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

Jianxin Hu, Guadalupe Reyes-Cruz, Wangzhong Chen, Kenneth A. Jacobson, Allen M. Spiegel

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Abstract

We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca2+ receptor (hCaR) in receptor activation by Ca2+ 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 Ca2+, and on NPS R-568 augmentation of sensitivity to Ca2+. Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp758, Glu759, and Glu767, increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca2+. 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 Ca2+. Mutation of Glu837 in exo-loop 3 to alanine did not alter Ca2+ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu837 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 Glu837 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.

Original languageEnglish (US)
Pages (from-to)46622-46631
Number of pages10
JournalJournal of Biological Chemistry
Volume277
Issue number48
DOIs
StatePublished - Nov 29 2002
Externally publishedYes

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N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine
Modulators
Chemical activation
Alanine
Mutation
Conformations
Substitution reactions
Cell Surface Receptors

ASJC Scopus subject areas

  • Biochemistry

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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. / Hu, Jianxin; Reyes-Cruz, Guadalupe; Chen, Wangzhong; Jacobson, Kenneth A.; Spiegel, Allen M.

In: Journal of Biological Chemistry, Vol. 277, No. 48, 29.11.2002, p. 46622-46631.

Research output: Contribution to journalArticle

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abstract = "We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca2+ receptor (hCaR) in receptor activation by Ca2+ 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 Ca2+, and on NPS R-568 augmentation of sensitivity to Ca2+. Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp758, Glu759, and Glu767, increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca2+. 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 Ca2+. Mutation of Glu837 in exo-loop 3 to alanine did not alter Ca2+ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu837 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 Glu837 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.",
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T1 - 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

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AU - Reyes-Cruz, Guadalupe

AU - Chen, Wangzhong

AU - Jacobson, Kenneth A.

AU - Spiegel, Allen M.

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AB - We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca2+ receptor (hCaR) in receptor activation by Ca2+ 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 Ca2+, and on NPS R-568 augmentation of sensitivity to Ca2+. Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp758, Glu759, and Glu767, increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca2+. 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 Ca2+. Mutation of Glu837 in exo-loop 3 to alanine did not alter Ca2+ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu837 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 Glu837 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.

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