title: Calcium-sensing receptor dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly.
abstract: Calcium-sensing receptor (CASR), expressed in parathyroid gland and kidney, is a critical regulator of extracellular calcium homeostasis. This G protein-coupled receptor exists at the plasma membrane as a homodimer, although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H, R66C or N583X-inactivating mutations identified in familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroid patients, which were transiently expressed in kidney cells. All mutants were deficient in cell signaling responses to extracellular CASR ligands relative to wild-type. All mutants, although as well expressed as wild-type, lacked mature glycosylation, indicating impaired trafficking from the endoplasmic reticulum (ER). Dimerized forms of wild-type, R66H and R66C mutants were present, but not of the N583X mutant. By immunofluorescence confocal microscopy of non-permeabilized cells, although cell surface expression was observed for the wild-type, little or none was seen for the mutants. In permeabilized cells, perinuclear staining was observed for both wild-type and mutants. By colocalization fluorescence confocal microscopy, the mutant CASRs were localized within the ER but not within the Golgi apparatus. By the use of photobleaching fluorescence resonance energy transfer microscopy, it was demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER, whereas the N583X mutant was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface.
authors.affiliation: Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada QC H3A 1A1.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Pidasheva
authors.forename: Michael
authors.initials: M
authors.lastname: Grant
authors.forename: Lucie
authors.initials: L
authors.lastname: Canaff
authors.forename: Oya
authors.initials: O
authors.lastname: Ercan
authors.forename: Ujendra
authors.initials: U
authors.lastname: Kumar
authors.forename: Geoffrey N
authors.initials: GN
authors.lastname: Hendy
mesh_terms: D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D006934:Hypercalcemia / Q000235:genetics / Q000378:metabolism; D006961:Hyperparathyroidism / Q000235:genetics / Q000378:metabolism; D007223:Infant; D007231:Infant, Newborn; D020935:MAP Kinase Signaling System; D008297:Male; D010375:Pedigree; D017354:Point Mutation; D020836:Protein Structure, Quaternary; D044169:Receptors, Calcium-Sensing / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins