U.S. patent application number 10/138787 was filed with the patent office on 2002-11-21 for oligomerized receptors which affect pathways regulated by transmembrane ligands for elk-related receptor tyrosine kinases.
This patent application is currently assigned to Mount Sinai Hospital Corporation. Invention is credited to Holland, Sacha, Mbamalu, Geraldine, Pawson, Tony.
Application Number | 20020172984 10/138787 |
Document ID | / |
Family ID | 21803293 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020172984 |
Kind Code |
A1 |
Holland, Sacha ; et
al. |
November 21, 2002 |
Oligomerized receptors which affect pathways regulated by
transmembrane ligands for elk-related receptor tyrosine kinases
Abstract
A method of modulating the biological activity of a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand, comprising forming a
complex between a purified and isolated oligomerized Elk-related
receptor tyrosine kinase, or an isoform or an extracellular domain
of the kinase, and a transmembrane ligand expressed on the cell,
thereby affecting a pathway in the cell which is regulated by the
transmembrane ligand.
Inventors: |
Holland, Sacha; (Toronto,
CA) ; Mbamalu, Geraldine; (Toronto, CA) ;
Pawson, Tony; (Toronto, CA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Mount Sinai Hospital
Corporation
Toronto
CA
|
Family ID: |
21803293 |
Appl. No.: |
10/138787 |
Filed: |
May 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10138787 |
May 3, 2002 |
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09214631 |
Mar 12, 1999 |
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6413730 |
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09214631 |
Mar 12, 1999 |
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PCT/CA97/00473 |
Jul 4, 1997 |
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60021272 |
Jul 5, 1996 |
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Current U.S.
Class: |
435/7.21 ;
424/94.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12N 9/1205 20130101; C07K 14/4702 20130101; C12Q 1/6897 20130101;
C07K 14/705 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/7.21 ;
424/94.5 |
International
Class: |
G01N 033/567; A61K
038/51 |
Claims
We claim:
1. A method of modulating the biological activity of a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand comprising forming a
complex between a purified and isolated oligomerized Elk-related
receptor tyrosine kinase, or an isoform or an extracellular domain
of the Elk-related receptor tyrosine kinase, and the transmembrane
ligand expressed on the cell, thereby modulating the biological
activity of the transmembrane ligand.
2. A method of affecting a pathway regulated by a transmembrane
ligand for an Elk-related receptor tyrosine kinase in a cell
expressing the transmembrane ligand, comprising forming a complex
between a purified and isolated oligomerized Elk-related receptor
tyrosine kinase, or an isoform or an extracellular domain of the
Elk-related receptor tyrosine kinase, and a transmembrane ligand
expressed on the cell, thereby affecting or modulating a pathway in
the cell which is regulated by the transmembrane ligand.
3. A method for evaluating a substance for its ability to modulate
the biological activity of a transmembrane ligand for an
Elk-related receptor tyrosine kinase in a cell expressing the
transmembrane ligand comprising the steps of: (a) contacting an
oligomerized Elk-related receptor tyrosine kinase, or an isoform or
an extracellular domain of the Elk-related receptor tyrosine
kinase; a transmembrane ligand which binds to the Elk-related
receptor tyrosine kinase to form a receptor-ligand complex, wherein
the transmembrane ligand is a transmembrane ligand for an
Elk-related receptor tyrosine kinase expressed on a cell; and, a
test substance, under conditions which permit the formation of
receptor-ligand complexes; (b) assaying for receptor-ligand
complexes, free Elk-related receptor tyrosine kinase, or
non-complexed transmembrane ligand, or for activation of the
transmembrane ligand; and (c) comparing to a control to determine
if the substance inhibits or enhances the binding of the
Elk-related receptor tyrosine kinase and transmembrane ligand, and
thereby modulates the biological activity of the transmembrane
ligand.
4. A method for identifying a substance which affects or modulates
a pathway regulated by a transmembrane ligand for an Elk-related
receptor tyrosine kinase in a cell expressing the transmembrane
ligand, comprising the steps of: (a) contacting an oligomerized
Elk-related receptor tyrosine kinase, or an isoform or an
extracellular domain of the Elk-related receptor tyrosine kinase; a
transmembrane ligand which binds to the Elk-related receptor
tyrosine kinase to form receptor-ligand complexes which activate a
pathway regulated by a transmembrane ligand for an Elk-related
receptor tyrosine kinase in a cell expressing the transmembrane
ligand; and a test substance under conditions which permit the
formation of receptor-ligand complexes; (b) assaying for
receptor-ligand complexes, free Elk-related receptor tyrosine
kinase, or non-complexed transmembrane ligand, or for activation of
the transmembrane ligand; and (c) comparing to a control to
determine if the substance inhibits or enhances the binding of the
Elk related receptor tyrosine kinase and transmembrane ligand and
thereby affects or modulates the pathway.
5. A method for evaluating a substance for its ability to inhibit
or enhance the interaction of an oligomerized Elk-related receptor
tyrosine kinase, or an isoform, or an extracellular domain of the
Elk-related receptor tyrosine kinase, and a transmembrane ligand
for an Elk-related receptor tyrosine kinase expressed on a cell
comprising: (a) providing a reporter gene operably linked to a DNA
binding site for a transcriptional activator; (b) providing a first
hybrid protein comprising the transmembrane ligand in polypeptide
linkage to a DNA binding domain of the transcriptional activator;
(c) providing a second hybrid protein comprising an oligomerized
Elk-related receptor tyrosine kinase or an isoform or an
extracellular domain of the Elk-related receptor tyrosine kinase in
polypeptide linkage to an activation domain of the transcriptional
activator; under conditions where the transmembrane ligand and
oligomerized Elk-related receptor tyrosine kinase can bind and
thereby reconstitute the transcriptional activator which induces
transcription of the reporter gene; (d) administering a test
substance; and (e) monitoring expression of the reporter gene,
wherein a decrease in expression is an indication that the
substance inhibits the interaction of the transmembrane ligand and
oligomerized Elk-related receptor tyrosine kinase, and an increase
in expression is an indication that the substance enhances the
interaction of the transmembrane ligand and oligomerized
Elk-related receptor tyrosine kinase.
6. A method for evaluating a substance for its ability to inhibit
or enhance the interaction of an oligomerized Elk-related receptor
tyrosine kinase, or an isoform, or an extracellular domain of the
Elk-related receptor tyrosine kinase, and a transmembrane ligand
for an Elk-related receptor tyrosine kinase expressed on a cell
comprising: (a) providing a reporter gene operably linked to a DNA
binding site for a transcriptional activator, (b) providing a first
hybrid protein comprising an oligomerized Elk-related receptor
tyrosine kinase or an isoform or an extracellular domain of the
Elk-related receptor tyrosine kinase in polypeptide linkage to a
DNA binding domain of the transcriptional activator; (c) providing
a second hybrid protein comprising a transmembrane ligand in
polypeptide linkage to an activation domain of the transcriptional
activator; under conditions where the transmembrane ligand and
oligomerized Elk-related receptor tyrosine kinase can bind and
thereby reconstitute the transcriptional activator which induces
transcription of the reporter gene; (d) administering a test
substance; and (e) monitoring expression of the reporter gene,
wherein a decrease in expression is an indication that the
substance inhibits the interaction of the transmembrane ligand and
oligomerized Elk-related receptor tyrosine kinase, and an increase
in expression is an indication that the substance enhances the
interaction of the transmembrane ligand and oligomerized
Elk-related receptor tyrosine kinase.
7. A method as claimed in any one of claims 1 to 6, wherein the
transmembrane ligand is Elk-L/LERK2/Efl-3/Cek5-L,
hHtk-L/ELF-2/Lerk5, or hElk-L3/Efl-6.
8. A method as claimed in any one of claims 1 to 7, wherein the
Elk-related receptor tyrosine kinase is Nuk, Hek5, Erk, Sek3, Cek5
, Elk, Cek6a, xEK, Hek2, Sek4, Cek10 , Htk, or Myk1.
9. A method as claimed in any one of claims 1 to 6 wherein the
transmembrane ligand is Elk-L and the Elk-related receptor tyrosine
kinase is Elk or Nuk.
10. A method as claimed in any one of claims 1 to 6 wherein the
transmembrane ligand is Htk-L and the Elk-related receptor tyrosine
kinase is Elk or Nuk.
11. An antibody preparation which specifically binds to a
receptor-ligand complex comprising an oligomerized Elk-related
receptor tyrosine kinase, or an isoform or an extracellular domain
of the kinase, and a transmembrane ligand for an Elk-related
receptor tyrosine kinase.
12. Use of a purified and isolated oligomerized Elk-related
receptor tyrosine kinase, or an isoform or an extracellular domain
thereof, in the preparation of a medicament for modulating the
biological activity of a transmembrane ligand for an Elk-related
receptor tyrosine kinase in a cell expressing the transmembrane
ligand.
13. Use of a purified and isolated oligomerized Elk-related
receptor tyrosine kinase, or an isoform or an extracellular domain
thereof, in the preparation of a medicament for modulating neuronal
development or regeneration in a subject.
14. Use of a purified and isolated oligomerized Elk-related
receptor tyrosine kinase, or an isoform or an extracellular domain
thereof, in the preparation of a medicament for modulating
axonogenesis in a subject .
15. A use as claimed in any one of claims 12 to 14, wherein the
transmembrane ligand is Elk-L/LERK2/Efl-3/Cek5-L,
hHtk-L/ELF-2/Lerk5, or hElk-L3/Efl6.
16. A use as claimed in any one of claims 12 to 15, wherein the
Elk-related receptor tyrosine kinase is Nuk, Hek5, Erk, Sek3, Cek5,
Elk, Cek6a, xEK, Hek2, Sek4, Cek10, Htk, or Myk1.
17. A use as claimed in any one of claims 12 to 14, wherein the
transmembrane ligand is Elk-L and the Elk-related receptor tyrosine
kinase is Elk or Nuk.
18. Use of a substance identified in accordance with a method as
claimed in any one of claims 1 to 10 in the preparation of a
medicament for modulating the biological activity of a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand.
19. A pharmaceutical composition which comprises a purified and
isolated oligomerized Elk-related receptor tyrosine kinase, or an
isoform or an extracellular domain thereof, in an amount effective
to stimulate or inhibit neuronal development or regeneration and a
pharmaceutically acceptable carrier, diluent or excipient.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a method of affecting or modulating
a pathway in a cell which is regulated by the binding of a
transmembrane ligand for an Elk-related receptor tyrosine kinase
and an oligomerized Elk-related receptor tyrosine kinase; a method
of identifying substances which affect the binding of a
transmembrane ligand for an Elk-related receptor tyrosine kinase
and an oligomerized Elk-related receptor tyrosine kinase; and to
methods and pharmaceutical compositions using oligomerized
Elk-related receptor tyrosine kinases, and substances identified
using a method of the invention.
