S49 CELLS ENDOGENOUSLY EXPRESS SUBTYPE-2 SOMATOSTATIN RECEPTORS WHICHCOUPLE TO INCREASE PROTEIN-TYROSINE-PHOSPHATASE ACTIVITY IN MEMBRANESAND DOWN-REGULATE RAF-1 ACTIVITY IN-SITU

S49 cells expressed type 2 somatostatin receptors (sstr(2)) by immunob lotting. Analysis by reverse transcription and polymerase chain reacti on (RT-PCR) methodologies, ies showed that S49 cells express predomina ntly sstr(2A) and sstr(2B) mRNAs; other subtypes were either not detec ted, in the case of sstr(1), sstr(3), sstr(4), or variably detected, i n the case of sstr(5). No mutations were present in S49 cells at codon 12, 13, or 61 of the N-, K-, or H-ras genes. Nevertheless, randomly g rowing S49 cells contained Raf-1 activity by specific immune complex k inase assays. Treatment of S49 cells with somatostatin transiently ina ctivated the basal activity of Raf-1, but not that of B-Raf. Addition of somatostatin plus guanyl-5'-yl imidodiphosphate (GMPPNP) to S49 mem branes stimulated PTPase activity. The concentration dependence for st imulation of PTPase activity correlated with high affinity binding of [I-125-Tyr(13)]somatostatin-14. Both the effect of somatostatin to sti mulate PTPase activity and to inactivate Raf-1 were abrogated by PTx. PTPase activity stimulated by somatostatin plus GMPPNP was recovered i n a peak of high apparent M-r, (670,000) after solubilisation with Tri ton X-100 and Superose 6 chromatography. Furthermore, addition of acti vated, brain G(alpha i/o), subunits to fractions from control membrane s stimulated PTPase activity in the high M, peak. Thus, S49 membranes contain a G-protein regulated PTPase (PTPase-G), and PTPase-G in these cells may reside in a high molecular weight complex. (C) 1997 Elsevie r Science Inc.