Endocrine Abstracts

Loss- and gain-of-function mutations of the calcium-sensing receptor (CaSR) cause familial hypocalciuric hypercalcaemia (FHH) and autosomal dominant hypocalcaemia (ADH), respectively. The CaSR is a homodimeric receptor that has a 612 amino acid extracellular domain (ECD), which binds extracellular calcium (Ca²⁺_e) and mediates dimer interactions upon ligand binding. The ECD consists of lobes 1 and 2, and a cysteine-rich domain (CRD). To elucidate the structure-function relationships of the ECD, we examined the location of CaSR ECD mutations reported to date in FHH and ADH probands using recently established CaSR crystal structures. These studies identified that 121 FHH and 65 ADH mutations affected ECD residues, with >50% of FHH mutations and >75% of ADH mutations being located at the dimer interface. Mutations predicted to disrupt key CaSR dimer-dimer interactions included: a lobe 1 Tyr161Cys mutation, which impaired an interprotomer interaction with the lobe 1 Pro55 residue; a Ser171Asn mutation predicted to disrupt a lobe 2 interprotomer salt bridge, which forms upon agonist binding; and a Gly553Arg mutation, which altered interprotomer hydrophobic interactions within the CRD. We characterized the effect of these mutations on CaSR function in HEK293 cells following stimulation with Ca²⁺_e, by measuring fold-change responses of nuclear factor of activated T-cells (NFAT), which is a downstream mediator of CaSR signaling. The Tyr161Cys, Ser171Asn and Gly553Arg dimer interface mutations were shown to markedly impair CaSR-mediated NFAT signalling. Indeed, cells expressing wild-type CaSR showed a >20-fold increase in NFAT responses following stimulation with 2.5 mM Ca²⁺_e, compared to a <5-fold increase in NFAT responses for mutant CaSR-expressing cells (P<0.01, N=4 biological replicates). Thus, these studies demonstrate that the majority of FHH1- and ADH1-causing CaSR ECD mutations are located at the dimer interface, and predicted to alter CaSR function through effects on dimer formation or agonist-induced conformational changes that occur at the dimer interface.