Ligand recognition of serine-cysteine amino acid exchanges in transmembrane domain 5 of α2-adrenergic receptors by UK 14,304

Ligand binding of UK 14,304 reveals notable species (i.e., human-rodent) and receptor-subtype differences of alpha(2)-adrenergic receptors (alpha(2)-ARs). To study the molecular basis of the selectivity of UK 14,304, we compared a series of conservative serine-cysteine exchange mutants at ligand-accessible positions in transmembrane domain 5 of the human and mouse alpha(2A)-ARs. UK 14,304 bound with similar to 200-fold higher affinity to the human alpha(2A)-AR wild-type receptor compared with the human alpha(2A)-ARSer(201) mutant, but only an approximately fivefold difference was seen with the corresponding mouse alpha(2A)-AR variant. These effects of cysteine-serine exchanges only involved the agonist low-affinity forms of the receptors, as the affinity of [H-3]UK 14,304 for the agonist high-affinity receptor populations was not influenced. The apparent affinities of a set of eight structurally diverse alpha(2)-AR ligands (six agonists and two antagonists) were not influenced significantly by the cysteine-serine exchanges (except for oxymetazoline and yohimbine, with up to nine- and eightfold differences in affinity, respectively). We conclude that position 201 (a) plays a primary role in determining observed subtype/species selectivity of UK 14,304 in competitive antagonist radio-ligand binding assays and (b) does not determine the subtype selectivity of chlorpromazine.