Endocrine Abstracts

The CaSR, a G-protein-coupled receptor that regulates extracellular calcium (Ca²⁺_o), predominantly signals via G-protein-α_q/11 (Gα_q/11), initiating IP₃-mediated intracellular calcium (Ca²⁺_i) accumulation, and mitogen-activated protein kinase (MAPK) signalling. CaSR also activates MAPK signalling via Gα_i/o, or by associating with the scaffolding protein β-arrestin. CaSR gain-of-function mutations cause autosomal dominant hypocalcaemia type-1 (ADH1). Mutational analysis of CASR in a seven-year-old male and his father with ADH1 identified a novel heterozygous mutation (p.Arg680Gly) in both patients. The variant was functionally characterised in HEK293 cells transiently expressing WT (Arg680) or mutant Gly680 CaSRs using: a flow cytometry assay to measure Ca²⁺_i; and a SRE luciferase reporter gene to assess MAPK signalling, in response to Ca²⁺_o elevations. In contrast to reported ADH1 mutations, Gly680 had no effect on Ca²⁺_i, but did significantly elevate MAPK responses. Measurements of the IP₃ breakdown product IP₁ showed no significant difference between WT and Gly680 expressing cells, confirming Gα_q/11- signalling was not responsible for elevated Gly680 MAPK responses. We hypothesized such differences could instead be due to biased signalling by Gα_i/o or a G-protein-independent β-arrestin-mediated pathway. To test the former, we assessed the effect of the Gα_i/o-blocking agent pertussis toxin (PTx) on SRE reporter responses. PTx reduced SRE responses in both WT and Gly680 expressing cells to similar levels, indicating Gα_i/o is not responsible for elevated MAPK signalling. To investigate G-protein-independent mechanisms we used a β-arrestin-1 siRNA, which had no effect on WT SRE reporter activity, but significantly reduced responses in Gly680 expressing cells. Structural homology modelling predicts Arg680 to form a salt bridge with CaSR transmembrane domain-7, which limits binding of β-arrestin; thus loss of this salt bridge would enhance β-arrestin binding and MAPK signalling. In conclusion, we report a CaSR mutation associated with hypocalcaemia that enhances MAPK signalling via a unique G-protein-independent mechanism involving β-arrestin-1.