Received for publication April 30, 2008.
Revised July 8, 2008.
Accepted for publication August 7, 2008.

Role of key transmembrane residues in agonist and antagonist actions at the two conformations of the human 1-adrenoceptor

Abstract

Functional studies with CGP 12177 at the human 1-adrenoceptor have provided evidence for two binding sites that have markedly different pharmacological properties. Here, key transmembrane residues (D104, D138, S228, S229, S232, F341, N344 and N363) have been mutated to provide structural insights into the nature of these sites. ³H-CGP 12177 binding and CRE-mediated reporter gene studies confirmed that CGP 12177 acted as a neutral antagonist (log K_D = -9.18) at the "catecholamine site" and as an agonist at the "CGP12177 site" (log EC₅₀ = -8.12). Agonist responses to isoprenaline and CGP12177 had different sensitivities to 1-antagonists (e.g. CGP 20712A; log K_D = -8.65 and -7.26 respectively). Site-directed mutagenesis showed that N363 and D138 were key residues for binding of both agonists and antagonists, and were also essential for the agonist actions of CGP 12177. S228A and S229A in TM5 reduced the binding of CGP 12177 and had an identical effect on its agonist and antagonist actions. Both N344A and F341A in TM6, however, abolished the ability of CGP20712A to discriminate between responses elicited by isoprenaline and CGP12177. The fact that both D138 and N363 are absolutely required for CGP12117 binding in both agonist and antagonist modes leads to the conclusion that the secondary agonist binding site for CGP12117 must overlap with the catecholamine binding site. Modelling 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 D138 and N363.

Key words: Adrenergic, Gs family, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches, Receptor binding studies