BACKGROUND OF THE INVENTION
[0002] Receptor tyrosine kinases play essential roles in cellular
signalling events. The largest known family of receptor tyrosine
kinases is the Eph subfamily of receptor tyrosine kinases. Eph
subfamily tyrosine kinases have been implicated in the control of
axon guidance and fasciculation.sup.1-7, in regulating cell
migration.sup.8, and in defining compartments in the developing
embryo.sup.9-11. Efficient activation of Eph receptors generally
requires that their ligands be anchored to the cell surface, either
through a transmembrane (TM) region or a glycosyl
phosphatidylinositol (GPI) group.sup.12. These observations have
suggested that Eph receptors can transduce signals initiated by
direct cell-cell interactions. Genetic analysis of Nuk, a mouse Eph
receptor that binds TM-ligands, has suggested that these ligands
have a signalling function.sup.6.
SUMMARY OF THE INVENTION
[0003] Challenging cells expressing the transmembrane (TM)-ligands,
Elk-L or Htk-L, with the clustered extracellular domain of Nuk was
found to induce phosphorylation of the ligands on tyrosine, a
process which is mimicked both in vitro and in vivo by an activated
Src tyrosine kinase. Co-culturing of cells expressing a TM-ligand
with cells expressing Nuk also was shown to lead to tyrosine
phosphorylation of both the ligand and Nuk. Therefore TM-ligands
are associated with a tyrosine kinase, and are inducibly
phosphorylated upon binding the Nuk receptor, in a fashion
reminiscent of cytokine receptors. Furthermore, it was shown that
TM-ligands, as well as Nuk, are phosphorylated on tyrosine in mouse
embryos, indicating that this is a physiological process. These
experimental results confirm that Eph receptors and their
TM-ligands mediate bi-directional cell signalling.
[0004] Therefore, the present invention provides a method of
modulating the biological activity of a transmembrane ligand for an
Elk-related receptor tyrosine kinase in a cell expressing the
transmembrane ligand comprising forming a complex between a
purified and isolated oligomerized Elk-related receptor tyrosine
kinase, or an isoform or an extracellular domain of the Elk-related
receptor tyrosine kinase, and the transmembrane ligand expressed on
the cell, thereby modulating the biological activity of the
transmembrane ligand.
[0005] The present invention also provides a method of affecting or
modulating a pathway regulated by a transmembrane ligand for an
Elk-related receptor tyrosine kinase in a cell expressing the
transmembrane ligand, comprising forming a complex between a
purified and isolated oligomerized Elk-related receptor tyrosine
kinase, or an isoform or an extracellular domain of the Elk-related
receptor tyrosine kinase, and a transmembrane ligand expressed on
the cell, thereby affecting or modulating a pathway in the cell
which is regulated by the transmembrane ligand.
[0006] The invention also provides a method for evaluating a
substance for its ability to modulate the biological activity of a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand comprising the steps
of:
[0007] (a) contacting an oligomerized Elk-related receptor tyrosine
kinase, or an isoform or an extracellular domain of the Elk-related
receptor tyrosine kinase; a transmembrane ligand which binds to the
Elk-related receptor tyrosine kinase to form a receptor-ligand
complex, wherein the transmembrane ligand is a transmembrane ligand
for an Elk-related receptor tyrosine kinase expressed on a cell;
and, a test substance, under conditions which permit the formation
of receptor-ligand complexes;
[0008] (b) assaying for receptor-ligand complexes, free Elk-related
receptor tyrosine kinase, or non-complexed transmembrane ligand, or
for activation of the transmembrane ligand; and
[0009] (c) comparing to a control to determine if the substance
inhibits or enhances the binding of the Elk-related receptor
tyrosine kinase and transmembrane ligand, and thereby modulates the
biological activity of the transmembrane ligand.
[0010] The invention also provides a method for identifying a
substance which affects or modulates a pathway regulated by a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand, comprising the steps
of:
[0011] (a) contacting an oligomerized Elk-related receptor tyrosine
kinase, or an isoform or an extracellular domain of the Elk-related
receptor tyrosine kinase; a transmembrane ligand which binds to the
Elk-related receptor tyrosine kinase to form receptor-ligand
complexes which activate a pathway regulated by a transmembrane
ligand for an Elk-related receptor tyrosine kinase in a cell
expressing the transmembrane ligand: and a test substance under
conditions which permit the formation of receptor-ligand
complexes;
[0012] (b) assaying for receptor-ligand complexes, free Elk-related
receptor tyrosine kinase, or non-complexed transmembrane ligand, or
for activation of the transmembrane ligand; and
[0013] (c) comparing to a control to determine if the substance
inhibits or enhances the binding of the Elk-related receptor
tyrosine kinase and transmembrane ligand, and thereby affects or
modulates the pathway.
[0014] The ability of a substance to inhibit or enhance the binding
of an oligomerized Elk-related receptor tyrosine kinase and
transmembrane ligand correlates with the ability of the substance
to inhibit or enhance the biological activity of the transmembrane
ligand, including the signal transduction activities of the ligand,
and in particular the activation of a pathway regulated by the
ligand.
[0015] The invention also contemplates a method for evaluating a
substance for its ability to inhibit or enhance the interaction of
an oligomerized Elk-related receptor tyrosine kinase, or an
isoform, or an extracellular domain of the Elk-related receptor
tyrosine kinase, and a transmembrane ligand for an Elk-related
receptor tyrosine kinase expressed on a cell comprising the steps
of:
[0016] (a) providing a reporter gene operably linked to a DNA
binding site for a transcriptional activator;
[0017] (b) providing a first hybrid protein comprising the
transmembrane ligand in polypeptide linkage to a DNA binding domain
of the transcriptional activator;
[0018] (c) providing a second hybrid protein comprising an
oligomerized Elk-related receptor tyrosine kinase or an isoform or
an extracellular domain of the Elk-related receptor tyrosine kinase
in polypeptide linkage to an activation domain of the
transcriptional activator; under conditions where the transmembrane
ligand and oligomerized Elk-related receptor tyrosine kinase can
bind and thereby reconstitute the transcriptional activator which
induces transcription of the reporter gene;
[0019] (d) administering a test substance; and
[0020] (e) monitoring expression of the reporter gene, wherein a
decrease in expression is an indication that the substance inhibits
the interaction of the transmembrane ligand and oligomerized
Elk-related receptor tyrosine kinase, and an increase in expression
is an indication that the substance enhances the interaction of the
transmembrane ligand and oligomerized Elk-related receptor tyrosine
kinase. In an alternate method, the oligomerized Elk-related
receptor tyrosine kinase is linked to the DNA binding domain, and
the transmembrane ligand is linked to the activation domain.
[0021] In another aspect, the invention features an antibody
preparation which specifically binds to a receptor-ligand complex
comprising an oligomerized Elk-related receptor tyrosine kinase, or
an isoform or an extracellular domain of the kinase, and a
transmembrane ligand for an Elk-related receptor tyrosine
kinase.
[0022] In another aspect, the invention features a method of
purifying a compound which inhibits or enhances the binding of an
oligomerized Elk-related receptor tyrosine kinase, or an isoform or
an extracellular domain of the kinase, and a transmembrane ligand
for an Elk-related receptor tyrosine kinase comprising contacting
the compound with one of the ligand or receptor; and, isolating the
compound by its binding affinity for the ligand or receptor.
[0023] The substances, and compounds obtained using the methods of
the invention and the antibodies specific for receptor-ligand
complexes may be used to modulate the biological activity of a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand, including inhibiting or
enhancing signal transduction activities of the transmembrane
ligand, and in particular modulating a pathway regulated by the
transmembrane ligand.
[0024] The invention still further provides a method for affecting
or modulating neuronal development or regeneration in a subject
comprising administering to a subject an effective amount of a
purified and isolated oligomerized Elk-related receptor tyrosine
kinase, or an isoform or an extracellular domain thereof, a
substance or compound identified using a method of the invention,
or an antibody specific for a receptor-ligand complex of the
invention.
[0025] In yet another aspect, the invention provides a method for
affecting or modulating axonogenesis in a subject comprising
administering to a subject an effective amount of a purified and
isolated oligomerized Elk-related receptor tyrosine kinase, or an
isoform or an extracellular domain thereof, a substance or compound
identified using a method of the invention, or an antibody specific
for a receptor-ligand complex of the invention.
[0026] The invention also relates to a pharmaceutical composition
which comprises a purified and isolated oligomerized Elk-related
receptor tyrosine kinase, or an isoform or an extracellular domain
thereof, a substance or compound identified using a method of the
invention, or an antibody specific for a receptor-ligand complex of
the invention, in an amount effective to stimulate or inhibit
neuronal development or regeneration and a pharmaceutically
acceptable carrier, diluent or excipient.
[0027] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples while indicating preferred
embodiments of the invention are given by way of illustration only,
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0028] The invention will be better understood with reference to
the drawings in which:
[0029] FIG. 1A is an alignment of human Elk-L, Htk-L and Elk-L3
cytoplasmic domains;
[0030] FIG. 1B is an immunoblot showing phosphorylation of
GST-hElk-L cytoplasmic domain fusion protein (GST-Elk-L cyt) in
vitro by v-Src;
[0031] FIG. 1C is an immunoblot showing expression of Elk-L and
Htk-L either alone or with v-Src in Cos-1 cells;
[0032] FIG. 1D is an immunoblot showing expression of TM-ligands
either alone or with v-Src in Cos-1cells;
[0033] FIG. 2A are immunoblots showing induction of tyrosine
phosphorylation of TM-ligands, expressed in Cos-1 cells, upon
stimulation with clustered Nuk-Fc;
[0034] FIG. 2B is an immunoblot showing stimulation of Elk-L
tyrosine phosphorylation by Nuk-Fc in CHP-100 cells, which express
endogenous Elk-L;
[0035] FIG. 2C are immunoblots showing bi-directional signalling
between Nuk-expressing NG 108 cells and TM-ligand-expressing cells
in co-culture;
[0036] FIG. 3 is an anti-phosphotyrosine immunoblot of TM-ligands
and Nuk receptor immunoprecipitated from E10.5 mouse body
tissue;
[0037] FIG. 4 is a schematic diagram showing bidirectional
signalling by Nuk and its TM-ligands;
[0038] FIG. 5A is a composite sequence of cDNAs encoding full
length human Elk-L3 and a segment of rat Elk-L3;
[0039] FIG. 5B shows amino acid sequences of all the known Eph
family ligands aligned with each other, using the human versions
with the exception of mouse ELF-1; and
[0040] FIG. 5C shows in matrix form the percent similarity between
ligands in FIG. 5B.
DETAILED DESCRIPTION OF THE INVENTION
[0041] As hereinbefore mentioned, the present invention provides a
method of modulating the biological activity of a transmembrane
ligand for an Elk-related receptor tyrosine kinase in a cell
expressing the transmembrane ligand comprising forming a complex
between a purified and isolated oligomerized Elk-related receptor
tyrosine kinase, or an isoform or an extracellular domain of the
kinase, and a transmembrane ligand expressed on the cell. The
biological activity of a transmembrane ligand may be modulated by
inhibiting or enhancing the signal transduction activities of the
ligand including affecting or modulating a pathway regulated by a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand.
[0042] A pathway which is regulated by a transmembrane ligand for
an Elk-related receptor tyrosine kinase in a cell expressing the
transmembrane ligand refers to a regulatory pathway in the cell
that affects, for example gene expression, cell division,
cytoskeletal architecture, cell metabolism, migration, cell-cell
interactions, and spatial positioning, and which is activated by
the binding of transmembane ligands with an oligomerized
Elk-related receptor tyrosine kinase, or an isoform, or an
extracellular domain thereof. Examples of such pathways are the
GAP/Ras pathway, the pathway that regulates the breakdown of the
polyphosphoinositides through phospholipase C (PLC) and the
Src/tyrosine kinase and Ras pathways.
