U.S. patent application number 09/894039 was filed with the patent office on 2002-01-31 for assay systems for leptin-enhancing agents.
Invention is credited to Carpenter, Laura R., Stahl, Neil, Yancopoulos, George D..
Application Number | 20020012949 09/894039 |
Document ID | / |
Family ID | 21958093 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020012949 |
Kind Code |
A1 |
Carpenter, Laura R. ; et
al. |
January 31, 2002 |
Assay systems for leptin-enhancing agents
Abstract
The present invention relates to methods for identifying
therapeutic agents that enhance the effect of leptin, an
adipocyte-derived cytokine that regulates food intake and body
weight. The invention further provides for use of agents identified
using this assay system to enhance the interaction between leptin
and its receptor, OB-R, thereby boosting leptin's weight-reducing
effects in obese individuals.
Inventors: |
Carpenter, Laura R.;
(Tuckahoe, NY) ; Stahl, Neil; (Carmel, NY)
; Yancopoulos, George D.; (Yorktown Heights, NY) |
Correspondence
Address: |
Joseph M. Sorrentino
Regeneron Pharmaceuticals, Inc.
777 Old Saw Mill River Road
Tarrytown
NY
10591
US
|
Family ID: |
21958093 |
Appl. No.: |
09/894039 |
Filed: |
June 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09894039 |
Jun 28, 2001 |
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09093814 |
Jun 9, 1998 |
|
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6270981 |
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60049108 |
Jun 9, 1997 |
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Current U.S.
Class: |
435/7.1 ;
514/5.3; 514/5.8; 514/7.7 |
Current CPC
Class: |
G01N 33/6842 20130101;
G01N 33/68 20130101; G01N 33/74 20130101; G01N 2500/00
20130101 |
Class at
Publication: |
435/7.1 ; 514/2;
514/12 |
International
Class: |
G01N 033/567; G01N
033/53; A61K 038/00 |
Claims
We claim:
1. A method of screening for a molecule capable of competing with
OB-R for binding to SHP2-SH2 comprising: a) contacting a
phosphopeptide comprising the Y986 domain of OB-R under conditions
in which the Y986-containing phosphopeptide is capable of binding
to a peptide comprising the SH2 domain of SHP2; b) determining the
amount of the Y986-containing domain that binds to the SHP2-SH2
containing peptide; c) contacting a known amount of the Y986
containing phosphopeptide to the SHP2-SH2 containing peptide, in
the presence of a sample suspected of containing the molecule
capable of competing with the Y986 containing phosphopeptide, under
the same conditions used in b); d) determining the amount of the
Y986-containing phosphopeptide that binds to the SHP2-SH2
containing peptide; e) comparing the amount from (b) with the
amount from (d), wherein a lesser amount in (d) indicates the
presence of a molecule capable of competing with the Y986
containing phosphopeptide for binding to the SHP2-SH2 domain
2. The method of claim 1, wherein the Y986 containing
phosphopeptide or the SHP2-SH2 containing peptide is bound to a
solid support.
3. The method of claim 2, wherein the Y986 containing
phosphopeptide or the SHP2-SH2 containing peptide is detectably
labeled.
4. The method of claim 1 wherein the Y986 containing phosphopeptide
is the cytoplasmic domain of OB-R.
5. The method of claim 1 wherein the Y986 containing phosphopeptide
is a dimer.
6. An assay system comprising a) an SH2 domain of an SH2 containing
phosphatase; b) a peptide containing the tyrosine phosphorylated
SH2 binding domain of a cytokine receptor; c) a means for detecting
the interaction between said SH2 domain and said SH2 binding
domain.
7. The assay system of claim 6 wherein said SH2 binding domain
containing peptide is the cytoplasmic domain of a cytokine receptor
that has a downstream signaling pathway that involves an
SH2-containing phosphatase.
8. The assays system of claim 7 wherein said SH2-containing
phosphatase is SHP1or SHP2.
9. The assays system of claim 7 wherein said cytokine receptor is
selected from the group consisting of OB-R and erythropoietin.
10. An agent identified according to the method of claim 1.
11. An agent that enhances the therapeutic effect of leptin
response comprising an agent according to claim 10.
Description
[0001] This application is a continuation-in-part of copending U.S.
application Ser. No. 60/049,108, filed Jun. 9, 1997, the contents
of which is hereby incorporated by reference. Throughout this
application various publications are referenced. The disclosures of
these publications in their entireties are hereby incorporated by
reference into this application.
BACKGROUND OF THE INVENTION
[0002] Leptin
[0003] Leptin, the product of the obese gene (1), is secreted by
adipocytes, and functions as a peripheral signal to the brain that
regulates food intake and energy metabolism. Leptin is thought to
exert its action in the hypothalamus through its receptor, OB-R
(2). Rodents with mutations that prevent normal expression of
either leptin or full-length OB-R are profoundly obese, diabetic,
and have a reduced metabolic rate (3). However, human obesity does
not appear to be associated with mutations in the genes encoding
leptin or OB-R (4, 5). Although mice with a mutant obese gene can
be returned to normal weight by administration of recombinant
leptin (6-8), it seems unlikely that this approach will succeed in
obese humans because their serum leptin levels are chronically
elevated (9-11). Obese humans, therefore, appear to be "leptin
resistant" (9, 12, 13) in that they do not generate a signal
commensurate with their serum leptin levels, perhaps because of
defective transport of leptin across the blood-brain barrier (14)
or an inadequate OB-R response. Analysis of OB-R signaling pathways
may reveal alternative therapeutic approaches of boosting OB-R
responses to overcome leptin resistance and reverse obesity.
[0004] Leptin and OB-R are members of the four-helical bundle
cytokine and receptor superfamilies respectively (2, 15). OB-R is
most closely related to the gp130 signal transducing receptor that
is activated by cytokines such as IL-6 (CNTF: ciliary neurotrophic
factor; IL-6: Interleukin-6; Jak: Janus kinase; NT-3;
neurotrophin-3; SH2: Src homology domain 2; STAT: Signal Transducer
and Activator of Transcription; TOBR: Trk-C-OB-R chimeric receptor)
and CNTF, whose signaling pathways have been intensively studied
(16, 17).
[0005] Ligand binding induces either homodimerization of gp130, or
heterodimerization of gp130 with related signal transducing
receptors, leading to activation of the receptor-associated Jaks
(18, 19). The Jaks then phosphorylate gp130 on cytoplasmic tyrosine
residues, forming phosphotyrosine motifs that recruit specific
SH2-containing signaling molecules such as STAT3, and the protein
tyrosine phosphatase SHP-2 (previously known as PTP1D, SH-PTP2 or
Syp) (20). Removal or mutation of the phosphotyrosine motifs in the
receptor eliminates activation of the corresponding SH2 target
molecule (20). Cytoplasmic deletions appear to affect OB-R in a
similar manner: there are multiple isoforms of OB-R corresponding
to alternatively spliced products with different cytoplasmic
domains (2, 21-24), but only one isoform with several potential
phosphotyrosine motifs, known as the long form or OB-Rb, appears
capable of mediating leptin's weight controlling effects (21, 22,
25, 26). Obese diabetic mice have a mutation in OB-R that prevents
expression of the long OB-R splice isoform, which renders them
incapable of appropriately mediating leptin's actions (21, 22). The
finding that only the long form of OB-R contains the sequence YXXQ
(2), which is a motif that specifies STAT3 activation (20), raised
the possibility that STAT3 is critical for mediating leptin
responses. Recent results verify that STAT3 is activated both in
cultured cells (25-27) and in vivo (28) by the long form of OB-R,
and not by a truncated OB-R or the long form of OB-R with a mutant
YXXQ motif (26, 29). Although leptin-induced activation of
overexpressed STAT1 and STAT5b is also observed in transfected
cells (25, 26), only activation of STAT3 has been detected in vivo
upon stimulation of hypothalamic OB-R by administration of leptin
(28). Thus it appears likely, but unproven, that transcriptional
activation of target genes by STAT3 in the hypothalamus is a
critical pathway that mediates leptin's regulation of food intake
and energy metabolism.
