α1-adrenergic receptor subtype determinants for 4-piperidyl oxazole antagonists

Mutational studies in conjunction with ligand binding assays were used to examine the basis of alpha(1)-adrenergic receptor subtype selectivity for a series of 4-piperidyloxazole antagonists. A set of chimeric alpha(1A) receptors were created by systematically substituting individual transmembrane domains from alpha(1D) adrenergic receptors. The oxazole antagonists exhibited significant reductions in affinity against the receptor construct alpha(1A/D)(TM2), and moderate reductions in affinity versus constructs alpha(1A/D)(TM5), alpha(1A/B)(TM5), and alpha(1A/D)(TM6) Antagonist affinities for these chimeras exceeded those found for wild type alpha(1D) and alpha(1B). Site-directed mutagenesis methods were then used to explore the role that individual residues in TM2 and TM5 play in ligand binding affinity and selectivity. These studies revealed that mutations at position 86 in the second transmembrane domain and position 185 in the fifth transmembrane domain of the alpha(1A) receptor have a major impact on receptor subtype selectivity.