[0043] "Transmembrane ligand or transmembrane ligands" refers to a
class of ligands which are anchored to the cell membrane through a
transmembrane domain, and bind to the extracellular domain of an
Eph subfamily receptor tyrosine kinase, facilitating dimerization
and autophosphorylation of the receptor. The transmembrane ligands
which are targeted or used in accordance with the methods of the
invention are those that bind to and preferably activate (i.e.
phosphorylate) an Elk-related receptor tyrosine kinase (see
discussion of the Elk-related receptor tyrosine kinases below).
Preferably the transmembrane ligands used in the methods of the
invention are Elk-L/LERK2/Efl-3/Cek5-L; hHtk-L/ELF-2/Lerk5
(Tessier-Lavigne, M., 1995, Cell 82:345-348) and hElk-L3/Efl-6
(Gale et al.). These transmembrane ligands have a highly conserved
cytoplasmic region with multiple potential sites for tyrosine
phosphorylation.sup.12-17. The amino acid sequences for the
transmembrane ligands hElk-L3, hHtk-L, and hElk-L, and the
cytoplasmic, transmembrane, and extracellular domains of the
ligands are shown in FIG. 5, which is FIG. 1 in Gale, N. W. et al.,
1996.
[0044] Transmembrane ligands which may be selected or targeted in
accordance with the present invention also include (i) proteins
having sequence identity with known transmembrane ligands such as
hElk-L3. hHtk-L, and hElk-L, and their homologs; (ii) proteins
which can bind with the extracellular domain of an Elk-related
receptor tyrosine kinase; and (iii) chimeric proteins of
transmembrane ligands, e.g. a protein containing a cytoplasmic
domain of one transmembrane ligand and an extracellular domain from
a different transmembrane ligand.
[0045] The term "Elk-related receptor tyrosine kinase" refers to a
particular subclass of the Eph subfamily of receptor tyrosine
kinases. The Eph subfamily receptor tyrosine kinases are a closely
related group of transmembrane receptor tyrosine kinases which
contain cell adhesion-like domains on their extracellular surface.
The Eph subfamily receptor tyrosine kinases are more specifically
characterised as encoding a structurally related cysteine rich
extracellular domain containing a single immunoglobulin (Ig)-like
loop near the N-terminus and two fibronectin III (FN III) repeats
adjacent to the plasma membrane. For example, Nuk contains 20
cysteine residues whose position is conserved in the extracellular
domain of Eph family members, an immunoglobulin-like domain near
the amino terminus (Ig-like), and two fibronectin type III repeats
(FN III: between Nuk amino acids residues 330-420 and 444-534). The
Ig-like domain of Nuk contains specific residues (Cys.sup.70,
Trp.sup.80, Cys.sup.115) known to be conserved in the Ig
superfamily (Williams and Barclay, Ann. Rev. Immunol. 6:381-405,
1988).
[0046] The Elk-related receptor tyrosine kinases bind to, and are
phosphorylated by transmembrane ligands, and include mouse Nuk and
its homologs namely, Hek5 and Erk in humans, Sek3 in mice, and Cek5
in chickens; rat Elk and its homologs including Cek6a in chickens
and xEK, human Hek2 and its homologs including Sek4 in mice and
Cek10 in chickens; and human Htk and its homologs including Myk1 in
mice. The amino acid sequences for representative Elk-related
receptor tyrosine kinases can be found in GenBank, for example
Accession Nos. L25890 (Nuk), X13411 (rat Elk), U07695 (human Htk),
M83941 (Hek), and the publications referred to therein.
[0047] Homologs of Elk-related receptor tyrosine kinases are
identified by aligning a sequence of a putative homolog with a
known Elk-related receptor tyrosine kinase and comparing the
positions in each sequence. When a position in the compared
sequence is occupied by the same nucleotide base or amino acid,
then the molecules are matching or have identical positions shared
by the sequences. Kinase proteins which may be used in the methods
and compositions of the invention may have over 60%, preferably
over 70%, most preferably over 80% identity with an Elk-related
receptor tyrosine kinase.
[0048] Isoforms of an Elk-related receptor tyrosine kinase may be
used in the methods and compositions of the invention. Generally,
an isoform contains the same number and kinds of amino acids and it
binds to a transmembrane ligand as described herein, but the
isoform has a different molecular structure.
[0049] An extracellular domain of an Elk-related receptor tyrosine
kinase may also be used in the methods and compositions of the
invention. The extracellular domain of an Elk-related receptor
tyrosine kinase is generally defined as the region extracellular to
the transmembrane domain. Specifically it is characterised by a
cysteine rich region, whose position is conserved in the
extracellular domain of Eph receptor family members, an
immunoglobulin-like domain near the amino terminus (Ig-like), and
two fibronectin type III repeats (FN III). An extracellular domain
of an Elk-related receptor tyrosine kinases may be selected based
on these characteristic features and by comparing the amino acid
sequences of the extracellular domains of known Elk-related
receptor tyrosine kinases.
[0050] An Elk-related receptor tyrosine kinase may be selected for
use in a method or composition of the invention based on the nature
of the transmembrane ligand which is targeted or selected. The
selection of a specific complementary ligand and Elk-related
receptor tyrosine kinase (e.g. Elk-L and Elk or Nuk; and Htk-L and
Elk or Nuk) in a method of the invention may allow for the
identification of a specific substance that affects a pathway
regulated by a specific transmembrane ligand.
[0051] An Elk-related receptor tyrosine kinase or extracellular
domain thereof, or a transmembrane ligand may be isolated from
cells which are known to express the proteins. Alternatively the
protein or part of the protein may be prepared using conventional
recombinant DNA methods (e.g. baculovirus expression in insect
cells). The proteins or parts thereof may also be prepared by
chemical synthesis using standard techniques such as solid phase
synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or
synthesis in homogenous solution (Houbenweyl, 1987, Methods of
Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme,
Stuttgart).
[0052] The Elk-related receptor tyrosine kinase protein or
extracellular domain thereof, or transmembrane ligands may also be
expressed on the surface of a cell (e.g. Cos- 1 cell) using
conventional methods.
[0053] An Elk-related receptor tyrosine kinase or extracellular
domain thereof, or transmembrane ligand may be conjugated with
other molecules, such as proteins or polypeptides. For example,
N-terminal fusion proteins may be synthesized by fusing, through
recombinant techniques, the N-terminal of an Elk-related receptor
tyrosine kinase or extracellular domain thereof, and the sequence
of a selected protein or marker protein with a desired biological
function, such as an oligomerization motif which facilitates
oligomerization of the receptor or ligand. Examples of
oligomerization motifs include immunoglobulins, and parts thereof
such as the constant region of an immunoglobulin; and motifs which
introduce reactive groups which provide for hydrophobic
interactions between receptors or ligands, for example, amphoteric
.alpha.-helices.
[0054] The transmembrane ligands and Elk-related receptor tyrosine
kinase or extracellular domain thereof, or fusions thereof, used in
the methods and compositions of the invention are oligomerized. An
Elk-related receptor tyrosine kinase or extracellular domain
thereof, or transmembrane ligands may be dimerized by preparing
fusion proteins as discussed above containing an oligomerization
motif such as a constant region of an immunoglobulin. Clusters of
receptors or ligands may then optionally be prepared by adding
antibodies specific for the constant region of the immunoglobulin.
For example, a Nuk extracellular domain-IgG constant chain fusion
protein may be clustered using anti-human IgG. If the receptor
tyrosine kinase or transmembrane ligand is associated with a cell,
interaction of the receptor or extracellular domain thereof with a
transmembrane ligand will result in dimerization of the receptor or
ligand.
[0055] The invention provides a method for evaluating a substance
for its ability to modulate the biological activity of a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand. The method involves
contacting an oligomerized Elk-related receptor tyrosine kinase, or
an isoform or an extracellular domain of the Elk-related receptor
tyrosine kinase; a transmembrane ligand for an Elk-related receptor
tyrosine kinase expressed on a cell that binds to the Elk-related
receptor tyrosine kinase to form a receptor-ligand complex; and, a
test substance, under conditions which permit the formation of
receptor-ligand complexes. Receptor-ligand complexes, free
Elk-related receptor tyrosine kinase, or non-complexed
transmembrane ligand, or activation of the transmembrane ligand are
assayed. The results are compared to a control to determine if the
substance inhibits or enhances the binding of the Elk-related
receptor tyrosine kinase and transmembrane ligand, and thereby
modulates the biological activity of the transmembrane ligand.
[0056] In an embodiment, the invention provides a method for
identifying a substance which affects or modulates a pathway
regulated by a transmembrane ligand for an Elk-related receptor
tyrosine kinase in a cell expressing the transmembrane ligand. An
oligomerized Elk-related receptor tyrosine kinase, or an isoform or
an extracellular domain of the Elk-related receptor tyrosine
kinase, is contacted with a test substance, and a transmembrane
ligand which binds to the receptor to form receptor-ligand
complexes which activate the pathway, under conditions which permit
the formation of receptor-ligand complexes. Receptor-ligand
complexes, free Elk-related receptor tyrosine kinase. or
non-complexed transmembrane ligands, or activation of the ligand
are assayed and the results are compared to a control to determine
the effect of the substance.
[0057] A substance identified using a method of the invention may
stimulate or inhibit the binding of an oligomerized Elk-related
receptor tyrosine kinase, or an isoform or an extracellular domain
thereof, and transmembrane ligands, or compete for a site on the
oligomerized Elk-related receptor tyrosine kinase which binds the
transmembrane ligands or a site on the transmembrane ligands which
binds to the oligomerized Elk-related receptor tyrosine kinase. The
substance may be an endogenous physiological substance, or it may
be a natural or synthetic drug.
[0058] Oligomerized Elk-related receptor tyrosine kinases that may
be used in the methods of the invention are described herein. In
particular, an oligomerized extracellular domain of an Elk-related
receptor tyrosine kinase, preferably Nuk, can be employed in the
method. The oligomerized extracellular domain may also be prepared
as a fusion protein as described herein. The Elk-related receptor
tyrosine kinase may be associated with a cell which either
exogenously expresses the kinase (e.g. transformed Cos-1 cells
which express the kinase) or a cell which endogenously expresses
the kinase (e.g. Cos-1 monkey kidney cells endogenously expressing
Nuk).
[0059] The transmembrane ligand used in the methods of the
invention may be a ligand which is native to the selected
oligomerized Elk-related receptor tyrosine kinase, or it may be a
ligand which is not native to the selected receptor tyrosine
kinase. The transmembrane ligand is preferably associated with a
cell which either exogenously expresses the ligand (e.g.
transformed Cos-1 cells) or a cell which endogenously expresses the
ligand (e.g. CHP-100 cells which express the transmembrane ligand
Elk-L).Where the transmembrane ligand used in a method of the
invention is not cell associated, it should be oligomerized using
the methods described herein.
