Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor
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Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor. / Elling, C E; Thirstrup, K; Holst, Birgitte; Schwartz, T W.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, No. 22, 1999, p. 12322-7.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor
AU - Elling, C E
AU - Thirstrup, K
AU - Holst, Birgitte
AU - Schwartz, T W
PY - 1999
Y1 - 1999
N2 - Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu(2+), and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure-activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.
AB - Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu(2+), and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure-activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.
KW - Adrenergic beta-Agonists
KW - Amino Acid Sequence
KW - Chelating Agents
KW - Metals
KW - Molecular Probes
KW - Molecular Sequence Data
KW - Mutagenesis, Site-Directed
KW - Receptors, Adrenergic, beta-2
KW - Signal Transduction
M3 - Journal article
C2 - 10535920
VL - 96
SP - 12322
EP - 12327
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 22
ER -
ID: 137292486