|
|
|
|||||||
|
|||||||||
(Received for publication, March 28, 1997, and in revised form, July 9, 1997)
From INSERM Unité 151, Institut Louis Bugnard, CHU Rangueil,
F 31403 Toulouse Cedex, France, ABSTRACT Activation of the somatostatin receptor sst2, a
member of the Gi protein-coupled receptor family,
results in the stimulation of a protein-tyrosine phosphatase activity
involved in the sst2-mediated growth inhibitory signal. Here, we report
that SHP-1, a cytoplasmic protein-tyrosine phosphatase containing two
Src homology 2 domains constitutively associated with sst2 as evidence
by coprecipitation of SHP-1 protein with sst2, in Chinese hamster ovary
cells coexpressing sst2 and SHP-1. Activation of sst2 by somatostatin
resulted in a rapid dissociation of SHP-1 from sst2 accompanied by an
increase of SHP-1 activity. SHP-1 was phosphorylated on tyrosine in
control cells and somatostatin induced a rapid and transient
dephosphorylation on tyrosine residues of the enzyme. Stimulation of
SHP-1 activity by somatostatin was abolished by pertussis toxin
pretreatment of cells. Gi INTRODUCTION Somatostatin is a widely distributed inhibitory hormone that
exhibits various biological effects, including neurotransmission, inhibition of exocrine and endocrine secretions, and cell
proliferation. The diverse biological effects of somatostatin are
mediated through somatostatin receptors that are coupled to a variety
of signal transduction pathways including adenylate cyclase, ionic
conductance channels, and protein phosphatases (1, 2). Recently five somatostatin receptors have been cloned. They belong to the family of G
protein-coupled receptors and can couple to diverse signal transduction
pathways (3-7).
The ability of somatostatin and its stable analogues to promote
inhibition of normal and tumor cell growth has been known for many
years (8, 9). In pancreatic tumor cells, we and others have previously
shown that somatostatin and analogues antagonize the mitogenic effect
of growth factors acting on tyrosine kinase receptors such as epidermal
growth factor (9, 10). Although the molecular events leading to the
inhibition of cell proliferation are still poorly understood, it has
been shown that, after binding to somatostatin receptors, somatostatin
analogues cause a rapid stimulation of a membrane protein-tyrosine
phosphatase (PTPase)1
activity and dephosphorylate phosphorylated epidermal growth factor
receptors (9, 11, 12) suggesting that a PTPase may participate in the
somatostatin-induced inhibition of growth factor-mediated mitogenic
signal. Recently, the expression of the sst2 somatostatin receptor
subtype in NIH3T3 and Chinese hamster ovary (CHO) cells led us to the
demonstration of the direct involvement of sst2 in both the
antiproliferative effect of somatostatin and its stimulatory effect on
PTPase activity (13, 14). Incubation of cells expressing sst2 with the
PTPase inhibitor, vanadate, prevented both effects suggesting that a
PTPase may be implicated in the negative growth signal induced by
activation of sst2. In addition, we demonstrated that a PTPase of 70 kDa, identified as SHP-1, copurified with membrane somatostatin
receptors (15) from pancreatic acinar cells that highly expressed sst2
receptor subtype (16). Taken together, these results suggest that SHP-1
may be a candidate for sst2-mediated early signaling events.
SHP-1 (also named SHPTP-1, SHP, or HCP) is a non-transmembrane PTPase
that contains two Src homology 2 (SH2) domains involved in its
association with multiple signaling molecules (17-20). SHP-1 associates in vivo with activated growth factor tyrosine
kinase receptors such as epidermal growth factor receptor (21). SHP-1 has also been shown to associate with activated cytokine receptors, interleukin-3 receptor Recent studies have suggested a role of SHP-1 in terminating growth
factor mitogenic signals by dephosphorylating critical molecules. SHP-1
dephosphorylates a variety of protein-tyrosine kinase receptors when
coexpressed in 293 cells (30) and has been shown to down-regulate
interleukin-3-induced tyrosine phosphorylation and mitogenesis in
hematopoietic cells (22). Association of SHP-1 with the
erythropoietin receptor causes inactivation of Janus kinase 2 and
termination of erythropoietin-induced proliferation signal (23).
Similarly, association of SHP-1 with interferon- If one of the role of SHP-1 is a negative regulation of growth factor
signaling, one might therefore speculate that SHP-1 could be activated
by factors that negatively regulate cell growth such as somatostatin.
In this study, the role of SHP-1 in signal transduction pathway of the
G protein-coupled sst2 somatostatin receptor was investigated. Our
results provide evidence of physical and functional association of
SHP-1 with sst2 and demonstrate that somatostatin is a physiological
modulator of SHP-1 that may be required in somatostatin-induced
tyrosine phosphatase activation and antiproliferative signals initiated
by sst2.
EXPERIMENTAL PROCEDURES SMS 201-995 (SMS) and somatostatin were generous
gifts of Dr. C. Bruns (Sandoz, Basel, Switzerland) and Dr. L. Moroder
(Munich, Germany), respectively. [Tyr11]somatostatin was
purchased from Bachem. [ The 2.1-kilobase HindIII/NotI
fragment of mouse SHP-1 cDNA (Dr. M. L. Thomas, Howard Hughes
Medical Institute, Washington University, St. Louis, MO) was subcloned
into the expression vector pcDNAneo vector (Invitrogen). CHO (DG44
variant) cells were grown to 50% confluency in 60-mm diameter dishes
and were cotransfected using Lipofectin reagent with 1 µg of SHP-1 or
SHP-1 (C453S) mutant in pcDNA I neo vector and 3 µg of
1.2-kilobase XbaI fragment of mouse sst2 gene in pCMV6c
vector (Dr. G. I. Bell, Howard Hughes Medical Institute, University of
Chicago, Chicago, IL) (3). SHP-1 (C453S) mutant was constructed with
the oligonucleotide primer 5 Polyclonal anti-sst2 antibodies were generated
in rabbits immunized with a peptide corresponding to amino acid
residues 191-206 of mouse sst2 as described previously (34). The
monoclonal anti-SHP-1 antibodies were obtained from Transduction
Laboratories (Medgene, France). Monoclonal antibodies raised against
phosphotyrosine (PY-20) were purchased from Santa Cruz Biotechnology
(Tebu, France). Go CHO cells and its derivatives
were cultured in For cell growth assay, cells were cultured in [Tyr11]somatostatin was
radioiodinated and purified by reverse phase-high performance liquid
chromatography as described previously (14). Cells were grown in
75-cm2 flasks for 48 h and lysed by freezing in liquid
nitrogen in 50 mM Tris-HCl (pH 7.8) containing soybean
trypsin inhibitor (0.3 mg/ml). After thawing, the cell lyzate was
centrifuged at 26,000 × g for 30 min at 4 °C, and
the pellet was resuspended in the same buffer. Binding studies were
performed on the resultant crude membranes as described previously
(14). Briefly, 5 µg of membrane proteins were incubated with 30 pM [125I-Tyr11]somatostatin at
25 °C for 90 min in 50 mM Tris-HCl (pH 7.8) containing bovine serum albumin (1 mg/ml), soybean trypsin inhibitor (0.3 mg/ml),
bacitracin (0.5 mg/ml), 5 mM MgSO4 (binding
buffer). Nonspecific binding was determined in the presence of 1 µM somatostatin. Samples were centrifuged at 10,000 × g for 10 min at 4 °C. The pellet was washed twice with
ice-cold binding buffer, and the radioactivity in the pellet was
measured. Specific binding was calculated as the difference between the
amount of radioactivity bound in the absence and the presence of 1 µM somatostatin.
