Pharmacochaperone-mediated rescue of calcium-sensing receptor loss-of-function mutants.
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.
White
Elissa
E
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2604, USA.
McKenna
Jennifer
J
Cavanaugh
Alice
A
Breitwieser
Gerda E
GE
D000494:Allosteric Regulation / Q000187:drug effects* / Q000235:genetics; D000495:Allosteric Site / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D008954:Models, Biological; D018832:Molecular Chaperones / Q000494:pharmacology*; D050505:Mutant Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020641:Polymorphism, Single Nucleotide / Q000502:physiology; D044169:Receptors, Calcium-Sensing / Q000819:agonists / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*
D018832:Molecular Chaperones; D050505:Mutant Proteins; D044169:Receptors, Calcium-Sensing