved in a4 integrin-specific signaling.
To identify biochemical bases for the signaling
properties of the a4 integrins, the binding of cellular
proteins to structural mimics of dimerized a4 integrin
cytoplasmic domains was analyzed. These structural mimics
were formed by fusing the cytoplasmic tail of the a4 or (31A
subunit to an N-terminal sequence containing 4 heptad
repeat sequences which form the coiled-coil dimers so that
the cytoplasmic domains are parallel dimerized and held in
a fixed vertical stagger.
Lysates of Jurkat T lymphoblasts were then incubated
with immobilized a4 cytoplasmic domain mimics. Bound
proteins were detected by immunoblotting for previously
identified integrin cytoplasmic domain binding proteins.
Within the bound fraction, it was found that paxillin was
enriched greater than 57 fold as compared to the cell
lysate. In contrast, while the (31A cytoplasmic domain
bound paxillin, there was no enrichment relative to the
cell lysate. The interactions with both the a4 and ~31A
tails were specific in that binding was not seen to resin
bearing no protein nor to the aIIb cytoplasmic domain.
Heterodimers of the a4~31A tails were also produced. These
heterodimers bound similar quantities of paxillin to the a4
tail alone. The a4 tail also bound small amounts of the
actin-binding proteins filamin and tails. However, these
proteins were not enriched relative to the cell lysate.
Further, the a4 tail did not bind to vinculin or a-actinin.
There are seven conserved N-terminal residues in the a
integrin subunit cytoplasmic tails (Hemler et al. Cold
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Spring Harbor Symposia on Quantitative Biology: The Cell
Surface 57, 213-220 (1992); Williams et al. Trends Cell
Biol. 4, 109-112 (1994); and Sastry S.K. and Horwitz, A.F.
Curr. Opin. Cell Biol. 5, 819-831 (1993)). Accordingly,
the specificity of the interaction of paxillin with the a4
cytoplasmic tails was determined by examining paxillin
binding to a series of a cytoplasmic domains. Paxillin
failed to bind to the aIIb, a3A, a5 or a6A tails. Thus,
conservation of seven residues of the N-terminal by
integrin a cytoplasmic domains does not appear to be
sufficient to mediate paxillin binding.
In addition to paxillin, experiments were also
performed to determine whether the paxillin related
proteins Hic-5 and leupaxin also bind to the a4 cytoplasmic
tails. In some experiments, a minor 55 K band, the size of
Hic-5, was observed bound to the a4 column. Using platelet
extracts as a source of Hic-5, a 7.4 fold enrichment
compared to cell lysate was observed. In similar
experiments in Jurkat cells, a 1.9 fold enrichment compared
to starting lysate was observed for leupaxin.
Experiments were also performed to confirm that
paxillin is also associated with intact a4 integrins and
intact a4~1 integrins. In these experiments, Jurkat cell
lysate was immunoprecipitated with monoclonal antibodies
reactive with a4, (31 or a5 integrin subunits or monoclonal
antibodies reactive to paxillin or an irrelevant IgG,
respectively. Paxillin was present in the a4 and (31
immunoprecipitates, but not in the immunoprecipitates
formed with a5 antibody or irrelevant IgG.
Immunoprecipitates of the surface biotin-labeled cells
confirmed the immunoprecipitation of x4(31 by the a4
antibody, x5(31 by the a5 antibody and a mixture of these
two plus a band with mobility of al by the anti (31
antibody. In cell lysate immunoprecipitated with monoclonal
antibody to paxillin or an irrelevant IgG, a4~1 integrin,
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but not a5(31 integrin co-precipitated with paxillin. x4(31
did not co-precipitate with the irrelevant IgG.
Paxillin's tight association with the a4 tails and its
ready isolation with a4 integrins is indicative of a
significant fraction of a4~1 being associated with paxillin
in the cells. To determine this fraction, surface biotin
labeled Jurkat cell lysate was sequentially
immunoprecipitated with anti-paxillin antibody or
irrelevant IgG. Western blotting with anti-paxillin
antibody confirmed depletion of virtually all paxillin.
