LAUSR

Abstract The calcium‐sensing receptor (CaSR) is a homodimeric G‐protein‐coupled receptor that signals via intracellular calcium (Ca2+i) mobilisation and phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK) to regulate extracellular calcium (Ca2+e) homeostasis. The central importance of the CaSR in Ca2+e homeostasis has been demonstrated by the identification of loss‐ or gain‐of‐function CaSR mutations that lead to familial hypocalciuric hypercalcaemia (FHH) or autosomal dominant hypocalcaemia (ADH), respectively. However, the mechanisms determining whether the CaSR signals via Ca2+i or ERK have not been established, and we hypothesised that some CaSR residues, which are the site of both loss‐ and gain‐of‐function mutations, may act as molecular switches to direct signalling through these pathways. An analysis of CaSR mutations identified in >300 hypercalcaemic and hypocalcaemic probands revealed five ‘disease‐switch’ residues (Gln27, Asn178, Ser657, Ser820 and Thr828) that are affected by FHH and ADH mutations. Functional expression studies using HEK293 cells showed disease‐switch residue mutations to commonly display signalling bias. For example, two FHH‐associated mutations (p.Asn178Asp and p.Ser820Ala) impaired Ca2+i signalling without altering ERK phosphorylation. In contrast, an ADH‐associated p.Ser657Cys mutation uncoupled signalling by leading to increased Ca2+i mobilization while decreasing ERK phosphorylation. Structural analysis of these five CaSR disease‐switch residues together with four reported disease‐switch residues revealed these residues to be located at conformationally active regions of the CaSR such as the extracellular dimer interface and transmembrane domain. Thus, our findings indicate that disease‐switch residues are located at sites critical for CaSR activation and play a role in mediating signalling bias