[0060] Conditions which permit the formation of receptor-ligand
complexes may be selected having regard to factors such as the
nature and amounts of the receptor and the ligand.
[0061] The receptor-ligand complex, free oligomerized receptor or
non-complexed transmembrane ligand may be isolated by conventional
isolation techniques, for example, salting out, chromatography,
electrophoresis, gel filtration, fractionation, absorption,
polyacrylamide gel electrophoresis, agglutination, or combinations
thereof.
[0062] Antibody against the ligand or the receptor, or a labelled
ligand, or a labelled oligomerized receptor may be utilized in the
methods of the invention to facilitate isolation of the complexes
etc. The antibodies, the oligomerized receptor, or substance may be
labelled with a detectable substance.
[0063] The receptor or ligand used in the method of the invention
may be insolubilized. For example, the receptor or ligand may be
bound to a suitable carrier including agarose, cellulose, dextran,
Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter
paper, ion-exchange resin, plastic film, plastic tube, glass beads,
polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid
copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The
carrier may be in the shape of, for example, a tube, test plate,
beads, disc, sphere etc. The insolubilized receptor or ligand may
be prepared by reacting the material with a suitable insoluble
carrier using known chemical or physical methods, for example,
cyanogen bromide coupling.
[0064] Where the ligand is expressed on the surface of a cell the
affect of a test substance may be determined by assaying for
activation of the ligand, or by assaying for a biological affect on
the cell. Activation of the ligand may be determined by assaying
for phosphorylation of the ligand.
[0065] The interaction of the receptor and ligand may be identified
using a two-hybrid expression system wherein the activity of a
transcriptional activator is reconstituted.(See for example, Chien
et al. 1991, Proc. Natl. Acad. Sci. (USA) 88:9578 re two-hybrid
systems). The system may comprise a reporter gene (e.g. genes
encoding gene products such as .beta.-galactosidase (e.g. lacZ),
luciferase, alkaline phosphatase, horseradish peroxidase), operably
linked to a DNA binding site for a transcriptional activator; a
first hybrid protein comprising a transmembrane ligand for an
Elk-related receptor tyrosine kinase in polypeptide linkage to a
DNA binding domain of a transcriptional activator; and a second
hybrid protein comprising an oligomerized Elk-related receptor
tyrosine kinase or an isoform or an extracellular domain of the
Elk-related receptor tyrosine kinase, in polypeptide linkage to an
activation domain of the transcriptional activator. The system is
carried out employing conditions that allow binding of the
transmembrane ligand and oligomerized Elk-related receptor tyrosine
kinase thereby reconstituting the transcriptional activator which
induces transcription of the reporter gene. A test substance is
added, and the expression of the reporter gene is monitored. A
decrease in expression is an indication that the substance inhibits
the interaction of the transmembrane ligand and oligomerized
Elk-related receptor tyrosine kinase, and an increase in expression
is an indication that the substance enhances the interaction of the
transmembrane ligand and oligomerized Elk-related receptor tyrosine
kinase. In an alternate method, the oligomerized Elk-related
receptor tyrosine kinase is linked to the DNA binding domain, and
the transmembrane ligand is linked to the activation domain.
[0066] The invention also provides host organisms (typically
unicellular organisms) which harbor a two-hybrid system as
described herein. Usually the host organism is a yeast cell such as
Saccharomyces cervisiae.
[0067] In a particular system, the yeast GAL4 protein which has a
domain responsible for DNA-binding and another domain for
transcriptional activation is employed. In the expression system,
plasmids encoding two hybrid proteins one containing the GAL4 DNA
binding domain fused to a first protein (a transmembrane ligand or
an oligomerized Elk-related receptor tyrosine kinase), and a second
plasmid containing the GAL4 activation domain fused to a second
protein (a transmembrane ligand or an oligomerized Elk-related
receptor tyrosine kinase which forms a complex with the first
protein) are introduced into the yeast. If the first and second
proteins interact with one another, the ability to activate
transcription from promoters containing GAL4-binding sites is
reconstituted leading to the expression of a reporter gene e.g.
lacZ.
[0068] The invention also features an antibody preparation which
specifically binds to a receptor-ligand complex comprising an
oligomerized Elk-related receptor tyrosine kinase, or an isoform or
an extracellular domain of the kinase, and a transmembrane ligand
for an Elk-related receptor tyrosine kinase. Antibodies (e.g.
monoclonal and polyclonal antibodies) may be prepared against
oligomerized receptor-ligand complexes. The invention can employ
not only intact monoclonal or polyclonal antibodies, but also
immunologically active fragments (e.g. a Fab or (Fab).sub.2,
fragment), an antibody heavy chain, and antibody light chain, a
genetically engineered single chain F.sub.v molecule (Ladner et al,
U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an
antibody which contains the binding specificity of a murine
antibody, but in which the remaining portions are of human origin.
Antibodies including monoclonal and polyclonal antibodies,
fragments and chimeras, may be prepared using methods known to
those skilled in the art.
[0069] The invention also features a method of purifying a compound
which inhibits or enhances the binding of an oligomerized
Elk-related receptor tyrosine kinase, or an isoform or an
extracellular domain of the kinase, and a transmembrane ligand for
an Elk-related receptor tyrosine kinase comprising contacting the
compound with one of the ligand or receptor, and, isolating the
compound by its binding affinity for the ligand or receptor.
Conventional affinity binding methods can be used to isolate a
compound.
[0070] The substances and compounds obtained using the methods of
the invention, and the antibodies specific for receptor-ligand
complexes may be used to modulate the biological activity of a
transmembrane ligand for an Elk-related receptor tyrosine kinase in
a cell expressing the transmembrane ligand, including inhibiting or
enhancing signal transduction activities of the transmembrane
ligand, and in particular modulating a pathway in a cell regulated
by a transmembrane ligand for an Elk-related receptor tyrosine
kinase, particularly those pathways involved in neuronal
development, axonal migration, pathfinding and regeneration. The
identification and isolation of substances and compounds will
permit studies of the role of the substances and compounds in the
developmental regulation of axonogenesis and neural regeneration,
and permit the development of substances which affect these roles,
such as functional or non-functional analogues of the oligomerized
extracellular domain of an Elk-related receptor tyrosine kinase.
The substances, compounds, and substances derived therefrom, and
antibodies, will be useful as pharmaceuticals to modulate
axonogenesis, nerve cell interactions and regeneration, to treat
conditions such as neurodegenerative diseases and conditions
involving trauma and injury to the nervous system, for example
Alzheimer's disease, Parkinson's disease, Huntington's disease,
demylinating diseases, such as multiple sclerosis, amyotrophic
lateral sclerosis, bacterial and viral infections of the nervous
system, deficiency diseases, such as Wernicke's disease and
nutritional polyneuropathy, progressive supranuclear palsy, Shy
Drager's syndrome, multistem degeneration and olivo ponto
cerebellar atrophy, peripheral nerve damage, trauma and ischemia
resulting from stroke.
[0071] The present invention thus provides a method for affecting
neuronal development or regeneration in a subject comprising
administering to a subject an effective amount of a purified and
isolated oligomerized Elk-related receptor tyrosine kinase, or an
isoform or an extracellular domain thereof, a substance or compound
identified using a method of the invention, or antibodies specific
for oligomerized receptor-ligand complexes. The invention also
contemplates a method for stimulating or inhibiting axonogenesis in
a subject comprising administering to a subject an effective amount
of a purified and isolated oligomerized Elk-related receptor
tyrosine kinase protein, or an isoform or an extracellular domain
thereof, a substance or compound identified using a method of the
invention, or antibodies specific for oligomerized receptor-ligand
complexes.
[0072] The invention still further relates to a pharmaceutical
composition which comprises a purified and isolated oligomerized
Elk-related receptor tyrosine kinase protein or an isoform or an
extracellular domain thereof, a substance or compound identified
using a method of the invention, or antibodies specific for
oligomerized receptor-ligand complexes, in an amount effective to
regulate neuronal development or regeneration and a
pharmaceutically acceptable carrier, diluent or excipient. The
pharmaceutical compositions may be used to stimulate or inhibit
neuronal development. regeneration and axonal migration associated
with neurodegenerative conditions and conditions involving trauma
and injury to the nervous system as described above.
[0073] The compositions of the invention are administered to
subjects in a biologically compatible form suitable for
pharmaceutical administration in vivo. By "biologically compatible
form suitable for administration in vivo" is meant a form of the
protein to be administered in which any toxic effects are
outweighed by the therapeutic effects of the protein. The term
subject is intended to include mammals and includes humans, dogs,
cats, mice, rats, and transgenic species thereof. Administration of
a therapeutically active amount of the pharmaceutical compositions
of the present invention is defined as an amount effective, at
dosages and for periods of time necessary to achieve the desired
result. For example, a therapeutically active amount of an
oligomerized Elk-related receptor tyrosine kinase protein may vary
according to factors such as the condition, age, sex, and weight of
the individual. Dosage regimes may be adjusted to provide the
optimum therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation.
[0074] The active compound (e.g., protein) may be administered in a
convenient manner such as by injection (subcutaneous, intravenous,
etc.), oral administration, inhalation, transdermal application, or
intracerebral administration. Preferably, the pharmaceutical
compositions of the invention are administered directly to the
peripheral or central nervous system, for example by administration
intracerebrally.
[0075] A pharmaceutical composition of the invention can be
administered to a subject in an appropriate carrier or diluent,
co-administered with enzyme inhibitors or in an appropriate carrier
such as microporous or solid beads or liposomes. The term
"pharmaceutically acceptable carrier" as used herein is intended to
include diluents such as saline and aqueous buffer solutions.
Liposomes include water-in-oil-in-water emulsions as well as
conventional liposomes (Strejan et al., (1984) J. Neuroimmunol
7:27). The active compound may also be administered parenterally or
intraperitoneally. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
may contain a preservative to prevent the growth of microorganisms.
Depending on the route of administration, the active compound may
be coated to protect the compound from the action of enzymes, acids
and other natural conditions which may inactivate the compound.
[0076] The pharmaceutical compositions may be administered locally
to stimulate axonogenesis and pathfinding, for example the
compositions may be administered in areas of local nerve injury or
in areas where normal nerve pathway development has not occurred.
The pharmaceutical compositions may also be placed in a specific
orientation or alignment along a presumptive pathway to stimulate
axon pathfinding along that line, for example the pharmaceutical
compositions may be incorporated on microcarriers laid down along
the pathway. In particular, the pharmaceutical compositions of the
invention may be used to stimulate formation of connections between
areas of the brain, such as between the two hemispheres or between
the thalamus and ventral midbrain. The pharmaceutical compositions
may be used to stimulate formation of the medial tract of the
anterior commissure or the habenular interpeduncle.
[0077] Therapeutic administration of polypeptides may also be
accomplished using gene therapy, A nucleic acid including a
promoter operatively linked to a heterologous polypeptide may be
used to produce high-level expression of the polypeptide in cells
transfected with the nucleic acid. DNA or isolated nucleic acids
may be introduced into cells of a subject by conventional nucleic
acid delivery systems. Suitable delivery systems include liposomes,
naked DNA, and receptor-mediated delivery systems, and viral
vectors such as retroviruses, herpes viruses, and adenoviruses.