Cells were washed
twice and collected in phosphate-buffered saline. After centrifugation
at 1000 × g for 5 min at 4 °C, cells were
solubilized with 50 mM Tris-HCl buffer (pH 7.5) containing 140 mM NaCl, 1 mM EDTA, 0.1 mg/ml soybean
trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride
(buffer A) in the presence of 1.5% CHAPS and 0.5 mM sodium
orthovanadate. The mixture was gently agitated for 30 min at 4 °C
and thereafter centrifuged at 13,000 × g for 20 min.
Soluble proteins (300-500 µg) were incubated for 3 h at 4 °C
with either anti-SHP-1, anti-sst2, anti-G protein antibodies, or
preimmune serum prebound to Sepharose-protein A beads prewashed in
buffer A. The beads were then washed twice with buffer A and
resuspended in either 50 µl of sample buffer for immunoblotting or
180 µl of PTPase buffer for PTPase assay.
For immunoblotting, solubilized proteins (90 µg) or
immunoprecipitated proteins (see above) were resolved through 7.5%
SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and
immunoblotted with anti-SHP-1, anti-sst2, anti-phosphotyrosine or
anti-G protein antibodies as described previously (35). Immunoreactive
proteins were visualized by the ECL immunodetection system and
quantified by image analysis using a Biocom apparatus (Biocom, Paris,
France).
Immunoprecipitated proteins were resuspended
in PTPase buffer containing 50 mM Tris-HCl (pH 7), 1 mg/ml
bovine serum albumin, and 5 mM dithiothreitol (PTPase
buffer). The substrate poly(Glu, Tyr) was phosphorylated with
[ Statistical comparison between
SMS-treated and nontreated cells was performed using Student's paired
t test.
RESULTS We
had previously reported that, in CHO cells expressing sst2 somatostatin
receptors, somatostatin analogues stimulated PTPase activity (14).
Furthermore, in pancreatic cells that highly expressed endogenous sst2,
SHP-1 copurified with somatostatin receptors (15). To investigate
whether SHP-1 interacts with sst2, we stably coexpressed SHP-1 and sst2
in CHO cells. CHO/sst2-SHP-1 clones expressed sst2 as a protein of 95 kDa detected by immunoblotting as previously reported (34) and SHP-1 as
a protein of 68 kDa, whereas these proteins were barely detectable in
wild CHO cells (Fig. 1). sst2
immunoprecipitates prepared from CHO/sst2-SHP-1 cells were examined by
immunoblotting for the presence of SHP-1. As shown in Fig.
2, the 68-kDa SHP-1 protein was
coprecipitated with the 95-kDa sst2 protein. Similarly, SHP-1
immunoprecipitation resulted in the coimmunoprecipitation of SHP-1 and
sst2. These protein bands were not seen when immunoprecipitations were
performed in the presence of preimmune serum instead of immune serum.
Quantification of immunoblots revealed that the amount of sst2 present
in the SHP-1 immunoprecipitates (lane ip SHP-1-blot sst2)
represents approximately 17-20% of immunoprecipitated sst2 (ip
sst2-blot sst2). Taking into account the efficiency of sst2
immunoprecipitation, this represents about 5% of total cellular sst2.
From these results, we conclude that SHP-1 physically associates with
sst2 and that sst2·SHP-1 complexes are preformed in resting
cells.
To
determine whether the interaction of SHP-1 with sst2 was affected by
somatostatin treatment, CHO/sst2-SHP-1 cells were incubated in the
presence of the somatostatin analogue, SMS, for various times prior to
solubilization and immunoprecipitation with anti-sst2 antibodies. The
amount of SHP-1 associated with sst2 was then analyzed by
immunoblotting with anti-SHP-1 antibodies and the blots were reprobed
with anti-sst2 antibodies to ensure that comparable amounts of sst2
molecules were immunoprecipitated at each time point of SMS treatment.
As observed in Fig. 3, somatostatin treatment resulted in a transient increase of the amount of SHP-1 immunoprecipitated with anti-sst2 antibodies within the first 30 s
after which the rate of immunodetected SHP-1 in sst2 immunoprecipitates decreased, about 80% of the preformed sst2·SHP-1 complexes being dissociated after 10 min of SMS treatment. Similarly, the amount of
immunodetected sst2 in SHP-1 immunoprecipitates was decreased by
60 ± 6% following SMS treatment of cells for 3 min.
We further investigated the effect of SMS on SHP-1 activity in
CHO/sst2-SHP-1 cells. Cells were incubated in the presence of 1 nM SMS for various times, after which they were
solubilized, and SHP-1 activity was measured in SHP-1
immunoprecipitates. As shown in Fig. 4,
SHP-1 activity was increased upon treatment with SMS. The stimulation
of SHP-1 activity was maximal after 30 s of SMS treatment and
slightly declined up to 10 min.
We then examined the effect of SMS treatment on the level of tyrosine
phosphorylation of SHP-1. SMS-treated or untreated cells were subjected
to immunoprecipitation with anti-SHP-1 antibodies and immunoblotted
with either anti-phosphotyrosine or anti-SHP-1 antibodies. The
immunoblot revealed that SHP-1 was tyrosine phosphorylated in untreated
cells and that SMS induced a rapid and transient dephosphorylation of
SHP-1 (Fig. 5). The dephosphorylation of SHP-1 was observed as early as 30 s after SMS treatment, was
maximal at 1 min and declined subsequently until control levels. All
these results indicate that SMS induced a transient increase and a
subsequent dissociation of preformed sst2·SHP-1 complexes, which was
associated with the stimulation and the transient dephosphorylation of
the enzyme.
In CHO cells
expressing sst2, we previously reported that stimulation of PTPase
activity by somatostatin was suppressed by pretreatment of cells with
pertussis toxin indicating that a pertussis toxin sensitive G protein
was involved in this effect (13). Furthermore, in these cells, sst2 has
been demonstrated to be coupled to pertussis toxin-sensitive G
proteins, Gi
To investigate whether the association of Gi To obtain direct
evidence of the role of SHP-1 in the inhibitory effect of somatostatin
on cell proliferation, we generated a catalytically inactive SHP-1 by
introducing a point mutation into the conserved catalytic residue,
cysteine 453, which is crucial for catalytic activity of the enzyme.
