Expression and Characterization of Inactivating and Activating Mutations in the Human Ca2+-sensing Receptor

Abstract

Nearly 30 mutations have been identified to date in the coding region of the extracellular calcium-sensing receptor (CaR) that are associated with inherited human hypo-and hypercalcemic disorders. To understand the mechanisms by which the mutations alter the function of the receptor may help to discern the structurefunction relationships in terms of ligand-binding and G protein coupling. In the present studies, we transiently expressed eight known CaR mutations in HEK293 cells. The effects of the mutations on extracellular calciumand gadolinium-elicited increases in the cytosolic calcium concentration were then examined. Seven inactivating mutations, which cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, show a reduced functional activity of the receptor because they may 1) reduce its affinity for agonists; 2) prevent conversion of the receptor from a putatively immature, high mannose form into the fully glycosylated and biologically active form of the CaR, in addition to lowering its affinity for agonists; or 3) fail to couple the receptor to and/or activate its respective G protein(s). Conversely, one activating mutation, which causes a form of autosomal dominant hypocalcemia, appears to increase the affinity of the receptor for its agonists.The recently cloned extracellular Ca 2ϩ (Ca 2ϩ o )-sensing receptor (CaR) 1 (1) has provided key insights into the pathogenesis of inherited human hypo-and hypercalcemic disorders (2, 3). The receptor, BoPCaR (bovine parathyroid Ca 2ϩ -sensing receptor) was first isolated from bovine parathyroid using expression cloning in Xenopus laevis oocytes and shows pharmacological properties nearly identical to those of the native receptor in its responses to extracellular divalent cations (i.e. Ca o ), and polyamines (e.g. neomycin) (1). Subsequently, cDNAs encoding the human homolog of the same receptor have been cloned from human parathyroid (4) and kidney (5), using a homology-based strategy. The human and bovine receptors share a high degree of homology at the amino acid level (93% identity). Stimulation of the CaR by agonists activates phospholipase C, with resultant increases in inositol phosphates and the cytosolic calcium concentration (Ca 2ϩ

Functional Calcium-sensing Receptors in Rat Fibroblasts Are Required for Activation of SRC Kinase and Mitogen-activated Protein Kinase in Response to Extracellular Calcium

“…Furthermore, we have used the nonfunctional CaR mutant R796W as a tool to disrupt the function of the endogenous CaR. In addition to its identification as the genetic mutation responsible for at least one form of hereditary severe neonatal hyperparathyroidism (15), CaR-R796W has been shown to function as an interfering mutant of the CaR when co-expressed with wild-type CaR in HEK293 cells and assayed by measurement of intracellular calcium release in response to Gd 3ϩ (16). We have shown that overexpression of CaR-R796W substantially inhibited the changes in SRC activity and ERK1 activation observed in response to agonists of the endogenous CaR.…”

“…In particular, a mutation of arginine 796 to tryptophan in the third intracellular loop of the CaR was found to cause neonatal severe hyperparathyroidism when homozygous and hypocalciuric hypercalcemia when heterozygous (15). This CaR-R796W mutant was nonfunctional when assayed for Gd 3ϩ -stimulated intracellular calcium release in Xenopus oocytes (15) and has been characterized as a "dominant negative" mutant when co-expressed with wild-type CaR in HEK293 cells or in human parathyroid cells (16).…”

Roles of intraloops‐2 and ‐3 and the proximal C‐terminus in signalling pathway selection from the human calcium‐sensing receptor

“…The wild-type (WT) human CaSR cDNA (cassette version, [20]) cloned between the Kpn I and Xba I sites of pcDNA3.1 (+) {pcDNA3.1 (+)-WTCaSR} was a kind gift from Dr. Mei Bai and Prof. Edward Brown (Endocrine-Hypertension Division and Membrane Biology Program, Brigham and Women's Hospital, Boston, MA, USA). All mutants were generated in pcDNA3.1(+)-WTCaSR and/ or pcDNA3.1(+)-WTCaSR(FLAG) plasmid, which contains the FLAG epitope DYKDDDDK between residues 371 and 372; insertion of the FLAG epitope at this position has been shown previously to have no impact on receptor function [21].…”

Pseudohypoparathyroidism type I‐b with neurological involvement is associated with a homozygous PTH1R mutation

“…Genetic forms of HP are rare and usually associated with mutations in four genes: calcium‐sensing receptor ( CASR ) (Bai et al 1996; Pearce et al 1996; Watanabe et al 1998), glial cells missing homolog 2 ( GCM2 ) (Ding et al 2001), guanine nucleotide‐binding protein alpha‐11 ( GNA11 ) (Mannstadt et al 2013), and PTH itself (Arnold et al 1990; Parkinson & Thakker 1992; Sunthornthepvarakul et al 1999). …”