The role of the seventh transmembrane region in high affinity binding of a beta 2-selective agonist TA-2005.

To determine the structural basis for binding subtype selective agonists in the beta-adrenergic receptor (beta AR), we examined the interaction of the mutant beta 2AR and chimeric beta 1/beta 2AR with a selective beta 2AR agonist, TA-2005 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxyphenyl)-1-methyle thy l] amino]ethyl] carbostyril hydrochloride). The beta 2AR 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-beta 2AR. In contrast with TA-2005, the EC50 values of compound I were reduced in the S204A-beta 2AR but not in the S207A-beta 2AR. 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 beta 2AR, the compounds lacking p-methoxyphenyl-ethyl (compound II) or p-methoxyphenyl-methylethyl groups (compound III) on the amine portion of TA-2005 lost beta 2AR subtype selectivity. When the second and seventh transmembrane (TM) region but not the TM1 region of the beta 2AR were replaced with the corresponding regions of the beta 1AR, the affinities of the chimeras for TA-2005 decreased compared with those of the wild type beta 2AR. Furthermore, substitution of the TM7 region of the beta 1AR with the corresponding region of the beta 2AR 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 beta 2AR plays an important role in beta 2-selective agonist binding. To determine the specific amino acid which confers this high affinity binding of TA-2005 to the beta 2AR, an alanine-scanning mutagenesis approach was employed. All amino acids that were different from those of the beta 1AR were individually changed to alanine. One mutant receptor (Y308A-beta 2AR) out of 10 point-mutated beta 2ARs showed a dramatically reduced affinity for TA-2005. These results indicate that Tyr308 is an essential amino acid for high affinity binding of the beta 2-selective agonist TA-2005.