A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin-biased signaling

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through G_q/11 and G_i/o to stimulate cytosolic calcium (Ca²⁺_i) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-function CASR mutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed that the CaSR signals in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca²⁺_i responses. We report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing CaSR^R680G in HEK 293 cells showed that this mutation increased MAPK signaling without altering Ca²⁺_i responses. Moreover, this gain of function in MAPK activity occurred independently of G_q/11 and G_i/o and was mediated instead by a noncanonical pathway involving β-arrestin proteins. Homology modeling and mutagenesis studies showed that the R680G CaSR mutation selectively enhanced β-arrestin signaling by disrupting a salt bridge formed between Arg⁶⁸⁰ and Glu⁷⁶⁷, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through β-arrestin and the importance of the Arg⁶⁸⁰-Glu⁷⁶⁷ salt bridge in mediating signaling bias.