[0006] Assay Systems
[0007] Ligand-receptor assays rely on the binding of ligands to
receptors to determine the presence and/or concentration of ligands
in a sample. Ligand-receptor assays can be described as either
competitive or non-competitive. Non-competitive assays generally
utilize receptors in substantial excess over the concentration of
ligand to be determined in the assay. Sandwich assays, in which the
ligand is detected by binding to two receptors, one receptor
labeled to permit detection and a second receptor frequently bound
to a solid phase to facilitate separation from unbound reagents,
such as unbound labeled first receptor, are examples of
non-competitive assays.
[0008] Competitive assays generally utilize ligand from the sample,
a ligand analogue labeled to permit detection, and the competition
of these species for a limited number of binding sites provided by
the ligand receptor. Those skilled in the art will appreciate that
many variations of this basic competitive situation have been
previously described. Examples of ligands which are commonly
measured by competitive ligand-receptor assays include haptens,
hormones and proteins. Antibodies or receptorbodies that can bind
these classes of ligands are frequently used in these assays as
ligand receptors.
[0009] Competitive ligand-receptor assays can be further described
as being either homogeneous or heterogeneous. In homogeneous
assays, all of the reactants participating in the competition are
mixed together and the quantity of ligand is determined by its
effect on the extent of binding between ligand receptor and labeled
ligand analogue. The signal observed is modulated by the extent of
this binding and can be related to the amount of ligand in the
sample. U.S. Pat. No. 3,817,837 describes such a homogeneous,
competitive immunoassay in which the labeled ligand analogue is a
ligand-enzyme conjugate and the ligand receptor is an antibody
capable of binding to either the ligand or the ligand analogue. The
binding of the antibody to the ligand-enzyme conjugate decreases
the activity of the enzyme relative to the activity observed when
the enzyme is in the unbound state. Due to competition between
unbound ligand and ligand-enzyme conjugate for antibody binding
sites, as the ligand concentration increases the amount of unbound
ligand-enzyme conjugate increases and thereby increases the
observed signal. The product of the enzyme reaction may then be
measured using a spectrophotometer.
[0010] In general, homogeneous assay systems require both an
instrument to read the result and calibration of the observed
signal by separate tests with samples containing known
concentrations of ligand. The development of homogeneous assays has
dominated competitive assay research and has resulted in several
commercially available systems.
[0011] Heterogeneous, competitive ligand-receptor assays require a
separation of bound labeled ligand or receptor from the free
labeled ligand or receptor and a measurement of either the bound or
the free fraction. Methods for performing such assays are described
in U.S. Pat. Nos. 3,654,090, 4,298,685, and 4,506,009. U.S. Pat.
Nos. 4,125,372, 4,200,690, 4,246,339, 4,366,241, 4,446,232,
4,477,576, 4,496,654, 4,632,901, 4,727,019, and 4,740,468 describe
devices and methods for ligand-receptor assays that develop colored
responses for visual interpretation of the results.
[0012] In the case of a competitive immunoassay, a labelled ligand
reagent is bound to a limited and known quantity of receptor
reagent. After that reaction reaches equilibrium, the ligand to be
detected is added to the mixture and competes with the labelled
ligand for the limited number of receptor binding sites. The amount
of labelled ligand reagent displaced, if any, in this second
reaction indicates the quantity of the ligand to be detected
present in the fluid sample.
[0013] Another approach, a non-competitive immunochromatographic
assay, is described in U.S. Pat. Nos. 4,168,146 and 4,435,504. This
assay provides a method for quantitatively determining the presence
of a single analyte in a sample in a visually interpreted
immunoassay. U.S. Pat. No. 5,089,391 describes a method for
performing competitive ligand-receptor assays so as to be able to
semiquantitatively or quantitatively determine the concentration of
the ligand.
SUMMARY OF THE INVENTION
[0014] The present invention relates to a novel method of screening
for agents that are capable of enhancing the weight reducing effect
of leptin. The invention is based on applicants' discovery that
SHP-2 can act as a negative regulator of certain cytokine
receptors, and specifically blocking activation of SHP-2 by leptin
could provide a possible mechanism to surmount leptin resistance in
obese individuals.
[0015] The present invention further comprises used of agents
discovered using the assay system described herein for the
treatment of conditions such as obesity, that are caused, in part,
by leptin resistance.
[0016] The invention further provides for agents that may be used
to enhance the effect of therapeutic doses of leptin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1. NT-3 stimulation of TrkC-OB-R chimeras leads to
phosphorylation of receptor, Jaks, STAT3, and SHP-2. Immunoblots
(Blot) of myc and P-Tyr immunoprecipitates (IP) from unstimulated
(-) and stimulated (+) cells transfected with TrkC-OB-R constructs.
Truncation constructs are designated by the number of the last
amino acid of the OB-R cytoplasmic domain present before the myc
epitope tag (indicated by a triangle), and the amino acids
surrounding each tyrosine are also indicated (bottom). The
phosphorylated form of each protein is indicated by a superscripted
P on the right side of each panel.
[0018] FIG. 2. SHP-2 phosphorylation is dependent on tyrosine 986.
Immunoblots (Blot) of myc, P-Tyr or SHP-2 immunoprecipitates (IP)
from unstimulated (-) and leptin stimulated (+) cells transfected
with the full-length OB-R constructs. Phenylalanine (F)
substitutions were made at the indicated tyrosines (Y).
[0019] FIG. 3. Majority of stimulation-dependent phosphorylation
occurs on tyrosines 986 and 1141. Immunoblots (Blot) of
immunoprecipitated proteins (IP) from unstimulated (-) and NT-3
stimulated (+) cells transfected with TrkC-OB-R constructs.
[0020] FIG. 4A. Mutation of Y986 in OB-R leads to an enhancement of
leptin-stimulated luciferase activity. The fold induction of
stimulated compared to non-stimulated cells is indicated as the
mean.+-.standard error.
[0021] FIG. 4B. Mutation of Y1078 and Y1079 (double mutation) in
OB-R leads to induction of similar luciferase levels as the native
receptor. The fold induction of stimulated compared to
non-stimulated cells is indicated as the mean.+-.standard
error.
[0022] FIG. 5. Timecourse of STAT3 tyrosine phosphorylation is
unchanged in cells expressing OB-RY986F. COS cells expressing
native OB-R or mutant OB-RY986F were either left unstimulated (0),
or stimulated with leptin for 10 minutes, washed 1.times. in PBS,
and either lysed immediately (10 minutes) or incubated in media
(without leptin) for the indicated times and then lysed ("Time"
equals total incubation interval from addition of leptin). Myc (A)
and STAT3 (B and C) immunoprecipitates (IF) were immunoblotted
(Blot) with antibodies to P-Tyr (A), P-STAT3 (B) and STAT3 (C). The
phosphorylated form of each protein is indicated by a superscrited
P on the right side of each panel.
[0023] FIG. 6. SHP-2 specifically binds OB-R and this binding is
dependent on Y986. (A). GST-SHP1-SH2 or GST-SHP2-SH2 were incubated
with either no phosphopeptide, a phosphopeptide containing the
sequence surrounding Y986 in OB-R, a phosphopeptide derived from
the sequence surrounding Y401 of the cytoplasmic domain of the EPO
receptor, a phosphopeptide derived from the sequence surrounding
Y583 of the cytoplasmic domain of the IL-4 receptor a, or a
phosphopeptide derived from the sequence surrounding Y440 of the
cytoplasmic domain of the IFN.gamma. receptor. Phosphopeptide was
immunoprecipitated with an antibody to the flag epitope tag and
co-precipitating GST-SHP1-SH2 or GST-SHP2-SH2 was visualized with
an anti-GST antibody.
