The calcium-sensing receptor (CASR) is a class C G-protein-coupled receptor (GPCR) that detects extracellular calcium concentrations, and modulates parathyroid hormone secretion and urinary calcium excretion to maintain calcium homeostasis. The CASR utilises multiple heterotrimeric G-proteins to mediate signalling effects including activation of intracellular calcium release; mitogen-activated protein kinase (MAPK) pathways; membrane ruffling; and inhibition of cAMP production. By studying germline mutations in the CASR and proteins within its signalling pathway that cause hyper- and hypocalcaemic disorders, novel mechanisms governing GPCR signalling and trafficking have been elucidated. This review focusses on two recently described pathways that provide novel insights into CASR signalling and trafficking mechanisms. The first, identified by studying a CASR gain-of-function mutation that causes autosomal dominant hypocalcaemia (ADH), demonstrated a structural motif located between the third transmembrane domain and the second extracellular loop of the CASR that mediates biased signalling by activating a novel β-arrestin-mediated G-protein-independent pathway. The second, in which the mechanism by which adaptor protein-2 σ-subunit (AP2σ) mutations cause familial hypocalciuric hypercalcaemia (FHH) was investigated, demonstrated that AP2σ mutations impair CASR internalisation and reduce multiple CASR-mediated signalling pathways. Furthermore, these studies showed that the CASR can signal from the cell surface using multiple G-protein pathways, whilst sustained signalling is mediated only by the Gq/11 pathway. Thus, studies of FHH- and ADH-associated mutations have revealed novel steps by which CASR mediates signalling and compartmental bias, and these pathways could provide new targets for therapies for patients with calcaemic disorders.
Caroline M Gorvin
Twenty-five years have elapsed since the calcium-sensing receptor (CaSR) was first identified in bovine parathyroid and the receptor is now recognized as a fundamental contributor to extracellular Ca2+ (Ca2+ e) homeostasis, regulating parathyroid hormone release and urinary calcium excretion. The CaSR is a class C G-protein-coupled receptor (GPCR) that is functionally active as a homodimer and couples to multiple G-protein subtypes to activate intracellular signalling pathways. The importance of the CaSR in the regulation of Ca2+ e has been highlighted by the identification of >400 different germline loss- and gain-of-function CaSR mutations that give rise to disorders of Ca2+ e homeostasis. CaSR-inactivating mutations cause neonatal severe hyperparathyroidism, characterised by marked hypercalcaemia, skeletal demineralisation and failure to thrive in early infancy; and familial hypocalciuric hypercalcaemia, an often asymptomatic disorder associated with mild-moderately elevated serum calcium concentrations. Activating mutations are associated with autosomal dominant hypocalcaemia, which is occasionally associated with a Bartter’s-like phenotype. Recent elucidation of the CaSR extracellular domain structure enabled the locations of CaSR mutations to be mapped and has revealed clustering in locations important for structural integrity, receptor dimerisation and ligand binding. Moreover, the study of disease-causing mutations has demonstrated that CaSR signals in a biased manner and have revealed specific residues important for receptor activation. This review presents the current understanding of the genetic landscape of CaSR mutations by summarising findings from clinical and functional studies of disease-associated mutations. It concludes with reflections on how recently uncovered signalling pathways may expand the understanding of calcium homeostasis disorders.
Hasnat Ali Abid, Asuka Inoue, and Caroline M. Gorvin
The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca2+ e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G-protein subfamilies (Gαq/11, Gαi/o, Gα12/13, Gαs), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messenger proteins or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G-protein activation directly, we took advantage of a recently described NanoBiT G-protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca2+ e stimulation activates all Gαq/11 family and several Gαi/o family proteins, although Gαz was not activated. CaSR stimulated dissociation of Gα12/13 and Gαs from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G-proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα11, Gαz, Gαi1 and Gα13 genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα11 and Gαi1 in parathyroids. In kidney and pancreas, the majority of G-proteins were similarly expressed, suggesting CaSR may activate multiple G-proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.