Calcium-sensing receptor biosynthesis includes a cotranslational conformational checkpoint and endoplasmic reticulum retention.
Metabolic labeling with [(35)S]cysteine was used to characterize early events in CaSR biosynthesis. [(35)S]CaSR is relatively stable (half-life approximately 8 h), but maturation to the final glycosylated form is slow and incomplete. Incorporation of [(35)S]cysteine is linear over 60 min, and the rate of [(35)S]CaSR biosynthesis is significantly increased by the membrane-permeant allosteric agonist NPS R-568, which acts as a cotranslational pharmacochaperone. The [(35)S]CaSR biosynthetic rate also varies as a function of conformational bias induced by loss- or gain-of-function mutations. In contrast, [(35)S]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 Ca(2+) (0.5 or 5 mm), suggesting that CaSR does not control its own release from the endoplasmic reticulum. A CaSR chimera containing the mGluR1alpha carboxyl terminus matures completely (half-time of approximately 8 h) and without a lag period, as does the truncation mutant CaSRDelta868 (half-time of approximately 16 h). CaSRDelta898 exhibits maturation comparable with full-length CaSR, suggesting that the CaSR carboxyl terminus between residues Thr(868) and Arg(898) 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 Ca(2+) and/or glutathione.
Cavanaugh
Alice
A
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, USA.
McKenna
Jennifer
J
Stepanchick
Ann
A
Breitwieser
Gerda E
GE
D000814:Aniline Compounds / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D050505:Mutant Proteins / Q000096:biosynthesis / Q000737:chemistry; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology; D010627:Phenethylamines; D011437:Propylamines; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021381:Protein Transport / Q000187:drug effects; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014162:Transfection
D000814:Aniline Compounds; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D050505:Mutant Proteins; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D049452:Green Fluorescent Proteins; D009355:Neomycin; D003545:Cysteine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D002118:Calcium