Somatostatin (SST) exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5) linked to multiple cellular effectors. The receptor structural domains involved in these effects have been only partially elucidated. The aim of the study was to investigate the molecular determinants mediating the interaction of the human SST5 with intracellular signaling in the pituitary cell line GH3, focusing on the BBXXB domain in the third intracellular loop and the DRY motif in the second intracellular loop. We analyzed the effects of the SST5 agonist BIM23206 on cAMP accumulation, intracellular calcium, GH secretion, cell proliferation, and ERK1/2 phosphorylation in cells expressing either wild-type SST5 or mutant receptors, in particular the naturally occurring mutant R240W in the BBXXB domain and the D136A and R137A mutants in the DRY motif. We found that residues D136 and R137 were critical for SST5 signaling because their substitutions abolished all the intracellular responses. Conversely, third intracellular loop mutations resulted in receptor that inhibited intracellular cAMP levels similar to the wild-type (50 +/- 9 vs. 53 +/- 12% inhibition) but failed to mediate the other responses elicited by wild-type SST5, i.e. reduction of intracellular calcium levels as well as inhibition of ERK1/2. These events resulted in an absent inhibition of GH release and an impaired reduction of cell proliferation (38 +/- 7 vs. 76 +/- 6% inhibition in wild type, P < 0.05). These data indicate that different regions of SST5 are required for the activation of different signaling pathways.