Biomedical subjects

C Susini

Publications and source records attributed to C Susini.

At least 37 records · Page 2Linked to original sources

PubMed1999

Inhibitory roles for SHP-1 and SOCS-3 following pituitary proopiomelanocortin induction by leukemia inhibitory factor.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that stimulates the hypothalamo-pituitary-adrenal (HPA) axis through JAK-STAT activation. We show here that LIF-induced JAK2 and STAT3 tyrosine phosphorylation is transient, disappearing within 20 and 40 minutes, respectively. LIF activates the SH2 domain-containing tyrosine phosphatase, SHP-1, with maximal stimulation observed at 30 minutes. SHP-1 is constitutively associated with JAK2, and LIF induces recruitment of phosphorylated STAT3 to this complex. Overexpression of wild-type or dominant negative forms of SHP-1 shows decreased or increased LIF-induced proopiomelanocortin (POMC) promoter activity, respectively. LIF-induced JAK2 and STAT3 dephosphorylation is delayed until after 60 minutes in cells that overexpress the mutant SHP-1. In addition, SOCS-3, a negative regulator of LIF signaling, binds to JAK2 after 60 minutes of LIF stimulation, after which the complex is degraded by the proteasome. SOCS-3 overexpression blocks LIF-induced JAK2 tyrosine phosphorylation, confirming a role for SOCS-3 in deactivating JAK2 by direct association. Using SOCS-3 fusion proteins, we also define regions of the SOCS-3 protein that are critical for inhibition of LIF-induced POMC promoter activity. Corticotrophic signaling by LIF is thus subject to 2 forms of negative autoregulation: dephosphorylation of JAK2 and STAT3 by the SHP-1 tyrosine phosphatase, and SOCS-3-dependent inactivation of JAK2.

Animals↗

PubMed1998

sst2 somatostatin receptor mediates negative regulation of insulin receptor signaling through the tyrosine phosphatase SHP-1.

We have previously reported in Chinese hamster ovary (CHO) cells expressing sst2 that activation of the sst2 somatostatin receptor inhibits insulin-induced cell proliferation by a mechanism involving stimulation of a tyrosine phosphatase activity. Here we show that the tyrosine phosphatase SHP-1 was associated with the insulin receptor (IR) at the basal level. Activation of IR by insulin resulted in a rapid and transient increase of tyrosine phosphorylation of IR, its substrates IRS-1 and Shc, and also of SHP-1. This was then followed by a rapid dephosphorylation of these molecules, which was related to the insulin-induced increase of SHP-1 association to IR and of SHP-1 activity. On the other hand, addition to insulin of the somatostatin analogue, RC160, resulted in a higher and faster increase of SHP-1 association to IR directly correlated with an inhibition of phosphorylation of IR and its substrates, IRS-1 and Shc. RC160 also induced a higher and more sustained increase in SHP-1 activity. Furthermore, RC160 completely suppressed the effect of insulin on SHP-1 phosphorylation. Finally, in CHO cells coexpressing sst2 and a catalytically inactive mutant SHP-1, insulin as well as RC160 could no longer stimulate SHP-1 activity. Overexpression of the SHP-1 mutant prevented the insulin-induced signaling to be terminated by dephosphorylation of IR, suppressed the inhibitory effect of RC160 on insulin-induced IR phosphorylation, and abolished the cell proliferation modulation by insulin and RC160. Our results suggest that SHP-1 plays a role in negatively modulating insulin signaling by association with IR. Furthermore, somatostatin inhibits the insulin-induced mitogenic signal by accelerating and amplifying the effect of SHP-1 on the termination of the insulin signaling pathway.

Animals↗

PubMed1997

The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signaling.

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. Gialpha3 was specifically immunoprecipitated by anti-sst2 and anti-SHP-1 antibodies, and somatostatin induced a rapid dissociation of Gialpha3 from sst2, suggesting that Gialpha3 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.

Animals↗

PubMed1997

Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sst5.

We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCKA receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCKA and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor.

Alkaloids↗

PubMed1997

Angiotensin II type 2 receptors mediate inhibition of mitogen-activated protein kinase cascade and functional activation of SHP-1 tyrosine phosphatase.

