Loss of residues required for β-arrestin recruitment in the calcium-sensing receptor C-terminal tail may contribute to hypocalcemia.

The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor (GPCR) with an important role in calcium homeostasis, activating mutations of which cause hypocalcemia. Despite the receptors clinical importance and evidence suggesting β-arrestin-1/2 can modulate CaSR signaling, the residues within CaSR required for β-arrestin recruitment and role of β-arrestin in desensitization, trafficking and signaling are ill defined. We confirmed that β-arrestin-1 and β-arrestin-2 are recruited to CaSR upon receptor activation and that depletion of GPCR kinases impairs β-arrestin recruitment. Deletion of the distal CaSR C-terminal tail, which replicates several mutations identified in individuals with hypocalcemia, reduced β-arrestin-1/2 recruitment and enhanced receptor signaling. Examination of the receptor cytoplasmic region (intracellular loops and C-terminal tail) identified three regions of serine and threonine residues that resemble phosphorylation codes in other GPCRs. Mutation of each of these residues to alanine demonstrated one region between amino acids 1003 to 1011 is important for both β-arrestin-1 and β-arrestin-2 recruitment, while disruption of a region between residues 976 to 981 impaired β-arrestin-2 recruitment. Alanine mutagenesis of these residues also enhanced CaSR signaling and impaired receptor internalization suggesting an important role in receptor desensitization. Three rare-coding variants reported in disorders of calcium homeostasis were identified in these residues and two, T1006M and T1008P, reduced β-arrestin recruitment and CaSR internalization and enhanced CaSR signaling. Thus, we identified two regions within the CaSR C-terminal tail that are important for β-arrestin recruitment, receptor desensitization and internalization. Mutation of residues within these regions may represent another mechanism contributing to hypocalcemia.