Functional Importance of the Ala116–Pro136 Region in the Calcium-sensing Receptor

Abstract

The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca 2؉ have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala 116 -Pro 136 region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening assay. Selected mutants were characterized further in an inositol phosphate assay. The vast majority of the mutants tested displayed an increased affinity for Ca 2؉ . Furthermore, 21 of the mutants showed increased basal activity in the absence of agonist. This constitutive activity was not diminished when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala 116 -Pro 136 region is of key importance for the maintenance of the inactive conformation of CaR.The calcium-sensing receptor (CaR) 1 belongs to family C of the 7 transmembrane (7TM) G-protein-coupled receptors (GPCRs) (1, 2). Besides CaR, the family comprises eight metabotropic glutamate receptors (mGluR1-8) (3, 4), two ␥-aminobutyric acid type B receptors (GABA B R1-2) (5), and a subfamily of putative pheromone receptors (6). The receptors in this family consist of a peptide chain longer than those of previously identified GPCRs and share no amino acid sequence similarity with any of these. Most notable is the unusually long amino-terminal domain (ATD) of ϳ500 -600 amino acids, which has been shown to contain the site of agonist binding of CaR (7,8), mGluR1 (9, 10), and GABA B R1a (11, 12).The ATDs of the family C receptors have limited amino acid sequence similarity with procaryotic periplasmic binding proteins (PBPs), a family of proteins involved in the transport of nutrients into bacteria (13). Based on the crystal structure of one of these PBPs, the leucine/isoleucine/valine-binding protein, we have previously proposed a tertiary molecular model of the ATD of mGluR1 (9). Recently, Pin and co-workers (11) have presented a model of the ATD of GABA B R1a based on the crystal structure of the leucine-binding protein. The ATD of the family C receptor is thought to be constituted by two globular lobes separated by a hinge region creating a cleft. Two pseudoconserved amino acids, corresponding to Ser 79 and Thr 102 in the leucine/isoleucine/valine-binding protein, have been identified as agonist binding residues in mGluR1 and GABA B R1 (9, 11). Fu...

Enhanced Ca 2+ -Sensing Receptor Function in Idiopathic Pulmonary Arterial Hypertension

“…One of the hallmarks of CaSR is the cysteine-rich large N-terminal extracellular domain (ECD, approximately 600 amino acids). The region between alanine 116 and proline 136 in the ECD is important for maintaining CaSR in an inactive conformation 28 and is associated with the activating mutations or single nucleotide polymorphism (SNPs) identified in the human CaSR gene. The ligands or activators of CaSR include polyvalent cations (e.g., Ca 2+ , Mg 2+ , Gd 3+ ), polypeptides (e.g., amyloid-β peptide), polyamines (e.g., spermine, spermidine, putrescine), aminoglycoside antibiotics (e.g., neomycin, kanamycin), and amino acids (e.g., phenylalanine, tyrosine, tryptophan, glutamate).…”

Human Ca2+ Receptor Extracellular Domain

“…Most of these mutations are located in the ECD, and loop II is by far the most frequent site with six mutations thus far identified between Ala 116 and Cys 129 (2). Random saturation mutagenesis of loop II also yielded multiple mutants with increased sensitivity to calcium (17).…”

Construction of a High Affinity Zinc Binding Site in the Metabotropic Glutamate Receptor mGluR1

“…The buffer was removed, and the cells were incubated for 40 min in the same buffer containing various concentrations of Glu, a fixed Glu concentration and various concentrations of ZnCl 2 or 100 M CPCCOEt, or containing a fixed ZnCl 2 concentration and various concentrations of Glu. The reactions were stopped by exchanging the buffer with 500 l of ice-cold 20 mM formic acid, and separation of total [ 3 H]inositol phosphates was carried out by ion exchange chromatography as described previously (23,25,26). The effect of ZnCl 2 on tsA cells transfected with CaR-pSI or m5-pCD was studied in the same manner, except that CaCl 2 and acetylcholine were used as agonists, respectively.…”