Angiotensin II type 2 (AT2) receptors are involved in the inhibition of cell proliferation as well as in apoptosis and neuronal differentiation, through intracellular signalling pathways that remain poorly defined. The present study examines the effect of AT2-receptor stimulation on growth-factor-induced pathways leading to the activation of mitogen-activated protein (MAP) kinases. In N1E-115 neuroblastoma cells, AT2 receptors inhibit the activity of MAP kinases induced by serum as well as by epidermal growth factor. The inhibitory effect of angiotensin II (Ang II) is rapid and transient, and affects both ERK1 and ERK2 (extracellular signal-related protein kinase) isoforms of the enzyme. AT2-mediated MAP kinase inactivation is not sensitive to pertussis toxin or okadaic acid, but involves a vanadate-sensitive protein tyrosine phosphatase (PTP). Expression of MAP kinase phosphatase-1 (MKP-1) is not significantly modified upon AT2-receptor activation, and insensitivity to actinomycin D also rules out transcriptional induction of other MKPs as a possible mechanism for AT2-mediated inactivation of MAP kinases. In addition, we report here that both in N1E-115 cells and in Chinese hamster ovary cells expressing recombinant human AT2 receptors, Ang II rapidly stimulates the catalytic activity of SHP-1, a soluble PTP that has been implicated in termination of signalling by cytokine and growth-factor receptors. These findings thus demonstrate functional negative cross-talk between heptahelical AT2 receptors and receptor tyrosine kinases, and suggest that SHP-1 tyrosine phosphatase is an early transducer of the AT2 receptor signalling pathway.

Angiotensin II↗

PubMed1997

Identification of a protein-tyrosine phosphatase (SHP1) different from that associated with acid phosphatase in rat prostate.

Using [32P]poly(Glu,Tyr) as substrate, we have identified, for the first time, in the rat prostatic gland a protein-tyrosine phosphatase activity different from that associated with prostatic acid phosphatase. Concanavalin A-Sepharose 4B was used to separate the two protein-tyrosyl phosphatases activities. The activity retained by the lectin had characteristics of the prostatic acid phosphatase. It was sensitive to inhibition by PNPP and the optimum pH shifted towards physiological values when [32P]poly(Glu,Tyr) was used as substrate. However, the major protein-tyrosine phosphatase activity was not retained by the lectin, and corresponded, at least in part, to SHP1 as probed by the presence of the protein, its mRNA and the loss of PTPase activity after immunodepletion of SHP1. This enzyme is localized within the epithelial cells. Thus, the coexistence of two protein-tyrosine phosphatase activities in rat prostate, one associated with the acid phosphatase and the other related to SHP1, makes it necessary to analyze the importance of both activities in vivo and their possible function regarding prostatic cell growth and its regulation.

Acid Phosphatase↗

PubMed1997

sst2 somatostatin receptor expression reverses tumorigenicity of human pancreatic cancer cells.

Among the five cloned somatostatin receptor subtypes (sst1 to sst5), sst2 mediates the antiproliferative effect of somatostatin analogues in vitro. Somatostatin analogues have been shown to inhibit cell growth in vitro and in vivo in pancreatic cancer models that expressed sst2. We recently demonstrated the loss of sst2 gene expression in human pancreatic adenocarcinomas and most of the derived pancreatic cancer cell lines. In the present study, we corrected the sst2 defect in human pancreatic cancer BxPC-3 and Capan-1 cells by stable transfection with human sst2 cDNA. In the absence of exogenous ligand, both BxPC-3 and Capan-1 cells expressing sst2 showed a significant reduction in cell growth. This inhibitory effect was blocked by treatment with antiserum to somatostatin. sst2-expressing cells produced somatostatin-like immunoreactivity that mainly corresponded to somatostatin 14, indicating the induction of a negative autocrine loop. In other respects, sst2 expression in Capan-1 cells induced a significant reduction of clonogenicity in soft agar. Moreover, a significantly reduced (Capan-1 cells) or suppressed (BxPC-3 cells) tumor growth in athymic nude mice was observed. The reversal of tumorigenicity induced by the restoration of sst2 expression suggests that the loss of sst2 contributes to the malignancy of human pancreatic cancers.

Animals↗

PubMed1997

Expression of somatostatin receptor SST4 in human placenta and absence of octreotide effect on human placental growth hormone concentration during pregnancy.