[0078] The following non-limiting example is illustrative of the
present invention:
EXAMPLE
[0079] The following is a detailed description of FIGS. 1 to 4
including a detailed description of the methods utilized in the
experiments illustrated in the Figures and outlined in the
Example:
[0080] Phosphorylation of Transmembrane Ligands by vSrc
[0081] (FIG. 1A)
[0082] Alignment of human Elk-L, Htk-L and Elk-L3 cytoplasmic
domains.
[0083] Black boxes indicate residues conserved in all TM-ligands;
grey boxes indicate residues conserved in two ligands. Conserved
tyrosines are marked by an asterisk. Mouse Htk-L cytoplasmic domain
is identical to human Htk-L except for a serine to glycine
substitution at position 278. El k-L is also referred to as
Lerk-2.sup.13 and Cek5-L.sup.14, Htk-L as Lerk-5.sup.25and
Elf-2.sup.15. (FIG. 1B) Phosphorylation of GST-hElk-L cytoplasmic
domain fusion protein (GST-Elk-L cyt) in vitro by v-Src. v-Src was
immunoprecipitated from v-Src transformed Rat-2 cells and incubated
with GST fusion proteins or enolase as exogenous substrates in the
presence of .sup.32P.gamma.ATP. (FIG. 1C) and (FIG. 1D) Tyrosine
phosphorylation of TM-ligands upon coexpression with v-Src. (FIG.
1C) Elk-L and Htk-L were expressed either alone or with v-Src in
Cos-1 cells and immunoprecipitated with anti-ligand antibody. Upper
panel: anti-phosphotyrosine blot; lower panel: anti-ligand blot
(reprobe). (FIG. 1D) TM-ligands were expressed as in (FIG. 1C),
precipitated using a Nuk extracellular domain IgG fusion protein
(Nuk-Fc) as an affinity reagent, and immunoblotted with
anti-phosphotyrosine serum. The band observed at .about.100 kDa
represents cross-reaction of Nuk-Fc with the protein A-HRP.
Methods: (FIG. 1B) v-Src was immunoprecipitated from v-Src
transformed Rat-2 cells using an anti-Src monoclonal antibody
(Oncogene Science) and immune complexes were incubated for 15
minutes at RT with 5 .mu.Ci of .sup.32P.gamma.ATP in Src-KRB.sup.26
alone or in buffer containing enolase, 10 .mu.g purified GST or
GST-Elk-L cyt (residues 262-343 of hElk-L.sup.12) as exogenous
substrates. Proteins were separated on a 10% SDS-PAGE gel and
.sup.32P labelled proteins were detected by autoradiography. (FIG.
1C) and (FIG. 1D) Cos-1 cells were transiently transfected as
indicated with 5 .mu.g of hElk-L, mHtk-L or v-Src cDNA expression
vectors, either alone or in combination, or with empty control
vector. Cells were serum starved for approximately 20 hours in
medium containing 0.5% foetal bovine serum (FBS) and lysed in PLC
Iysis buffer.sup.2 at approximately 60 hours post transfection.
TM-ligands were precipitated using (FIG. 1C) anti-ligand serum
(raised against residues 326-343 of hElk-L, which also recognises
Htk-L; Santa Cruz) or (FIG. 1D) 10 .mu.g of Nuk-Fc fusion
protein.sup.11 plus protein A sepharose. Precipitated proteins were
washed three times in HNTG.sup.2, separated on a 10% SDS-PAGE gel,
transferred to PVDF membrane (Millipore) and immunoblotted with
(FIG. 1C) monoclonal (4G10) or (FIG. 1D) polyclonal
anti-phosphotyrosine antibodies. Detection was by Enhanced
Chemiluminescence (Pierce). In (FIG. 1C) the filter was stripped
using 0.1 M glycine pH 2.5 and reprobed with anti-ligand serum.
[0084] Stimulation of Tyrosine Phosphorylation of TM-Ligands by Nuk
Extracellular Domain and Nuk-Expressing Cells
[0085] FIG. 2A
[0086] Induction of Tyrosine Phosphorylation of TM-Ligands,
Expressed in Cos-1 Cells, upon Stimulation with clustered
Nuk-Fc.
[0087] Cos-1 cells were transiently transfected with Elk-L, Htk-L.
or control expression vectors and treated with 2 .mu.g/ml clustered
Nuk-Fc fusion protein or Fc tag alone (c) for the indicated periods
of time. Cells were lysed, immunoprecipitated with anti-ligand
serum and blotted with antibodies to phosphotyrosine (upper
panels). Filters were stripped and reprobed with anti-ligand serum
(lower panels). As a control, excess immunizing peptide was
included as indicated (+pep). Elk-L and Htk-L phosphorylated by
cotransfection with v-Src were included on these gels to indicate
the mobility of tyrosine phosphorylated ligands; as less protein
was loaded in these lanes than the Nuk-Fc stimulated lanes, no
bands were detected upon reprobing with anti-ligand serum.
[0088] FIG. 2B
[0089] Stimulation of Elk-L Tyrosine Phosphorylation by Nuk-Fc in
CHP-100Cells, which Express Endogenous Elk-L
[0090] Cells were stimulated with 2 .mu.g/ml clustered Nuk-Fc or Fc
alone (c) for the indicated periods of time, lysed and
immunoblotted as in FIG. 2A. Upper panel: anti-phosphotyrosine
blot; lower panel: anti-ligand blot (reprobe).+Pep=+competing
immunizing peptide.
[0091] FIG. 2C
[0092] Bi-Directional Signalling between Nuk-Expressing and
TM-Ligand-Expressing Cells in Co-Culture.
[0093] Cos-1 cells transiently transfected with Elk-L or Htk-L were
co-cultured with parental or Nuk-expressing NG108 cells for the
indicated times. Left panels: anti-ligand IP; right panels:
anti-Nuk IP from pooled, cocultured cells. Upper panels:
anti-phosphotyrosine blots; lower panels: anti-ligand blots
(reprobe). Methods: In FIG. 2A and FIG. 2B, Cos- 1 cells were
transiently transfected and serum starved as in FIG. 1. Human
neuroepithelial CHP-100 cells were serum starved for 8 hours in
medium containing 0.5% FBS. Nuk-Fc.sup.11 or Fc tag (c) was
clustered using anti-human IgG (Jackson Immunoresearch) for 1-2
hours at 4.degree. C., diluted to a final concentration of 2
.mu.g/ml in serum free medium, and applied to cells for the
indicated periods of time. Cells were lysed in PLC lysis buffer and
immunoprecipitated with anti-ligand antibodies. Immunoprecipitation
of TM-ligands was inhibited where indicated by addition of 100-fold
excess immunizing Elk-L C-terminal peptide (+pep; residues 326-343
of hElk-L, Santa Cruz). Immune complexes were separated and
transferred as in FIG. 1 and immunoblotted with monoclonal
anti-phosphotyrosine antibodies (4G 10; upper panel). In FIG. 2C,
NG 108-15 cells (NG 108: mouse neuroblastoma .times.rat glioma
fusion.sup.20 ) were stably transfected with an expression vector
containing full-length Nuk, and indvidual G418 resistant clones
were isolated (NG108-Nuk). Parental or Nuk-expressing NG108 cells
were removed from the plate by tituration and resuspended in
PBS+magnesium and calcium. Cell suspensions were placed on top of
serum starved Cos- 1 cells transiently expressing Elk-L (left
panels) or Htk-L (right panels). Cells were cocultured for 30 or 60
minutes at 37.degree. C., 5% CO.sub.2 , and lysed together in PLC
lysis buffer. Cleared lysates were divided in two and
immunoprecipitated with either anti-ligand or anti-Nuk serum.sup.2
as indicated. Proteins were separated and transferred as in FIG. 2
and immunoblotted with monoclonal anti-phosphotyrosine antibodies
(upper panels). Blots were stripped and reprobed with anti-ligand
antibodies (lower panel).
[0094] FIG. 3
[0095] Both Transmembrane Ligands and Nuk are Phosphorylated on
Tyrosine in the Mouse Embryo
[0096] Anti-phosphotyrosine immunoblot of TM-ligands (left panel)
and Nuk receptor (right panel) immunoprecipitated from E10.5 mouse
body tissue. Lysed tissue was immunoprecipitated with anti-Nuk or
pre-immune serum (PI), or anti-ligand antibodies with or without
addition of excess competing immunizing peptide (ligand pep). The
mobility of tyrosine phosphorylated Elk-L and Htk-L is indicated by
inclusion of v-Src phosphorylated TM-ligands on the gel
(Elk-L+v-Src and Htk-L+v-Src). Methods: E10.5 mouse embryos from
wild-type matings were harvested and divided into heads and bodies.
Tissue was lysed in PLC lysis buffer by Dounce homogenisation,
cleared and precleared by incubation with protein A sepharose.
Supematants were immunoprecipitated with anti-Nuk or pre-immune
serum, or anti-ligand antibodies with or without addition of
100-fold excess competing ligand C-terminal peptide. Proteins were
separated, transferred and immunoblotted with monoclonal
antiphosphotyrosine antibodies. Results for E10.5 head tissue were
essentially identical.
[0097] FIG. 4
[0098] Model for Bi-Directional Signalling by Nuk and Its
TM-Ligands.
[0099] Interaction of the ligand-expressing cell (right) with the
receptor-expressing cell (left) promotes aggregation and
autophosphorylation of the receptor. This is followed by
recruitment of SH2 domain-containing proteins to phosphorylated
tyrosines e.g. in the juxtamembrane region.sup.27, 28, and tyrosine
phosphorylation of cellular proteins. Concomitantly, interaction of
the receptor with TM-ligands causes ligand clustering and
phosphorylation by an associated tyrosine kinase, leading to
propagation of signals in the ligand presenting cell.
[0100] DESCRIPTION OF RESULTS
[0101] Nuk belongs to a subclass of Eph receptors that bind
specifically to the TM-subgroup of Eph receptor ligands.sup.11, 14,
19. Genetic analysis of Nuk in the mouse has revealed a
physiological role for this receptor in pathfinding of specific
anterior commissure axons, and has raised the possibility that the
TM-ligands might themselves possess a signalling function, which is
activated by binding of the Nuk extracellular domain.sup.6 . The
three known TM-ligands have highly conserved cytoplasmic domains,
and are virtually identical over their C-terminal 33 amino
acids.sup.12-17. These sequences contain five potential tyrosine
phosphorylation sites (FIG. 1A). A GST fusion protein containing
the cytoplasmic domain of human Elk-L (residues 262-343) was
tyrosine phosphorylated by v-Src in vitro (FIG. 1B), whereas GST
alone or a fusion protein containing the Elk-L extracellular domain
were not (FIG. 1B and data not shown).
