A Double Mutation in the Extracellular Ca2+-sensing Receptor's Venus Flytrap Domain That Selectively Disables l-Amino Acid Sensing

Abstract

The extracellular Ca 2؉ -sensing receptor is activated allosterically by L-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca 2؉ -sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca 2؉ sensing intact, as determined by receptor-dependent activation of intracellular Ca 2؉ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to L-Phe, and T145A also exhibited markedly impaired L/D selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca 2؉ but was resistant to L-Phe at concentrations up to 100 mM. We conclude that T145A/S170T selectively disables L-amino acid sensing and that the Ca 2؉ and Lamino acid-sensing functions of the CaR can be dissociated.

Allosteric Modulation of the Calcium-sensing Receptor by γ-Glutamyl Peptides

“…5C) whereas HEK-293 cells expressing the T145A/S170T double mutant CaR exhibited no change in Ca 2ϩ o sensitivity (Fig. 5D) as reported previously (15).…”

“…S1). The results indicate that ␥-glutamyl peptides and S-methylgluta- (14) and glutathione (21) binding sites to the VFT domain and we previously identified T145A/S170T as a double mutant form of the VFT domain with near-normal Ca 2ϩ o -sensitivity but markedly impaired L-amino acid sensing (15). In the current study, we first confirmed that this mutant exhibited normal or near-normal Ca 2ϩ o sensitivity in the absence of L-Phe or S-methylglutathione (Fig.…”

“…Cell Culture of Control and Stably Transfected HEK-293 cells-Human embryonic kidney (HEK)-293 cells stably expressing the CaR (HEK-CaR cells), untransfected HEK-293 cells, and HEK-293 cells that stably expressed a double mutant form of the CaR (HEK-CaR-T145A/S170T cells) were thawed from frozen stocks and cultured in DMEM (Invitrogen)/10% fetal bovine serum as described previously (15). In general, cultured cells were studied between passage numbers 4 and 20.…”

“…1). Based on chimeric receptor and mutational analyses, L-amino acids bind in the receptor N-terminal Venus Fly Trap (VFT) domain (14) and the effects of L-amino acids are selectively impaired by a double mutant (T145A/S170T), which exhibits normal Ca 2ϩ o -sensing (15). Comparative molecular modeling of the mGlu-1 and CaR VFT domain ligand binding surfaces indicates that, whereas the amino acid side-chain binding region is tightly defined by a cluster of positively charged residues in mGlu-1 and other mGlus (16,17) it is relatively unrestricted in the CaR and closely related class C GPCRs including GPRC6A and T1R1 (18).…”

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

“…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]. The Quikchange II sitedirected mutagenesis protocol was used to introduce the point mutations F706A, L797A and E803A.…”