In pregnancy, the human placenta GH acts as a growth-promoting hormone and appears to be the main stimulator of insulin-like growth factor I (IGF-I) secretion. In a woman with a TSH-secreting macroadenoma, successful treatment with the somatostatin analog octreotide was conducted during the first month and the second half of pregnancy without side-effects on placental and fetal development. As observed in normal pregnancy, both serum placental GH and IGF-I levels increased throughout pregnancy and dropped sharply after delivery. In placental membranes from both treated and healthy untreated patients, we demonstrated the presence of high affinity binding sites for somatostatin-14 (Kd, 4.6 and 5.3 nmol/L; binding capacity, 1.53 and 1.35 pmol/mg protein, respectively). These receptors displayed low affinity for octreotide (IC50, 1.2-2 mumol/L), suggesting the presence of SST1 and/or SST4 receptors. We found that messenger ribonucleic acids of these two subtypes were expressed in both human placental tissue and purified human cytotrophoblast cells. Finally, the SST1-selective analog, des-AA1,2,5[D-Trp8,IAmp9]S-14 had low affinity for placental somatostatin receptors. These results argue in favor of the presence of the SST4 subtype in human placenta. At the doses administered, octreotide did not bind to placental somatostatin receptors. Our results may explain the absence of changes in both human placental GH and IGF-I concentrations that we observed during octreotide treatment.

Adenoma↗

PubMed1996

Induction of a negative autocrine loop by expression of sst2 somatostatin receptor in NIH 3T3 cells.

The somatostatin receptor subtype sst2 mediates both activation of a tyrosine phosphatase activity and inhibition of cell proliferation induced by somatostatin analogues. In the absence of exogenous ligand, expression of sst2 in NIH 3T3 cells resulted in inhibition of cell growth. Polymerase chain reaction coupled to reverse transcription demonstrated that expression of sst2 in NIH 3T3 cells stimulated the expression of preprosomatostatin mRNA accompanied by a production of immunoreactive somatostatin-like peptide which corresponded predominantly to somatostatin 14. Moreover anti-somatostatin antibodies suppressed sst2-promoted inhibition of cell proliferation. Inhibition of cell proliferation associated with increased secretion of somatostatin-like immunoreactivity was also observed after expression of sst2 in human pancreatic tumor cells BxPC3 devoid of endogenous receptors. In addition, expression of sst2 in NIH 3T3 cells was associated with constitutive activation of tyrosine phosphatase PTP1C that resulted from enhanced expression of the protein. Blocking of PTP1C tyrosine phosphatase activity with orthovanadate or that of PTP1C protein with antisense PTP1C oligonucleotides decreased the sst2-induced inhibition of cell proliferation. These results, taken together, show that expression of sst2 in NIH 3T3 cells generated a negative autocrine loop by stimulating sst2 ligand production and amplifying PTP1C sst2-transducer. Sst2/ligand may function as a determinant factor involved in the negative growth control of cells.

3T3 Cells↗

PubMed1996

Loss of sst2 somatostatin receptor gene expression in human pancreatic and colorectal cancer.

Five somatostatin receptor subtypes (sst1 to sst5) have been cloned. We demonstrated previously that sst2 and sst5 mediate the antiproliferative effect of the somatostatin analogues octreotide and vapreotide. Using reverse transcription-PCR, we investigated gene expression of the five receptors in 47 human normal and cancerous tissues or cell lines from pancreatic and colorectal origin. mRNAs of somatostatin receptor subtypes were detected in 98% of samples, with more than two mRNA subtypes being expressed in 55% of cases. sst1, sst4, and sst5 were heterogeneously expressed in both normal and cancerous tissues; sst3 was rarely or not expressed. sst2 was present in normal pancreatic tissues but was absent in exocrine pancreatic carcinomas and their metastases. sst2 mRNAs were detected in normal colon, sporadic polyadenomas, and 50% of Dukes' stage B and 20% of Dukes' stage C carcinomas but were undetectable in Dukes' stage D carcinomas, hepatic metastases, and adenomas from familial adenomatous polyposis. The loss of sst2 expression could represent a growth advantage in these tumors and provide an explanation for the lack of therapeutic effect of somatostatin analogues in such adenocarcinomas. A subtyping of somatostatin receptors should be carried out before considering a somatostatin analogue treatment in patients with colorectal or pancreatic cancer.

Cell Line↗

PubMed1996

Molecular mechanisms of antiproliferative effect of somatostatin: involvement of a tyrosine phosphatase.