[0024] FIG. 7. GST-SHP1-SH2 or GST-SHP2-SH2 binding only to
phosphorylated OB-R cytoplasmic domain. GST-SHP1-SH2 or
GST-SHP2-SH2 was incubated with native or Y986F mutant OB-R
cytoplasmic domain-GST fusions, either unphosphorylated (OB-R) or
phosphorylated (OB-R-P). OB-R cytoplasmic domain fusion proteins
were immunoprecipitated with an antibody to the myc epitope tag,
and co-precipitating GST-SHP1-SH2 or GST-SHP2-SH2 was visualized
with an anti-GST antibody (Sigma, St. Louis, Mo.). The position of
the GST-SHP2-SH2 is indicated, and GST-SHP1-SH2 lies just
below.
[0025] FIG. 8. GST-SHP1-SH2 or GST-SHP2-SH2 binding only to
phosphorylated EPOR cytoplasmic domain. GST-SHP1-SH2 or
GST-SHP2-SH2 was incubated with EPOR cytoplasmic domain-Trx
fusions, either unphosphorylated or phosphorylated (EPOR-cyto-P).
GST-SHP1-SH2 or GST-SHP2-SH2 were immunoprecipitated with an
antibody to GST and co-precipitating EPOR cytoplasmic domain fusion
protein was visualized with an anti-Trx antibody (Sigma, St. Louis,
Mo.).
[0026] FIG. 9. Competition for GST-SHP1-SH2 or GST-SHP2-SH2 binding
to OB-R-cyto-P by a phosphopeptide containing the sequence
surrounding Y986 in OB-R. 200 nM GST-SHP1-SH2 or GST-SHP2-SH2, 15
nM OB-R-cyto-P, with and without 4 .mu.M peptide were incubated
together. OB-R-cyto-P was immunoprecipitated with an antibody to
the myc epitope tag, and co-precipitating GST-SHP1-SH2 or
GST-SHP2-SH2 was visualized with an anti-GST antibody.
[0027] FIG. 10. Competition for GST-SHP1-SH2 or GST-SHP2-SH2
binding to EPOR-cyto-P by a phosphopeptide derived from the
sequence surrounding Y401 of the cytoplasmic domain of the EPO
receptor. 200 nM GST-SHP1-SH2 or GST-SHP2-SH2, 15 nM EPOR-cyto-P,
with and without 4 .mu.M peptide were incubated together.
GST-SHP1-SH2 or GST-SHP2-SH2 was immunoprecipitated with
Glutathione Sepharose (Pharmacia), and co-precipitating EPOR-cyto-P
was visualized with an anti-Trx antibody.
[0028] FIG. 11. Mutation of Y986 in OB-R leads to an enhancement of
leptin-stimulated STAT3-mediated luciferase gene induction.
Neuroblastoma (NBFL) cells were transiently transfected with the
indicated TrkC-OB-R receptor constructs and 3(G3)-Cy6-LUC reporter.
The mean fold induction of luciferase activity.+-.standard error
(n=3) is indicated.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention is based on applicants' discovery
regarding certain signaling molecules in the signal transduction
pathway activated upon the binding of leptin to its receptor OB-R.
Applicants have discovered that the interaction of the OB-R
cytoplasmic domain with the signaling molecule SHP-2 results in
down-regulation of STAT-mediated gene induction, which in turn
results in down regulation of the effect of leptin on weight
reduction.
[0030] Accordingly, based on this discovery, Applicants have
developed assay systems designed to identify agents that are
capable of interfering with the interaction between the OB-R and
the SHP-2 molecule, thereby preventing the down-regulation of the
STAT3 signaling molecule. Agents identified using this assay system
should enhance the weight reducing effect of leptin.
[0031] According to the invention, a set of reagents which were
used to directly demonstrate that the SH2 domains of SHP-2 directly
interacts with the region encompassing tyrosine-phosphorylated
Y986, provide a means to screen for potential therapeutic agents
that block this interaction.
[0032] In one embodiment, these reagents are created as follows:
the tandem SH2 domains of SHP-2 are produced in bacteria as a
fusion to GST, which are expressed and purified by standard
methods. Two distinct phosphotyrosine containing reagents are used
as targets of SHP-2 binding. The first is a synthetic
phosphopeptide encompassing Y986 containing a C-terminal FLAG
epitope. The second reagent, is a GST fusion protein containing the
entire cytoplasmic domain of human OB-R, which was phosphorylated
on tyrosine residues in E. coli by secondarily inducing the ectopic
expression of a mammalian tyrosine kinase following the primary
induction of expression of the GST-fused OB-R cytodomain.
[0033] As demonstrated in Example 1, both
phosphotyrosine-containing reagents show specific binding to the
SH2 domains of SHP-2. Specificity is demonstrated by the fact that
other phosphopeptides do not bind to GST-SHP2-SH2, and a
tyrosine-phosphorylated OB-R cytoplasmic domain construct
containing the Y986F mutation no longer binds to GST-SHP2-SH2.
[0034] The present invention also contemplates the use of the
entire cytoplasmic domains of cytokine receptors for screening
purposes, wherein an assay system is being utilized in which the
aim is to measure the interaction between the cytoplasmic domain
and downstream signaling molecules such as SHP-1 and SHP-2.
Unexpectedly, tyrosine-phosphorylated GST-OBR apparently binds with
much higher affinity to GST-SHP2-SH2 reagent, as indicated by the
inability of a large excess of the phosphopeptide to block binding,
than does the phosphopeptide. One possible explanation of this
result is that the simple phosphopeptide is "floppy" in that it can
undertake many different conformations, of which only a limited
subset can effectively bind to the SH2 domains. In contrast, the
corresponding region imbedded in the context of the
tyrosine-phosphorylated cytoplasmic domain may be constrained to a
particular conformation that is in the proper orientation to bind
the SH2 domain, which thus results in a higher observed energy of
interaction. A similar result was observed for the interaction of
the SH2 domains of SHP-1 with its target phosphotyrosine sequence
in the cytoplasmic domain of the erythropoietin receptor (EPOR).
Very poor binding was observed for GST-SHP1-SH2 binding to a
synthetic phosphopeptide from EPOR, while the
tyrosine-phosphorylated cytoplasmic domain bound well. These
examples provide evidence supporting the existence of a previously
unknown general principle that utilization of larger cytoplasmic
domains containing tyrosine-phosphorylated target sequences that
bind SH2 domains may provide superior reagents for screening for
therapeutic agents to block that interaction.
[0035] In addition, the use of the entire cytoplasmic domain of the
cytokine receptor provides an additional advantage. Both of the
tyrosine phosphatases SHP-1 and SHP-2 are involved in the
downstream signaling pathways of a wide variety of receptors. In
preferred embodiments, agents are discovered that bind to the
cytokine receptor cytoplasmic domains to block interaction of the
SHP molecule with their tyrosine-phosphorylated targets, giving
greater specificity than therapeutic agents that block by binding
to the phosphatase. Accordingly, the present invention further
provides for use of tyrosine-phosphorylated cytoplasmic domains to
provide a much higher likelihood of obtaining therapeutic agents
that block by binding to the cytoplasmic domain, since they present
a more precise mimic and larger molecular surface for interaction
than does a simple unstructured phosphopeptide.
[0036] According to the invention, assay systems are utilized which
are designed to measure the interaction between the SH2 binding
domain of the cytokine receptor and the phosphatase comprising the
SH2 domain. In a preferred embodiment, the SH2 binding domain of
the cytokine receptor, which must contain a phosphorylated tyrosine
residue, is presented in the assay as the full cytoplasmic domain,
which, as described above, appears to present the binding domain in
an appropriate conformation and which provides a larger surface
area in which a competing agent might interact.
[0037] The present invention therefore involves a method of
screening for a molecule capable of competing with OB-R for binding
to SHP2-SH2 comprising:
[0038] a) contacting a phosphopeptide comprising the Y986 domain of
OB-R under conditions in which the Y986-containing phosphopeptide
is capable of binding to a peptide comprising the SH2 domain of
SHP2;
[0039] b) determining the amount of the Y986-containing domain that
binds to the SHP2-SH2 containing peptide;
[0040] c) contacting a known amount of the Y986 containing
phosphopeptide to the SHP2-SH2 containing peptide, in the presence
of a sample suspected of containing the molecule capable of
competing with the Y986 containing phosphopeptide, under the same
conditions used in b);
[0041] d) determining the amount of the Y986-containing
phosphopeptide that binds to the SHP2-SH2 containing peptide;
[0042] e) comparing the amount from (b) with the amount from (d),
wherein a lesser amount in (d) indicates the presence of a molecule
capable of competing with the Y986 containing phosphopeptide for
binding to the SHP2-SH2 domain.