[0102] To investigate whether full-length Elk-L or Htk-L could be
phosphorylated on tyrosine in vivo, these TM-ligands were expressed
in Cos-1 cells either alone or in combination with v-Src. The
ligands were then precipitated from the transfected cells using
either an antibody to the common C-terminal region of Elk-L and
Htk-L (anti-ligand) or a fusion protein containing the
extracellular domain of Nuk fused to the Fc region of the Ig heavy
chain (Nuk-Fc.sup.11 ), which binds with high affinity to the
extracellular domain of TM-ligands. When such precipitates were
blotted with ligand antibody, a diffuse band of approximately 45-48
kDa was specifically detected in cells transfected with Elk-L,
whereas a protein of between 38 and 46 kDa was identified in cells
transfected with Htk-L (FIG. 1C lower panel). The predicted
molecular weights of Elk-L and Htk-L are 38 and 37 kDa
respectively, and their slow electrophoretic mobility is apparently
due to glycosylation (data not shown). Immunoblotting of
anti-ligand immunoprecipitates from transfected Cos-1 cells with
antibodies to phosphotyrosine (FIG. 1C upper panel) showed that
both Elk-L and Htk-L were basally phosphorylated on tyrosine at low
levels. Co-transfection of Elk-L with v-Src led to the appearance
of a highly tyrosine phosphorylated .about.48 kDa form of Elk-L in
both anti-ligand and Nuk-Fc precipitates (FIG. 1C upper panel and
FIG. 1D). In addition a tyrosine phosphorylated protein of 130-140
kDa was observed to co-precipitate with Elk-L from cells
co-expressing ligand and v-Src. v-Src also induced strong tyrosine
phosphorylation of Htk-L, which migrated as a broad band of 38-48
kDa in the phosphorvlated form. Immunoprecipitation of both
Elk-L/Htk-L and the 130 kDa protein was markedly reduced by
addition of the immunizing peptide which competes for antibody
binding (data not shown). In v-Src co-transfected cells the total
amount of either ligand detected in western blots by the
anti-ligand antibodies was reduced. It is possible that these
antibodies, which were raised against the C-terminal part of Elk-L,
are less efficient in recognising highly tyrosine phosphorylated
forms of the denatured ligands in an immunoblot. These results show
that Elk-L and Htk-L are potent in vivo substrates for an activated
Src tyrosine kinase, and can be detected in association with other
phosphotyrosine-containing proteins in cells expressing both ligand
and v-Src.
[0103] The phosphorylation of TM-ligands on tyrosine may provide a
mechanism by which signals are transmitted into ligand-presenting
cells and such a signal might be activated by the clustering of
ligands on the cell surface. To address this issue, Cos-1 cells
expressing Elk-L or Htk-L were exposed to the Nuk extracellular
domain, in the form of a Nuk-Fc fusion protein clustered with
anti-Ig. Under these conditions Nuk-Fc induced a several-fold
increase in the tyrosine phosphorylation of both Elk-L and Htk-L,
whereas no stimulation of ligand tyrosine phosphorylation was
induced by Fc alone (FIG. 2A). The tyrosine phosphorylated band
immunoprecipitated by the anti-ligand serum was markedly reduced by
addition of excess ligand C-terminal peptide to the
immunoprecipitates. This experiment indicates that the binding of
clustered Nuk-Fc to the TM-ligands induces the activation of an
endogenous tyrosine kinase in Cos-1 cells that can subsequently
phosphorylate Elk-L and Htk-L. These results, whilst provocative,
were performed using exogenously overexpressed ligand. To
corroborate these observations in a more physiologically relevant
cell type, the human neuroepithelioma cell line CHP-100, previously
shown to express endogenous Elk-L.sup.12 was employed. Incubation
of CHP-100 cells with clustered Nuk-Fc led to a striking increase
in the tyrosine phosphorylation of Elk-L and to coprecipitation of
several tyrosine phosphorylated polypeptides (FIG. 2B). Thus, the
binding of the Nuk extracellular domain to a cell that normally
expresses Elk-L also leads to tyrosine kinase activation and
concomitant Elk-L phosphorylation.
[0104] These findings raised the possibility that the interaction
of a cell expressing TM-ligands on its surface with a second cell
expressing Nuk might lead to both the activation of the Nuk
receptor, and subsequent signalling within the Nuk-expressing cell,
and also to the activation of a ligand-associated kinase and
consequent ligand phosphorylation. To test this notion, Cos-1 cells
expressing Elk-L or Htk-L were co-cultured with the neuronal cell
line NG108-15.sup.20 (NG 108), that does not express endogenous Eph
receptors which bind TM-ligands, or with a transfected NG 108 clone
which stably expresses high levels of the 130 kDa mouse Nuk protein
(NG108-Nuk). In cocultures of ligand-expressing cells with
NG108-Nuk cells, both the induction of Nuk tyrosine
phosphorylation, reflecting activation of the Nuk catalytic domain,
and also tyrosine phosphorylation of Elk-L or Htk-L, were observed
which is consistent with stimulation of a ligand-associated
tyrosine kinase in the ligand-expressing cells (FIG. 2C). Parental
NG 108 cells lacking Nuk were without effect and conversely, no
phosphorylation of either TM-ligands or Nuk was induced using
untransfected Cos- 1 cells (FIG. 2C and data not shown).
[0105] The observation that Elk-L and Htk-L are inducibly
phosphorylated on tyrosine in cultured cells upon exposure to
clustered Nuk-Fc, or Nuk-expressing cells, suggests that this may
be a physiological event. To test this possibility, protein lysates
from mouse embryos at 10.5 days of development were
immunoprecipitated with antibodies to either TM-ligands or Nuk, and
the immune complexes were immunoblotted with antibodies to
phosphotyrosine (FIG. 3). Nuk immunoprecipitated from embryonic
body or head tissue was phosphorylated on tyrosine (FIG. 3 and data
not shown). Furthermore, anti-ligand antibodies specifically
precipitated phosphotyrosine-containing polypeptides from these
embryonic lysates that co-migrated with authentic TM-ligands. The
intensity of the tyrosine phosphorylated band immunoprecipitated by
the anti-ligand antibodies was markedly reduced by addition of
excess ligand C-terminal peptide. These data demonstrate that not
only Eph receptors such as Nuk, but also their TM-ligands, are
phosphorylated on tyrosine in the developing mouse embryo.
[0106] Eph receptors and their ligands are expressed in reciprocal,
mutually exclusive domains in the developing embryo.sup.11. Such
expression patterns support data implicating Eph receptors in
establishing boundaries between two distinct cell types, for
example in the rhombomeres of the hindbrain and in development of
the forebrain.sup.9, 10. To achieve this purpose, it would be
advantageous if cell-cell contact initiated a bi-directional
signal, thereby regulating the phenotype of both receptor- and
ligand-expressing cells. The experiments described above
demonstrated a biochemical mechanism through which such
bi-directional signalling can be achieved (FIG. 4). In the neuronal
cell line NG 108, activation of Nuk by TM-ligands leads not only to
Nuk autophosphorylation, but also the phosphorylation of potential
receptor targets (FIG. 2C). The data also indicate that binding of
Nuk to TM-ligands activates a tyrosine kinase in the
ligand-expressing cell, leading to phosphorylation of the conserved
C-terminal region of the ligand itself. The TM-ligands contain
several tyrosine residues in a favourable sequence context for
phosphorylation by Src-like kinases.sup.21. One scheme consistent
with the results is that phosphorylation of the ligands by Src-like
kinases induces the binding of SH2-containing proteins which then
transmit signals within the ligand-expressing cell. However, the
finding that TM-ligands are highly phosphorylated on tyrosine in
mouse embryos suggests that ligand signalling is a significant
event in the intact organism.
[0107] Having illustrated and described the principles of the
invention in a preferred embodiment, it should be appreciated to
those skilled in the art that the invention can be modified in
arrangement and detail without departure from such principles. We
claim all modifications coming within the scope of the following
claims.
[0108] All publications, patents and patent applications referred
to herein are incorporated by reference in their entirety to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety.
[0109] Below full citations are set out for the references referred
to in the footnotes in the specification.
FULL CITATIONS FOR REFERENCES REFERRED TO IN THE FOOTNOTES IN THE
SPECIFICATION
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H. J. Neuroscience 12: 3956-3967 (1992)
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P., Conlon R. A., Holmyard D. P., Letwin K., Pawson T. Oncogene 9:
1001-1014 (1994)
[0112] 3. Winslow J. W., Moran P., Valverde J., Shih A., Yuan J.
G., Wong S. C., Tsai S. P., Goddard A., Henzel W. J., Hefti F.,
Beck K. D., Caras I. W. Neuron 14:973-981 (1995)
[0113] 4. Drescher U., Kremoser C., Handwerker C., Loschinger J.,
Noda M., Bonhoeffer F. Cell 82: 359-370 (1995)
[0114] 5. Cheng H-J., Nakamoto M., Bergemann A. D., Flanagan J. G.
Cell 82: 371-381 (1995)
[0115] 6. Henkemeyer M., Orioli D., Henderson J. T., Saxton T. M.,
Roder J., Pawson T., Klein R. Cell, in press
[0116] 7. Tessier-Lavigne, M. Cell 82: 345-348 (1995)
[0117] 8. Pandey A., Shao H., Marks R. M., Polverini P. J., Dixit
V. M. Science 268: 587-569 (1995)
[0118] 9. Xu Q., Alldus G., Holder N., Wilkinson D. G. Development
121: 4005-4016 (1995)
[0119] 10. Xu Q., Alldus G., Macdonald R., Wilkinson D. G., Holder
N. Nature 381: 319-322 (1996)
[0120] 11. Gale N. W., Holland S. J., Valenzuela D. M., Flenniken
A., Pan L., Ryan T. E., Henkemeyer M., Hirai, H., Strebhardt K.,
Wilkinson D. G., Pawson T., Davis S., Yancopoulos G. D. Neuron, in
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Lhotak V., Pawson T., Goldfarb, M., Yancopoulos G. D. Science 26:
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James L., Farrah T., Kozlosky, C., Hollingsworth T., Shilling H.,
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EMBO J. 13: 3757-3762 (1994)
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J. Biol. Chem. 269: 26606-26609 (1994)
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Flanagan J. G. Mol. Cell Biol. 15: 4921-4929 (1995)
[0125] 16. Bennett B. D., Zeigler F. C., Gu Q., Fendly B., Goddard
A. D., Gillet N., Matthews W. Proc. Natl. Acad. Sci. USA 92:
1886-1870 (1995)
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Davis S., Wilkinson D. G., Yancopoulos G. D. Oncogene, in press
[0127] 18. Taniguchi T. Science 268: 251-255 (1995)
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Bergemann A. D., Flanagan J. G., Pasquale E. B., Klein R. EMBO J.
14: 3116-3126 (1995)
[0129] 20. Nelson P., Christian C., Nirenberg M. Proc. Nat. Acad.
Sci. USA 73: 123-127 (1976)
[0130] 21. Songyang Z., Carraway K. L. III , Eck M. J., Harrison S.
