Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor

Previously metal-ion sites have been used as structural and functional prob es in seven transmembrane receptors (7TM), but as yet ail the engineered si tes 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-bin ding Asp-113 in TM-III-or a His residue introduced at this position-and a C ys residue substituted for Asn-312 in TM-VII. No increase in constitutive a ctivity was observed in the mutant receptors. Signal transduction was activ ated in the mutant receptors not by normal catecholamine ligands but instea d 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 hydro phobic chelators such as phenanthroline or bipyridine protected the cells f rom the toxic effect of, for example Cu2+. and in several cases increased t he affinity of the ions for the agonistic site. Wash-out experiments and st ructure-activity analysis indicated, that the high-affinity chelators and t he metal ions bind and activate the mutant receptor as metal ion guided lig and 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 th e future be used as generic, pharmacologic tools to switch 7TM receptors wi th engineered metal-ion sites on or off at will.