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

Abstract

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.

The Tyrosine Phosphatase SHP-1 Associates with the sst2 Somatostatin Receptor and Is an Essential Component of sst2-mediated Inhibitory Growth Signaling

“…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.…”

“…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.…”

A somatostatin analogue inhibits MAP kinase activation and cell proliferation in human neuroblastoma and in human small cell lung carcinoma cell lines

“…Our data, showing that BIM inhibits MAP kinase activity stimulated by both IGF-1 and carbachol, indicate that BIM acts at a very early post-receptor level and at some step common to the signalling cascade initiated by both tyrosine kinase and Gprotein coupled receptors leading to MAP kinase activation. Having excluded an involvement of CAMP, Ca2+ and pH, a tyrosine phosphatase activity, which has recently been found to be associated with SST receptors [23,24], might be suggested as responsible for the analogue's action.…”

Somatostatin Interferes with Thyrotropin-induced G1-S Transition Mediated by cAMP-dependent Protein Kinase and Phosphatidylinositol 3-Kinase

“…This suggests a mechanism for somatostatin action upstream of PKA and PI 3-kinase and indicates the inhibitory potential of somatostatin, which is able to counteract PKA-dependent and -independent proliferative effects. FRTL-5 thyroid cells express the somatostatin receptor SSTR2 (8), and this receptor can be coupled to several downstream effectors such as adenylyl cyclase or protein-tyrosine phosphatases (55,56). Moreover, TSH increases intracellular cAMP levels (28,43).…”