ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2008) 16 P658

G protein coupling and adenylyl cyclase inhibition are mediated by the DRY motif in the second intracellular domain of the SST5 receptor

Erika Peverelli, Andrea Lania, Giovanna Mantovani, Sara Bondioni, Paolo Beck-Peccoz & Anna Spada

Endocrine Unit, Department of Medical Sciences, University of Milan, Fondazione Policlinico IRCCS, Milan, Italy.

Somatostatin exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1–SST5) linked to effector systems via G proteins. The receptor structural domains mediating these effects have not been identified. Since SS is the hypothalamic peptide that physiologically inhibits GH secretion, aim of this study was to investigate the molecular determinants mediating the interaction of the human SST5 with G proteins and subsequent signal transduction after stimulation with BIM23206, the SST agonist with highest affinity for SST5, in the rat pituitary cell line GH3. The most critical regions important for signal transduction in different GPCRs are the second and the third intracellular loops (i2 and i3). To investigate the role of these regions in SST5 receptor, we focused on the highly conserved DRY motif (Asp-Arg-Tyr) in the i2 loop and the BBXXB domain in the i3 loop. We introduced mutations into SST5 replacing D136 and R137 residues in the DRY motif with Ala, and we used a naturally occurring mutant (R240W) previously found in one acromegalic patient resistant to somatostatin analogues in which the BBXXB domain is altered. By analyzing adenylyl cyclase inhibition mediated by BIM23206 in cells transfected with wt or mutated SST5, we have found that residues D136 and R137 are important for adenylyl cyclase inhibition (maximum 15% inhibition versus 53.4% of wt), whereas substitution of basic residue R240 resulted in mutant receptor that normally activates effector (maximum 50.5% inhibition), but shows a persistent inhibition of cAMP after agonist pretreatment. Our data suggest that an intact BBXXB domain is not crucial in mediating G protein activation but it is involved in desensitization processes, whereas the DRY motif within the i2 loop is crucial in G protein interaction and adenylyl cyclase inhibition, thus suggesting a specific role of i2 loop in SST5 function.

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