Novel Mutants of the Human β1-Adrenergic Receptor Reveal Amino Acids Relevant for Receptor Activation.

Activation of G protein-coupled receptors like the β1-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 β1-adrenergic receptor. Activation patterns of mutant receptors were analyzed using two structurally different ligands for β1-adrenergic receptors that both are mixed agonist/antagonists. Broxaterol and terbutaline are agonists at β2 and β3-receptors; however, they act as antagonists at the β1-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 β1-adrenergic receptor for residues that were identical in the β2- and β3-subtypes but different in the β1-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 (1185V 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 β1-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. [ABSTRACT FROM AUTHOR]