DEVELOPMENT AND USE OF A RECEPTOR ANTIBODY TO CHARACTERIZE THE INTERACTION BETWEEN SOMATOSTATIN RECEPTOR SUBTYPE-1 AND G-PROTEINS

The signal transduction pathways regulated by somatostatin receptor su btype 1 (sst1) have been difficult to define because of the variabilit y observed when this receptor is expressed in different cell types by transfection and because pharmacological approaches are inadequate to distinguish sst1 receptor function in tissues or cells that express mu ltiple sst receptor subtypes. To study the sst1 receptor in its endoge nous environment, we developed a polyclonal antibody to a 15-amino aci d peptide corresponding to a unique sequence in the receptor carboxyl terminus. The peptide antibody routinely precipitated 70% of the solub le [I-125-Tyr(11)]somatostatin/receptor complex prepared from Chinese hamster ovary-K1 cells expressing the sst1 receptor but precipitated < 1% of the complex from cells expressing other sst receptor subtypes. P hotoaffinity-labeled sst1 receptor was also specifically immunoprecipi tated and migrated as a broad 60-kDa band on sodium dodecyl sulfate po lyacrylamide gels. The observation that sst receptors from GH(4)C(1) p ituitary cells were immunoprecipitated by the antibody and that recept ors from AR4-2J pancreatic acinar cells were not indicated that only t he former expressed sst1 receptor protein. Because reverse transcripti on-polymerase chain reaction showed that GH(4)C(1) cells contained bot h sst1 and sst2 receptor mRNA, immunoprecipitation permitted the sst1 receptor to be separated from the other receptors present. Two observa tions showed that G proteins were coprecipitated with sst1 receptors f rom GH(4)C(1) cells. First, pertussis toxin pretreatment markedly decr eased hormone binding in the immunoprecipitate. Second, the addition o f 20 mu M guanosine-5'-(gamma-thio)triphosphate to the immunoprecipita ted [I-125-Tyr(11)]somatostatin/receptor complex stimulated the rate o f dissociation of bound ligand by 10-fold. Interestingly, however, the dissociation rate of similar to 30% of the ligand/receptor complex wa s unaffected by guanosine-5'-(gamma-thio)triphosphate. In summary, we have developed an sst1 receptor-specific antibody and used it to show that sstl receptors endogenously expressed in GH(4)C(1) pituitary cell s couple primarily to pertussis toxin-sensitive G proteins. Furthermor e, these receptors exist in two distinct high affinity states distingu ished by their GTP sensitivity.