[0043] As used herein, the phosphopeptide comprising the Y986
comprising domain is any peptide or protein that is derived from
OB-R and which comprises the Y986 site of the protein. Accordingly,
this domain may be either a phosphopeptide or the entire
cytoplasmic domain of OB-R. In a preferred embodiment, the Y986
phosphopeptide is the entire cytoplasmic domain, which, as
described above, may have enhanced properties which make it more
useful in screening systems.
[0044] In alternative embodiments, the phosphopeptide is derived
from the SH2 binding site derived from the cytoplasmic domains of
other cytokine receptors, such as EPOR, for which SH2 phosphatases
are negative regulators of receptor signaling. For example, SHP-1
has been shown to suppress signaling from the IL-3 receptor (35).
Accordingly, the present invention contemplates an assay system
using the SHP-1-SH2 domain and a phophopeptide comprising the SH2
binding domain of the IL-3 receptor.
[0045] Thus, assay systems are developed in which the interaction
between the SH2 binding domain of a ctyokine receptor and the SH2
domain of an SH2 phosphatase that acts as a negative regulator in
signaling may be measured, and agents identified with interfere
with this interaction. Such assay systems would contain
[0046] a) a peptide comprising an SH2 domain of an SH2
phosphatase;
[0047] b) a phosphopeptide comprising the tyrosine phosphorylated
SH2 binding domain of a cytokine receptor; and
[0048] c) a means for detecting the interaction between said SH2
domain and said SH2 binding domain.
[0049] The present invention also provides for assay systems that
may be used according to the methods described supra. Such assay
systems may comprise in vitro preparations of the Y986 containing
phosphopeptide and or SH2 continuing peptide, e.g. affixed to a
solid support, or may, preferably, comprise cells that express one
or both of these peptides.
[0050] The present invention further comprises uses of agents
identified using the assay systems described herein to enhance the
therapeutic effect of endogenous leptin, as well as exogenously
supplied leptin or leptin derivatives or chimeras or ligandbodies
in accordance, for example, with published applications WO97/11192
(published Mar. 12, 1997; WO97/02004 (published Jan. 23, 1997);
WO97/06816 (published Feb. 27, 1997); and WO97/00319 (published
Jan. 3, 1997).
EXAMPLES
Example 1
Demonstration that SHP-2 Interaction with OB-R Downregulates Leptin
Effect
[0051] Materials and Methods
[0052] Expression Constructs and Cell Transfection
[0053] The cytoplasmic domain and full-length human OB-R was cloned
by PCR using human fetal liver and adipocyte cDNA (Clontech) and
cloned into the expression vectors pCMX and pMT21. Chimeric
receptors were constructed by fusing the extracellular domain and
transmembrane domain of TrkC (amino acids 1 to 458) to the
cytoplasmic domain of OB-R (amino acids 865-1165). A triple myc
epitope tag was added at the C-terminus of all OB-R constructs
(18). Truncation and point mutant constructs were generated by PCR
and the sequences of all constructs were verified. Expression
constructs were transfected into COS-7 cells using lipofectamine
(GIBCO-BRL); 36-48 hours after transfection, cells were starved for
either 2 hour,s for chimeric receptors or overnight for full-length
receptors prior to stimulation.
[0054] Immunoprecipitation and Western Blotting
[0055] Cells were stimulated with 50 ng/ml of human NT-3 for 15 min
or 100 ng/ml of human leptin (R&D Systems) for 10 min. Cells
were then lysed in a 1% Brij 96 buffer and receptor was
immunoprecipitated with anti-myc antibody 9E10 (18) followed by
protein G-sepharose (Pharmacia). Unbound lysate was then
immunoprecipitated with anti-P-Tyr antibody (UBI, Lake Placid) or
agarose-conjugated anti-SHP-2 antibody (Santa Cruz Biotechnology).
For analysis of the STAT3 tyrosine phosphorylation state, STAT3 was
first immunoprecipitated with anti-STAT3 antibody (Transduction
Labs), and the unbound lysate was then immunoprecipitated with
anti-myc antibody. Immunoprecipitated proteins were separated on
SDS-PAGE and proteins were immunoblotted with antibodies to SHP-2,
STAT3, Jak1 (all from Transduction Laboratories), Jak2, P-Tyr
(UBI), or myc and visualized by chemiluminescence (DuPont NEN).
Endogenous STAT3, SHP-2, Jak1 and Jak2 is being detected in all
immunoblotting experiments.
[0056] Luciferase Reporter Gene Assay
[0057] Expression constructs (1 .mu.g) for OB-R, OB-RY1141F,
OB-RY986F, OB-RY1078, 1079F, and OB-RY986, 1141F were
co-transfected with 1 .mu.g of 3(G3)-Cy6-LUC and 0.1 .mu.g of
pCMX.LacZ into COS-7 cells (6-well dishes) using lipofectamine. The
day after transfection, media was changed to serum-free OptiMEM
(GIBCO-BRL) and then treated with 100 ng/ml murine leptin
(PeproTech) or human leptin (R&D Systems) for 24 hours.
Expression constructs for TOBR and TOBRY986F were co-transfected
with 3(G3)-Cy6-LUC and pCMX.LacZ into neuroblastoma (NBFL) cells by
calcium-phosphate precipitation for 6 hours. Cells were
subsequently treated with neurotrophin-3 (NT-3) at 20 ng/ml for 36
hours. Luciferase and .beta.-galactosidase activities were measured
in cell lysates (30) and luciferase activity was normalized to
.beta.-galactosidase activity to control for transfection
differences.
[0058] Results
[0059] We first examined OB-R signaling by creating a series of
epitope-tagged chimeric receptors consisting of the extracellular
and transmembrane domains of the receptor tyrosine kinase TrkC
fused to the cytoplasmic domain of the long form of human OB-R,
thus allowing the use of a surrogate ligand to specifically
activate the OB-R signaling pathway. Analogous chimeric receptors
were used previously to investigate gp130 signaling, and showed
activation of the identical pathways as native gp130 (20).
Following transient transfection of COS cells with a vector
expressing the TrkC--OB-R chimera (TOBR), receptor dimerization
with NT-3 resulted in ligand-dependent tyrosine phosphorylation of
TOBR and downstream signaling molecules (FIG. 1). As first observed
for gp130 (18), and subsequently noted for many other cytokine
receptors, preassociation of Jak1 with the OB-R cytoplasmic domain
was observed in the absence of ligand (FIG. 1C), and receptor
activation resulted in tyrosine phosphorylation of the associated
Jak1 (FIG. 1B). The receptor-associated Jak is most likely Jak1 as
immunoblots of myc immunoprecipitates show co-migration of Jak1,
but not Jak2, with the tyrosine phosphorylated Jak (FIG. 1B and C,
lane 4 and data not shown). Jak2 also becomes tyrosine
phosphorylated upon receptor activation (FIG. 1D), but is not found
associated with OB-R following detergent lysis of the cells. Direct
immunoprecipitation of Jak1 followed by immunoblotting with
anti-phosphotyrosine revealed that Jak1 becomes tyrosine
phosphorylated upon NT-3 stimulation. In addition, receptor was
found to co-immunoprecipitate with Jak1 independent of ligand
stimulation. Ligand-dependent tyrosine phosphorylation of STAT3 was
observed (FIG. 1E, lane 4), consistent with previous observations
that STAT3 is activated by OB-R (25-28). We also found that
activation of OB-R mediates tyrosine phosphorylation of the protein
tyrosine phosphatase SHP-2 (FIG. 1F, lane 4), which has not been
previously reported to be a target of leptin action.
