G-protein-coupled receptors (GPCRs) are a target for over 34% of current drugs. The calcium-sensing receptor (CaSR), a family C GPCR, regulates systemic calcium (Ca²⁺) homeostasis that is critical for many physiological, calciotropical, and noncalciotropical outcomes in multiple organs. However, the mechanisms by which extracellular Ca²⁺ (Ca²⁺_ex) and the CaSR mediate networks of intracellular Ca²⁺-signaling and players involved throughout the life cycle of CaSR are largely unknown. Here we report the first CaSR protein-protein interactome with 94 novel putative and 8 previously published interactors using proteomics. Ca²⁺_ex promotes enrichment of 66% of the identified CaSR interactors, pertaining to Ca²⁺ dynamics, endocytosis, degradation, trafficking, and primarily to protein processing in the endoplasmic reticulum (ER). These enhanced ER-related processes are governed by Ca²⁺_ex-activated CaSR which directly modulates ER-Ca²⁺ (Ca²⁺_ER), as monitored by a novel ER targeted Ca²⁺-sensor. Moreover, we validated the Ca²⁺_ex dependent colocalizations and interactions of CaSR with ER-protein processing chaperone, 78-kDa glucose regulated protein (GRP78), and with trafficking-related protein. Live cell imaging results indicated that CaSR and vesicle-associated membrane protein-associated A (VAPA) are inter-dependent during Ca²⁺_ex induced enhancement of near-cell membrane expression. This study significantly extends the repertoire of the CaSR interactome and reveals likely novel players and pathways of CaSR participating in Ca²⁺_ER dynamics, agonist mediated ER-protein processing and surface expression.

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