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 K , of 0.46 pM and an apparent V,,, of 0.9 nmol . mg protein-. min-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 70000 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 [1251-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.Protein tyrosine phosphorylation plays a crucial role in cellular regulation of various functions such as proliferation, differentiation and transformation and is controlled by two sets of enzymes; protein tyrosine kinases (PTK) and protein tyrosine phosphatase (PTPase). Whereas the field of PTK has grown explosively over the last 10 years, less is known about PTPase.Recently, a cytosolic PTPase from human placenta (PTPIB) was purified as a 37-kDa soluble protein [l, 21 and its amino acid sequence showed an unexpected similarity to cytoplasmic domains I and I1 of human leukocyte commun antigen CD45 [3], and leukocyte-commun-antigen-related molecule (LAR) [4]. CD45 and LAR have been subsequently shown to have intrinsic PTPase activity in their cytoplasmic domain [5, 61. The two proteins resemble receptor molecules since they have a transmembrane domain, a cytoplasmic domain composed of two repeated PTPase domains and contain different extracellular domains. More recently, a novel recep-

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.

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The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of '"I-[Tyr' ']somatostatin or its analogue '251-[Leu8,~Trp22,Tyr25]somatostatin 28, to pancreatic plasma membranes.The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammoniol-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1 -propanesulfonate) and octyl P-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol.In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent MI = 93 000 which interacted with the two radioligands.In The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars.We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent M I % 85000-90000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.Somatostatin, a tetradecapeptide (SI4) initially isolated from hypothalamus as an inhibitor of growth hormone secretion, has been found to exert inhibitory actions on various cellular processes in the central nervous system, gastrointestinal tract and pancreas [l]. High-affinity binding sites for somatostatin have been detected on plasma membranes or intact cells from various tissues including brain [2, 31, pituitaries [4, 51, adrenal cortex 16, 71 and endocrine pancreas [8]. We have recently demonstrated that pancreatic acinar cells possess specific somatostatin receptors [9, 101 which are involved in the inhibitory effect of the peptide on secretinstimulated cellular cyclic AMP [ll].

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