[0060] To identify regions of the OB-R that are required to direct
phosphorylation of downstream signaling molecules, we created a
series of chimeras containing C-terminal truncations of TOBR that
successively eliminate tyrosine residues from the receptor
cytoplasmic domain (FIG. 1).
[0061] For gp130 and other cytokine receptors, phosphorylated
tyrosines along with their surrounding amino acids constitute
modular tyrosine-based motifs that recruit specific downstream
targets through interaction with their SH2 domains (20, 31).
Stimulation-dependent STAT3 phosphorylation is not observed for the
construct TOBR.DELTA.1140, in which the sequence YMPQ, a
tyrosine-based motif capable of specifying STAT3 activation (20),
has been deleted (FIG. 1E, lane 6). Tyrosine phosphorylation of the
receptor, Jak and SHP-2 are still observed for TOBR.DELTA.1140
(FIG. 1B, D, F; lane 6); however, receptor phosphorylation is
dramatically reduced. Deletion of the tandem tyrosine residues at
1078 and 1079 has no effect on phosphorylation of the Jaks or SHP-2
(FIG. 1B, D, F; lane 8). In contrast, cells expressing the
construct TOBR.DELTA.985 cannot mediate SHP-2 phosphorylation (FIG.
1F, lane 10), although this truncated receptor can still confer
phosphorylation of Jak2 and receptor-bound Jak1 (FIG. 1B, D; lane
10). TOBR.DELTA.985 lacks the sequence YATL, which is similar to
other tyrosine-based motifs that interact with the SH2 domains of
SHP-2 (32, 33), and may specify SHP-2 interaction with OB-R.
[0062] To confirm our assertion that the specific tyrosine residues
within OB-R are critical for specifying STAT3 and SHP-2
phosphorylation, we created epitope-tagged versions of OB-R
containing mutations that substitute phenylalanine for tyrosine
within the YMPQ or YATL motifs, and examined their ability to
activate STAT3 and SHP-2. Leptin stimulation of COS cells
expressing OB-R results in phosphorylation of the receptor, STAT3
and SHP-2 (FIG. 2B, C, D; lane 3), which corroborates the
observations made with the chimeric receptors, and confirms that
leptin indeed induces tyrosine phosphorylation of wildtype OB-R in
COS cells. Mutation of the tyrosine within the YMPQ motif in
OB-R.sub.Y1141F not only eliminates STAT3 activation upon leptin
stimulation as previously reported (26), but also markedly reduces
the level of receptor phosphorylation (FIG. 2B, C; lane 5).
Nonetheless, phosphorylation of SHP-2 is still observed (FIG. 2D;
lane 5). However, mutation of the YATL motif in OB-R.sub.Y986F
eliminates leptin-stimulated SHP-2 phosphorylation (FIG. 2D; lane
7), but does not affect STAT3 phosphorylation (FIG. 2C; lane 7). In
contrast, mutation of the tyrosine within the YMPQ motif in
OB-RY1141F results in SHP-2 phosphorylation upon leptin stimulation
(FIG. 2D; lane 5), however, as expected, STAT3 phosphorylation is
no longer observed (FIG. 2C, lane 5). Stimulation of OB-R
possessing phenylalanine substitutions at Y1078,1079 results in
phosphorylation of both STAT3 and SHP-2, while OR-R carrying a
double mutation, OB-RY986,1141F cannot direct phosphorylation of
either STAT3 or SHP-2 (data not shown). This result demonstrates
that Y986 within OB-R is required to specifically mediate the
phosphorylation of SHP-2. We have also observed direct binding of
recombinant SH2 domains derived from SHP-2 with a synthetic peptide
corresponding to the phosphorylated YATL sequence from.
[0063] Mutant OB-R with phenylalanine substitution at either
residue Y1141 or Y986 resulted in decreased receptor
phosphorylation after leptin addition despite undiminished levels
of receptor expression and phosphorylation of SHP-2 or STAT3
respectively (FIG. 2). One interpretation of this result is that
residues 1141 and 986 are the only major sites of tyrosine
phosphorylation in OB-R in COS cells, so that mutation of either
leads to a 50% decrease in the observed signal. To assess this
possibility, we analyzed the level of tyrosine phosphorylation of
various phenylalanine substitution mutants of the chimeric receptor
TOBR, which is expressed at higher levels than OB-R and gives a
more robust tyrosine phosphorylation response (FIG. 1 vs. FIG. 2).
Consistent with the above hypothesis, point mutants simultaneously
eliminating tyrosines 1078 and 1079 produced a receptor with levels
of tyrosine phosphorylation comparable to parental TOBR, while
mutation of either tyrosine at position 1141 or 986 gave a
substantial, but similar abrogation of tyrosine phosphorylation
relative to TOBR (FIG. 3). Moreover, a double mutant substituting
phenylalanine at positions 1141 and 986 showed no detectable
receptor tyrosine phosphorylation upon activation, but displayed
undiminished levels of Jak phosphorylation (FIG. 3). These results
indicate that tyrosine 986 and 1141 are the major sites of tyrosine
phosphorylation upon ligand stimulation of OB-R in COS cells.
[0064] The role that SHP-2 and its relative SHP-1 (previously known
as PTP1C, SH-PTP1 or HCP) play in signaling has been examined for
several receptors. SHP-1 generally functions as a negative
regulator of receptor signaling in hematopoietic cells where it is
expressed. Recruitment of SHP-1 to the activated erythropoietin
receptor causes inactivation of receptor-bound Jak2 and dampens
proliferation (34). SHP-1 has been shown to similarly suppress
signaling from the IL-3 receptor (35) and the Fc.gamma.RIIB1
receptor (36). The absence of SHP-1 in mutant motheaten mice leads
to dramatic increases in the total number of myeloid precursor
cells of multiple lineages resulting from loss of the suppressive
function of SHP-1 (37). In contrast, the ubiquitously expressed
SHP-2 generally acts to positively regulate signaling, as found for
the prolactin (38), PDGF (39, 40), insulin (41), interferon (42),
and EGF (39, 40) receptor systems. For the PDGF receptor, tyrosine
phosphorylated SHP-2 acts as an adaptor molecule that recruits GRB2
and Sos, members of the signaling pathway leading to activation of
Ras and the MAP kinase ERK (40). Likewise, gp130 apparently uses
SHP-2 to activate ERK2 by coupling to Grb2 and Sos, leading to
stimulation of mitogenesis in BAF cells (43). SHP-2 is not
exclusively a positive regulator of signaling as it associates with
CTLA and functions to downregulate T cell receptor signaling
(44).
[0065] To assess the role of SHP-2 in regulating leptin-stimulated
gene induction, we co-transfected COS cells with constructs
encoding OB-R and the luciferase reporter 3(G3)-Cy6-LUC, which
contains 3 copies of a STAT3 responsive DNA element from the
promoter that drives expression of the neuropeptide VIP (30). A
6-fold induction of luciferase activity was observed upon leptin
stimulation of cells expressing wild type OB-R (FIG. 4A). There was
no induction of luciferase activity upon stimulation of cells
expressing the mutant construct OB-RY1141F, in which the mutated
tyrosine motif is no longer competent to allow activation of STAT3
(FIG. 4A), while activation of the double mutant OB-RY1078,1079F
induced similar luciferase levels as the native receptor (FIG. 4B).
Surprisingly, leptin stimulation of cells expressing the mutant
receptor OB-RY986F, which can mediate phosphorylation of STAT3 but
not SHP-2, results in a 20-fold increase in luciferase activity
(FIG. 4A). Thus SHP-2, in contrast to its role in most other
receptor systems, is a negative regulator of STAT3-mediated gene
induction following OB-R activation. An analogous finding is
observed for gp130, where phenylalanine substitution of Y759, which
prevents phosphorylation of SHP-2 upon activation of gp130, also
gives rise to a much greater level of gene induction by STAT3 (51).