A protein of 66 kd immunoreactive to anti-tyrosine phosphatase (PTP1C) antibodies coeluted with, and so may be associated with, somatostatin receptors (ssts) from rat pancreatic membranes. Also, anti-PTP1C antibodies immunoprecipitated functional ssts from pancreatic membranes, suggesting a PTP1C protein can associate with ssts at the membrane level. Somatostatin analog RC 160 had good affinity for sst2,3 and sst5 (IC50 = 0.2, 0.1, and 21 nmol/L) and low affinity for sst1 and sst4 (IC50 = 200 and 620 nmol/L), and induced rapid dose-dependent stimulation of PTP activity (maximal at 1 nmol/L and half maximal at 5 pmol/L) in NIH3T3 and CHO cells expressing sst2, with similar results for sst1, but no stimulation with sst3,4 or sst5. Treatment of cells expressing sst2 with RC 160 for 24 hours inhibited serum- or growth factor-induced cell proliferation dose-dependently (maximal at 1 nmol/L, half maximal at 6 to 53 pmol/L RC 160). In cells expressing sst1, weak inhibition of fibroblast growth factor 2-induced NIH3T3 cell proliferation was provoked by somatostatin analogs (> 10 nmol/L). The good correlation between inhibition of somatostatin binding, stimulation of PTP activity, and inhibition of cell proliferation implicates a PTP in growth inhibition mediated by sst2 and sst1.

3T3 Cells↗

PubMed1995

Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and SSTR5 through different mechanisms.

Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO cells expressing the five somatostatin receptor subtypes SSTR1 to -5. Binding experiments were performed on crude membranes by using [125I-labeled Tyr11] somatostatin-14; RC-160 exhibited moderate-to-high affinities for SSTR2, -3, and -5 (IC50, 0.17, 0.1 and 21 nM, respectively) and low affinity for SSTR1 and -4 (IC50, 200 and 620 nM, respectively). Cell proliferation was induced in CHO cells by 10% (vol/vol) fetal calf serum, 1 microM insulin, or 0.1 microM cholecystokinin (CCK)-8; RC-160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (EC50, 53 and 150 pM, respectively) but had no effect on growth of cells expressing SSTR1, -3, or -4. In SSTR2-expressing cells, orthovanadate suppressed the growth inhibitory effect of RC-160. This analogue inhibited insulin-induced proliferation and rapidly stimulated the activity of a tyrosine phosphatase in only this cellular clone. This latter effect was observed at doses of RC-160 (EC50, 4.6 pM) similar to those required to inhibit growth (EC50, 53 pM) and binding to the receptor (IC50, 170 pM), implicating tyrosine phosphatase as a transducer of the growth inhibition signal in SSTR2-expressing cells. In SSTR5-expressing cells, the phosphatase pathway was not involved in the inhibitory effect of RC-160 on cell growth, since this action was not influenced by tyrosine and serine/threonine phosphatase inhibitors. In addition, in SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium mobilization at doses (EC50, 0.35 nM) similar to those necessary to inhibit somatostatin-14 binding (IC50, 21 nM) and CCK-induced cell proliferation (EC50, 1.1 nM). This suggests that the inositol phospholipid/calcium pathway could be involved in the antiproliferative effect of RC-160 mediated by SSTR5 in these cells. RC-160 had no effect on the basal or carbachol-stimulated calcium concentration in cells expressing SSTR1 to -4. Thus, we conclude that SSTR2 and SSTR5 bind RC-160 with high affinity and mediate the RC-160-induced inhibition of cell growth by distinct mechanisms.

Amino Acid Sequence↗

PubMed1995

A tyrosine phosphatase is associated with the somatostatin receptor.

Regulation of tyrosine phosphorylation is thought to be an essential step in signal transduction mechanisms that mediate cellular responses. In pancreatic tumour cells we demonstrated that somatostatin analogues inhibited cell proliferation and stimulated a membrane protein tyrosine phosphatase (PTP) activity at concentrations at which they bind to the somatostatin receptor. To elucidate the role of PTP in the signal transduction pathway activated by somatostatin receptors we first studied the interaction of PTP with the somatostatin receptor at the membrane. We purified somatostatin receptors by immunoaffinity from pancreatic membranes that strongly expressed the type 2 somatostatin receptor sstr2. We identified the receptor as an 87 kDa protein. We demonstrated that a PTP activity co-purified with somatostatin receptors. The PTP was identified as a 66 kDa protein immunoreactive to antibodies against SHPTP1. These antibodies immunoprecipitated somatostatin receptors either occupied or unoccupied by ligand indicating that SHPTP1 is associated with somatostatin receptors. We then expressed sstr2A in monkey kidney COS-7 cells and mouse NIH/3T3 fibroblasts and demonstrated that somatostatin analogues (RC 160, octreotide and BIM 23014) which exhibited high affinity for sstr2 stimulated a PTP activity and inhibited cell proliferation in proportion to their affinities for sstr2. Under the same conditions these analogues have no effect on the growth of cells expressing sstr1. All these results suggest that a PTP related to SHPTP1 is associated with somatostatin receptors and may be involved in the negative growth signal promoted by sstr2.