Paxillin depletion resulted in almost complete loss of a4
in the lysate. In contrast, there was little depletion of
a5. Immunoprecipitation with an irrelevant IgG did not
result in significant loss of either a4~1 or a5~1.
Accordingly, a majority of or all of the a4 appears to
physically associate with paxillin.
A chimera consisting of the aIIb extracellular and
transmembrane domain and the a4 cytoplasmic domain was then
constructed to determine whether the a4 cytoplasmic tail
alone is sufficient to connect paxillin to an integrin. To
provide appropriate (3 tail partners, the extracellular and
transmembrane domains of ~i3 were joined to the (31A or ~7
cytoplasmic domain. The aIIba4(33(31A and aIIba4(33(37
chimeric integrins were expressed in CHO cells. A chimera
in which the a6A cytoplasmic domain was joined to aIIb and
expressed as aIIba6A~i3~ilA chimera in CHO cells was used
(Hughes et al. Cell 88, 521-530 (1997)). When lysates from
these cells were immunoprecipitated with antibodies against
the extracellular domain of aIIb(33, similar quantities of
recombinant integrin were precipitated from each cell line.
Only cells containing the a4 tail-bearing chimeric integrin
manifested substantial paxillin co-immunoprecipitation.
Thus, the a4 cytoplasmic domain must mediate the
association of intact integrins with paxillin.
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The functional effect of the a4 tails was then
examined by assaying cells adhesion and spreading on the
aIIb~3 ligand, fibrinogen. The a4 tail did not alter
aIIb~3-dependent cell adhesion. However, the a4 tail
opposed aIIb(33-dependent cell spreading. These two cell
lines adhered and spread equally well on a ligand for
endogenous a5~1, fibronectin, confirming that the effect
was specific to the recombinant integrin. In assaying the
paxillin-binding site within the a4 tail, an amino acid
residue was identified, Y991A, that disrupted binding of
paxillin. This mutation was introduced into a4 chimera and
aIIba4(Y991A)~33(31A was expressed in CHO cells. This
mutation restored aIIb(33-dependent cell spreading, but did
not alter either aIIb~3-dependent cell adhesions or cell
spreading on fibronectin. Thus, interaction of a4 tail
with paxillin results in diminished cell spreading.
To confirm that paxillin is required for a4-inhibition
of cell spreading, the a4 subunit was expressed in primary
fibroblasts derived from wild-type or paxillin-deficient
mice and cell spreading on VCAM-1 , an a4 integrin-
specific ligand, was assayed. Primary mouse embryonic
fibroblasts from two paxillin-null embryos spread where
those from littermate wild-type embryos failed to spread.
To determine whether other cytosolic proteins may be
mediating the observed binding of paxillin to the a4
complex in whole cell extracts, a recombinant human
paxillin-GST fusion protein was prepared. Purified
recombinant paxillin-GST fusion protein quantitatively
bound to the a4 cytoplasmic domain. In contrast, paxillin
binding was not detectable on the aIIb tail. Further,
there was no binding of GST to the a4 tail. Since both
binding partners are recombinant bacterial proteins, a
requirement far tyrosine phosphorylation in the direct
interaction of paxillin with the a4 tail can be excluded.
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Paxillin binding to the a4 tails was saturable and of high
affinity.
These experiments with the structural mimics of the
present invention demonstrate that paxillin binds directly
and tightly to the a4 cytoplasmic tail. Paxillin is
therefore believed to play an important role in the
signaling properties of a4 integrins. In particular, it is
believed that direct binding of paxillin to a4 tail opposes
a4-dependent cell spreading. Thus, blockade of the binding
of a4 to paxillin should inhibit a4-mediated cell
migration. Since a major function of a4 is the migration
and trafficking of leukocytes, inhibitors of the binding of
paxillin to a4 are expected to be useful in blocking immune
responses. a4 integrin activation has also been associated
with atherosclerosis. Accordingly, agents which inhibit
activation will also be useful in inhibiting
atherosclerosis. Further activation of a4 integrin occurs
during wound healing. More specifically, a4 integrin
activation signals monocytes to aggregate at the wound
site. However, this aggregation can lead to scarring.
