Novel aromatic residues in transmembrane domains IV and V involved in agonist binding at α(1a)-adrenergic receptors

Abstract

We examined the role that aromatic residues located in the transmembrane helices of the α(1a)-adrenergic receptor play in promoting antagonist binding. Since α1-antagonists display low affinity binding at β2- adrenergic receptors, two phenylalanine residues, Phe-163 and Phe-187, of the α(1a)-AR were mutated to the corresponding β2-residue. Neither F163Q nor F187A mutations of the α(1a) had any effect on the affinity of the α1- 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 α(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 β2 to the phenylalanine residues in the α(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 α(1a) relative to the wild type receptor. Therefore, Phe-163 and Phe-187 represent novel binding contacts in the agonist binding pocket of the α(1a)-AR, but are not involved directly in receptor activation.