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
Highlights
The calcium (Ca2+)-sensing receptor (CaSR), a member of the class C subfamily of G-protein-coupled receptors (GPCRs), is highly expressed in the parathyroid glands and kidneys and plays an essential role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone (PTH) release and urinary Ca2+ excretion [1]
An analysis of calcium-sensing receptor (CaSR) mutations and variants identified in >300 index cases of familial hypocalciuric hypercalcaemia type 1 (FHH1) or autosomal dominant hypocalcaemia type 1 (ADH1) that had been referred to our centre in Oxford (UK) since 2005 together with a review of previously reported CaSR mutations [17] identified five diseaseswitch residues, which are the location of both FHH1- and ADH1associated mutations
The Gln27 residue is affected by a novel FHH1-associated p.Gln27Pro variant and a reported FHH1-associated p.Gln27Arg mutation [18], and a novel ADH1-associated p.Gln27Glu variant (Table 1 and Figs. 1 and S1); whereas the Asn178 residue is affected by a reported FHH1-associated p.Asn178Asp mutation [19] and by a novel ADH1-associated p.Asn178Tyr variant (Table 1 and Figs. 1 and S1)
Summary
The calcium (Ca2+)-sensing receptor (CaSR), a member of the class C subfamily of G-protein-coupled receptors (GPCRs), is highly expressed in the parathyroid glands and kidneys and plays an essential role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone (PTH) release and urinary Ca2+ excretion [1]. The importance of the CaSR for the regulation of Ca2+e has been highlighted by the identification of >230 different germline loss- and gain-of-function CaSR mutations that give rise to disorders of Ca2+e homeostasis known as familial hypocalciuric hypercalcaemia type 1 (FHH1) and autosomal dominant hypocalcaemia type 1 (ADH1), respectively [11]. To gain further insights into the role of CaSR disease-switch residues, we sought for additional residues affected by loss- and gain-of-function mutations, by analysis of familial hypocalciuric hypercalcaemia and autosomal dominant hypocalcaemia patients who had been referred to our centre for diagnostic genetic testing of the CASR gene. We determined the effect of disease-switch residue mutations on CaSR-mediated Ca2+i and ERK signalling and analysed the structural consequences of these mutations, using the recently determined crystal structures of the CaSR ECD [3,4]
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