Identification of acidic residues in the extracellular loops of the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+ and a positive allosteric modulator.
We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca(2+) receptor (hCaR) in receptor activation by Ca(2+) and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca(2+), and on NPS R-568 augmentation of sensitivity to Ca(2+). Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp(758), Glu(759), and Glu(767), increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca(2+). Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca(2+). Mutation of Glu(837) in exo-loop 3 to alanine did not alter Ca(2+) sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu(837) drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu(837) at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR.
Hu
Jianxin
J
Molecular Pathophysiology Section, NIDCD, and the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. jianxinh@ intra.niddk.nih.gov
Reyes-Cruz
Guadalupe
G
Chen
Wangzhong
W
Jacobson
Kenneth A
KA
Spiegel
Allen M
AM
D000409:Alanine / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D002135:Calcium-Binding Proteins; D000409:Alanine; D002118:Calcium