Such an effect was not observed with catalytically inactive SHP-1 (our unpublished data). an inhibitory G protein-coupled receptor. INTRODUCTION Somatostatin is a widely expressed hormone that exerts pleiotropic biological actions, including neurotransmission, inhibition of hormonal and hydroelectrolytic secretions, and cell proliferation. This neuropeptide acts by AM 0902 interacting with specific AM 0902 receptors that belong AM 0902 to the G protein-coupled seven-transmembrane domain receptor (GPCR) superfamily. Five subtypes of somatostatin receptors have been thus far cloned (sst1C5). They mediate a variety of signal transduction pathways, including inhibition of adenylate cyclase and guanylate cyclase, modulation of ionic conductance channels and protein phosphorylation, activation of mitogen-activating protein kinase and phospholipase C (Meyerhof, 1998 ). Among somatostatin receptors, sst2 has been found to play a critical role in the negative control of cell growth and to act as a tumor suppressor gene for pancreatic cancer (Benali (CHO/sst2 cells) as described previously (gift from G.I. Bell, Howard Hughes Medical Institute, University of Chicago, Chicago, IL; T. Reisine, University of Pennsylvania, School of Medicine, Philadelphia, PA) or with the catalytically inactive SHP-2 (C459S) mutant [CHO/sst2-SHP-2(C/S) cells] (Yamada SDH5 with 1 mM isopropyl–d-thiogalactopyranoside. Expressed fusion proteins were isolated by procedures described previously (Vely test: *p 0.05, **p 0.01. RESULTS SHP-2 Directly Associates with sst2 via Phosphorylated ITIMs To assess whether the endogenously expressed SHP-2 tyrosine phosphatase interacts with sst2, we first analyzed SHP-2 association with sst2 in CHO/sst2 cells after sst2 immunoprecipitation with anti-sst2 antibody. CHO/sst2 cells expressed sst2 as a protein of 95 kDa detected AM 0902 by immunoblotting as reported previously (Lopez GST-SHP-2-SH2 ????ITIM-pY228 0.99 0.01 1.75 0.09 18.6 1.9 ????ITIM-pY312 1.74 0.71 1.68 0.38 14.9 2.2 GST-SHP-1-SH2 ????ITIM-pY228 ND ND ITIM-pY312 ND ND Open in a separate window ND, not detectable. Binding constants were calculated from SPR data over all tested concentrations of fusion proteins as Slc7a7 described in MATERIALS AND METHODS. Mean SE of three experiments. To evaluate the possibility of in vivo involvement of both tyrosines (Y228 and Y312) in sst2 interaction with SHP-2 SH2 domains, we constructed sst2 mutants harboring substitution of Y228 and Y312 with phenylalanine residues (Y228F, Y312F and Y228/312F). We transiently transfected COS-7 cells with the wild-type or mutated sst2. Binding analysis using 125I-[Tyr11]somatostatin 14 as a tracer revealed that sst2 Y312F and Y228/312F mutants displayed similar constant affinity and maximal binding capacity as wild-type sst2, whereas Y228F exhibited a decrease in affinity for somatostatin and an increase in binding capacity compared with wild-type sst2 (Table 2). Binding analysis was also performed in CHO cells transfected with wild-type and sst2 Y228/312F mutant. In these cells also, constant affinities of [125Tyr11]somatostatin 14 and maximal binding capacities were similar for wild-type and mutated sst2 ((nM) sst2 1.71 0.79 0.52 0.09 Y228F 8.72 2.89* 1.33 0.33* Y312F 4.13 1.92 0.9 0.39 Y228/312F 3.43 0.4 0.56 0.16 Open in a separate window AM 0902 Data represent mean SE obtained from three separate experiments performed in duplicate. Because SHP-2 binding to sst2 is thought to be mediated through interaction with phosphorylated Y228 and Y312 residues, we then examined the level of sst2 tyrosine phosphorylation in sst2 immunoprecipitates derived from wild-type or mutated sst2 expressing cells. Wild-type sst2 was highly phosphorylated, whereas the phosphorylation of sst2 Y312F was partially decreased (71 2.5% of wild-type) and the phosphorylation of sst2 Y228F and Y228/312F.