The calcium-sensing receptor (CaSR) is a class C GPCR essential for calcium homeostasis, and its dysregulation underlies inherited disorders of hyper- and hypocalcaemia. Following G protein signalling at the plasma membrane, CaSR is understood to undergo desensitisation and internalisation, which can be both constitutive and agonist-mediated, and can involve β-arrestin proteins. CaSR can also internalise by β-arrestin-independent mechanisms, and these internalised receptors can produce sustained Gq/11 signals from intracellular membranes. This talk presents two complementary lines of work defining the molecular determinants that govern these processes elucidated in part by studying human disease.
First, I will describe the identification of two clusters of serine/threonine residues within the CaSR C-terminal domain that function as phosphorylation-dependent codes controlling β-arrestin-1 and β-arrestin-2 recruitment. Alanine substitution within these regions impairs β-arrestin binding, enhances receptor signalling, and disrupts internalisation, implicating these motifs in receptor desensitisation. Disease-associated variants identified in ClinVar recapitulate this phenotype, suggesting defective desensitisation as a mechanism underlying hypocalcaemia.
Second, I will expand upon our previous findings that CaSR does not simply recycle or degrade but is actively routed through spatially and temporally distinct endosomal compartments to sustain receptor signalling. A dileucine endocytic motif within the CaSR C-terminal domain directs this trafficking by engaging the adaptor protein-2 σ-subunit, a protein mutated in patients with hypercalcemia. From early endosomes, CaSR is further partitioned into recycling and late endosomal pathways, each associated with distinct signaling outputs. The allosteric modulator cinacalcet biases signalling to preferentially enhance early endosomal-to-recycling pathways, demonstrating the veracity of targeting discrete endosomal pathways.
Together, these findings reveal that CaSR employs discrete structural motifs - phosphorylation codes and a dileucine sorting signal - to coordinate arrestin recruitment, desensitization, and multidimensional endosomal signaling. These studies provide new mechanistic insight into the pathogenesis of calcium homeostasis disorders and new opportunities for compartment-selective pharmacology.