A POLYMORPHISM OF THE HUMAN BETA(2)-ADRENERGIC RECEPTOR WITHIN THE 4TH TRANSMEMBRANE DOMAIN ALTERS LIGAND-BINDING AND FUNCTIONAL-PROPERTIESOF THE RECEPTOR

We have recently identified several naturally occurring variants of th e human beta2-adrenergic receptor (beta2AR). One of these polymorphism s, which is relatively uncommon, is a mutation occurring in the fourth transmembrane spanning domain, with Ile substituted for Thr at amino acid 164 within the proposed ligand binding pocket. This mutation is a djacent to Ser165 which has been predicted to interact with the beta-c arbon hydroxyl group of adrenergic ligands. To determine the functiona l significance of this variant, we constructed by site-directed techni ques a mutated beta2AR (Ile164) with this substitution and expressed i t in CHW-1102 cells. In the presence of guanine nucleotide, Ile164 dis played a lower binding affinity for epinephrine as compared with the w ild-type beta2AR (K(i) = 1450 +/- 79 versus 368 +/- 39 nM; p < 0.001). A similarly decreased affinity was found with the catecholamines isop roterenol and norepinephrine, but not with dobutamine or dopamine whic h lack hydroxyl groups on their beta-carbons. In addition, antagonists without aromatic ring polar substituents displayed a decreased affini ty for the mutated receptor. In agonist competition experiments conduc ted in the absence of guanine nucleotide, Ile164 failed to exhibit det ectable high affinity binding, suggesting an impairment in the formati on of the agonist-receptor-Gs complex. Consistent with this finding, f unctional coupling to Gs as determined in adenylyl cyclase assays was significantly (approximately 50%) depressed with Ile164 under both bas al and agonist-stimulated conditions. Beta2AR sequestration, which is also triggered by agonist binding, was also found to be approximately 65% reduced in the Ile164 polymorphism. This study represents the firs t characterization of a naturally occurring mutation of a human adrene rgic receptor. Our findings generally support the hypothesized role of this region of the receptor for ligand binding and receptor activatio n, as well as for establishing critical interactions for overall recep tor conformation.