Pharmacochaperone-mediated rescue of calcium-sensing receptor loss-of-function mutants.

[familial hypocalciuric hypercalcemia]

The calcium sensing receptor (CaSR ) is a Family C /3 G protein-coupled receptor that translates changes in extracellular Ca (2 +) into diverse intracellular signals . Loss -of-function mutations in human CaSR cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism . CaSR must navigate a number of endoplasmic reticulum quality control checkpoints during biosynthesis , including a conformational /functional checkpoint . Here we examine the biosynthesis of 25 CaSR mutations causing familial hypocalciuric hypercalcemia /neonatal severe hyperparathyroidism using immunoprecipitation , biotinylation , and functional assays . We define classes of CaSR mutants based on their biosynthetic profile . Class I CaSR mutants are not rescued to the plasma membrane . To dissect the organellar compartments that class I mutants can access , we engineered a cleavage site for the proprotein convertase furin into the extracellular domain of wild-type CaSR and class I mutants . Based on absence or presence of cleavage fragments , we find most mutants are degraded from the endoplasmic reticulum (no furin-mediated cleavage ), whereas others access the Golgi (furin-mediated cleavage ) before degradation . Class II CaSR mutants show increased expression and /or enhanced plasma membrane localization upon treatment with MG 132 or the pharmacochaperone NPS R-568 , permitting assay of functional activity . Of the 10 CaSR mutants that exhibit plasma membrane localization , only two did not show enhanced functional activity after rescue with NPS R-568 . The established approaches can be used with current and newly identified CaSR mutations to identify the location of biosynthetic block and to determine the likelihood of rescue by allosteric agonists .