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.

[familial hypocalciuric hypercalcemia]

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 .T he aim of the study was to characterize the CaSR mutations causing neonatal severe hyperparathyroidism in a consanguineous family .Parathyroid glands from the index patient were stained for CaSR protein . The CaSR gene was sequenced , mutations were recreated in CaSR cDNA , and HEK 293 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 .Immunocytochemistry showed reduced intracellular CaSR in patient parathyroids . An in -frame homozygous deletion /insertion mutation , c .1031 > 1034 (delACAAinsT ), replaced His 344 -Asn 345 with a single Leu in CaSR loop III . The mutant reduced cell surface expression of CaSR in transfected HEK 293 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 .Deletion of His 344 -Asn 345 in the ECD loop III region affects cell surface targeting of CaSR in transfected cells and in affected parathyroid glands . Absence of conserved Asn 345 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 .