C., Feldman R. A., Mohammadi, M., Schlessinger J., Hubbard S. R.,
Smith D. P., Eng C., Lorenzo M. J., Ponder B. A. J., Mayer B. J.,
Cantley L. C. Nature 373: 536-539 (1995)
[0131] 22. Pandey A., Lindberg R. A., Dixit V. M. Current Biology
5: 986-989 (1995)
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Stockinger H. Science 254: 1016-1019 (1991)
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Reddy P., Maraskovsky E., Park S., Lyman S. D., Copeland N. G.,
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Sequence CWU 1
1
13 1 1120 DNA Homo sapiens 1 ccggggggtg ggcgactttg ggggagttgg
tgccccgccc cccaggcctt ggcggggtca 60 tggggccccc ccattctggg
ccggggggcg tgcgagtcgg ggccctgctg ctgctggggg 120 ttttggggct
ggtgtctggg ctcagcctgg agcctgtcta ctggaactcg gcgaataaga 180
ggttccaggc agagggtggt tatgtgctgt accctcagat cggggaccgg ctagacctgc
240 tctgcccccg ggcccggcct cctggccctc actcctctcc taattatgag
ttctacaagc 300 tgtacctggt agggggtgct cagggccggc gctgtgaggc
accccctgcc ccaaacctcc 360 ttctcacttg tgatcgccca gacctggatc
tccgcttcac catcaagttc caggagtata 420 gccctaatct ctggggccac
gagttccgct cgcaccacga ttactacatc attgccacat 480 cggatgggac
ccgggagggc ctggagagcc tgcagggagg tgtgtgccta accagaggca 540
tgaaggtgct tctccaagtg ggacaaagtc cccgaggagg ggctgtcccc cgaaaacctg
600 tgtctgaaat gcccatggaa agagaccgag gggcagccca cagcctggag
cctgggaagg 660 agaacctgcc aggtgacccc accagcaatg caacctcccg
gggtgctgaa ggccccctgc 720 cccctcccag catgcctgca gtggctgggg
cagcaggggg gctggcgctg ctcttgctgg 780 gcgtggcagg ggctgggggt
gccatgtgtt ggcggagacg gcgggccaag ccttcggaga 840 gtcgccaccc
tggtcctggc tccttcggga ggggagggtc tctgggcctg gggggtggag 900
gtgggatggg acctcgggag gctgagcctg gggagctagg gatagctctg cggggtggcg
960 gggctgcaga tccccccttc tgcccccact atgagaaggt gagtggtgac
tatgggcatc 1020 ctgtgtatat cgtgcaggat gggccccccc agagccctcc
aaacatctac tacaaggtat 1080 gagggctcct ctcacgtggc tatcctgaat
ccagcccttc 1120 2 29 DNA Rattus norvegicus 2 tatcgacttt acgcaccaac
aaatggaag 29 3 340 PRT Homo sapiens 3 Met Gly Pro Pro His Ser Gly
Pro Gly Gly Val Arg Val Gly Ala Leu 1 5 10 15 Leu Leu Leu Gly Val
Leu Gly Leu Val Ser Gly Leu Ser Leu Glu Pro 20 25 30 Val Tyr Trp
Asn Ser Ala Asn Lys Arg Phe Gln Ala Glu Gly Gly Tyr 35 40 45 Val
Leu Tyr Pro Gln Ile Gly Asp Arg Leu Asp Leu Leu Cys Pro Arg 50 55
60 Ala Arg Pro Pro Gly Pro His Ser Ser Pro Asn Tyr Glu Phe Tyr Lys
65 70 75 80 Leu Tyr Leu Val Gly Gly Ala Gln Gly Arg Arg Cys Glu Ala
Pro Pro 85 90 95 Ala Pro Asn Leu Leu Leu Thr Cys Asp Arg Pro Asp
Leu Asp Leu Arg 100 105 110 Phe Thr Ile Lys Phe Gln Glu Tyr Ser Pro
Asn Leu Trp Gly His Glu 115 120 125 Phe Arg Ser His His Asp Tyr Tyr
Ile Ile Ala Thr Ser Asp Gly Thr 130 135 140 Arg Glu Gly Leu Glu Ser
Leu Gln Gly Gly Val Cys Leu Thr Arg Gly 145 150 155 160 Met Lys Val
Leu Leu Gln Val Gly Gln Ser Pro Arg Gly Gly Ala Val 165 170 175 Pro
Arg Lys Pro Val Ser Glu Met Pro Met Glu Arg Asp Arg Gly Ala 180 185
190 Ala His Ser Leu Glu Pro Gly Lys Glu Asn Leu Pro Gly Asp Pro Thr
195 200 205 Ser Asn Ala Thr Ser Arg Gly Ala Glu Gly Pro Leu Pro Pro
Pro Ser 210 215 220 Met Pro Ala Val Ala Gly Ala Ala Gly Gly Leu Ala
Leu Leu Leu Leu 225 230 235 240 Gly Val Ala Gly Ala Gly Gly Ala Met
Cys Trp Arg Arg Arg Arg Ala 245 250 255 Lys Pro Ser Glu Ser Arg His
Pro Gly Pro Gly Ser Phe Gly Arg Gly 260 265 270 Gly Ser Leu Gly Leu
Gly Gly Gly Gly Gly Met Gly Pro Arg Glu Ala 275 280 285 Glu Pro Gly
Glu Leu Gly Ile Ala Leu Arg Gly Gly Gly Ala Ala Asp 290 295 300 Pro
Pro Phe Cys Pro His Tyr Glu Lys Val Ser Gly Asp Tyr Gly His 305 310
315 320 Pro Val Tyr Ile Val Gln Asp Gly Pro Pro Gln Ser Pro Pro Asn
Ile 325 330 335 Tyr Tyr Lys Val 340 4 333 PRT Homo sapiens 4 Met
Ala Val Arg Arg Asp Ser Val Trp Lys Tyr Cys Trp Gly Val Leu 1 5 10
15 Met Val Leu Cys Arg Thr Ala Ile Ser Lys Ser Ile Val Leu Glu Pro
20 25 30 Ile Tyr Met Asn Ser Ser Asn Ser Lys Phe Leu Pro Gly Gln
Gly Leu 35 40 45 Val Leu Tyr Pro Gln Ile Gly Asp Lys Leu Asp Ile
Ile Cys Pro Lys 50 55 60 Val Asp Ser Lys Thr Val Gly Gln Tyr Glu
Tyr Tyr Lys Val Tyr Met 65 70 75 80 Val Asp Lys Asp Gln Ala Asp Arg
Cys Thr Ile Lys Lys Glu Asn Thr 85 90 95 Pro Leu Leu Asn Cys Ala
Lys Pro Asp Gln Asp Ile Lys Phe Thr Ile 100 105 110 Lys Phe Gln Glu
Phe Ser Pro Asn Leu Trp Gly Leu Glu Phe Gln Lys 115 120 125 Asn Lys
Asp Tyr Tyr Ile Ile Ser Thr Ser Asn Gly Ser Leu Glu Gly 130 135 140
Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr Arg Ala Met Lys Ile 145
150 155 160 Leu Met Lys Val Gly Gln Asp Ala Ser Ser Ala Gly Ser Thr
Pro Asn 165 170 175 Lys Asp Pro Thr Arg Arg Pro Glu Leu Glu Ala Gly
Thr Asn Gly Arg 180 185 190 Ser Ser Thr Thr Ser Pro Phe Val Lys Pro
Asn Pro Gly Ser Ser Thr 195 200 205 Asp Gly Asn Ser Ala Gly His Ser
Gly Asn Asn Ile Leu Gly Ser Glu 210 215 220 Val Ala Leu Phe Ala Gly
Ile Ala Ser Gly Cys Ile Ile Phe Ile Val 225 230 235 240 Ile Ile Ile
Thr Leu Val Val Leu Leu Leu Lys Tyr Arg Arg Arg His 245 250 255 Arg
Lys His Ser Pro Gln His Thr Thr Thr Leu Ser Leu Ser Thr Leu 260 265
270 Ala Thr Pro Lys Arg Ser Gly Asn Asn Asn Gly Ser Glu Pro Ser Asp
275 280 285 Ile Ile Ile Pro Leu Arg Thr Ala Asp Ser Val Phe Cys Pro
His Tyr 290 295 300 Glu Lys Val Ser Gly Asp Tyr Gly His Pro Val Tyr
Ile Val Gln Glu 305 310 315 320 Met Pro Pro Gln Ser Pro Ala Asn Ile
Tyr Tyr Lys Val 325 330 5 345 PRT Homo sapiens 5 Met Ala Arg Pro
Gly Gly Arg Trp Leu Gly Lys Trp Leu Tyr Ala Met 1 5 10 15 Val Val
Trp Ala Leu Cys Arg Leu Ala Thr Pro Leu Ala Lys Asn Leu 20 25 30
Glu Pro Val Ser Trp Ser Ser Leu Asn Pro Lys Phe Leu Ser Gly Lys 35
40 45 Gly Leu Val Ile Tyr Pro Lys Ile Gly Asp Lys Leu Asp Ile Ile
Cys 50 55 60 Pro Pro Ala Glu Ala Gly Arg Pro Tyr Glu Tyr Tyr Lys
Leu Tyr Leu 65 70 75 80 Val Arg Pro Glu Gln Ala Ala Ala Cys Ser Thr
Val Leu Asp Pro Met 85 90 95 Val Leu Val Thr Cys Asn Arg Pro Glu
Gln Glu Ile Arg Phe Thr Ile 100 105 110 Lys Phe Gln Glu Phe Ser Pro
Asn Tyr Met Gly Leu Glu Phe Lys Lys 115 120 125 His His Asp Tyr Tyr
Ile Thr Ser Thr Ser Asn Gly Ser Leu Glu Gly 130 135 140 Leu Glu Asn
Arg Glu Gly Gly Val Cys Arg Thr Arg Thr Met Lys Ile 145 150 155 160
Ile Met Lys Val Gly Gln Asp Pro Asn Ala Val Thr Pro Glu Gln Leu 165
170 175 Thr Thr Ser Arg Pro Ser Lys Glu Ala Asp Asn Thr Val Lys Met
Ala 180 185 190 Thr Gln Ala Pro Gly Ser Arg Gly Ser Leu Gly Asp Ser
Asp Gly Lys 195 200 205 His Glu Thr Val Asn Gln Glu Glu Lys Ser Gly
Pro Gly Ala Ser Gly 210 215 220 Gly Ser Ser Gly Asp Pro Asp Gly Phe
Phe Asn Ser Lys Val Ala Leu 225 230 235 240 Phe Ala Ala Val Gly Ala
Gly Cys Val Ile Phe Leu Leu Ile Ile Ile 245 250 255 Phe Leu Thr Val
Leu Leu Leu Lys Leu Pro Lys Arg His Arg Lys His 260 265 270 Thr Gln
Arg Ala Ala Ala Leu Ser Leu Ser Thr Ile Ala Ser Pro Lys 275 280 285
Gly Gly Ser Gly Thr Ala Gly Thr Glu Pro Ser Asp Ile Ile Ile Pro 290
