Phe-308 and Phe-312 in transmembrane domain 7 are major sites of alpha(1)-adrenergic receptor antagonist binding - Imidazoline agonists bind like antagonists

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there a re few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-prote in coupled receptor. It has been speculated that antagonist binding is quit e diverse depending upon the chemical structure of the antagonist, which ca n be quite different from agonists, We now report the identification of two phenylalanine residues in transmembrane domain 7 of the alpha (1a)-adrener gic receptor (Phe-312 and Phe-308) that are a major site of antagonist affi nity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4-1200-fold) for the antagonists prazosin, WB4101, BMY7378, () niguldipine, and 5-methylurapidil, with no changes in affinity for phenet hylamine-type agonists such as epinephrine, methoxamine, or phenylephrine, Interestingly, both residues are involved in the binding of all imidazoline -type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than pheneth ylamine-type agonists and may be more antagonist-like, which may explain th eir partial agonist properties. In modeling these interactions with previou s mutagenesis studies and using the current backbone structure of rhodopsin , we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed towar d transmembrane domain 7.