Calcium-sensing receptor biosynthesis includes a cotranslational conformational checkpoint and endoplasmic reticulum retention.

Abstract Metabolic labeling with [35S]cysteine was used to characterize early events in CaSR biosynthesis. [35S]CaSR is relatively stable (half-life ∼8 h), but maturation to the final glycosylated form is slow and incomplete. Incorporation of [35S]cysteine is linear over 60 min, and the rate of [35S]CaSR biosynthesis is significantly increased by the membrane-permeant allosteric agonist NPS R-568, which acts as a cotranslational pharmacochaperone. The [35S]CaSR biosynthetic rate also varies as a function of conformational bias induced by loss- or gain-of-function mutations. In contrast, [35S]CaSR maturation to the plasma membrane was not significantly altered by exposure to the pharmacochaperone NPS R-568, the allosteric agonist neomycin, or the orthosteric agonist Ca2+ (0.5 or 5 mm), suggesting that CaSR does not control its own release from the endoplasmic reticulum. A CaSR chimera containing the mGluR1α carboxyl terminus matures completely (half-time of ∼8 h) and without a lag period, as does the truncation mutant CaSRΔ868 (half-time of ∼16 h). CaSRΔ898 exhibits maturation comparable with full-length CaSR, suggesting that the CaSR carboxyl terminus between residues Thr868 and Arg898 limits maturation. Overall, these results suggest that CaSR is subject to cotranslational quality control, which includes a pharmacochaperone-sensitive conformational checkpoint. The CaSR carboxyl terminus is the chief determinant of intracellular retention of a significant fraction of total CaSR. Intracellular CaSR may reflect a rapidly mobilizable “storage form” of CaSR and/or may subserve distinct intracellular signaling roles that are sensitive to signaling-dependent changes in endoplasmic reticulum Ca2+ and/or glutathione.