Moreover, overexpression of the N-terminal SH2 domain of SHP-2,
which acts as dominant negative regulator that binds to
phosphorylated gp130 and blocks native SHP-2 phosphorylation, can
enhance the CNTF-mediated upregulation of neuropeptides during
cholinergic differentiation of sympathetic neurons (52). Thus SHP-2
plays a dual regulatory role for gp130, and possibly for OB-R, in
that it is required to mediate activation of ERK (and mitogenesis
in a BAF cell), but functions as a negative regulator to suppress
STAT3-mediated gene induction.
[0066] The mechanism by which SHP-2 acts to downregulate
STAT3-mediated signaling is unclear but may differ from that
observed for SHP-1. Overall levels of tyrosine phosphorylation of
STAT3 and receptor-associated Jak appear equal (FIG. 5) in native
and mutant OB-RY986F which cannot recruit and activate SHP-2. In
addition, there was also no difference in the duration of tyrosine
phosphorylation of the receptor, STAT3, or receptor-associated Jak
between native and mutant OB-RY986F (FIG. 5). Therefore, in
contrast to the role of SHP-1 in EPOR signaling, SHP-2 may signal
through a downstream effector, perhaps lying in the pathway leading
to ERK activation, that can influence STAT3-mediated gene
transcription. Alternatively, it is also possible that SHP-2
signaling involves the cytokine-inducible inhibitors of STAT
signaling (46-48) to regulate levels of transcription upon ligand
stimulation.
[0067] Activation of OB-R and gp130 result in the recruitment and
activation of common signaling molecules, most notably STAT3 (25,
26, 28) and SHP-2. Thus, for both receptor systems STAT3 appears to
be critical for mediating the individual ligand's biological
actions. The receptor for CNTF, a neurocytokine, consists of the
signal transducing components gp130 and LIFR.beta., along with a
specificity determining component CNTFR.alpha.. Interestingly,
functional CNTF receptors are found in the hypothalamus, and
administration of CNTF to humans mimics leptin's ability to reduce
food intake and cause weight loss (49). Activation of CNTPR by
systemic administration of CNTF to obese mice that lack either
functional leptin (ob/ob) or OB-R (db/db) results in the reversal
of their obesity phenotypes, including reduction of adiposity and
hyperphagia (50). Both CNTF and leptin were shown to similarly
activate STAT3 in a neuronal cell line and moreover, mediate the
induction of the STAT3 responsive tis-11 gene in the hypothalamus
of ob/ob mice (50). The potent and similar effects of CNTF and
leptin on the reduction of food intake and the increase in energy
expenditure in mutant obese mice are most likely due to common
signaling pathways, ie. STAT3 activation, in the brain. Indeed,
only STAT3 was activated upon leptin stimulation of hypothalamic
ObR in vivo (28). It remains to be established whether the
downregulation of STAT-mediated signaling occurs through a
downstream effector, perhaps lying in the pathway leading to ERK
activation, or whether SHP-2 phosphatase activity is directly
involved in STAT deactivation.
[0068] An exciting, but speculative, implication of this finding is
that specifically blocking the interaction of SHP-2 with OB-R may
enhance leptin's STAT3-mediated effects, and possibly overcome
leptin resistance by increasing the effectiveness of the endogenous
leptin signal. Enhancing leptin-induced gene responses could
possibly overcome leptin resistance by increasing the effectiveness
of the endogenous leptin signal, thereby boosting leptin's
weight-reducing effects in obese individuals. This possibility is
analogous to one existing naturally for another cytokine: humans
that express a truncated mutant of the erythropoietin receptor,
which eliminates the tyrosine-based motif that recruits SHP-1,
display enhanced erythropoietin action and increased levels of red
blood cells that result from the failure to down-regulate the
activated receptor (45).
Example 2
Assay System for Measuring the Binding of OB-R Cytoplasmic Domain
to SHP2-SH2.
[0069] Materials and Methods
[0070] The GST-SHP2-SH2 construct consists of the two SH2 domains
of SHP2fused downstream of glutathione-S-transferase (GST) from
Schistosoma japonicum. The fusion has aa 2-224 of SHP2 cloned as an
EcoRI/BamHI fragment into pGEX-3X and was expressed in E. coli. The
SHP2-SH2-Fc construct consists of the same amino acids as the above
fusion fused to human IgG-Fc as a BlpI/SrfI fragment in pMT21 and
was expressed in COS-7 cells.
[0071] The GST-SHP1-SH2 construct consists of the two SH2 domains
of SHP1fused downstream of GST. Amino acids 2-221 of SHP1 were
cloned as a EcoRI/BglII fragment into pGEX-3X and expressed in E.
coli.
[0072] The SHP1-SH2-Fc construct consists of the same amino acids
as the above fusion fused to human IgG-Fc as a BlpI/SrfI fragment
in pMT21 and expressed in COS-7 cells.
[0073] The OB-R phosphopeptide came from Research Genetics and has
the sequence QPFVKp-YATLISNDYKDDDDK. The last eight amino acids are
a flag tag.
[0074] The EPOR phosphopeptide came from Research Genetics and has
the sequence CPHLKp-YLYLVVSDDYKDDDDK. The last eight amino acids
are a flag tag.
[0075] The OB-R cytoplasmic domain was expressed in E. coli as a
fusion with GST. The fusion was to amino acids 865-1165 and
contains the full cytoplasmic region. It was cloned as a SalI/NotI
fragment from pCMX-Tob (10) into pGEX-5X-1. Both triple myc tagged
and triple flag tag versions were made (fusion to COOH end of
OB-R). THE EPOR cytoplasmic domain was expressed in E. coli as a
fusion with E. coli thioredoxin (Trx) and as fusion to GST. The
latter was tagged with a triple flag tag. EPOR-cyto was cloned as a
BglII/NotI fragment from pMT21-huEPOR, including amino acids
258-485 of the mature protein into pGEX-5X-1 and pET32(a).
[0076] To get phosphorylated protein the receptor cytoplasmic
domain clones were expressed in an E. coli that carried another
plasmid encoding ELK Stratagene, La Jolla, Calif.). The gene for
this kinase is under the control of the trp promoter and is induced
by Trp starvation. The cytoplasmic domain fusions to GST are under
the control of the IPTG inducible tac promoter, and the EPOR-cyto
fusion to Trx is under the control of T7 polymerase. To get
phosphorylated protein, the tac promoter or the T7 promoter is
induced and cytoplasmic domain fusions are expressed for two hours.
The cells are then centrifuged and resuspended in minimal medium
plus a Trp competitor, indoleacrylic acid, inducing the synthesis
of ELK and the subsequent phosphorylation of cytoplasmic
domains.
[0077] Bindings were done as immunoprecipitations. Binding
concentrations were typically 200 nM GST-SHP2-SH2 or GST-SHP1-SH2
and 15 nM phosphopeptide or cytoplasmic domain fusion in TBST (20
mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4) at 4.degree. C.
overnight. After binding either anti-flag mAb, M2, was added (for
the phosphopeptide and flag tagged GST-OB-R-cyto) or anti-myc mAb,
9E10, was added (for the myc tagged GST-OB-R-cyto) and the proteins
were precipitated with protein G-sepharose. Proteins were then
detected on western blots using anti-GST polyclonal antibodies. For
the EPOR-cyto-Trx fusion, the protein complexes were precipitated
with glutathione-sepharose and the EPOR-cyto-Trx fusion was
detected with anti-Trx polyclonal antibodies.
[0078] Results
[0079] The above assays revealed the following results.
GST-SHP2-SH2 bound specifically to the phosphopeptide and not to
other, nonspecific peptides (FIG. 6). GST-SHP1-SH2 bound to both
the OB-R and EPOR phosphopeptides, indicating SHP1 has the ability
to bind both OB-R and EPOR (FIG. 6). It did not bind to the
nonspecific IFN.gamma.R.alpha. or IL-4R.alpha. phosphopeptides
(FIG. 6).
[0080] GST-SHP2-SH2 and GST-SHP1-SH2 bound to the phosphorylated
GST-OB-R-cyto and not to unphosphorylated GST-OB-R-cyto (FIG. 7). A
mutation of the second tyrosine (amino acid 986 of OB-R) in
GST-OB-R-cyto/myc significantly decreased the amount of
GST-SHP2-SH2 and GST-SHP1-SH2 that coprecipitated with the
phosphorylated GST-OB-R-cyto/myc (FIG. 7). Both the mutant and
wild-type forms of GST-OB-R-cyto were phosphorylated similarly.
