title: Identification by site-directed mutagenesis of residues involved in ligand recognition and activation of the human A3 adenosine receptor.
abstract: Ligand recognition has been extensively explored in G protein-coupled A(1), A(2A), and A(2B) adenosine receptors but not in the A(3) receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A(3) adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His(95), Trp(243), Leu(244), Ser(247), Asn(250), and Lys(152). The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by approximately 3-38-fold, suggesting that these residues were critical for the high affinity of A(3) adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A(3) agonist 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5'-gamma-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp(243) was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A(3) receptor interactions.
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Zhan-Guo
authors.initials: ZG
authors.lastname: Gao
authors.forename: Aishe
authors.initials: A
authors.lastname: Chen
authors.forename: Dov
authors.initials: D
authors.lastname: Barak
authors.forename: Soo-Kyung
authors.initials: SK
authors.lastname: Kim
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D058906:Purinergic P1 Receptor Agonists; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D008024:Ligands; D010716:Phosphatidylinositols; D058906:Purinergic P1 Receptor Agonists; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D016244:Guanosine 5'-O-(3-Thiotriphosphate)