The role of the seventh transmembrane region in high affinity binding of a β₂-selective agonist TA-2005

To determine the structural basis for binding subtype selective agonists in the β-adrenergic receptor (βAR), we examined the interaction of the mutant β₂AR and chimeric β₁/β₂AR with a selective β₂AR agonist, TA- 2005 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxyphenyl)-1- methylethyl]amino]ethyl] carbostyril hydrochloride). The β₂AR mutant with Ala substituted for Ser204 (S204A) significantly decreased the affinities for TA-2005, des-8-hydroxy-TA-2005 derivative (compound I), and isoproterenol. In contrast, a S207A mutation slightly decreased the affinities for TA-2005 and compound I, although the affinity for isoproterenol was decreased dramatically. The EC₅₀ values of TA-2005 to activate adenylyl cyclase were not changed in either the S204A- or S207A-β₂AR. In contrast with TA-2005, the EC₅₀ values of compound I were reduced in the S204A-β₂AR but not in the S207A-β₂AR. These results suggest that Ser204 is important for high affinity binding but not necessary to activate adenylyl cyclase. Although TA- 2005 was highly selective at the β₂AR, the compounds lacking p- methoxyphenyl-ethyl (compound II) or p-methoxyphenyl-methylethyl groups (compound III) on the amine portion of TA-2005 lost β₂AR subtype selectivity. When the second and seventh transmembrane (TM) region but not the TM1 region of the β₂AR were raplaced with the corresponding regions of the β₁AR, the affinities of the chimeras for TA-2005 decreased compared with those of the wild type β₂AR. Furthermore, substitution of the TM7 region of the β₁AR with the corresponding region of the β₂AR significantly increased the affinities for TA-2005. The affinities for isoproterenol and compounds II and III were not affected in the chimeras. These data suggest that the TM7 region of the β₂AR plays an important role in β₂-selective agonist binding. To determine the specific amino acid which confers this high affinity binding of TA-2005 to the β₂AR, an alanine- scanning mutagenesis approach was employed. All amino acids that were different from those of the β₁AR were individually changed to alanine. One mutant receptor (Y308A-β₂AR) out of 10 point-mutated β₂ARs showed a dramatically reduced affinity for TA-2005. These results indicate that Tyr308 is an essential amino acid for high affinity binding of the β₂-selective agonist TA-2005.