This indicated that both SHP1-SH2 and SHP2-SH2 required the second
phospho-tyrosine of OB-R for binding.
[0081] Both GST-SHP2-SH2 and GST-SHP1-SH2 bound with a higher
affinity to the phosphorylated EPOR-cyto-Trx fusion than to the
unphosphorylated fusion (FIG. 8).
[0082] We attempted to compete binding of GST-OB-R-cyto/3myc and
GST-SHP2-SH2 or GST-SHP1-SH2 with 4 .mu.M phosphopeptide and saw no
competition (the cytoplasmic domain concentration was 15 nM, 266
fold less than the peptide) (FIG. 9). In this experiment the SH2
domain, the cytoplasmic domain, and the peptide were mixed and
allowed to reach equilibrium. The receptor cytoplasmic domain was
precipitated with anti-myc mAb and complexed GST-SHP1-SH2 and
GST-SHP2-SH2 were detected by western blot with an anti-GST
antibody.
[0083] A similar competition of EPOR-cyto-Trx with EPOR or OB-R
phosphopeptide for GST-SHP1-SH2 binding showed a small amount of
competition, but the phosphopeptides were not able to fully compete
for binding (FIG. 10). In this competition the complexes were
precipitated with glutathione-sepharose and the EPOR-cyto-Trx was
detected by western blot using anti-Trx antibodies. The results
from these experiments indicate that there may be higher binding
affinity of the SH2 domains with the full receptor cytoplasmic
domains than with the phosphopeptides, possibly because of
secondary and tertiary structures the proteins have that the
phosphoproteins lack.
[0084] Similar results were obtained utilizing the TrkC-OB-R
chimeric receptors transiently transfected into neuroblastoma cells
(NBFL). Activation of TOBR resulted in .about.8-fold increase in
luciferase activity whereas expression of TOBRY986F gave
.about.45-fold increase (FIG. 11). Thus, abrogation of SHP-2
activation by mutating tyrosine Y986 in OB-R results in an
upregulation of STAT3-mediated signaling upon OB-R activation.
[0085] Upregulation of STAT3-mediated gene transcription in cells
expressing OB-RY986F could be explained if SHP-2 normally
functioned to directly dephosphorylate STAT3, and the receptor
mutation preventing SHP-2 activation resulted in elevated or
prolonged levels of STAT3 tyrosine phosphorylation. We therefore
examined a timecourse of STAT3 tyrosine phosphorylation, using an
antibody specific for phosphotyrosine at amino acid 705, following
leptin activation of either native OB-R or mutant OB-RY986F. At 10
minutes, STAT3 is tyrosine phosphorylated to similar levels in
cells expressing either native OB-R or mutant OB-RY986F (FIG. 5B,
lane 4 and 10, respectively), and shows comparable rates of
disappearance over 5 hours for both receptors (FIG. 5B, lane 8 and
14, respectively). The total amount of receptor tyrosine
phosphorylation is lower for OB-RY986F than OB-R despite similar
levels of receptor expression (data not shown) due to the
replacement of tyrosine 986 with phenylalanine. However, the
relative rates of disappearance of receptor tyrosine
phosphorylation for OB-R and OB-RY986F were similar, with a maximum
at 10 minutes (FIG. 5A, lanes 4 and 11, respectively) and detection
of phosphorylated receptor for up to 1 hour (FIG. 5A, lanes 6 and
12, respectively). Tyrosine phosphorylation of receptor-associated
Jak was also observed (FIG. 5A), and followed a similar rate of
disappearance as receptor phosphorylation, though the overall
amount appears slightly lower at 30 minutes for OB-RY986F (FIG. 5A,
compare lanes 5 and 11). These results indicate that there is no
large difference in the tyrosine phosphorylation state or duration
for either receptor, Jak or STAT3 between cells expressing native
OB-R and mutant OB-RY986F. Thus, direct dephosphorylation of
receptor, receptor-associated Jak, or STAT3 does not appear to be
the mechanism by which SHP-2 downregulates STAT3-mediated gene
transcription upon leptin stimulation of OB-R.
[0086] References
[0087] 1. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold,
L. & Friedman, J. M. (1994) Nature 372, 425-431
[0088] 2. Tartaglia, L. A., Dembski, M., Weng, X., Deng, N.,
Culpepper, J., Devos, R., Richards, G. J., Campfield, L. A., Clark,
F. T., Deeds, J., Muir, C., Sanker, S., Moriarty, A., Moore, K. J.,
Smutko, J. S., Mays, G. G., Woolf, E. A., Monroe, C. A. &
Tepper, R. I. (1995) Cell 83, 1263-1271
[0089] 3. Coleman, D. L. (1978) Diabetologia 14, 141-148
[0090] 4. Considine, R. V., Considine, E. L., Williams, C. J.,
Nyce, M. R., Magosin, S. A., Bauer, T. L., Rosato, E. L., Colberg,
J. & Caro, J. F. (1995) J. Clin. Invest. 95, 2986-2988
[0091] 5. Considine, R. V., Considine, E. L., Williams, C. J.,
Hyde, T. M. & Caro, J. F. (1996) Diabetes 19, 992-994
[0092] 6. Pelleymounter, M. A., Cullen, M. J., Baker, M. B., Hecht,
R., Winters, D., Boone, T. & Collins, F. (1995) Science 269,
540-543
[0093] 7. Halaas, J. L., Gajiwala, K. S., Maffei, M., Cohen, S. L.,
Chait, B. T., Rabinowitz, D., Lallone, R. L., Burley, S. K. &
Friedman, J. M. (1995) Science 269, 543-546
[0094] 8. Campfield, L. A., Smith, F. J., Guisez, Y., Devos, R.
& Burn, P. (1995) Science 269, 546-549
[0095] 9. Maffei, M., Halaas, J., Ravussin, E., Pratley, R. E.,
Lee, G. H., Zhang, Y., Fei, H., Kim, S., Lallone, R., Ranganathan,
S., Kern, P. A. & Friedman, J. M. (1995) Nature Med. 1,
1155-1161
[0096] 10. Considine, R. V., Sinha, M. K., Heiman, M. L.,
Kriauciunas, A., Stephens, T. W., Nyce, M. R., Ohannesian, J. P.,
Marco, C. C., McKee, L. J., Bauer, T. L. & Caro, J. F. (1996)
N. Engl. J. Med. 334, 292-295
[0097] 11. Sinha, M. K., Opentabova, I., Ohannesian, J. P.,
Kolaczynski, J. W., Heiman, M. L., Hale, J., Becker, G. W.,
Bowsher, R. R., Stephens, T. W. & Caro, J. F. (1996) J. Clin.
Invest. 98, 1277-1282
[0098] 12. Flier, J. S. & Elmquist, J. K. (1997) Nature
Biotech. 15, 20-21
[0099] 13. Campfield, L. A., Smith, F. J. & Burn, P. (1996)
Horm. Metab. Res. 28, 619-632
[0100] 14. Caro, J. F., Kolaczynski, J. W., Nyce, M. R.,
Ohannesian, J. P., Opentanova, I., Goldman, W. H., Lynn, R. B.,
Zhang, P.-L., Sinha, M. K. & Considine, R. V. (1996) Lancet
348, 159-161
[0101] 15. Madej, T., Boguski, M. S. & Bryant, S. H. (1995)
FEBS Lett. 373, 13-18
[0102] 16. Kishimoto, T., Akira, S. & Taga, T. (1992) Science
258, 593-597
[0103] 17. Stahl, N. & Yancopoulos, G. D. (1994) J.
