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

We examined the role that aromatic residues located in the transmembrane he lices of the alpha(1a)-adrenergic receptor play in promoting antagonist bin ding. Since alpha(1)-antagonists display low affinity binding at beta(2)-ad renergic receptors, two phenylalanine residues, Phe-163 and Phe-187, of the alpha(1a)-AR were mutated to the corresponding beta(2)-residue. Neither F1 63Q 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 e pinephrine was reduced 12.5- and 8-fold by the F163Q and F187A mutations, r espectively. An additive loss in affinity (150-fold) for epinephrine was ob served at an alpha(1a) containing both mutations. The loss of agonist affin ity scenario could be reversed by a gain of affinity with mutation of the c orresponding residues in the beta(2) to the phenylalanine residues in the a lpha(1a). We propose that both Phe-163 and Phe-187 are involved in independ ent aromatic interactions with the catechol ring of agonists, The potency b ut not the efficacy of epinephrine in stimulating phosphatidylinnostto hydr olysis was reduced 35-fold at the F163Q/F187A alpha(1a) relative to the wil d type receptor. Therefore, Phe-163 and Phe-187 represent novel binding con tacts in the agonist binding pocket of the alpha(1a)-AR, but are not involv ed directly in receptor activation.