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.

钙敏感受体 (CaSR) 是一种 G 蛋白偶联受体 (GPCR)，通过 G _q/11和 G _i/o发出信号以刺激胞质钙 (Ca ²⁺ _i ) 和丝裂原激活蛋白激酶 (MAPK) 信号传导控制细胞外钙稳态。对分别导致家族性低钙尿性高钙血症 1 型 (FHH1) 和常染色体显性低钙血症 1 型 (ADH1) 的功能丧失和获得功能的CASR突变的研究表明，CaSR 信号存在偏差。因此，一些与 FHH1 相关的突变主要通过 MAPK 途径导致信号传导，而与 ADH1 相关的突变优先增强 Ca ²⁺ _i反应。我们报告了一个先前未鉴定的 ADH1 相关 R680G CaSR 突变，该突变导致鉴定出介导偏向信号传导的 CaSR 结构基序。在 HEK 293 细胞中表达 CaSR ^R680G表明，这种突变增加了 MAPK 信号传导，但不改变 Ca ²⁺ _i反应。此外，MAPK 活性的这种功能获得独立于 G _q/11和 G _i/o发生，而是由涉及 β-arrestin 蛋白的非经典途径介导。同源建模和诱变研究表明，R680G CaSR 突变通过破坏分别位于 CaSR 跨膜结构域 3 和细胞外环 2 的 Arg ⁶⁸⁰和 Glu ⁷⁶⁷之间形成的盐桥来选择性增强 β-arrestin 信号传导。因此，我们的结果证明了通过 β-arrestin 的 CaSR 信号传导以及 Arg ⁶⁸⁰ -Glu ⁷⁶⁷盐桥在介导信号偏差中的重要性。