Activating Mutations of the G-protein Subunit α (11) Interdomain Interface Cause Autosomal Dominant Hypocalcemia Type 2

CONTEXT: Autosomal dominant hypocalcemia types 1 and 2 (ADH1 and ADH2) are caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and its signaling partner, the G-protein subunit α (11) (Gα (11)), respectively. More than 70 different gain-of-function CaSR mutations, but only 6 different gain-of-function Gα (11) mutations are reported to date. METHODS: We ascertained 2 additional ADH families and investigated them for CaSR and Gα (11) mutations. The effects of identified variants on CaSR signaling were evaluated by transiently transfecting wild-type (WT) and variant expression constructs into HEK293 cells stably expressing CaSR (HEK-CaSR), and measuring intracellular calcium (Ca(2+)(i)) and MAPK responses following stimulation with extracellular calcium (Ca(2+)(e)). RESULTS: CaSR variants were not found, but 2 novel heterozygous germline Gα (11) variants, p.Gly66Ser and p.Arg149His, were identified. Homology modeling of these revealed that the Gly66 and Arg149 residues are located at the interface between the Gα (11) helical and GTPase domains, which is involved in guanine nucleotide binding, and this is the site of 3 other reported ADH2 mutations. The Ca(2+)(i) and MAPK responses of cells expressing the variant Ser66 or His149 Gα (11) proteins were similar to WT cells at low Ca(2+)(e), but significantly increased in a dose-dependent manner following Ca(2+)(e) stimulation, thereby indicating that the p.Gly66Ser and p.Arg149His variants represent pathogenic gain-of-function Gα (11) mutations. Treatment of Ser66- and His149-Gα (11) expressing cells with the CaSR negative allosteric modulator NPS 2143 normalized Ca(2+)(i) and MAPK responses. CONCLUSION: Two novel ADH2-causing mutations that highlight the Gα (11) interdomain interface as a hotspot for gain-of-function Gα (11) mutations have been identified.