Pharmacochaperone-Mediated Rescue of Calcium-Sensing Receptor Loss-of-Function Mutants

Abstract

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 MG132 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.

GENETICS IN ENDOCRINOLOGY: Gain and loss of function mutations of the calcium-sensing receptor and associated proteins: current treatment concepts

“…This supports the proposed notion that even if the CASR mutant itself may not be responsive to calcimimetics, the remaining normal CASR allele in FHH or in heterozygous NHPT could confer sensitivity to calcimimetic treatment in vivo (86). In addition, calcimimetics may act as pharmacological chaperones to rescue misfolded inactivating mutants (87,88,89). The polymorphic variants A986S and R990G occur in about 24% and 4% of healthy adults respectively and lead to slight changes in ionized serum calcium levels (90).…”

Calcium-sensing Receptor Biosynthesis Includes a Cotranslational Conformational Checkpoint and Endoplasmic Reticulum Retention

“…19). We have previously shown that NPS R-568 can rescue both WT CaSR and some loss-of-function mutants identified in familial hypocalciuric hypercalcemia/ neonatal severe primary hyperparathyroidism patients, increasing both total and plasma membrane-targeted levels of receptor protein and function (21,22). Allosteric agonists may have similar effects in vivo because uremic rats treated with cinacalcet (54) as well as first/second generation allosteric agonists (calcimimetics) NPS R-568 (55), Amgen R-568 (56, 57), or AMG-641 (58) show reduced parathyroid gland hyperplasia, vascular calcification, and remodeling as a result of both enhanced activation and expression of CaSR in relevant tissues (54 -58).…”

“…Point mutations in the WT FLAG-CaSR background (E837I, A843E, and L849P) were generated as described previously (22). The CaSR/mGluR1␣ CT chimera was generated by incorporating a silent PvuI restriction site at the proposed junction in both CaSR and mGluR1␣ constructs, digesting each vector with PvuI, followed by ligation.…”

“…Many CaSR loss-of-function mutations interfere with proper trafficking of CaSR through the secretory pathway and can be rescued in functional form to the plasma membrane by overnight treatment with the allosteric agonist NPS R-568 (21,22). Conversely, some gain-of-function mutants are resistant to ERAD, but their degradation in the ER can be promoted by the allosteric antagonist NPS 2143 (21).…”

Characterization of Highly Efficacious Allosteric Agonists of the Human Calcium-Sensing Receptor

“…Several studies reported that the calcimimetic 3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine (NPS R-568) improves the signal transduction characteristics of loss-of-function polymorphic variants of the human CaSR associated with human disease (Rus et al, 2008;Lu et al, 2009;White et al, 2009). NPS R-568 is structurally very similar to cinacalcet, and therefore one would expect cinacalcet to have similar effects on those polymorphic receptors, and, vice versa, one would expect NPS R-568 to have effects similar to those of cinacalcet and calindol on the polymorphic receptors studied here.…”