Accordingly, inhibition of a4 integrin activation is also
useful in inhibiting scarring during wound healing.
This structural model was used to identify a 15 mer
peptide, SILQEENRRDSWSYI (SEQ ID N0:15) derived from the a4
cytoplasmic domain as an inhibitor of the binding of
paxillin and the a4 tail. The IC50 of inhibition of the
interaction of paxillin and the a4 tail by this peptide was
150 ~.M. Similar experiments with additional 15 mer
peptides, KAGFFKRQYKSILQE (SEQ ID N0:16) and
RRDSWSYINSKSNDD (SEQ ID N0:17), showed no inhibition.
Further substitution of various single amino acids within
SEQ ID N0:15 with alanine also abolished inhibitory
activity. Thus, inhibition by the 15 mer peptide
SILQEENRRDSWSYI (SEQ ID N0:15) is structurally specific.
The core active sequence of this peptide has been
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determined to comprise the 9 amino acid sequence ENRRDSV~ISY
(SEQ ID N0:18). Knowledge of this core sequence and its
structure are useful in the rational design of therapeutic
agents which inhibit a4 integrin biological responses.
As demonstrated by these experiments, the structural
models of the present invention provide a novel
experimental tool for the analysis of various proteins
associations with integrin tails in vitro and the
structural aspect of the cytoplasmic face of integrins.
The structural models of the present invention thus have a
number of applications based upon their ability to maintain
the cytoplasmic tails of the construct in a configuration
that is equivalent or similar to the configuration
predominating in vivo while maintaining solubility and
stability in an aqueous system, namely in staggered,
parallel, and proximal topology. As demonstrated herein,
these models can be used to detect intracellular molecules
capable of binding to integrins and modulating signals by
inside-out signaling. Alternatively, these molecules can
be used in vivo to disrupt or modulate inside-out signaling
by binding to the cells in a manner such that the
cytoplasmic domains of these recombinant models compete for
intracellular molecules with the natural integrins.
Because these structural models do not contain the
extracellular ligand-binding sites of integrins, they would
then disrupt inside-out signaling. This would be
particularly useful in conditions in which overactivity of
integrins is involved, such as inflammation, thrombosis,
and malignancy. This would provide a new method of
treating such conditions or their sequelae; because these
molecules mimic the orientation of the natural integrins
within the membrane, they would not disrupt membrane
structure and would therefore be better tolerated and avoid
side effects. Additionally, structural models of the
present invention can be used to detect molecules capable
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of binding to the intracellular or cytoplasmic domain of
integrins and other transmembrane molecules in vivo, such
as by affinity chromatography. Accordingly, these models
are useful in identifying various therapeutic compounds for
selected cytoplasmic domains. By "therapeutic compounds"
it is meant to include, but is not limited to, molecules
which are found to bind to a selected cytoplasmic domain of
the model, molecules which bind to proteins that bind to
the cytoplasmic domain of the model, and the models
themselves. For example, in one embodiment, a structural
model or mimic comprising an a4 integrin cytoplasmic tail
can be used in a high throughput screening assay to
identify agents which inhibit binding of paxillin to the a4
cytoplasmic tails. In this assay, the structural model
comprising an a4 integrin cytoplasmic tail is exposed to
paxillin or a paxillin related molecule in the presence or
absence of a test agent. Binding of paxillin or the
paxillin related molecule to the structural model in the
presence and absence of the test agent is then determined.
A test agent which decreases binding of paxillin or the
paxillin related molecule to the structural model as
compared to binding of paxillin or paxillin related
molecules to the structural model in the absence of the
test agent can inhibit biological responses relating to a4
integrins. For example, these agents may be useful in
inhibiting normal wound healing response of a4 integrin
which can lead to scarring. These agents can also be used
in the inhibition of pathological responses of a4 integrin
such as those involved in atherosclerosis and immune
responses in conditions such as inflammatory bowel disease,
arthritis, multiple sclerosis, and asthma. Compositions
comprising such agents and a known pharmaceutically
acceptable vehicle are believed to be useful
therapeutically to inhibit biological responses of a4
integrins.
