Loss of phosphorylation sites required for β-arrestin recruitment in the calcium-sensing receptor cytoplasmic region may contribute to hypocalcemia

The calcium-sensing receptor (CaSR) is a class 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 the 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. Deletion of the distal cytoplasmic region of CaSR, 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 identified three regions of serine and threonine residues that resemble phosphorylation codes identified in other GPCRs. Mutation of each of these residues to alanine demonstrated one region between amino acids 1003-1011 is important for both β-arrestin-1 and β-arrestin-2 recruitment, while disruption of a second region between residues 976-981 impaired β-arrestin-2 recruitment. Alanine mutagenesis of these residues also reduced CaSR signaling and impaired receptor internalization suggesting an important role in receptor desensitization. Three variants in the ClinVar dataset reported in disorders of calcium homeostasis were identified and two, T1006M and T1008P, were shown to reduce β-arrestin recruitment and enhance CaSR signaling. Further analysis of the T1006M variant showed it reduced CaSR internalization. Thus, we have identified two regions within the CaSR cytoplasmic region that are important for β-arrestin recruitment, receptor desensitization and internalization. Mutation of residues within these regions may represent another mechanism for the pathogenesis of hypocalcemia.