3T3 Cells↗

PubMed1995

Somatostatin binding capacity, guanylate cyclase and tyrosine phosphatase activities during pancreatic proliferation in the rat induced by gastrectomy.

Gastrectomy increased pancreatic growth and this effect was associated with an increase in the number of somatostatin-14 (SS) receptors (146% of control) without altering their affinity. SS increased guanylate cyclase activity twofold in pancreatic acinar membranes from gastrectomized rats. The gastrectomy decreased pancreatic SS-like immunoreactivity (SS-LI) content (55% of control levels) and tyrosine phosphatase activity (74% of control levels). Administration of proglumide (20 mg/kg, IP), a gastrin/cholecystokinin (CCK) receptor antagonist, suppressed the inhibitory effect of gastrectomy on basal tyrosine phosphatase activity and SS-LI content, which returned to control levels. Furthermore, proglumide suppressed the increase of the number of SS receptors and of SS-stimulated guanylate cyclase activity induced by gastrectomy. All this suggests that pancreatic acinar cell growth is associated with upregulation of SS receptors, which could represent a mechanism promoted by the cell to negatively regulate the mitogenic activity of pancreatic growth factors such as CCK. In addition, the results also suggest that the negative regulation of tyrosine phosphatase activity may be important in the events involved in the pancreatic hyperplasia observed after gastrectomy.

Animals↗

PubMed1995

Somatostatin receptor-effector system in rat pancreatic acinar membranes after subtotal enterectomy.

In the present study we found that exocrine pancreatic hyperplasia observed after proximal small bowel resection is accompanied by an increase in pancreatic somatostatin (SS) content at 1 mo and an increase in the number of SS receptors at 2 wk and 1 mo after intestinal surgery. At 6 mo after small bowel resection SS content and SS receptors had returned to control values. However, the original increase in SS receptor number was accompanied by a decrease in the ability of SS to inhibit forskolin-stimulated adenylyl cyclase (AC) activity. In addition, the ability of 5'-guanylylimidodiphosphate (a nonhydrolyzable GTP analogue) to inhibit SS receptor binding was decreased in pancreatic acinar membranes from enterectomized rats at 2 wk and 1 mo after jejunoileal resection. These data suggest that there is an abnormality in the integrity of SS receptor binding site-G protein interactions and would explain the decreased inactivation of AC by SS at 2 wk and 1 mo after proximal small bowel resection.

Adenylyl Cyclase Inhibitors↗

PubMed1994

Co-purification of a protein tyrosine phosphatase with activated somatostatin receptors from rat pancreatic acinar membranes.

We have previously shown that somatostatin promotes the stimulation of a membrane tyrosine phosphatase activity in pancreatic cells. To gain insight into the mechanism of somatostatin action, we purified somatostatin-receptor complexes from somatostatin 28-prelabelled rat pancreatic plasma membranes by immunoaffinity chromatography using immobilized antibodies raised against the N-terminal part of somatostatin 28, somatostatin 28 (1-14), which is not involved in receptor-binding-site recognition. After SDS gel electrophoresis a band with a molecular mass of 87 kDa was identified in the affinity-purified material as the somatostatin receptor. The 87 kDa protein was not observed when the membrane receptors were solubilized in a free unoccupied or somatostatin 14-occupied form, or when nonimmune serum replaced the anti-[somatostatin 28 (1-14)] anti-serum. Somatostatin 14 inhibited the appearance of the 87 kDa protein in the same range of concentrations that inhibit radioligand binding on pancreatic membranes. After somatostatin 28 treatment of membranes, purified somatostatin receptor preparations exhibited an elevated tyrosine phosphatase activity that dephosphorylated phosphorylated epidermal growth factor receptor and poly(Glu,Tyr). This activity was related to the presence of somatostatin receptors in purified material. It was increased by dithiothreitol and inhibited by orthovanadate. In purified material containing somatostatin receptors, anti-[Src homology 2 domains (SH2)]-containing tyrosine phosphatase SHPTP1 polyclonal antibodies identified a protein of 66 kDa which was not detected in the absence of somatostatin receptor. Furthermore, the anti-SHPTP1 antibodies immunoprecipitated specific somatostatin receptors from somatostatin-prelabelled pancreatic membranes and from untreated membranes. These results indicate that a 66 kDa tyrosine phosphatase related to SHPTP1 co-purifies with the pancreatic somatostatin receptors, and suggest that this protein is associated with somatostatin receptors at the membrane level.