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The following examples are provided for illustrative
purposes only and are not intended to limit the invention.
EXAMPLES
Example 1 Antibodies and cDNAs
Antibodies for the analysis of proteins bound to
cytoplasmic domain model proteins on western blots
included: goat serum against filamin (Sigma Chemical Co.,
St. Louis, MO), rabbit serum against a-actinin (Sigma
Chemical Co.), mAbs against talin (clone 8d4) (Sigma
Chemical Co.), vinculin (clone hVIN-1) (Sigma Chemical
Co.), pacillin (clone 2035) (Zymed Laboratories Inc., S.
San Francisco, CA), filamin (MAB1680) (Chemicon
International Inc. Temecula, CA), a-actinin (MB75.2) (Sigma
Chemical Co.), actin (clone C4) (Boehringer-Mannheim Corp.,
Indianapolis, IN), mAb against pp60sr~ (clone 327),
polyclonal rabbit serum against pplzsFAK (BC3) and rabbit
anti-pp72sY'', mAb against human (31 integrin (B-D15,
BioSource, International), mAb against human a4 integrin
(HP2/1, ImmunoTech), mAb human against human a5 integrin
(PharMingen), mAb against HA-tag (12C5, ATCC), mAb against
paxillin (clone 349, Transduction Laboratories), and mAb
against GST (B-14, Santa Cruz). Polyclonal antibody
against FAK (C-20, Santa Cruz was also used. Biotin
labeled anti-paxillin antibody was prepared by labeling
commercial anti-paxillin (clone 349) with NHS-Biotin
(Pierce) according to the manufacturer's instructions.
Rabbit polyclonal anti-leupaxin was raised against the N-
terminal 14 amino acids of human leupaxin (Lipsky et al. J.
Biol. Chem. 273 11709-11713 (1998))._
Human cDNA used in these experiments included: ~1C
cDNA; (31 cDNA with the point mutation, Y788A1; a cDNA for
the cytoplasmic domain of human integrin (31D obtained by
RT-PCT of heart muscle total RNA; cDNA of human integrin
(37; and a cDNA coding for the human (31B subunit cytoplasmic
domain synthesized in PCR reactions using a human (31A
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vector with a partially overlapping reverse-oligonucleotide
containing the human ~1B sequence.
Example 2 Recombinant cytoplasmic domain models
Oligonucleotides were synthesized and used in PCR
reactions to create a cDNA for the a-helical heptad repeat
protein sequence KLEALEGRLDALEGKLEALEGKLDALEG (SEQ ID N0:
6) G1-([heptad]4). Variants containing 1 to 3 additional
Gly residues (G2-4-([heptad]4)) at the C-terminus were
synthesized by modification of the antisense
oligonucleotide. These cDNAs were ligated into a NdeI-
HindIII restricted modified pETlSb vector (Novagen,
Madison, WI). Integrin cytoplasmic domains were joined to
the helix as a HindIII-BamHI fragments. The final
constructs coded for the N-terminal sequence
GSSHHHHHHSSGLVPRGSHMCG (SEQ ID NO: 5) [heptad]~ linked to
the cytoplasmic domains of integrins. Different
cytoplasmic domain cDNAs were cloned via PCR from
appropriate cDNAs using forward oligonucleotides
introducing a 5'-HindIII site and reverse oligonucleotide
creating a 3'-BamHI site directly after the Stop-codon.
PCR products were first ligated into the pCRT"" vector using
the TA cloning~ kit (Invitrogen Corp., San Diego, CA).
