title: Novel aromatic residues in transmembrane domains IV and V involved in agonist binding at alpha(1a)-adrenergic receptors.
abstract: We examined the role that aromatic residues located in the transmembrane helices of the alpha(1a)-adrenergic receptor play in promoting antagonist binding. Since alpha(1)-antagonists display low affinity binding at beta(2)-adrenergic receptors, two phenylalanine residues, Phe-163 and Phe-187, of the alpha(1a)-AR were mutated to the corresponding beta(2)-residue. Neither F163Q nor F187A mutations of the alpha(1a) had any effect on the affinity of the alpha(1)-antagonists. However, the affinity of the endogenous agonist epinephrine was reduced 12.5- and 8-fold by the F163Q and F187A mutations, respectively. An additive loss in affinity (150-fold) for epinephrine was observed at an alpha(1a) containing both mutations. The loss of agonist affinity scenario could be reversed by a gain of affinity with mutation of the corresponding residues in the beta(2) to the phenylalanine residues in the alpha(1a). We propose that both Phe-163 and Phe-187 are involved in independent aromatic interactions with the catechol ring of agonists. The potency but not the efficacy of epinephrine in stimulating phosphatidylinositol hydrolysis was reduced 35-fold at the F163Q/F187A alpha(1a) relative to the wild type receptor. Therefore, Phe-163 and Phe-187 represent novel binding contacts in the agonist binding pocket of the alpha(1a)-AR, but are not involved directly in receptor activation.
authors.affiliation: Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Waugh
authors.forename: M M
authors.initials: MM
authors.lastname: Zhao
authors.forename: M J
authors.initials: MJ
authors.lastname: Zuscik
authors.forename: D M
authors.initials: DM
authors.lastname: Perez
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004809:Ephedrine / Q000378:metabolism; D004837:Epinephrine / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D000316:Adrenergic alpha-Agonists; D007295:Inositol Phosphates; D018340:Receptors, Adrenergic, alpha-1; D010649:Phenylalanine; D004809:Ephedrine; D004837:Epinephrine