Somatostatin receptor SST5 is an inhibitory
G protein-coupled receptor that exerts a strong
cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of
mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells),
somatostatin inhibited cell proliferation induced by
nitric oxide donors and overexpression of the
neuronal nitric-oxide synthase (nNOS)
protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the
somatostatin analog
RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following
RC-160 treatment.
RC-160 induced SST5-p60(src)
kinase complex formation and subsequent p60(
src) kinase activation. Coexpression of an inactive p60(
src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(
src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following
RC-160 treatment. Using
recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the
RC-160 anti-proliferative effect on pancreatic endocrine
tumor-derived cells. We therefore identified nNOS as a new p60(
src) kinase substrate essential for SST5-mediated anti-proliferative action.