After sequencing, HindIII/BamHI inserts were ligated into a
modified pETl5b vector. Recombinant expression in
BL21(DE3)pLysS cells (Novagen) and purification of the
recombinant products were performed according to the pET
System Manual (Novagen) with an additional final
purification step on a reverse phase C18 HPLC column
(Vydac, Hesperia, CA). Products were analyzed by
electrospray mass spectrometry on an API-III quadruple
spectrometer (Sciex, Toronto, Ontario, Canada).
Example 3 Ultraviolet circular dichroism spectroscopy
Far UV CD spectra were recorded on an AVIV 60DS
spectropolarimeter with peptides dissolved in 50 mM boric
acid pH 7Ø Data were corrected for the spectrum obtained
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with buffer only and related to protein concentrations
determined from identical samples by quantitative amino
acid analysis. From these values, the percentage of
helical secondary structure was calculated in accordance
with procedures described by Muir et al. Biochemistry
33:7701 (1994).
Example 4 Cells and cell lysates
Human platelets were obtained by centrifugation of
freshly drawn blood samples at 1000 rpm for 20 minutes and
sedimentation of the resulting platelet-rich plasma at 2600
rpm for 15 minutes. They were washed twice with 0.12 M
NaCl, 0.0129 M trisodium citrate, 0.03 M glucose, pH 6.5,
and once in Hepes-Saline (3.8 mM Hepes, 137 mM NaCl, 2.7 mM
KC1, 5.6 mM D-Glucose, 3.3 mM NazHP04, pH 7.3-7.4). Human
Jurkat and HT1080 cells and mouse C2C12 cells were obtained
from the American Type Culture Collection (Rockville, MD)
and cultured in RPMI1680 (Jurkat) or DMEM with 10% fetal
calf serum. For differentiation to myotubes, C2C12
myoblasts were kept confluent in DMEM with 5o horse serum
for 6 days. Cultured cells were washed twice in phosphate-
buffered saline (PBS) and biotinylated with 1 mM NHS-biotin
(Pierce) in PBS during 30 minutes at room temperature.
Platelets were biotinylated in Hepes-Saline. After two
additional washes with TBS, cells were lysed on ice with
buffer A (1 mM Na3V04, 50 mM NaF, 40 mM NaPyrophosphate, 10
mM Pipes, 50 mM NaCl, 150 mM sucrose, pH 6.8) containing to
TRITON X-100, 0.5o sodium deoxycholate, 1 mM EDTA and
protease inhibitors (1/100th volume of aprotinin (Sigma A-
6379), 5 ~.g/ml leupeptin, 1 mM PMSF). To platelet lysates
0.1 mM of the calpain inhibitor E-64 (Boehringer Mannheim)
were added in addition. Lysates were sonicated 5 times on
ice for 10 seconds at a setting of 3 using an Astrason
Ultrasonic Processor (Heart Systems, Farmingdale, NY).
After 30 minutes, lysates were clarified by centrifugation
at 12,000 g for 30 minutes.
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Example 5 Affinity chromatography experiments with integrin
cytoplasmic domain mimics
Purified recombinant cytoplasmic domain proteins (500
fig) were dissolved in a mixture of 5 ml 20 mM Pipes, 50 mM
NaCl, pH 6.8 and 1 ml 0.1 M sodium acetate, pH 3.5 and
bound overnight to 80 ~l of Ni2' saturated His-bind resin
(Novagen). In control experiments, it was found that this
leads to approximate saturation of the resin with peptide.
Resins were washed twice with 20 mM Pipes, 50 mM NaCl, pH
6.8, and stored at 4°C with O.lo sodium azide as suspensions
with one volume of this buffer. Fifty microliters of such
a suspension were added to 4.5 ml of cell lysates which had
been diluted tenfold with buffer A containing 0.05% TRITON
X-100, 3 mM MgClZ and protease-inhibitors. After incubation
overnight at 4°C, resins were washed five times with this
buffer and finally heated in 50 ~.1 of reducing sample
buffer for SDS PAGE. Samples were separated on 4-20o SDS
polyacrylamide gels (NOVEX) and either stained with
Coomassie or transferred to Immobilon P membranes (Amersham
Corp., Arlington Hts, IL). Membranes were blocked with
TBS, 5o nonfat-mild powder and stained with streptavidin-
peroxidase (VECTASTAIN) or specific antibodies. Bound
peroxidase was detected with an enhanced chemiluminescence
kit (Amersham).
