COUPLING SPECIFICITY BETWEEN SOMATOSTATIN RECEPTOR SST2A AND G-PROTEINS - ISOLATION OF THE RECEPTOR G-PROTEIN COMPLEX WITH A RECEPTOR ANTIBODY

Somatostatin initiates its actions via a family of seven-transmembrane domain receptors. Of the five somatostatin receptor genes cloned, sst 2 exists as two splice variants with the sst2A isoform being predomina ntly expressed. This receptor is widely distributed in endocrine, exoc rine, and neuronal cells, as well as in hormonally responsive tumors, and leads to inhibition of secretion, electrical excitability, and cel l proliferation. To investigate the specificity of signal transduction by the sst2A receptor, we developed antibodies against two overlappin g peptides located within the C terminus of the receptor protein: pept ide 2C(SG), containing amino acids 334-348, and peptide 2C(ER), contai ning amino acids 339-359. Although antibodies to both peptides bound t he inducing antigen with high affinity, only the antibodies against pe ptide 2C(ER) precipitated the receptor. The best antibody, R2-88, prec ipitated about 80% of the sst2A receptor-ligand complex solubilized fr om transfected CHO cells and was specific for the sst2A receptor isoty pe. Addition of GTP gamma S (10 mu M) to the immunoprecipitated ligand -sst2A receptor complex markedly accelerated ligand dissociation, indi cating that G proteins remained functionally associated with the recep tor in the immunoprecipitate. Analysis of the G proteins coprecipitate d with the sst2A receptor by immunoblotting with G protein antibodies showed that both G(alpha) and G(beta) subunits were bound to the hormo ne-receptor complex. Immunoprecipitation of the receptor was not affec ted by the presence of bound ligand. However, G protein subunits were coprecipitated only with the hormone-occupied receptor. Thus, the unoc cupied receptor has low affinity for G proteins, and hormone binding s tabilizes the receptor-G protein complex. Use of subtype-specific G pr otein antisera further showed that G alpha(i1), G alpha(i2), and G alp ha(i3) were complexed with the sst2A receptor whereas G alpha(o,) G al pha(z), and G alpha(q) were not. Together, these studies demonstrate t hat the sst2A receptor interacts selectively with G alpha(i) proteins in a hormone-dependent manner. The finding that this receptor couples to all three G alpha(i) subunits may help explain how somatostatin can regulate multiple signaling pathways.