title: Naturally-occurring mutation in the calcium-sensing receptor reveals the significance of extracellular domain loop III region for class C G-protein-coupled receptor function.
abstract: BACKGROUND
  Inactivating mutations of the calcium-sensing receptor (CaSR) cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Most mutations are clustered in the N-terminal and Cys-rich regions of the extracellular domain (ECD) and seven-transmembrane domain. Disease-causing mutations are uncommon in the C terminus of ECD.
  
  
  OBJECTIVE
  The aim of the study was to characterize the CaSR mutations causing neonatal severe hyperparathyroidism in a consanguineous family.
  
  
  METHODS
  Parathyroid glands from the index patient were stained for CaSR protein. The CaSR gene was sequenced, mutations were recreated in CaSR cDNA, and HEK293 cells were transfected with the CaSR mutant expression vector. Cellular CaSR targeting was detected by immunoblotting and immunocytochemistry; CaSR activity was assayed by inositol phosphate accumulation, MAPK activation, and single-cell microfluorimetry.
  
  
  RESULTS
  Immunocytochemistry showed reduced intracellular CaSR in patient parathyroids. An in-frame homozygous deletion/insertion mutation, c.1031 > 1034 (delACAAinsT), replaced His344-Asn345 with a single Leu in CaSR loop III. The mutant reduced cell surface expression of CaSR in transfected HEK293 cells. Inositol phosphate accumulation, MAPK activation, and single-cell microfluorimetry revealed blunted signaling responses of the mutant receptor to changes in extracellular Ca(2+) concentration.
  
  
  CONCLUSIONS
  Deletion of His344-Asn345 in the ECD loop III region affects cell surface targeting of CaSR in transfected cells and in affected parathyroid glands. Absence of conserved Asn345 may interfere with CaSR folding or glycosylation, leading to poor protein targeting to the cell membrane. This loss-of-function mutant indicates that the ECD loop III is required for CaSR activity.
authors.affiliation: Department of Pediatrics, University of California, San Francisco, San Francisco, California 94143-0434, USA. dongq@peds.ucsf.edu
authors.forename: Qing
authors.initials: Q
authors.lastname: Dong
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Cheng
authors.forename: Wenhan
authors.initials: W
authors.lastname: Chang
authors.forename: Brigitte E
authors.initials: BE
authors.lastname: Blackman
authors.forename: Felix A
authors.initials: FA
authors.lastname: Conte
authors.forename: Jianxin
authors.initials: J
authors.lastname: Hu
authors.forename: Dolores
authors.initials: D
authors.lastname: Shoback
authors.forename: Walter L
authors.initials: WL
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003241:Consanguinity; D005260:Female; D017353:Gene Deletion; D006801:Humans; D006934:Hypercalcemia / Q000235:genetics; D006961:Hyperparathyroidism / Q000151:congenital / Q000235:genetics; D007223:Infant; D009154:Mutation / Q000502:physiology; D010375:Pedigree; D017434:Protein Structure, Tertiary / Q000235:genetics; D044169:Receptors, Calcium-Sensing / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology
chemical_list: D044169:Receptors, Calcium-Sensing; D043562:Receptors, G-Protein-Coupled; D002118:Calcium