ECE2015 Eposter Presentations Endocrine tumours (69 abstracts)
Somatostatin exerts its inhibitory effects on hormone secretion and cell proliferation via five receptors subtypes (SST1-SST5). After agonist binding, receptor residues mainly located in the carboxyl terminal (CT) and in the third intracellular loop (IC3) are phosphorylated and β-arrestins are recruited to drive SSTRs internalization.
Aim of the study is to characterize the intracellular mechanisms responsible for SST2 internalization and identify the molecular determinants mediating its intracellular signaling in pancreatic neuroendocrine QGP1 cell line. To this purpose we created SST2 receptor mutants, the first lacking 20 aminoacids in CT portion (del349), the second a IC3 mutant with a point mutation (Ser-245-Ala) in a phosphorylation site. We evaluated SST2 mutants ability to associate with β-arrestins and to internalize after SST2 selective somatostatin analog stimulation (BIM23120). In cells transfected with the SST2 IC3 mutant, no differences in β-arrestins recruitment and receptor internalization were observed after SST2 activation in comparison with cells bearing wild type SST2. Conversely, the truncated SST2 failed to recruit β-arrestins and to internalize after BIM23120 incubation. Then, we analyzed the effect of BIM23120 on cell proliferation, cyclin D1 expression and ERK1/2 phosphorylation in the presence of either IC3 mutant and truncated SST2. As expected, BIM23120 induced a reduction in cell proliferation (−30±7%, P<0.01 vs untreated) and in CD1 expression (−23±8%, P<0.001 vs untreated) in SST2 wild type QGP1 transfected cells, this effect being completely lost in the presence of both SST2 mutants. To further analyze the antiproliferative actions of SST2, we analyzed the effects of SST2 activation on ERK1/2 phosphorylation and we found the both SST2 mutants fail to affect ERK1/2 phosphorylation status after BIM23120 incubation. Taken together these data suggest that: i) CT region is crucial for β-arrestins/SST2 interaction and SST2 internalization; ii) antiproliferative intracellular signaling mediated by SST2 require the integrity of both CT and IC3 domains.
Disclosure: This work was supported by an AIRC (Associazione Italiana Ricerca Cancro - Milan) grant KFR062-2 to A.G.L.