Development and use of a receptor antibody to characterize the interaction between somatostatin receptor subtype 1 and G proteins

YZ Gu, PJ Brown, DS Loose-Mitchell, PJ Stork and A Schonbrunn

Department of Pharmacology, University of Texas Medical School, Houston 77225, USA.

The signal transduction pathways regulated by somatostatin receptor subtype 1 (sst1) have been difficult to define because of the variability observed when this receptor is expressed in different cell types by transfection and because pharmacological approaches are inadequate to distinguish sst1 receptor subtypes. To study the sst1 receptor in its endogenous environment, we developed a polyclonal antibody to a 15-amino acid peptide corresponding to a unique sequence in the receptor carboxyl terminus. The peptide antibody routinely precipitated 70% of the soluble [125I-Tyr11]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. Photoaffinity-labeled sst1 receptor was also specially immunoprecipitated and migrated as a broad 60-kDa band on sodium dodecyl sulfate polyacrylamide gels. The observation that sst receptors from GH4C1 pituitary cells were immunoprecipitated by the antibody and that receptors from AR4-2J pancreatic acinar cells were not indicated that only the former expressed sst1 receptor protein. Because reverse transcription- polymerase chain reaction showed that GH4C1 cells contained both sst1 and sst2 receptor mRNA, immunoprecipitation permitted the sst1 receptor to be separated from the other receptors present. Two observations showed that G proteins were coprecipitated with sst1 receptors from GH4C1 cells. First, pertussis toxin pretreatment markedly decreased hormone binding in the immunoprecipitate. Second, the addition of 20 microM guanosine-5'-(gamma-thio)triphosphate to the immunoprecipitated [125I-Tyr11]somatostatin/receptor complex stimulated the rate of dissociation of bound ligand by 10-fold. Interestingly, however, the dissociation rate of approximately 30% of the ligand/receptor complex was unaffected by guanosine-5'-(gamma-thio)triphosphate. In summary, we have developed an sst1 receptor-specific antibody and used it to show that sst1 receptors endogenously expressed in GH4C1 pituitary cells couple primarily to pertussis toxin-sensitive G proteins. Furthermore, these receptors exist in two distinct high affinity states distinguished by their GTP sensitivity.

Volume 48, Issue 6, pp. 1004-1014, 12/01/1995
Copyright © 1995 by American Society for Pharmacology and Experimental Therapeutics

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