The third intracellular loop of human SST5 is crucial for receptor internalization after SS28 stimulation
Erika Peverelli1, Giovanna Mantovani1, Andrea G Lania1, Davide Calebiro1, Andrea1 Doni2, Sara Bondioni1, Paolo Beck-Peccoz1 & Anna Spada1
Somatostatin (SS) is a widely distributed polypeptide that exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5), that display important differences in tissue distribution, coupling to second messengers, affinity for SS and intracellular trafficking. SS analogues currently used in the treatment of acromegaly inhibit hormone secretion and cell proliferation by binding to SST2 and 5. Beta-arrestins have been implicated in regulating SST internalization but the structural domains mediating this effect are largely unknown. The aim of this study was to characterize the intracellular mechanisms responsible for internalization of human SST5 in the rat pituitary cell line GH3. To this purpose we evaluated by fluorescence microscopy SS28-mediated trafficking of receptor fused to DsRed and beta-arrestin2 fused to GFP. To identify the SST5 structural domains involved in these processes, we evaluated progressive C-terminal truncated proteins, SST5 mutants in which serine or threonine residues within the third cytoplasmic domain were mutated (S242A, T247A) and a naturally occurring R240W mutant in the third loop previously found in one acromegalic patient resistant to somatostatin analogues. We tested the ability of these mutants to associate with beta-arrestin2 and to internalize under agonist stimulation. The truncated mutants are comparable to the wild-type receptor with respect to beta-arrestin recruitment and internalization, whereas third cytoplasmic loop mutants show a significantly reduced internalization and arrestin translocation upon SS28 stimulation. Surprisingly, SST5 with both C-terminal truncation and third loop mutation exhibits normal internalization and beta-arrestin recruitment. Our results indicate SST5 third intracellular loop as an important mediator of beta-arrestin/receptor interaction and receptor internalization, while the role of the C-terminal tail would be to sterically prevent beta-arrestin/receptor interaction in basal conditions. Further elucidation of the molecular signals underlying SST5 intracellular trafficking will provide a better understanding of its function during prolonged agonist treatment.