Role of key transmembrane residues in agonist and antagonist actions at the two conformations of the human beta1-adrenoceptor.

Studies with 4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP 12177) at the human beta1-adrenoceptor have provided evidence for two binding modes or conformations that have markedly different pharmacological properties. Here, key transmembrane residues (Asp104, Asp138, Ser228, Ser229, Ser232, Phe341, Asn344 and Asn363) have been mutated to provide structural insights into the nature of these conformations. [(3)H]CGP 12177 binding and cAMP response element-mediated reporter gene studies confirmed that CGP 12177 was a neutral antagonist (log K(D) = -9.18) at the "catecholamine site" and an agonist at the "CGP 12177 site" (log EC(50) = -8.12). Agonist responses to isoprenaline and CGP 12177 had different sensitivities to beta1-antagonists (e.g., CGP 20712A; log K(D) = -8.65 and -7.26, respectively). Site-directed mutagenesis showed that Asn363 and Asp138 were key residues for binding of agonists and antagonists, and they were also essential for the agonist actions of CGP 12177. S228A and S229A in transmembrane-spanning region (TM) 5 reduced the binding of CGP 12177 and had an identical effect on its agonist and antagonist actions. Both N344A and F341A in TM6 abolished the ability of CGP 20712A to discriminate between responses elicited by isoprenaline and CGP 12177. The fact that both Asp138 and Asn363 are absolutely required for CGP 12117 binding in both agonist and antagonist modes leads to the conclusion that the secondary agonist binding site for CGP 12117 must overlap with the catecholamine binding site. Modeling studies provide a basis for these overlapping sites with either the tert-butylamino group or the hydroxyethyloxy and imidazolone portions of CGP 12177 capable of forming polar interactions with Asp138 and Asn363.