IDENTIFICATION OF CRITICAL DETERMINANTS OF ALPHA(1)-ADRENERGIC RECEPTOR SUBTYPE-SELECTIVE AGONIST BINDING

alpha(1)-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. The three cloned subtypes (alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR), although structurally similar, bind a ser ies of ligands with different relative potencies. This is particularly true for the alpha(1a)-AR, which recognizes a number of agonists and antagonists with 5-50-fold higher affinity than the alpha(1b)- or alph a(1d)-subtypes. Since ligands bind to receptor-residues that are locat ed in the transmembrane spanning domains, we hypothesize that subtype differences in ligand recognition are due to differences in the bindin g properties of nonconserved transmembrane residues, Using site-direct ed mutagenesis, selected putative ligand-binding residues in the alpha (1b)-AR were converted, either individually or in combination, to the corresponding residues in the alpha(1b)-AR. Mutation of two such resid ues (of approximately 172 amino acids in the transmembrane domains) co nverted the agonist binding profile entirely to that of the alpha(1a)- AR. Over 80% of this conversion was due to an Ala(204) --> Val substit ution; the remainder was due to the additional substitution of Leu(314 ) --> Met. To confirm that Ala(204) and Leu(314) are indeed critical f or agonist subtype-selectivity, the equivalent residues in the alpha(1 a)-AR (Val(185) and Met(293)) were reversed of that of the alpha(1b)-A R. Correspondingly, the agonist-binding profile of this double alpha(1 a)-AR mutant reverted to that of the alpha(1b)-AR. From these data, in conjunction with macromolecular modeling of the ligand-binding pocket , a model has been developed, which indicates that the determinants of these two residues for agonist subtype-selectivity are due not only t o interactions between their side chains and specific ligand moieties but also to a critical interaction between these two amino acids.