Specific roles of Gi protein family members revealed by dissecting SST5 coupling in human pituitary cells

Abstract

SummaryDespite intensive investigation over the past 20 years, the specific role played by individual Gi protein family members in mediating complex cellular effects is still largely unclear. Therefore, we investigated the role of specific Gi proteins in mediating somatostatin (SS) effects in somatotroph cells. Because our previous data showed that SS receptor type 5 (SST5) carrying a spontaneous R240W mutation in the third intracellular loop had a similar ability to inhibit intracellular cAMP levels to the wild-type protein but failed to mediate inhibition of growth hormone (GH) release and cell proliferation, we used this model to check specific receptor-G-protein coupling by a bioluminescent resonance energy transfer analysis. In HEK293 cells, wild-type SST5 stimulated the activation of Ga i1-3 and Ga oA, B , whereas R240W SST5 maintained the ability to activate Ga i1-3 and Ga oB , but failed to activate the splicing variant Ga oA . To investigate the role of the selective deficit in Ga oA coupling, we co-transfected human adenomatous somatotrophs with SST5 and a pertussis toxin (PTX)-resistant Ga oA (Ga oA(PTX-r) ) protein. In PTX-treated cells, Ga oA(PTX-r) rescued the ability of the selective SST5 analog BIM23206 to inhibit extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation, GH secretion and intracellular cAMP levels. Moreover, we demonstrated that silencing of Ga oA completely abolished SST5-mediated inhibitory effects on GH secretion and ERK1/2 phosphorylation, but not on cAMP levels. In conclusion, by analysing the coupling specificity of human SST5 to individual Ga i and Ga o subunits, we identified a crucial role for Ga oA signalling in human pituitary cells.

Somatostatin system: molecular mechanisms regulating anterior pituitary hormones

“…In somatotroph cells, SRIF regulation of K C currents are mediated by Ga i3 (Chen 1997), while Ca 2C currents are mediated by Ga o2 (Chen 1997, Degtiar et al 1997), b1, b3 (Kleuss et al 1992), and g3 (Kleuss et al 1993. In human GH-secreting tumor cultures, sst5-specific signaling is dependent on Ga o1 (Peverelli et al 2013). SRIF-mediated inhibition of corticotropinreleasing hormone (CRH)-stimulated Ca 2C levels in human corticotropin-secreting pituitary adenomas is Petersenn et al (1999) and van der Hoek et al…”

“…In addition, PTX attenuated SRIFdependent inhibition of GHRH-induced GH in MtT/SGL somatotroph cells (Morishita et al 2003). sst5-specific inhibition of forskolin-stimulated cAMP accumulation was dependent on Ga o1 in human GH-secreting tumor primary cultures (Peverelli et al 2013). In AtT-20 cells, SRIF inhibition of adenylate cyclase and ACTH secretion is Ga i1 -dependent (Tallent & Reisine 1992).…”

“…The role of SRIF-mediated regulation of protein phosphatase activity and growth arrest in non-pituitary cells has been described (Florio 2008 2007). In human GH-secreting tumor cultures, sst5-dependent inhibition of ERK phosphorylation was dependent on Ga o1 (Peverelli et al 2013). Octreotide activated PI3K/Akt and MAPK pathways through sst2 and sst5 in GH-secreting cells, inducing histone methyltransferases associated with menin, resulting in the upregulation of p27 Kip and cell cycle arrest (Horiguchi et al 2009).…”

“…Cortistatin (CST), a ligand with SSTR binding affinity similar to that of SRIF, is expressed in the cerebral cortex and hippocampus, but not in the hypothalamus (Spier & de Lecea 2000); hence, it is not a major endocrine regulator of pituitary signaling and function.regulate GH and ACTH production (Ben-Shlomo et al 2009, 2013. The SRIF ligand and the five SRIF receptor subtypes (sst1-sst5) regulate pituitary function at two levels, via ligand exposure and potentially via selective receptors, independently of the ligand.…”

cAMP in the pituitary: an old messenger for multiple signals

“…coupled to inhibitory G proteins that reduce cAMP formation by inhibiting adenylyl cyclase activity, it has recently been demonstrated that SST5, the most expressed SS receptor subtype in pituitary together with SST2, exerts its antiproliferative effects in somatotrophs by coupling with the inhibitory G protein Go A , independently of cAMP reduction (Peverelli et al 2009b(Peverelli et al , 2013.…”