Activation of G protein-coupled receptors like the ␤ 1 -adrenergic receptor results in conformational changes that ultimately lead to signal propagation through a G protein to an effector like adenylyl cyclase. In this study we identified amino acids that seem to be critical for activation of the human ␤ 1 -adrenergic receptor. Activation patterns of mutant receptors were analyzed using two structurally different ligands for ␤-adrenergic receptors that both are mixed agonist/antagonists.

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... rol and terbutaline are agonists at ␤ 2and ␤ 3 -receptors; however, they act as antagonists at the ␤ 1 -subtype. We reasoned that this functional selectivity may be reflected by a corresponding sequence pattern in the receptor subtypes. Therefore, we exchanged single amino acids of the ␤ 1 -adrenergic receptor for residues that were identical in the ␤ 2 -and ␤ 3 -subtypes but different in the ␤ 1 -receptor. Pharmacological characterization of such receptor mutants revealed that binding of a panel of agonists and antagonists including broxaterol and terbutaline was unaltered. However, two of the mutants (I185V and D212N) were activated by broxaterol and terbutaline, which acted as antagonists at the wild-type receptor. Two additional mutants (V120L and K253R) could be activated by terbutaline alone, which is structurally more closely related to endogenous catecholamines like epinephrine than to broxaterol. A model of the human ␤ 1 -adrenergic receptor showed that the four gain-of-function mutations are outside of the putative ligand-binding domain substantiating the lack of an effect of the mutations on binding characteristics. These results support the notion that Val-120, Ile-185, Asp-212, and Lys-253 are critically involved in conformational changes occurring during receptor activation. . 3 The abbreviations used are: GPCR, G protein-coupled receptor; [ 125 I]CYP, (Ϫ)-3-[ 125 I]iodocyanopindolol; TM, transmembrane domain; E2 loop, second extracellular loop.