Human Molecular Genetics Advance Access published online on June 1, 2006
Human Molecular Genetics, doi:10.1093/hmg/ddl145
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Medicine, Royal Victoria Hospital, McGill University, Montreal QC H3A 1A1, Canada; Department of Calcium Research Laboratory, Royal Victoria Hospital, McGill University, Montreal QC H3A 1A1, Canada
* To whom correspondence should be addressed. CASR, expressed in parathyroid gland and kidney, is a critical regulator of extracellular calcium homeostasis. This GPCR 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, that 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 nonpermeabilized cells, while 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 the Golgi apparatus. By the use of photobleaching fluorescence resonance energy transfer (pbFRET) 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.
Received April 6, 2006
Revised May 30, 2006
Accepted May 30, 2006
Article
The calcium-sensing receptor (CASR) dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly
Svetlana Pidasheva 1,
Michael Grant 2,
Lucie Canaff 1,
Oya Ercan 3,
Ujendra Kumar 2,
and
Geoffrey N. Hendy 4 *
2 Department of Medicine, Royal Victoria Hospital, McGill University, Montreal QC H3A 1A1, Canada; Fraser Laboratories, McGill University, Montreal QC H3A 1A1, Canada
3 Cerrahpasa Medical Faculty Department of Paediatrics, Istanbul University, Istanbul, Turkey
4 Department of Medicine, Royal Victoria Hospital, McGill University, Montreal QC H3A 1A1, Canada; Department of Physiology, Royal Victoria Hospital, McGill University, Montreal QC H3A 1A1, Canada; Department of Human Genetics, Royal Victoria Hospital, McGill University, Montreal QC H3A 1A1, Canada; Department of Calcium Research Laboratory, Royal Victoria Hospital, 687 Pine Ave. West, Rm. H4.67, McGill University, Montreal QC H3A 1A1, Canada; Department of Hormones and Cancer Research Unit, Royal Victoria Hospital, McGill University, Montreal QC H3A 1A1, Canada
Geoffrey N. Hendy, E-mail: geoffrey.hendy{at}mcgill.ca
Abstract 
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. White, J. McKenna, A. Cavanaugh, and G. E. Breitwieser Pharmacochaperone-Mediated Rescue of Calcium-Sensing Receptor Loss-of-Function Mutants Mol. Endocrinol., July 1, 2009; 23(7): 1115 - 1123. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E C Cole, F. H J Yun, B. Y L Wong, A. Y Shuen, R. A Booth, A. Scillitani, S. Pidasheva, X. Zhou, L. Canaff, and G. N Hendy Calcium-sensing receptor mutations and denaturing high performance liquid chromatography J. Mol. Endocrinol., April 1, 2009; 42(4): 331 - 339. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Guan, X. Feng, X. Wu, M. Zhang, X. Zhang, T. E. Hebert, and D. L. Segaloff Bioluminescence Resonance Energy Transfer Studies Reveal Constitutive Dimerization of the Human Lutropin Receptor and a Lack of Correlation between Receptor Activation and the Propensity for Dimerization J. Biol. Chem., March 20, 2009; 284(12): 7483 - 7494. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Rus, C. Haag, C. Bumke-Vogt, V. Bahr, B. Mayr, M. Mohlig, E. Schulze, K. Frank-Raue, F. Raue, and C. Schofl Novel Inactivating Mutations of the Calcium-Sensing Receptor: The Calcimimetic NPS R-568 Improves Signal Transduction of Mutant Receptors J. Clin. Endocrinol. Metab., December 1, 2008; 93(12): 4797 - 4803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Canaff, X. Zhou, and G. N. Hendy The Proinflammatory Cytokine, Interleukin-6, Up-regulates Calcium-sensing Receptor Gene Transcription via Stat1/3 and Sp1/3 J. Biol. Chem., May 16, 2008; 283(20): 13586 - 13600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kern, D. Hubbard, A. Amano, and G. D. Bryant-Greenwood Cloning, Expression, and Functional Characterization of Relaxin Receptor (Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 7) Splice Variants from Human Fetal Membranes Endocrinology, March 1, 2008; 149(3): 1277 - 1294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Zajickova, J. Vrbikova, L. Canaff, P. D. Pawelek, D. Goltzman, and G. N. Hendy Identification and Functional Characterization of a Novel Mutation in the Calcium-Sensing Receptor Gene in Familial Hypocalciuric Hypercalcemia: Modulation of Clinical Severity by Vitamin D Status J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2616 - 2623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang and G. E. Breitwieser Rescue of Calcium-sensing Receptor Mutants by Allosteric Modulators Reveals a Conformational Checkpoint in Receptor Biogenesis J. Biol. Chem., March 30, 2007; 282(13): 9517 - 9525. [Abstract] [Full Text] [PDF]
|
|