Activating Mutations of the G-protein Subunit [alpha] 11 Interdomain Interface Cause Autosomal Dominant Hypocalcemia Type 2

Context: Autosomal dominant hypocalcemia types 1 and 2 (ADH1 and AD[H.sub.2]) are caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and its signaling partner, the G-protein subunit [alpha] 11 (G[alpha] 11), respectively. More than 70 different gain-of-function CaSR mutations, but only 6 different gain-of-function G[alpha] 11 mutations are reported to date. Methods: We ascertained 2 additional ADH families and investigated them for CaSR and G[alpha] 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.sup.2+] i) and MAPK responses following stimulation with extracellular calcium ([Ca.sup.2+] e). Results: CaSR variants were not found, but 2 novel heterozygous germline G[alpha] 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 G[alpha] 11 helical and GTPase domains, which is involved in guanine nucleotide binding, and this is the site of 3 other reported AD[H.sub.2] mutations. The [Ca.sup.2+] i and MAPK responses of cells expressing the variant Ser66 or His149 G[alpha] 11 proteins were similar to WT cells at low [Ca.sup.2+] e, but significantly increased in a dose-dependent manner following [Ca.sup.2+] e stimulation, thereby indicating that the p.Gly66Ser and p.Arg149His variants represent pathogenic gain-of-function G[alpha] 11 mutations. Treatment of Ser66- and His149-G[alpha] 11 expressing cells with the CaSR negative allosteric modulator NPS 2143 normalized [Ca.sup.2+] i and MAPK responses. Conclusion: Two novel AD[H.sub.2]-causing mutations that highlight the G[alpha] 11 interdomain interface as a hotspot for gain-of-function G[alpha] 11 mutations have been identified. (J Clin Endocrinol Metab 105: 952-963, 2020) Key Words: G-protein, calcium-sensing receptor, parathyroid hormone