Cys453 was mutated to Ser. This mutation completely
abolished the phosphatase activity of the SHP-1 mutant transiently
expressed in COS-7 cells.2 We
stably coexpressed the SHP-1 mutant and sst2 in CHO cells and we
selected clones (CHO/sst2-SHP-1(C453S)) that expressed the two proteins
at a similar level with that observed in CHO/sst2-SHP-1 cells as
demonstrated by immunoblotting (Fig. 7).
CHO/sst2-SHP-1(C453S) cells were incubated with SMS after which SHP-1
was immunoprecipitated with anti-SHP-1 antibodies, and PTPase activity
was measured. SMS no more stimulated SHP-1 activity in cells expressing
the SHP-1 mutant (Fig. 8). Expression of
sst2 in CHO cells did not modify the serum-stimulated cell growth
whereas coexpression of sst2 and SHP-1 inhibited by 39 ± 6%
(n = 3) serum-activated cell growth after 3 days of
culture when compared with control cells. These results are consistent
with the negative role of SHP-1 on CHO cell growth (37). Incubation of
wild CHO cells for 24 h in the presence of 1 nM SMS
did not modify the serum-stimulated cell growth, whereas SMS inhibited
by 36% the growth of CHO/sst2-SHP-1 cells (Fig.
9), in agreement with the growth
inhibitory effect of somatostatin analogues in cells expressing sst2
(13, 14). In contrast, the SMS-induced inhibition of cell proliferation was abolished in CHO cells coexpressing sst2 and the SHP-1 mutant. This
indicates that the expression of the catalytic inactive SHP-1 blocks
the negative regulation of cell growth induced by SMS and demonstrates
that SHP-1 may play an important role in the transduction of the
negative growth signal promoted by activation of sst2.
DISCUSSION Somatostatin acts as a growth inhibitory factor in a variety of
normal and tumor cells (2, 9, 10). We and others have demonstrated that
somatostatin and analogues induce the stimulation of a membrane PTPase,
which may be involved in the inhibitory effect of these peptides on
cell proliferation (9, 11, 12, 38). More recently, we have established
the role of the somatostatin receptor sst2 in mediating the stimulatory
effect of somatostatin on PTPase activity and its negative effect on
cell growth (13, 14). The identification of involved PTPase is
therefore important for the understanding of the negative growth signal
transduction promoted by sst2. In the present study, we have shown that
the phosphotyrosine phosphatase SHP-1 associates with the sst2
somatostatin receptor subtype and established that SHP-1 is involved in
the growth inhibitory signal transduction pathway of sst2.
SHP-1 is a cytoplasmic PTPase containing two SH2 domains that enable it
to bind specific tyrosine residues of phosphorylated proteins. SHP-1
has been reported to associate with a variety of activated growth
factor tyrosine kinase receptors, cytokine receptors, and also with the
B cell Fc Furthermore, we demonstrated that the occupancy of sst2 promotes a
rapid and transient increase (at 0.5 min) of the recruitment of SHP-1
and to a lesser extent that of Gi We also demonstrated that, concomitantly with the increase of SHP-1
activity, somatostatin induces a transient dephosphorylation of SHP-1
on tyrosine residues that is maximal at 1 min and returns to basal
level at 10 min. These results suggest that activated SHP-1 undergoes a
rapid tyrosine autodephosphorylation due to the enzyme activation.
This hypothesis is strengthened by the observation that SHP-1 was
rapidly autodephosphorylated in vitro, whereas the
catalytically inactive mutant SHP-1 was stably tyrosine-phosphorylated (44, 45). In contrast, SHP-1 has been reported to be
tyrosine-phophorylated in response to various growth factors and
cytokines receptors as well as mitogenic factors acting on G
protein-coupled receptors (22, 26, 27, 29, 46). The role of tyrosine
phosphorylation/dephosphorylation of SHP-1 has to be elucidated.
Different sites of SHP-1 tyrosine phosphorylation have been identified
and may play specific role (44, 47).
SHP-1 was found to play a major role in negatively regulating signaling
pathways. For instance, SHP-1 has also been demonstrated to terminate
IL-3 and EPO growth signals, its recruitment to the activated receptors
causing dephosphorylation and inactivation of specifically associated
signaling molecules (22, 23). In CHO cells overexpressing the insulin
receptor, a negative effect of overexpressed SHP-1 on cell
proliferation has been also reported (37). The demonstration that the
negative effect of somatostatin on CHO cell growth can be suppressed in
cells coexpressing sst2 and the inactive SHP-1 mutant argues in favor
of the role of SHP-1 in the negative growth signal induced by
somatostatin-activated sst2. Further, the rapid activation of SHP-1
following somatostatin addition in CHO cells coexpressing sst2 and
SHP-1 raises the possibility that activation of SHP-1 is the initiating
step for sst2 signal transduction leading to inhibition of cell
proliferation. These hypothesis are strengthened by the observation
that rat pancreatic tumor AR42J cells are sensitive to the
antiproliferative effect of somatostatin analogs and highly express
sst2 receptors and SHP-1 (10, 16, 35). Examining SHP-1 expression in
cells sensitive to somatostatin analogs would add significantly to
these conclusions. It is well known that growth factors transduce cell proliferation signal through activation of receptor tyrosine kinases that phosphorylate downstream enzymes and/or adaptator proteins leading
to phosphorylation and activation of mitogen-activated protein kinase.
Recent data provide evidence that mitogen peptides acting on G
protein-coupled receptors can also promote phosphorylation and
activation of mitogen-activated protein kinase via activation of
tyrosine kinase (48). Conversely, one could be expected that growth
inhibitory peptides acting via G protein-coupled receptors may induce
growth inhibition through activation of PTPase resulting in tyrosine
dephosphorylation of mitogen-induced tyrosine phosphorylation of
signaling molecules. Such a stimulation of PTPase by growth inhibitory
peptides acting via G protein-coupled receptors has been previously
reported for angiotensin II AT2 receptors (49). The effector molecules
that act downstream of sst2·SHP-1 complexes are not known. However,
it has been shown that somatostatin dephosphorylates tyrosine
phosphorylated EGF receptors (9), suggesting that growth factor
receptors could be one of the substrates of SHP-1. sst1 and sst3
somatostatin receptor subtypes expressed in heterologous cells have
been also demonstrated to mediate somatostatin stimulation of PTPase
activity (14, 50, 51). Whether SHP-1 binds to other somatostatin
receptor subtypes has yet to be investigated. If this was the case,
then activation of SHP-1 could be an early signal pathway shared by the
somatostatin receptor family.
FOOTNOTES ACKNOWLEDGEMENTS We are grateful to Dr. M. Thomas and Dr. G. I. Bell for kindly providing SHP-1 cDNA and mouse sst2 cDNA,
respectively and to Dr. F. R. McKenzie and Dr. N. Rivard for
advice regarding SHP-1 expression.
