Endocrine Abstracts

Mutations of the calcium-sensing receptor (CaSR), G-protein alpha-11 subunit (Gα11), and adaptor protein-2 sigma subunit (AP2σ) resulting in a loss-of-function, cause familial hypocalciuric hypercalcemia types 1-3 (FHH1-3), respectively. We investigated a family with FHH (four affected and two unaffected members) for CaSR, Gα11 and AP2σ mutations, and identified a heterozygous Gα11 missense mutation, Phe220Ser, which is predicted to disrupt a cluster of hydrophobic residues that are important for G-protein/G-protein coupled receptor interactions. Gα11, which acts downstream of the CaSR, activates phospholipase C (PLC) leading to two predominant signal outputs: intracellular calcium (Ca2+i) release; and activation of the extracellular-signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We assessed the effects of the Gα11 mutation on signalling by expressing Gα11-wild-type Phe220, and three Gα11-mutants: Ser220, detected in the FHH2 kindred; and engineered mutants, Ala220 (a hydrophobic residue); and, Glu220 (a non-hydrophobic residue), in HEK293 cells stably expressing CaSR. Ca2+i responses to extracellular calcium (Ca2+e) were assessed using a Fluo-4 fluorescent assay and an NFAT-response element-containing luciferase reporter that measures Ca2+i–induced gene expression; and MAPK responses assessed using a phospho-ERK (pERK) AlphaScreen assay and a serum-response element (SRE)-containing luciferase reporter that measures ERK-induced gene expression. Mutation of Phe220 to the non-hydrophobic Ser220 and Glu220 residues, but not the hydrophobic residue, Ala220, significantly impaired Gα11 signalling via PLC-Ca2+i and ERK/MAPK pathways. Thus, Ser220 and Glu220, when compared to wild-type (Phe220) and Ala220 led to: a rightward shift of the dose-response curves of Ca2+i responses to Ca2+e with increased mean half-maximal concentration (EC50) values; reductions in NFAT reporter responses; impaired pERK responses; and reductions in SRE reporter activity. Thus, we have identified a novel Gα11 mutation, Phe220Ser, causing FHH2, and have demonstrated the importance of the hydrophobic Phe220 residue, which forms part of a cluster, for G-protein signaling via PLC-Ca2+i and ERK/MAPK pathways.