Amino Acid Sequence↗

PubMed1994

Up-regulation of somatostatin receptors by epidermal growth factor and gastrin in pancreatic cancer cells.

Interactions between growth factor receptor systems may be important in the regulation of cell growth. The proliferation of pancreatic tumor AR42J cells has been shown to be stimulated by Epidermal growth factor (EGF) and gastrin and inhibited by somatostatin. To analyze the interaction between these different peptides, we explored the influence of EGF and gastrin on the somatostatin receptors. Treatment of AR42J cells with 10 nM EGF or gastrin for 24 hr increased specific binding of [125I] Tyr3SMS to 131 and 147% of that in control cells, respectively. The effect of peptides on [125I]Tyr3SMS binding was time- and dose-dependent, with half-maximal effect at 0.2 +/- 0.03 nM EGF and 0.3 +/- 0.15 nM gastrin. Scatchard plots revealed an increase in somatostatin receptor number of 27 and 80% after 48 hr of treatment with EGF and gastrin, respectively, without any change in receptor affinity. The increase in somatostatin receptor density was accompanied by the enhancement of biological responses to somatostatin. In cells pretreated with EGF or gastrin, the potency of somatostatin for inhibiting vasoactive intestinal peptide-stimulated cAMP content was increased 2-fold as that of somatostatin analog, SMS, for inhibiting cell proliferation. Furthermore, the efficiency of SMS as antiproliferative agent was greatly increased. Vasoactive intestinal peptide or forskolin did not modify [125I]Tyr3SMS binding of control or treated cells. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not affect [125I]Tyr3SMS binding. On the other hand, cycloheximide completely blocked the increase in [125I]Tyr3SMS binding induced by EGF and gastrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1992

Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells.

A phosphoryl protein tyrosine phosphatase (PTPase) activity has been characterized in rat pancreatic acinar membranes using 32P-labeled poly(Glu,Tyr) as substrate. Acinar membranes exhibited a high affinity for the substrate, with an apparent Km of 0.46 microM and an apparent Vmax of 0.9 nmol.mg protein-1.min-1. Acinar membrane PTPase activity displayed specific characteristics of other PTPases; it was inhibited by the inhibitors Zn2+, orthovanadate and by the divalent cations Mn2+ and Mg2+, and was stimulated by the reducing-agent dithiothreitol. It was also inhibited by soybean trypsin inhibitor and stimulated by trypsin. Gel permeation of pancreatic acinar membranes gave a single peak of enzyme activity with an apparent molecular mass of 70 000 Da. Further purification by HPLC on DEAE revealed two peaks of PTPase activity at 120 mM and 180 mM NaCl. These two peaks reacted in a Western-blot procedure with anti-(peptide) serum directed towards conserved domain of PTPase as a common 67-kDa form associated with lower-molecular-mass proteolytic fragments (31-56 kDa). Incubation of pancreatic acini with somatostatin analogues, SMS 201-995 or BIM 23014, resulted in a stimulation of membrane PTPase activity. The stimulation was rapid and transient, with a maximal level reached within 15 min of addition. The two analogs stimulated PTPase activity in a dose-dependent manner with half-maximal activation occurring at 7 pM and 37 pM and maximal activation at 0.1 nM and 0.1-1 nM for SMS 201-995 and BIM 23014, respectively. The stimulated-membrane PTPase activity also eluted at an apparent molecular mass of 70 kDa in gel-permeation chromatography. The two analogs inhibited the binding of [125I-Tyr3]SMS 201-995 to pancreatic acinar membranes with similar relative potencies to that observed on stimulation of PTPase activity. We conclude that pancreatic acinar membranes possess a low-molecular-mass PTPase which is stimulated by somatostatin analogs at concentrations involving activation of membrane somatostatin receptors.

Amino Acid Sequence↗