Neurobiology 25, 1454-1466
[0104] 18. Stahl, N., Boulton, T. G., Farruggella, T., Ip, N. Y.,
Davis, S., Witthuhn, B., Quelle, F. W., Silvennoinen, O., Barbieri,
G., Pellegrini, S., Ihle, J. N. & Yancopoulos, G. D. (1994)
Science 263, 92-95
[0105] 19. Lutticken, C., Wegenka, U. M., Yuan, J., Buschmann, J.,
Schindler, C., Ziemiecki, A., Harpur, A. G., Wilks, A. F.,
Yasukawa, K., Taga, T., Kishimoto, T., Barbieri, G., Pellegrini,
S., Sendtner, M., Heinrich, P. C. & Horn, F. (1994) Science
263, 89-92
[0106] 20. Stahl, N., Farrugella, T. J., Boulton, T. G., Zhong, Z.,
Darnell, J. E. & Yancopoulos, G. D. (1995) Science 267,
1349-1353
[0107] 21. Lee, G.-H., Proenca, R., Montez, J. M., Carroll, K. M.,
Darvishzadeh, J. G., Lee, J. I. & Friedman, J. M. (1996) Nature
379, 632-635
[0108] 22. Chen, H., Charlat, O., Tartaglia, L. A., Woolf, E. A.,
Weng, X., Ellis, S. J., Lakey, N. D., Culpepper, J., Moore, K. J.,
Breitbart, R. E., Duyk, G. M., Tepper, R. T. & Morgenstern, J.
P. (1996) Cell 84, 491-495
[0109] 23. Cioffi, J. A., Shafer, A. W., Zupancic, T. J.,
Smith-Gbur, J., Mikhail, A., Platika, D. & Snodgrass, H. R.
(1996) Nature Med. 2, 585-593
[0110] 24. Wang, M. Y., Zhou, Y. T., Newgard, C. B. & Unger, R.
H. (1996) FEBS Left. 392, 87-90
[0111] 25. Ghilardi, N., Ziegler, S., Wiestner, A., Stoffel, R.
& Heim, M. H. (1996) Proc. Natl. Acad. Sci. USA 93,
6231-6235
[0112] 26. Baumann, H., Morell, K. K., White, D. W., Dembski, M.,
Bailon, P. S., Kim, H., Lai, C.-F. & Tartaglia, L. A. (1996)
Proc. Natl. Acad. Sci. USA 93, 8374-8378
[0113] 27. Rosenblum, C. I., Tota, M., Cully, D., Smith, R.,
Collum, R., Qureshi, S., Hess, J. F., Phillips, M. S., Hey, P. J.,
Vongs, A., Fong, R. M., Xu, L., Chen, H. Y., Smith, R. G.,
Schindler, C. & Van der Ploeg, L. H. T. (1996) Endocrinology
137, 5178-5181
[0114] 28. Vaisse, C., Halaas, J. L., Horvath, C. M., Darnell, J.
E., Stoffel, M. & Friedman, J. M. (1996) Nature Gen. 14,
95-97
[0115] 29. White, D. W., Kuropatwinski, K. K., Devos, R., Baumann,
H. & Tartaglia, L. A. (1997) J. Biol. Chem 272, 4065-4071
[0116] 30. Symes, A., Lewis, S., Corpus, L., Rajan, P., Hyman, S.
E. & Fink, J. S. (1994) Mol. Endocrin. 8, 1750-1763
[0117] 31. Baumann, H., Symes, A. J., Comeau, M. R., Morella, K.
K., Wang, Y., Friend, D., Ziegler, S. F., Fink, J. S. &
Gearing, D. P. (1994) Mol. Cell. Biol. 14,138-146
[0118] 32. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G.,
Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S.,
Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou,
M. M., Hanafusa, H., Schaffhausen, B. & Cantley, L. C. (1993)
Cell 72, 767-778.
[0119] 33. Tauchi, T., Damen, J. E., Toyama, K., Feng, G.-S.,
Broxmeyer, H. E. & Krystal, G. (1996) Blood 87, 4495-4501
[0120] 34. Klingmuller, U., Lorenz, U., Cantley, L. C., Neel, B. G.
& Lodish, H. F. (1995) Cell 80, 729-738
[0121] 35. Yi, T., Mui, A. L.-F., Krystal, G. & Ihle, J. N.
(1993) Mol. Cell. Biol. 13, 7577-7586
[0122] 36. D'Ambrosio, D., Hippen, K. L., Minskoff, S. A., Mellman,
I., Pani, G., Siminovitch, K. A. & Cambier, J. C. (1995)
Science 268, 293-297
[0123] 37. Shultz, L. D., Schweitzer, P. A., Rajan, T. V., Yi, T.,
Ihle, J. N., Matthews, R. J., Thomas, M. L. & Beier, D. R.
(1993) Cell 73, 1445-1454
[0124] 38. Ali, S., Chen, Z., Lebrun, J.-J., Vogel, W.,
Kharitonenkov, A., Kelly, P. A. & Ullrich, A. (1996) EMBO J.
15, 135-142
[0125] 39. Lechleider, R. J., Freeman, R. M. & Neel, B. G.
(1993) J. Biol. Chem. 268, 13434-13438
[0126] 40. Li, W., Nishimura, R., Kashishian, A., Batzer, A. G.,
Kim, W. J., Cooper, J. A. & Schlessinger, J. (1994) Mol. Cell.
Biol. 14, 509-517
[0127] 41. Kharitonenkov, A., Schnekenburger, J., Chen, Z.,
Knyazev, P., Sli, S., Zwick, E., White, M. & Ullrich, A. (1995)
J. Biol. Chem. 270, 29189-29193
[0128] 42. David, M., Zhou, G., Pine, R., Dixon, J. E. &
Larner, A. C. (1996) J. Biol. Chem. 271, 15862-15865
[0129] 43. Fukuda, T., Hibi, M., Yamanaka, Y., Takahasi-Tezuka, M.,
Fujitani, Y., Tamaguchi, T., Nakajima, K. & Hirano, T. (1996)
Immunity 5, 449-460
[0130] 44. Marengere, L. E. M., Waterhouse, P., Duncan, G. S.,
Mittrucker, H.-W., Feng, G.-S. & Mak, T. W. (1996) Science 272,
1170-1173
[0131] 45. de la Chapelle, A., Traskelin, A.-L. & Juvonen, E.
(1993) Proc. Natl. Acad. Sci. USA 90, 4495-4499
[0132] 46. Endo, T. A., Masuhara, M., Yokouchi, M., Suzuki, R.,
Sakamoto, H., Mitsui, K., Matsumoto, A., Tanimura, S., Ohtsubo, M.,
Misawa, H., Miyazaki, T., Leonor, N., Taniguchi, T., Fujita, T.,
Kanakura, Y., Komiya, S. &
[0133] 47. Naka, T., Narazaki, M., Hirata, M., Matsumoto, T.,
Minamoto, S., Aono, A., Nishimoto, N., Kajita, T., Taga, T.,
Yoshizaki, K., Akira, S. & Kishimoto, T. (1997) Nature 387,
924-928.
[0134] 48. Starr, R., Willson, T. A., Viney, F. M., Murray, L. J.
L., Rayner, J. R., Jenkins, B. J., Gonda, T. J., Alexander, W. S.,
Metcalf, D., Nicola, N. A. & Hilton, D. J. (1997) Nature 387,
917-921.
[0135] 49. ALS CNTF Treatment Study Group (1996) Neurology 46,
1244-1249.
[0136] 50. Gloagen, I., Costa, P., Demartis, A., Lazzaro, D., Di
Marco, A., Graziani, R., Paonessa, G., Chen, F., Rosenblum, C. I.,
Van der Ploeg, L. H. T., Cortese, R., Ciliberto, G. & Laufer,
R. (1997) Proc. Natl. Acad. Sci (USA) 94, 6456-6461.
[0137] 51. Symes, A., Stahl, N., Reeves, S. A., Farruggella, T.,
Servidei, T., Gearan, T., Yancopoulos, G. D. & Fink, J. S.
(1997) Curr. Biol. 7, 697-700.
[0138] 52. Servidei, T., Aoki, Y., Lewis, S. E., Symes, A., Fink,
J. S. & Reeves, S. A. (1998) J. Biol. Chem, 273, 6233-6241.
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