A polymorphism of the human beta 2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor
Green, S.A.; Cole, G.; Jacinto, M.; Innis, M.; Liggett, S.B.
Journal of Biological Chemistry 268(31): 23116-23121
1993
ISSN/ISBN: 0021-9258 PMID: 7901205 Accession: 045094457
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We have recently identified several naturally occurring variants of the human beta 2-adrenergic receptor (beta 2AR). One of these polymorphisms, 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 adjacent to Ser165 which has been predicted to interact with the beta-carbon hydroxyl group of adrenergic ligands. To determine the functional significance of this variant, we constructed by site-directed techniques a mutated beta 2AR (Ile164) with this substitution and expressed it in CHW-1102 cells. In the presence of guanine nucleotide, Ile164 displayed a lower binding affinity for epinephrine as compared with the wild-type beta 2AR (Ki = 1450 +/- 79 versus 368 +/- 39 nM; p < 0.001). A similarly decreased affinity was found with the catecholamines isoproterenol and norepinephrine, but not with dobutamine or dopamine which lack hydroxyl groups on their beta-carbons. In addition, antagonists without aromatic ring polar substituents displayed a decreased affinity for the mutated receptor. In agonist competition experiments conducted in the absence of guanine nucleotide, Ile164 failed to exhibit detectable high affinity binding, suggesting an impairment in the formation of the agonist-receptor-Gs complex. Consistent with this finding, functional coupling to Gs as determined in adenylyl cyclase assays was significantly (approximately 50%) depressed with Ile164 under both basal and agonist-stimulated conditions. beta 2AR sequestration, which is also triggered by agonist binding, was also found to be approximately 65% reduced in the Ile164 polymorphism. This study represents the first characterization of a naturally occurring mutation of a human adrenergic receptor. Our findings generally support the hypothesized role of this region of the receptor for ligand binding and receptor activation, as well as for establishing critical interactions for overall receptor conformation.