REFERENCES
Volume 272, Number 39,
Issue of September 26, 1997
pp. 24448-24454
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
Laboratoire de
Génétique et Physiologie du développement, Luminy
CASE 907, 13288 Marseille Cedex 9, France, and § Institut
Cochin de Génétique Moléculaire, 75014 Paris, France
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
3 was specifically
immunoprecipitated by anti-sst2 and anti-SHP-1 antibodies, and
somatostatin induced a rapid dissociation of Gi3 from
sst2, suggesting that Gi3 may be involved in the
sst2·SHP-1 complexes. Finally, somatostatin inhibited the proliferation of cells coexpressing sst2 and SHP-1, and this effect was
suppressed in cells coexpressing sst2 and the catalytic inactive SHP-1
(C453S mutant). Our data identify SHP-1 as the tyrosine phosphatase
associated with sst2 and demonstrate that this enzyme may be an initial
key transducer of the antimitogenic signaling mediated by sst2.
chain (22), erythropoietin receptor (23),
interferon-
/ receptor (24), and also with B cell Fc
RIIB receptor (25). As a result of these interactions, SHP-1 becomes tyrosine-phosphorylated by these factors (22, 26, 27). SHP-1 is also
tyrosine-phosphorylated in response to activation of G protein-coupled
receptors (28, 29).
/ receptor
negatively regulates interferon-/-induced Janus kinase 1/Stat1
signaling pathway (24). SHP-1 has been also implicated as the mediator
used by the FcRIIB1 receptor in B lymphocytes to turn off B cell
antigen receptor signaling (25). The role of SHP-1 in the negative
regulation of hematopoiesis is consistent with the various
hematopoietic abnormalities and hypersensitivity to growth factors in
mice carrying the lethal mutation in the SHP-1 gene that results in
motheaten phenotype (31, 32).
-33P]ATP (3,000 Ci/mmol) was
purchased from Isotopchim (France). Enhanced chemiluminescence (ECL)
immunodetection system and Hybond ECL nitrocellulose membrane were from
Amersham Corp. Poly(Glu, Tyr), cholesterol hemisuccinate, geneticin
(G418), and Sepharose-protein A beads were from Sigma. Minimal
essential medium (MEM), fetal calf serum (FCS), and Lipofectin
reagent were from Life Technologies, Inc. CHAPS was from Serva.
-GAT GCC AGC GCT GGA ATG CAC AAT-3 by
using the method of Kunkel et al. (33). The mutation was
confirmed by dideoxynucleotide sequencing. Stable colonies obtained by
selection with G418 (600 µg/ml) were screened for somatostatin
binding using [125I-Tyr11]somatostatin as
described below. Cellular clones expressing somatostatin binding sites
at a similar level were screened for the presence of SHP-1 or SHP-1
(mutant) using Western blot analysis as described below.
1-2, Gi1-2,
Gi2, and Gi3 antibodies were from Gramsch
Laboratories (Germany).
MEM containing 10% FCS and G418 (200 µg/ml) as
described previously (14). For cell treatment, cells were plated in
100-mm dishes (106 cells/dish) in MEM containing 10%
FCS, and after an overnight attachment phase, cells were cultured for
24 h, washed, and cultured overnight in serum-free MEM. Cells
were rinsed and then incubated for indicated times in the presence of
the somatostatin analogue, SMS, at 1 nM.
MEM containing 10%
FCS and plated in 35-mm dishes at 55 × 103 cells/ml
(2 ml/dish). After an overnight attachment phase (time 0), cells were
cultured for 24 h in serum-free MEM or in MEM containing
10% FCS with or without 1 nM SMS. Cell growth was measured at the indicated times by counting cells with a Coulter counter model
ZM (Coulter Electronics) as described elsewhere (13).
-33P]ATP as described elsewhere (13). The reaction
was initiated by the addition of 30,000 cpm of 33P-labeled
poly(Glu, Tyr) and allowed to proceed for 10 min at 30 °C as
described previously (13). PTPase activity was expressed in picomoles
of inorganic phosphate released per min at 30 °C from radiolabeled
substrate.
Fig. 1.
Detection of sst2 and SHP-1 in CHO/sst2-SHP-1
cells. CHAPS-solubilized cell proteins (90 µg/lane) from
CHO/sst2-SHP-1 cells (lanes 1, 3, and
5) and wild CHO cells (lanes 2 and 4)
were subjected to a 7.5% SDS-PAGE and immunoblotted with polyclonal anti-sst2 (lanes 2 and 3) or monoclonal
anti-SHP-1 antibody (lanes 4 and 5) or preimmune
serum (PI) (lane 1). The position of molecular mass markers are shown on the side and are indicated in kilodaltons. Arrows indicate the positions of SHP-1 and sst2.
[View Larger Version of this Image (42K GIF file)]
Fig. 2.
Association of SHP-1 with sst2 in
CHO/sst2-SHP-1 cells. CHO/sst2-SHP-1 cells (106
cells/dish) were cultured for 24 h in MEM containing 10% FCS and solubilized with 1.5% CHAPS. Soluble proteins were
immunoprecipitated (ip) with either anti-sst2
(sst2) or anti-SHP-1 (SHP-1) antibodies. Immune
complexes were fractionated by a 7.5% SDS-PAGE and subjected to
sequential immunoblotting (blot) with anti-sst2 or
anti-SHP-1 antibodies or preimmune serum (PI).
Arrows indicate the position of sst2 and SHP-1. Size markers
(kDa) are indicated to the left of the immunoblot.
[View Larger Version of this Image (46K GIF file)]
Fig. 3.
SHP-1-sst2 association following SMS
treatment of CHO/sst2-SHP-1 cells. A, CHO/sst2-SHP-1 cells
(106 cells/dish) were cultured for 24 h in MEM
containing 10% FCS and in serum-free MEM overnight. Cells were then
incubated at 37 °C for the indicated times with 1 nM SMS
and solubilized with 1.5% CHAPS as described under "Experimental
Procedures." Soluble proteins (500 µg) were immunoprecipitated
(ip) with anti-sst2 (sst2) antibody. sst2
immunoprecipitates were analyzed by SDS-PAGE and immunoblotted
(blot) with anti-SHP-1 (SHP-1) antibody. The blot
was reprobed with anti-sst2 antibody. Arrows indicate the position of SHP-1 and sst2. B, immunoblots were
densitometrically analyzed, and the data were plotted as percentage of
control values obtained from cells at time 0. Data from four separate
experiments are presented as means ± S.E. (Statistical comparison
between treated and untreated cells, *p < 0.05.)
[View Larger Version of this Image (40K GIF file)]
Fig. 4.
Time course of SMS-induced stimulation of
SHP-1 activity in CHO/sst2-SHP-1 cells. CHO/sst2-SHP-1 cells were
cultured for 24 h in MEM containing 10% FCS and in serum-free
MEM overnight. Cells were incubated at 37 °C for the indicated
times with 1 nM SMS prior to solubilization and
immunoprecipitation with anti-SHP-1 antibodies. Immunuprecipitates were
assayed for PTPase activity in the presence of 33P-labeled
poly(Glu, Tyr) for 10 min at 30 °C. Results are expressed as percent
of PTPase activity obtained from cells at time 0. Basal SHP-1 activity
was 0.37 ± 0.04 pmol/min. Results are means ± S.E. of three
experiments in duplicate. (Statistical comparison between treated and
untreated cells, *p < 0.05.)
