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Coupling Specificity between Somatostatin Receptor sst2A and G Proteins: Isolation of the Receptor-G Protein Complex with a Receptor Antibody

Department of Integrative Biology, Pharmacology, and Physiology University of Texas Medical School Houston, Texas 77225

Somatostatin initiates its actions via a family of seven-transmembrane domain receptors. Of the five somatostatin receptor genes cloned, sst2 exists as two splice variants with the sst2A isoform being predominantly expressed. This receptor is widely distributed in endocrine, exocrine, and neuronal cells, as well as in hormonally responsive tumors, and leads to inhibition of secretion, electrical excitability, and cell proliferation. To investigate the specificity of signal transduction by the sst2A receptor, we developed antibodies against two overlapping peptides located within the C terminus of the receptor protein: peptide 2C_SG, containing amino acids 334–348, and peptide 2C_ER, containing amino acids 339–359. Although antibodies to both peptides bound the inducing antigen with high affinity, only the antibodies against peptide 2C_ER precipitated the receptor. The best antibody, R2–88, precipitated about 80% of the sst2A receptor-ligand complex solubilized from transfected CHO cells and was specific for the sst2A receptor isotype. Addition of GTPS (10 µM) to the immunoprecipitated ligand-sst2A receptor complex markedly accelerated ligand dissociation, indicating that G proteins remained functionally associated with the receptor in the immuno-precipitate. Analysis of the G proteins coprecipitated with the sst2A receptor by immunoblotting with G protein antibodies showed that both G and G_ß subunits were bound to the hormone-receptor complex. Immunoprecipitation of the receptor was not affected by the presence of bound ligand. However, G protein subunits were coprecipitated only with the hormone-occupied receptor. Thus, the unoccupied receptor has low affinity for G proteins, and hormone binding stabilizes the receptor-G protein complex. Use of subtype-specific G protein antisera further showed that G_i1, G_i2, and G_i3 were complexed with the sst2A receptor whereas G_o, G_z, and G_q were not. Together, these studies demonstrate that the sst2A receptor interacts selectively with G_i proteins in a hormone-dependent manner. The finding that this receptor couples to all three G_i subunits may help explain how somatostatin can regulate multiple signaling pathways.

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