Heterotrimeric G-proteins are important molecular switches that transduce extracellular ligand-binding at G-protein-coupled receptors (GPCRs) to intracellular signals. G-protein alpha-subunits (Gα) have two domains, a helical and GTPase domain, which provide structural stability and mediate GTPase activity, respectively. Gain-of-function Gα mutations cause endocrine conditions including McCune-Albright Syndrome, due to Gαs mutations, and autosomal dominant hypocalcemia type-2 (ADH2) due to Gα11 mutations. ADH2 arises as Gα11 mediates signalling by calcium-sensing receptor, a GPCR that regulates calcium homeostasis. Gα11 signalling activates phospholipase-C, inducing intracellular calcium (Ca2+i) release and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway activation. Studies of ADH2 mutations can provide important structure-function insights into G-protein roles in endocrinopathies. We therefore investigated two novel heterozygous ADH2-causing Gα11 missense mutations, Gly66Ser and Arg149His, that alter highly-conserved residues located between the helical and GTPase domains (the interdomain interface), and are predicted to disrupt interdomain contacts, thereby increasing G-protein flexibility and activating GDP-to-GTP exchange. We assessed the effect of Gα11 mutations on Ca2+i and MAPK pathways by expressing Gα11-wild-type, and Ser66 or His149 Gα11-mutants, in HEK293 cells stably-expressing CaSR. Ca2+i responses to extracellular calcium (Ca2+e) were assessed using a Fluo-4 fluorescent assay and NFAT-response element-containing luciferase reporter (measuring Ca2+iinduced gene expression); and MAPK responses assessed using a phospho-ERK (pERK) AlphaScreen assay and serum-response element (SRE)-containing luciferase reporter (measuring ERK-induced gene expression). The Ser66 and His149 mutants, when compared to Gα11-wild-type, led to: a leftward shift of the Ca2+i dose-response curves, with decreased mean half-maximal concentration (EC50) values, and elevated NFAT, pERK, and SRE responses. Treatment of Ser66- and His149-expressing cells with the CaSR negative allosteric modulator NPS-2143 normalised Ca2+i and MAPK responses. Thus, the Gα11 interdomain interface plays an important role in calcium homeostasis, and interdomain interface mutations can be normalised by CaSR allosteric modulators, indicating effective treatments for symptomatic hypocalcaemia.
06 Nov 2017 - 08 Nov 2017