[View Larger Version of this Image (35K GIF file)]
Fig. 5.
Time-dependent tyrosine
dephosphorylation of SHP-1 in response to SMS in CHO/sst2-SHP-1
cells. A, CHO/sst2-SHP-1 cells were cultured for 24 h
in MEM containing 10% FCS and in serum-free MEM overnight. Cells
were incubated for indicated times at 37 °C with 1 nM
SMS and solubilized with 1.5% CHAPS. Cell lysates were subjected to
immunoprecipitation (ip) with anti-SHP-1 (SHP-1) antibodies. Immunoprecipitates were resolved by a 7.5% SDS-PAGE and
analyzed by immunoblotting with anti-phosphotyrosine antibodies (blot P-Tyr). The same filter was then reprobed with
anti-SHP-1 antibodies (blot SHP-1). B,
immunoblots were densitometrically analyzed, and data were plotted as a
percentage of control values obtained from cells at time 0. Data from
three separate experiments are presented as means ± S.E.
(Statistical comparison between treated and untreated cells,
*p < 0.05.)
[View Larger Version of this Image (39K GIF file)]
3
3 and Go2, but not to
Gi1 and Gi2 (36). Preincubation of
CHO/sst2-SHP-1 cells with pertussis toxin for 18 h at 100 ng/ml abolished the stimulatory effect of SMS on SHP-1 activity in SHP-1 immunoprecipitates (not shown) suggesting that sst2-coupled activation of SHP-1 was mediated by a pertussis toxin-sensitive G protein. To
identify the G protein involved in the sst2·SHP-1 complexes, SHP-1
and sst2 immunoprecipitates from CHO/sst2-SHP-1 cells were analyzed by
immunoblotting with antibodies directed against
Gi1, 2, 3 and Go1-2 subunits.
Gi3 was immunoprecipitated with anti-sst2 antibodies as
well as anti-SHP-1 antibodies, suggesting that Gi3 was
present in the sst2·SHP-1 complexes. In contrast, Gi1,
Gi2, and Go were never detected in the
sst2·SHP-1 immunoprecipitates (Fig. 6).
In addition PTPase activity can be immunoprecipitated by
anti-Gi3 antibodies but not by Go antibodies (not shown). All these results argue in favor of a role for
Gi3 in the formation of the sst2·SHP-1 complexes.
Fig. 6.
Identification of the G protein subunits in
sst2 and SHP-1 immunoprecipitates from CHO/sst2-SHP-1 cells.
CHO/sst2-SHP-1 cells were cultured for 24 h in MEM containing
10% FCS and in serum-free MEM overnight. Cells were incubated at
37 °C for the indicated times with 1 nM SMS and
solubilized with 1.5% CHAPS. Cell lysates were subjected to
immunoprecipitation (ip) with anti-sst2 (sst2)
(A) or anti-SHP-1 (SHP-1) (B)
antibodies. Immunoprecipitates were resolved by a 7.5% SDS-PAGE
and sequentially analyzed by immunoblotting with antibodies directed
against Gi3 and sst2 (A) or SHP-1
(B). C and D, Immunoblots were
densitometrically analyzed, and data were plotted as the percentage of
control values obtained from cells at time 0. Data are presented as
means ± S.E. of three separate experiments. (Statistical
comparison between treated and untreated cells, *p < 0.05.) E, cell lysates were subjected to immunoprecipitation
(ip) with anti-sst2 (sst2) or anti-SHP-1
(SHP-1) antibodies. Immunoprecipitates were sequentially analyzed by immunoblotting with antibodies directed against
Go1-2 (o), Gi3
(i3), Gi2 (i2),
Gi1-2 (i1/2), sst2, SHP-1, or preimmune
serum (PI).
[View Larger Version of this Image (49K GIF file)]
3 with sst2
can be modified by somatostatin, CHO/sst2-SHP-1 cells were treated for
various times with 1 nM SMS and Gi3 was
identified in sst2 and SHP-1 immunoprecipitates. As observed in Fig. 6,
SMS treatment induced a transient increase at 30 s of the amount
of Gi3 immunoprecipitated either by anti-sst2 or
anti-SHP-1 antibodies which was followed by a rapid decrease of the
amount of immunodetected Gi3. Only 30% of
sst2-associated Gi3 was detected after 10 min of SMS
treatment. The time course of Gi3 dissociation from sst2
and SHP-1 paralleled that of dissociation of SHP-1 from sst2,
suggesting that these events can be linked.
Fig. 7.
Detection of sst2 and SHP-1 in CHO/sst2-SHP-1
and CHO/sst2-SHP-1(C453S) cells. Cell lysates from CHO/sst2-SHP-1
cells (lanes 1 and 3) and CHO/sst2-SHP-1(C453S)
cells expressing the catalytic inactive SHP-1 (C453S) (lanes
2 and 4) were subjected to SDS-PAGE and immunoblotted
with anti-sst2 (lanes 1 and 2) or anti-SHP-1
antibody (lanes 3 and 4). Size markers (kDa) are
indicated to the left of the immunoblot. Arrows indicate the
position of sst2 and SHP-1.
[View Larger Version of this Image (43K GIF file)]
Fig. 8.
Effect of SMS on SHP-1 activity in
CHO/sst2-SHP-1 and CHO/sst2-SHP-1(C453S) cells. CHO/sst2-SHP-1
cells and CHO/sst2-SHP-1(C453S) cells were cultured for 24 h in
MEM containing 10% FCS and in serum-free MEM overnight. Cells
were treated (hatched bar) or not (hollow bar)
with 1 nM SMS for 1 min prior to solubilization and
immunoprecipitation with anti-SHP-1 antibodies. Immunoprecipitates were
assayed for PTPase activity in the presence of 33P-labeled
poly(Glu, Tyr) for 10 min at 30 °C as described under "Experimental Procedures." Results are expressed as the percent of
PTPase activity obtained from cells at time 0. Results are means ± S.E. of four separate experiments made in duplicate. (Statistical comparison between treated and untreated cells, *p < 0.05.)
[View Larger Version of this Image (28K GIF file)]
Fig. 9.
Effect of SMS on proliferation of
CHO/sst2-SHP-1 and CHO/sst2-SHP-1(C453S) cells. Wild CHO cells,
CHO/sst2-SHP-1 cells, or CHO/sst2-SHP-1(C453S) cells were cultured for
24 h in MEM containing 10% FCS with (hatched bar)
or without (hollow bar) 1 nM SMS as indicated
under "Experimental Procedures." Results are expressed as
percentage of control values obtained from untreated cells. Values are
mean ± S.E. of three separate experiments made in
triplicate. (Statistical comparison between treated and untreated cells, *p < 0.05.)