Example 6 Binding to purified talin and filamin
Human uterus filamin (ABP-280) was prepared as a 1.5
mg/ml solution in 0.6 M KC1, 0.5 mM ATP, 0.5 mM DTT, 10 mM
imidazole, pH 7.5. For binding assays performed as
described in Example 5, this solution was diluted 1/12 with
buffer A, 0.05% TRITON X-100, 3 mM MgCl2, 2 mg/ml BSA,
protease-inhibitors (see Example 5), omitting the 50 mM
NaCl (see Example 5), and resins with bound model proteins
were added. Washing was performed in this buffer without
BSA and with additional 50 mM Kcl.
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Talin was purified from human platelets in accordance
with well known procedures with an additional purification
step using chromatography on phosphocellulose and stored at
1 mg/ml in 10 mM NaCl, 50% glycerol. This solution was
diluted to either 87 or 17 ~,g/ml talin with buffer A, 0.050
TRITON X-100, 3 mM MgCl2, 2 mg/ml BSA and protease
inhibitors (see Example 5, including 0.1 mM E-64) and
processed as indicated in the binding assays with cell
lysates. For densitometric analysis, scans of Coomassie-
stained gels were processed using the program NIH-Image
(NIH, Bethesda, MD). Equal loading of gels was controlled
in Coomassie-stained gels of the recombinant cytoplasmic
domain polypeptides coeluted with the ligand from the
resins.
Example 7 Chimera formation
The aIIba4 and aIIba4*Y991A) chimeras were formed by
connecting human aIIb extracellular and transmembrane
domains to human a4 or a4(Y991A) cytoplasmic domain. (33(31A
or X33(37 chimeras were formed by connecting human X33
extracellular and transmembrane domains to human ~31A or (37
cytoplasmic domains. CHO cells stably expressing
aIIba4(33(31A, aIIba4(Y991A)~33(31A, or cell lines expressing
these chimeras were transfected and isolated as described
by Hughes et al. Cell 88, 521-530 (1997). Primary mouse
embryonic fibroblasts from paxillin-null and littermate
matched wild-type embryos were isolated by standard methods
such as those described by Thomas et al. Nature 376 (6537),
267-71 (1995)).
Example 8 Immunoprecipitation and Western blot analysis
Jurkat T cells or CHO cells were cell surface-labeled
with sulfo-NHS-Biotin (Pierce) in accordance with the
manufacturer's instructions. Cell lysate was prepared and
immunoprecipitation was performed as described by Chen et
al. Blood 84, 1857-1865 (1994). Precipitated cell surface
biotin-labeled polypeptides were separated under non-
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reducing conditions and detected with streptavidin-
peroxidase followed by ECL (Amersham). Immunoprecipitation
for detection of co-precipitated paxillin was performed as
above except cells were not surface-labeled with biotin;
and immunoprecipitated proteins were separated under
reducing conditions and paxillin co-precipitation was
detected with biotin-labeled anti-paxillin. For co-
precipitation of a4(31 with paxillin, surface biotin-labeled
Jurkat cell lysate was precipitated with antibodies
reactive to paxillin, a4, a5 or irrelevant IgG.
Immunoprecipitates were separated on 6o SDS-PAGE under non-
reducing conditions and surface polypeptides were detected
with streptavidin-peroxidase and ECL. For paxillin-
depletion assay, aliquots of cell surface biotinylated
Jurkat T cell lysate were subjected to varying rounds of
immunoprecipitation using anti-paxillin antibody or
irrelevant IgG. The degree of paxillin-depletion in the
cell lysate was assessed by Western blot analysis. Cell
lysates with or without paxillin-depletion, as well as with
the irrelevant IgG precipitation, were then
immunoprecipitated with either anti-a4 or a5 antibody.
Immunoprecipitates of surface proteins were separated on 6%
SDS-PAGE under non-reducing conditions and polypeptides
were detected with streptavidin-peroxidase and ECL.
Example 9 Cell Adhesion and Spreading Assays
Assays of cell adhesion and spreading on fibrinogen or
fibronectin for different CHO cell lines were performed in
accordance with procedures described by Ylanne et al. J.
Cell Biol. 122, 223-233 (1993). For cell spreading assay
of mouse fibroblasts, paxillin knock-out as well as wild-
type cells were transfected with human a4 integrin subunit
using retroviral infection. Forty-eight hours after
transfection, equal expression of a4 integrin in wild-type
and knock-out cells was observed by FRCS using anti-a4
antibody. Cells resuspended in DMEM plus 1 mg/ml of BSA
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were plated on coverslips coated with 10 ~g/ml of either
VCAM-1 (Biogen Inc. Cambridge MA) or fibronectin (Sigma
Chemical Co.) and incubated at 37°C for 1 hour. Unattached
cells were washed away with PBS. Attached cells were fixed
with 3.7% paraformaldehyde and examined by phase
microscopy. Photo images were taken with a Nikon Diaphot
microscope equipped with a Sensys cooled CCD video camera.
Example 10 Production and Binding of Recombinant Paxillin
Recombinant human paxillin was expressed and isolated
in accordance with procedures described by Salgia et al. J.
Biol. Chem. 270, 5039-5047 (1995). Aliquots of recombinant
GST-paxillin or GST alone were mixed with 300 ~1 of buffer
A plus 20 ~g/ml of aprotinin, 5 ~g/ml of leupeptin, 1 mM
PMSF, 0.1% Triton X-100, 3 mM MgCl2, and 1 mg/ml of BSA,
added to model protein-loaded resins, and incubated at room
temperature with rotation for 2 hours. Both bound and
unbound proteins were collected and detected with
antibodies specific for HA-tag or GST. For determination
of EC50 of paxillin binding to a4 tail, different amounts
of recombinant paxillin were added to a4 or aIIb tail-
loaded resins and bound paxillin was assayed as described
above.
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SEQUENCE LISTING
<110> GINSBERG, MARK H.
PFAFF, MARTIN
LIU, SHOUCHUN
THE SCRIPPS RESEARCH INSTITUTE
<120> STRUCTURAL MODELS FOR CYTOPLASMIC DOMAINS OF
TRANSMEMBRANE RECEPTORS
<130> SRI-0010
<140> 09/187,236
<141> 1998-11-05
<150> 09/323,447
<151> 1999-06-Ol
<160> 18
<170> PatentIn Ver. 2.0
<210> 1
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
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Lys Gln Ala Gly Asp Val
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Asp Gly Glu Ala
1
1
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Gly Ser His Met
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<223> Description of Artificial Sequence: Synthetic
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Glu Ala Leu Glu Gly Lys Leu Asp Ala Leu Glu Gly
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Lys Glu Lys Met Asn Ala Lys Trp Asp Thr Gly Glu Asn Pro Ile Ala
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4
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Arg Phe Gln Gln Phe Ser Cys Leu Ser Leu Pro Ser Thr Trp Asp Tyr
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Arg Val Lys Ile Leu Phe Ile Arg Val Pro
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5
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Ser Pro Thr Leu
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Gly Xaa Leu Xaa Xaa Leu Xaa Gly
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Ser Ile Leu Gln Glu Glu Asn Arg Arg Asp Ser Trp Ser Tyr Ile
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Lys Ala Gly Phe Phe Lys Arg Gln Tyr Lys Ser Ile Leu Gln Glu
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<210> 17
<211> 15
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6
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Arg Arg Asp Ser Trp Ser Tyr Ile Asn Ser Lys Ser Asn Asp Asp
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<210> 18
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<213> Artificial Sequence
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Glu Asn Arg Arg Asp Ser Trp Ser Tyr
1 5
7
