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

Hideo Kikkawa, Masafumi Isogaya, Taku Nagao, Hitoshi Kurose

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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 β12AR 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.

Original languageEnglish
Pages (from-to)128-134
Number of pages7
JournalMolecular Pharmacology
Issue number1
Publication statusPublished - Jan 1998
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology


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