295 300 Leu Phe Thr Thr Glu Asn Asn Tyr Cys Pro His Tyr Glu Lys Val
Ser 305 310 315 320 Gly Asp Tyr Gly His Pro Val Tyr Ile Val Gln Glu
Met Pro Pro Gln 325 330 335 Ser Pro Ala Asn Ile Tyr Tyr Lys Val 340
345 6 209 PRT Mus musculus 6 Met Ala Arg Ala Gln Arg Pro Leu Leu
Pro Leu Leu Leu Leu Leu Leu 1 5 10 15 Pro Leu Arg Ala Arg Asn Glu
Asp Pro Ala Arg Ala Asn Ala Asp Arg 20 25 30 Tyr Ala Val Tyr Trp
Asn Arg Ser Asn Pro Arg Phe Gln Val Ser Ala 35 40 45 Val Gly Asp
Gly Gly Gly Tyr Thr Val Glu Val Ser Ile Asn Asp Tyr 50 55 60 Leu
Asp Ile Tyr Cys Pro His Tyr Gly Ala Pro Leu Pro Pro Ala Glu 65 70
75 80 Arg Met Glu Arg Tyr Ile Leu Tyr Met Val Asn Gly Glu Gly His
Ala 85 90 95 Ser Cys Asp His Arg Gln Arg Gly Phe Lys Arg Trp Glu
Cys Asn Arg 100 105 110 Pro Ala Ala Pro Gly Gly Pro Leu Lys Phe Ser
Glu Lys Phe Gln Leu 115 120 125 Phe Thr Pro Phe Ser Leu Gly Phe Glu
Phe Pro Pro Gly His Glu Tyr 130 135 140 Tyr Tyr Ile Ser Ala Thr Pro
Pro Asn Leu Val Asp Arg Pro Cys Leu 145 150 155 160 Arg Leu Lys Val
Tyr Val Arg Pro Thr Asn Glu Thr Leu Tyr Glu Ala 165 170 175 Pro Glu
Pro Ile Phe Thr Ser Asn Ser Ser Cys Ser Gly Leu Gly Ala 180 185 190
Cys His Leu Phe Leu Thr Thr Val Pro Val Leu Trp Ser Leu Leu Gly 195
200 205 Ser 7 233 PRT Homo sapiens 7 Met Ala Ala Ala Pro Leu Leu
Leu Leu Leu Leu Leu Val Pro Val Pro 1 5 10 15 Leu Leu Pro Leu Leu
Ala Gly Gly Pro Ala Gly Ala Leu Gly Asn Arg 20 25 30 His Ala Val
Tyr Trp Asn Ser Ser Asn Gln His Leu Arg Arg Glu Gly 35 40 45 Tyr
Thr Val Gln Val Asn Val Asn Asp Tyr Leu Asp Ile Tyr Cys Pro 50 55
60 His Tyr Asn Ser Ser Gly Ala Gly Pro Gly Pro Gly Gly Gly Ala Glu
65 70 75 80 Tyr Val Leu Tyr Met Val Ser Arg Asn Gly Tyr Arg Thr Cys
Asn Ala 85 90 95 Ser Gln Gly Phe Lys Arg Trp Glu Cys Asn Arg Pro
His Ala Pro His 100 105 110 Ser Pro Ile Lys Phe Ser Glu Lys Phe Gln
Arg Tyr Ser Ala Phe Ser 115 120 125 Leu Gly Tyr Glu Phe His Ala Gly
His Glu Tyr Tyr Tyr Ile Ser Thr 130 135 140 Pro Thr His Asn Leu His
Trp Lys Cys Leu Arg Met Lys Val Phe Val 145 150 155 160 Cys Cys Ala
Ser Thr Ser His Ser Gly Glu Lys Pro Val Pro Thr Leu 165 170 175 Pro
Gln Phe Thr Met Gly Pro Asn Val Lys Ile Asn Val Leu Glu Asp 180 185
190 Phe Glu Gly Glu Asn Pro Gln Val Pro Lys Leu Glu Lys Ser Ile Ser
195 200 205 Gly Thr Ser Pro Lys Arg Glu His Leu Pro Ala Leu Val Gly
Ile Ala 210 215 220 Phe Phe Leu Met Thr Phe Leu Ala Ser 225 230 8
201 PRT Homo sapiens 8 Met Arg Leu Leu Pro Leu Leu Ile Thr Val Leu
Trp Ala Ala Phe Leu 1 5 10 15 Gly Ser Pro Leu Arg Gly Gly Ser Ser
Leu Arg His Val Val Tyr Trp 20 25 30 Asn Ser Ser Asn Pro Arg Ser
Leu Arg Gly Asp Ala Val Val Glu Leu 35 40 45 Gly Leu Asn Asp Tyr
Leu Asp Ile Val Cys Pro His Tyr Glu Gly Pro 50 55 60 Gly Pro Pro
Glu Gly Pro Glu Thr Phe Ala Leu Tyr Met Val Asp Trp 65 70 75 80 Pro
Gly Tyr Glu Ser Cys Gln Ala Glu Gly Pro Arg Gly Tyr Lys Arg 85 90
95 Trp Val Cys Ser Leu Pro Phe Gly His Val Gln Phe Ser Glu Lys Ile
100 105 110 Gln Arg Phe Thr Pro Phe Ser Leu Gly Phe Glu Phe Leu Pro
Gly Glu 115 120 125 Thr Tyr Tyr Tyr Ile Ser Val Pro Thr Pro Glu Ser
Ser Gly Gln Cys 130 135 140 Leu Arg Leu Gln Val Ser Val Cys Cys Lys
Glu Arg Lys Ser Glu Ser 145 150 155 160 Ala His Pro Val Gly Ser Pro
Gly Glu Ser Gly Thr Ser Gly Trp Arg 165 170 175 Gly Gly Asp Thr Pro
Ser Pro Leu Cys Leu Leu Leu Leu Leu Leu Leu 180 185 190 Leu Ile Leu
Arg Leu Leu Arg Ile Leu 195 200 9 228 PRT Homo sapiens 9 Met Leu
His Val Glu Met Leu Thr Leu Val Phe Leu Val Leu Trp Met 1 5 10 15
Cys Val Phe Ser Gln Asp Pro Gly Ser Lys Ala Val Ala Asp Arg Tyr 20
25 30 Ala Val Tyr Trp Asn Ser Ser Asn Pro Arg Phe Gln Arg Gly Asp
Tyr 35 40 45 His Ile Asp Val Cys Ile Asn Asp Tyr Leu Asp Val Phe
Cys Pro His 50 55 60 Tyr Glu Asp Ser Val Pro Glu Asp Lys Thr Glu
Arg Tyr Val Leu Tyr 65 70 75 80 Met Val Asn Phe Asp Gly Tyr Ser Ala
Cys Asp His Thr Ser Lys Gly 85 90 95 Phe Lys Arg Trp Glu Cys Asn
Arg Pro His Ser Pro Asn Gly Pro Leu 100 105 110 Lys Phe Ser Glu Lys
Phe Gln Leu Phe Thr Pro Phe Ser Leu Gly Phe 115 120 125 Glu Phe Pro
Pro Gly Arg Glu Tyr Phe Tyr Ile Ser Ser Ala Ile Pro 130 135 140 Asp
Asn Gly Arg Arg Ser Cys Leu Lys Leu Lys Val Phe Val Arg Pro 145 150
155 160 Thr Asn Ser Cys Met Lys Thr Ile Gly Val His Asp Arg Val Phe
Asp 165 170 175 Val Asn Asp Lys Val Glu Asn Ser Leu Glu Pro Ala Asp
Asp Thr Val 180 185 190 His Glu Ser Ala Glu Pro Ser Arg Gly Glu Asn
Ala Ala Gln Thr Pro 195 200 205 Arg Ile Pro Ser Arg Leu Leu Ala Ile
Leu Leu Phe Leu Leu Ala Met 210 215 220 Leu Leu Thr Leu 225 10 205
PRT Homo sapiens 10 Met Glu Phe Leu Trp Ala Pro Leu Leu Gly Leu Cys
Cys Ser Leu Ala 1 5 10 15 Ala Ala Asp Arg His Thr Val Phe Trp Asn
Ser Ser Asn Pro Lys Phe 20 25 30 Arg Asn Glu Asp Tyr Thr Ile His
Val Gln Leu Asn Asp Tyr Val Asp 35 40 45 Ile Ile Cys Pro His Tyr
Glu Asp His Ser Val Ala Asp Ala Ala Met 50 55 60 Glu Gln Tyr Ile
Leu Tyr Leu Val Glu His Glu Glu Tyr Gln Leu Cys 65 70 75 80 Gln Pro
Gln Ser Lys Asp Gln Val Arg Trp Gln Cys Asn Arg Pro Ser 85 90 95
Ala Lys His Gly Pro Glu Lys Leu Ser Glu Lys Phe Gln Arg Phe Thr 100
105 110 Pro Phe Thr Leu Gly Lys Glu Phe Lys Glu Gly His Ser Tyr Tyr
Tyr 115 120 125 Ile Ser Lys Pro Ile His Gln His Glu Asp Arg Cys Leu
Arg Leu Lys 130 135 140 Val Thr Val Ser Gly Lys Ile Thr His Ser Pro
Gln Ala His Val Asn 145 150 155 160 Pro Gln Glu Lys Arg Leu Ala Ala
Asp Asp Pro Glu Val Arg Val Leu 165 170 175 His Ser Ile Gly His Ser
Ala Ala Pro Arg Leu Phe Pro Leu Ala Trp 180 185 190 Thr Val Leu Leu
Leu Pro Leu Leu Leu Leu Gln Arg Pro 195 200 205 11 82 PRT Homo
sapiens 11 Leu Arg Lys Arg His Arg Lys His Thr Gln Gln Arg Ala Ala
Ala Leu 1 5 10 15 Ser Leu Ser Thr Leu Ala Ser Pro Lys Gly Gly Ser
Gly Thr Ala Gly 20 25 30 Thr Glu Pro Ser Asp Ile Ile Ile Pro Leu
Arg Thr Thr Glu Asn Asn 35 40 45 Tyr Cys Pro His Tyr Glu Lys Val
Ser Gly Asp Tyr Gly His Pro Val 50 55 60 Tyr Ile Val Gln Glu Met
Pro Pro Gln Ser Pro Ala Asn Ile Tyr Tyr 65 70 75 80 Lys Val 12 82
PRT Homo sapiens 12 Tyr Arg Arg Arg His Arg Lys His Ser Pro Gln His
Thr Thr Thr Leu 1 5 10 15 Ser Leu Ser Thr Leu Ala Thr Pro Lys Arg
Ser Gly Asn Asn Asn Gly 20 25 30 Ser Glu Pro Ser Asp Ile Ile Ile
Pro Leu Arg Thr Ala Asp
Ser Val 35 40 45 Phe Cys Pro His Tyr Glu Lys Val Ser Gly Asp Tyr
Gly His Pro Val 50 55 60 Tyr Ile Val Gln Glu Met Pro Pro Gln Ser
Pro Ala Asn Ile Tyr Tyr 65 70 75 80 Lys Val 13 89 PRT Homo sapiens
13 Arg Arg Arg Arg Ala Lys Pro Ser Glu Ser Arg His Pro Gly Pro Gly
1 5 10 15 Ser Phe Gly Arg Gly Gly Ser Leu Gly Leu Gly Gly Gly Gly
Gly Met 20 25 30 Gly Pro Arg Glu Ala Glu Pro Gly Glu Leu Gly Ile
Ala Leu Arg Gly 35 40 45 Gly Gly Ala Ala Asp Pro Pro Phe Cys Pro
His Tyr Glu Lys Val Ser 50 55 60 Gly Asp Tyr Gly His Pro Val Tyr
Ile Val Gln Asp Gly Pro Pro Gln 65 70 75 80 Ser Pro Pro Asn Ile Tyr
Tyr Lys Val 85
* * * * *