[View Larger Version of this Image (38K GIF file)]
RIIB receptor (21-25). We have established that SHP-1
associates with another class of receptor, the G protein-coupled
receptor sst2. Results from coprecipitation of SHP-1 with sst2 in CHO
cells coexpressing sst2 and SHP-1 provide evidence that SHP-1
associates with sst2 in basal conditions. Specific components mediating
this association remain to be determined. However, the demonstration
that the Gi3 subunit, which is known to couple sst2
receptors at the resting level (36), is present in the sst2·SHP-1
complex and can immunoprecipitate PTPase activity strongly suggests
that Gi3 could achieve a direct coupling between sst2
and SHP-1. Such a receptor-G protein coupling which is evident in
overexpression system (39) has been recently reported for the
5-HT1A receptor which interacts with Gz in the absence of agonist (40). The molecular base of interaction between Gi3 and SHP-1 is not known, but preliminary results
suggest that Gi3 could be tyrosyl-phosphorylated and
therefore interact with the SHP-1 SH2
domains.3 However, our
results do not preclude the possibility that another protein may
contribute to the interaction of SHP-1 with sst2.
3 to sst2, which is
followed by a rapid dissociation of these molecules from sst2. The weak
increase of the interaction between sst2 and either Gi3 or SHP-1 following sst2 stimulation may be related to the known transient nature of the association of receptors with G proteins driven
by receptor occupancy (41). A similar dynamic interaction was recently
observed with IL-8 receptors that interacted with Gi2 in
a time-dependent manner, attaining a maximal level by 1 min
and then declining until control levels after 10 min of IL-8
stimulation (42). Moreover, the observation that the interaction of
sst2 with Gi3 and SHP-1 was decreased following sst2
activation indicates that somatostatin stimulates the dissociation of
the majority of preformed complexes. We also demonstrated that somatostatin activation of sst2 leads to a rapid increase of SHP-1 activity, which is maximal at 0.5 min, maintained as a plateau until 3 min, and then decreased at 10 min. These results argue in favor of the
role of SHP-1 in the early events of somatostatin action. One might
speculate that, following exposure of cells to SMS, the transient
increase of association of SHP-1 to sst2 via Gi3 was
accompanied by an activating conformational change of the enzyme, the
activation of the enzyme being able to result from the relief of
autoinhibition by the SH2 domains (43). The fact that
somatostatin-induced stimulation of the enzyme was inhibited by
pertussis toxin emphasizes the implication of a
Gi/o-like protein in this effect and supports the idea
that stimulation of SHP-1 by somatostatin results from activation of
Gi3. Consistent with these results, we and others have
previously reported that the activation of PTPase activity by
somatostatin involved a pertussis toxin-sensitive G protein (11, 14).
On the other hand, we found that somatostatin induces a rapid
dissociation (at 1 min) of sst2·Gi3·SHP-1 complexes.
When dissociation of the complexes start, the enzyme is still active.
The dissociation may allow SHP-1 to associate with specific substrates,
such as phosphorylated growth factor receptors and downstream
molecules, with this probably leading to negative regulation of
mitogenic signals by dephosphorylation.
¶
To whom correspondence should be addressed. Tel.:
33-5-61-32-2407; Fax: 33-5-61-32-2403; E-mail:
susinich{at}rangueil.inserm.fr.
1
The abbreviations used are: PTPase,
protein-tyrosine phosphatase; CHO, Chinese hamster ovary; sst,
somatostatin receptor; MEM, modified Eagle's medium; FCS, fetal calf
serum; PAGE, polyacrylamide gel electrophoresis; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio] -1-propanesulfonate; SMS,
somatostatin analogue SMS 201-995.
2
C. Nahmias, unpublished results.
3
F. Lopez, unpublished results.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Peverelli, A. G. Lania, G. Mantovani, P. Beck-Peccoz, and A. Spada Characterization of Intracellular Signaling Mediated by Human Somatostatin Receptor 5: Role of the DRY Motif and the Third Intracellular Loop Endocrinology, July 1, 2009; 150(7): 3169 - 3176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Perry and G. I. Sandle Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin Am J Physiol Gastrointest Liver Physiol, July 1, 2009; 297(1): G159 - G167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Barbieri, A. Pattarozzi, M. Gatti, C. Porcile, A. Bajetto, A. Ferrari, M. D. Culler, and T. Florio Somatostatin Receptors 1, 2, and 5 Cooperate in the Somatostatin Inhibition of C6 Glioma Cell Proliferation in Vitro via a Phosphotyrosine Phosphatase-{eta}-Dependent Inhibition of Extracellularly Regulated Kinase-1/2 Endocrinology, September 1, 2008; 149(9): 4736 - 4746. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Florio, F. Barbieri, R. Spaziante, G. Zona, L. J Hofland, P. M van Koetsveld, R. A Feelders, G. K Stalla, M. Theodoropoulou, M. D Culler, et al. Efficacy of a dopamine-somatostatin chimeric molecule, BIM-23A760, in the control of cell growth from primary cultures of human non-functioning pituitary adenomas: a multi-center study Endocr. Relat. Cancer, June 1, 2008; 15(2): 583 - 596. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Li, M. Mogi, K. Tsukuda, H. Tomochika, J. Iwanami, L.-J. Min, C. Nahmias, M. Iwai, and M. Horiuchi Angiotensin II-Induced Neural Differentiation via Angiotensin II Type 2 (AT2) Receptor-MMS2 Cascade Involving Interaction between AT2 Receptor-Interacting Protein and Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatase 1 Mol. Endocrinol., February 1, 2007; 21(2): 499 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Arena, A. Pattarozzi, A. Massa, J.-P. Esteve, R. Iuliano, A. Fusco, C. Susini, and T. Florio An Intracellular Multi-Effector Complex Mediates Somatostatin Receptor 1 Activation of Phospho-Tyrosine Phosphatase {eta} Mol. Endocrinol., January 1, 2007; 21(1): 229 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C Susini and L Buscail Rationale for the use of somatostatin analogs as antitumor agents Ann. Onc., December 1, 2006; 17(12): 1733 - 1742. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Roosterman, T. Goerge, S. W. Schneider, N. W. Bunnett, and M. Steinhoff Neuronal control of skin function: the skin as a neuroimmunoendocrine organ. Physiol Rev, October 1, 2006; 86(4): 1309 - 1379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Cordelier, J.-P. Esteve, S. Najib, L. Moroder, N. Vaysse, L. Pradayrol, C. Susini, and L. Buscail Regulation of Neuronal Nitric-oxide Synthase Activity by Somatostatin Analogs following SST5 Somatostatin Receptor Activation J. Biol. Chem., July 14, 2006; 281(28): 19156 - 19171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Tagliati, M. C. Zatelli, A. Bottoni, D. Piccin, A. Luchin, M. D. Culler, and E. C. degli Uberti Role of Complex Cyclin D1/Cdk4 in Somatostatin Subtype 2 Receptor-Mediated Inhibition of Cell Proliferation of a Medullary Thyroid Carcinoma Cell Line in Vitro Endocrinology, July 1, 2006; 147(7): 3530 - 3538. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Theodoropoulou, J. Zhang, S. Laupheimer, M. Paez-Pereda, C. Erneux, T. Florio, U. Pagotto, and G. K. Stalla Octreotide, a Somatostatin Analogue, Mediates Its Antiproliferative Action in Pituitary Tumor Cells by Altering Phosphatidylinositol 3-Kinase Signaling and Inducing Zac1 Expression Cancer Res., February 1, 2006; 66(3): 1576 - 1582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Barrett, S. M. Black, H. Todor, R. K. Schmidt-Ullrich, K. S. Dawson, and R. B. Mikkelsen Inhibition of Protein-tyrosine Phosphatases by Mild Oxidative Stresses Is Dependent on S-Nitrosylation J. Biol. Chem., April 15, 2005; 280(15): 14453 - 14461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Massa, F. Barbieri, C. Aiello, S. Arena, A. Pattarozzi, P. Pirani, A. Corsaro, R. Iuliano, A. Fusco, G. Zona, et al. The Expression of the Phosphotyrosine Phosphatase DEP-1/PTP{eta} Dictates the Responsivity of Glioma Cells to Somatostatin Inhibition of Cell Proliferation J. Biol. Chem., July 9, 2004; 279(28): 29004 - 29012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Turner, A. K. Gelasco, and J. R. Raymond Calmodulin Interacts with the Third Intracellular Loop of the Serotonin 5-Hydroxytryptamine1A Receptor at Two Distinct Sites: PUTATIVE ROLE IN RECEPTOR PHOSPHORYLATION BY PROTEIN KINASE C J. Biol. Chem., April 23, 2004; 279(17): 17027 - 17037. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wu, M. Iwai, Z. Li, T. Shiuchi, L.-J. Min, T.-X. Cui, J.-M. Li, M. Okumura, C. Nahmias, and M. Horiuchi Regulation of Inhibitory Protein-{kappa}B and Monocyte Chemoattractant Protein-1 by Angiotensin II Type 2 Receptor-Activated Src Homology Protein Tyrosine Phosphatase-1 in Fetal Vascular Smooth Muscle Cells Mol. Endocrinol., March 1, 2004; 18(3): 666 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Lahlou, N. Saint-Laurent, J.-P. Esteve, A. Eychene, L. Pradayrol, S. Pyronnet, and C. Susini sst2 Somatostatin Receptor Inhibits Cell Proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 Activation J. Biol. Chem., October 10, 2003; 278(41): 39356 - 39371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Ferjoux, F. Lopez, J.-P. Esteve, A. Ferrand, E. Vivier, F. Vely, N. Saint-Laurent, L. Pradayrol, L. Buscail, and C. Susini Critical Role of Src and SHP-2 in sst2 Somatostatin Receptor-mediated Activation of SHP-1 and Inhibition of Cell Proliferation Mol. Biol. Cell, September 1, 2003; 14(9): 3911 - 3928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. SASSON, A. DANTES, K. TAJIMA, and A. AMSTERDAM Novel genes modulated by FSH in normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action FASEB J, July 1, 2003; 17(10): 1256 - 1266. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hortala, G. Ferjoux, A. Estival, C. Bertrand, S. Schulz, L. Pradayrol, C. Susini, and F. Clemente Inhibitory Role of the Somatostatin Receptor SST2 on the Intracrine-regulated Cell Proliferation Induced by the 210-Amino Acid Fibroblast Growth Factor-2 Isoform: IMPLICATION OF JAK2 J. Biol. Chem., May 30, 2003; 278(23): 20574 - 20581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Florio, M. Morini, V. Villa, S. Arena, A. Corsaro, S. Thellung, M. D. Culler, U. Pfeffer, D. M. Noonan, G. Schettini, et al. Somatostatin Inhibits Tumor Angiogenesis and Growth via Somatostatin Receptor-3-Mediated Regulation of Endothelial Nitric Oxide Synthase and Mitogen-Activated Protein Kinase Activities Endocrinology, April 1, 2003; 144(4): 1574 - 1584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Banihashemi and P. R. Albert Dopamine-D2S Receptor Inhibition of Calcium Influx, Adenylyl Cyclase, and Mitogen-Activated Protein Kinase in Pituitary Cells: Distinct G{alpha} and G{beta}{gamma} Requirements Mol. Endocrinol., October 1, 2002; 16(10): 2393 - 2404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-H. Feng, Y. Sun, and J. G. Douglas Gbeta gamma -independent constitutive association of Galpha s with SHP-1 and angiotensin II receptor AT2 is essential in AT2-mediated ITIM-independent activation of SHP-1 PNAS, September 17, 2002; 99(19): 12049 - 12054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Leach, S. M. Black, R. K. Schmidt-Ullrich, and R. B. Mikkelsen Activation of Constitutive Nitric-oxide Synthase Activity Is an Early Signaling Event Induced by Ionizing Radiation J. Biol. Chem., May 3, 2002; 277(18): 15400 - 15406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. Zapata, R. M. Ropero, A. M. Valencia, L. Buscail, J. I. Lopez, R. M. Martin-Orozco, J. C. Prieto, J. Angulo, C. Susini, P. Lopez-Ruiz, et al. Autocrine Regulation of Human Prostate Carcinoma Cell Proliferation by Somatostatin through the Modulation of the SH2 Domain Containing Protein Tyrosine Phosphatase (SHP)-1 J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 915 - 926. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Takeda-Matsubara, H. Nakagami, M. Iwai, T.-X. Cui, T. Shiuchi, M. Akishita, C. Nahmias, M. Ito, and M. Horiuchi Estrogen Activates Phosphatases and Antagonizes Growth-Promoting Effect of Angiotensin II Hypertension, January 1, 2002; 39(1): 41 - 45. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Goddard, S. Bauer, A. Gougeon, F. Lopez, N. Giannetti, C. Susini, M. Benahmed, and S. Krantic Somatostatin Inhibits Stem Cell Factor Messenger RNA Expression by Sertoli Cells and Stem Cell Factor-Induced DNA Synthesis in Isolated Seminiferous Tubules Biol Reprod, December 1, 2001; 65(6): 1732 - 1742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Florio, S. Arena, S. Thellung, R. Iuliano, A. Corsaro, A. Massa, A. Pattarozzi, A. Bajetto, F. Trapasso, A. Fusco, et al. The Activation of the Phosphotyrosine Phosphatase {eta} (r-PTP{eta}) Is Responsible for the Somatostatin Inhibition of PC Cl3 Thyroid Cell Proliferation Mol. Endocrinol., October 1, 2001; 15(10): 1838 - 1852. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Hennige, R. Lammers, W. Hoppner, D. Arlt, V. Strack, R. Teichmann, F. Machicao, A. Ullrich, H.-U. Haring, and M. Kellerer Inhibition of Ret Oncogene Activity by the Protein Tyrosine Phosphatase SHP1 Endocrinology, October 1, 2001; 142(10): 4441 - 4447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Yoshida, T. Ishibashi, and M. Nishio Growth-inhibitory Effect of a Streptococcal Antitumor Glycoprotein on Human Epidermoid Carcinoma A431 Cells: Involvement of Dephosphorylation of Epidermal Growth Factor Receptor Cancer Res., August 1, 2001; 61(16): 6151 - 6157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Sharp, K. McAllen, G. Gekker, N. A. Shahabi, and P. K. Peterson Immunofluorescence Detection of {{delta}} Opioid Receptors (DOR) on Human Peripheral Blood CD4+ T Cells and DOR-Dependent Suppression of HIV-1 Expression J. Immunol., July 15, 2001; 167(2): 1097 - 1102. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-X. Cui, H. Nakagami, M. Iwai, Y. Takeda, T. Shiuchi, L. Daviet, C. Nahmias, and M. Horiuchi Pivotal role of tyrosine phosphatase SHP-1 in AT2 receptor-mediated apoptosis in rat fetal vascular smooth muscle cell Cardiovasc Res, March 1, 2001; 49(4): 863 - 871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Charland, M.-J. Boucher, M. Houde, and N. Rivard Somatostatin Inhibits Akt Phosphorylation and Cell Cycle Entry, But Not p42/p44 Mitogen-Activated Protein (MAP) Kinase Activation in Normal and Tumoral Pancreatic Acinar Cells Endocrinology, January 1, 2001; 142(1): 121 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Boudin, P. Sarret, J. Mazella, A. Schonbrunn, and A. Beaudet Somatostatin-Induced Regulation of SST2A Receptor Expression and Cell Surface Availability in Central Neurons: Role of Receptor Internalization J. Neurosci., August 15, 2000; 20(16): 5932 - 5939. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Elbaz, K. Bedecs, M. Masson, M. Sutren, A. D. Strosberg, and C. Nahmias Functional Trans-inactivation of Insulin Receptor Kinase by Growth-Inhibitory Angiotensin II AT2 Receptor Mol. Endocrinol., June 1, 2000; 14(6): 795 - 804. [Abstract] [Full Text]
|
|
|
|
|
|
A. Schwarzler, H.-J. Kreienkamp, and D. Richter Interaction of the Somatostatin Receptor Subtype 1 with the Human Homolog of the Shk1 Kinase-binding Protein from Yeast J. Biol. Chem., March 24, 2000; 275(13): 9557 - 9562. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Ghahremani, C. Forget, and P. R. Albert Distinct Roles for Galpha i2 and Gbeta gamma in Signaling to DNA Synthesis and Galpha i3 in Cellular Transformation by Dopamine D2S Receptor Activation in BALB/c 3T3 Cells Mol. Cell. Biol., March 1, 2000; 20(5): 1497 - 1506. [Abstract] [Full Text]
|
|
|
|
|
|
P. CORDELIER, J.-P. ESTÈVE, N. RIVARD, M. MARLETTA, N. VAYSSE, C. SUSINI, and L. BUSCAIL The activation of neuronal NO synthase is mediated by G-protein {beta}{gamma} subunit and the tyrosine phosphatase SHP-2 FASEB J, November 1, 1999; 13(14): 2037 - 2050. [Abstract] [Full Text]
|
|
|
|
|
|
J. Y. A. Lehtonen, L. Daviet, C. Nahmias, M. Horiuchi, and V. J. Dzau Analysis of Functional Domains of Angiotensin II Type 2 Receptor Involved in Apoptosis Mol. Endocrinol., July 1, 1999; 13(7): 1051 - 1060. [Abstract] [Full Text]
|
|
|
|
|
|
L. A. Sellers, W. Feniuk, P. P. A. Humphrey, and H. Lauder Activated G Protein-coupled Receptor Induces Tyrosine Phosphorylation of STAT3 and Agonist-selective Serine Phosphorylation via Sustained Stimulation of Mitogen-activated Protein Kinase. RESULTANT EFFECTS ON CELL PROLIFERATION J. Biol. Chem., June 4, 1999; 274(23): 16423 - 16430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Pages, N. Benali, N. Saint-Laurent, J.-P. Esteve, A. V. Schally, J. Tkaczuk, N. Vaysse, C. Susini, and L. Buscail sst2 Somatostatin Receptor Mediates Cell Cycle Arrest and Induction of p27Kip1. EVIDENCE FOR THE ROLE OF SHP-1 J. Biol. Chem., May 21, 1999; 274(21): 15186 - 15193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Douziech, E. Calvo, Z. Coulombe, G. Muradia, J. Bastien, R. A. Aubin, A. Lajas, and J. Morisset Inhibitory and Stimulatory Effects of Somatostatin on Two Human Pancreatic Cancer Cell Lines: A Primary Role for Tyrosine Phosphatase SHP-1 Endocrinology, February 1, 1999; 140(2): 765 - 777. [Abstract] [Full Text]
|
|
|
|
|
|
T. Florio, H. Yao, K. D. Carey, T. J. Dillon, and P. J. S. Stork Somatostatin Activation of Mitogen-Activated Protein Kinase via Somatostatin Receptor 1 (SSTR1) Mol. Endocrinol., January 1, 1999; 13(1): 24 - 37. [Abstract] [Full Text]
|
|
|
|
|
|
K. Sharma, Y. C. Patel, and C. B. Srikant C-Terminal Region of Human Somatostatin Receptor 5 Is Required for Induction of Rb and G1 Cell Cycle Arrest Mol. Endocrinol., January 1, 1999; 13(1): 82 - 90. [Abstract] [Full Text]
|
|
|
|
|
|
C. Bousquet, N. Delesque, F. Lopez, N. Saint-Laurent, J.-P. Esteve, K. Bedecs, L. Buscail, N. Vaysse, and C. Susini sst2 Somatostatin Receptor Mediates Negative Regulation of Insulin Receptor Signaling through the Tyrosine Phosphatase SHP-1 J. Biol. Chem., March 20, 1998; 273(12): 7099 - 7106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Puente, N. Saint-Laurent, J. Torrisani, C. Furet, A. V. Schally, N. Vaysse, L. Buscail, and C. Susini Transcriptional Activation of Mouse sst2 Somatostatin Receptor Promoter by Transforming Growth Factor-beta . INVOLVEMENT OF Smad4 J. Biol. Chem., April 13, 2001; 276(16): 13461 - 13468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Benali, P. Cordelier, D. Calise, P. Pages, P. Rochaix, A. Nagy, J.-P. Esteve, P. M. Pour, A. V. Schally, N. Vaysse, et al. Inhibition of growth and metastatic progression of pancreatic carcinoma in hamster after somatostatin receptor subtype 2 (sst2) gene expression and administration of cytotoxic somatostatin analog AN-238 PNAS, August 1, 2000; 97(16): 9180 - 9185. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |