Binding Pockets of the β₁- and β₂-Adrenergic Receptors for Subtype-Selective Agonists

Abstract

We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.

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

“…The mutation of Val-120 to Ala shows an indirect contribution to subtype selectivity of selected agonists (8). Although ligand binding was not affected in our study we also found that activation by terbutaline, but not by broxaterol, was changed by mutation of Val-120 to Leu in our case, confirming a significant role of this position.…”

“…Isogaya et al (8) describe a number of mutations including mutation of Val-120, which is one of the positions that led to a gain-of-function mutation in our study. The mutation of Val-120 to Ala shows an indirect contribution to subtype selectivity of selected agonists (8).…”

“…3B some amino acids thought to be involved in agonist recognition are shown. These amino acids were identified previously as critical for agonist binding in models for both ␤ 1 -(30) and ␤ 2 -receptors (7,8). In addition, terbutaline is docked into the putative binding site of the receptor.…”

“…For mutation of such positions functionally different regions were selected. As the putative ligand-binding domain is embedded in the transmembrane domains (7,8) several mutations were introduced in transmembrane regions. The two mutants L154V and K253R are at the interface between transmembrane domains and intracellular loops two and three, respectively, which are thought to be involved in receptor-G protein coupling (9).…”

Beta-Blockers Show Inverse Agonism to a Novel Constitutively Active Mutant of β1-Adrenoceptor

“…As with our findings, these investigations reported that amino acid substitutions were introduced within transmembrane domains. Another investigation confirmed that replacement of eight amino acids of transmembrane domain 2 of the β 1 -AR had shown that this transmembrane domain is a major determinant of the highaffinity binding of the β 1 -AR selective agonists (28). However, an amino acid in the same domain was not considered in the present study.…”