The Role of the Seventh Transmembrane Region in High Affinity Binding of a β₂-Selective Agonist TA-2005

Abstract

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.

The Third Intracellular Loop and the Carboxyl Terminus of β2-Adrenergic Receptor Confer Spontaneous Activity of the Receptor

“…Thus, the third intracellular loop and the C terminus of ␤-ARs are not essentially involved in ligand binding, although those domains play important roles in interacting with G proteins and adenylyl cyclase (O'Dowd et al, 1988;Green et al, 1992). This is consistent with previous reports that the binding domain of ␤-ARs is mainly located in a pocket in the transmembrane domains 3, 5, and 6 (Dixon et al, 1987;Dohlman et al, 1988;Wong et al, 1988;Hockerman et al, 1996) and that other transmembrane domains, such as 2, 6, and 7, may also play a role in determining ␤-AR subtype-selectivity for antagonists (Marullo et al, 1990;Kurose et al, 1998;Isogaya et al, 1998Isogaya et al, , 1999Kikkawa et al, 1998). Thus, the inhibitory effects of ICI 118,551 on the chimera-induced elevations of myocyte contraction and cAMP production is mediated by its inverse agonist functionality, rather than by enhanced binding affinity of ICI 118,551 to those chimeras.…”

β1-Selective Agonist (−)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(−)-RO363] Differentially Interacts with Key Amino Acids Responsible for β1-Selective Binding in Resting and Active States

“…We have studied the binding domains for ␤ 1 -and ␤ 2 -selective agonists and have reported that transmembrane domains 2 and 7 of ␤ 1 -and ␤ 2 -adrenergic receptors form a binding pocket, and that Leu 110 , Thr 117 , and Val 120 in transmembrane domain 2 of ␤ 1 -adrenergic receptor or Tyr 308 in transmembrane domain 7 of ␤ 2 -adrenergic receptor are major determinants for ␤ 1 -or ␤ 2 -selective agonists, respectively (Isogaya et al, , 1999Kikkawa et al, 1998). However, ␤ 1 -selectivity of T-0509 and denopamine [K i (␤ 2 )/K i (␤ 1 )] used in the previous study was at most 10-fold.…”

Efficacy and safety of long-acting beta-2-agonists

“…Кармотерол ведет себя как очень сильный и се лективный агонист β2 АР, обладает в 53 раза бóльшей аффинностью в отношении β 2 АР по сравнению с β 1 АР [44] и приблизительно в 5 раз более селекти вен в отношении β 2 АР, присутствующих в препара те трахеи, чем в тех, которые регулируют хронотроп ный ответ в правом предсердии [45]. В результате воздействия кармотерола продемонстрирована наи большая мощность среди всех LABA при оценке на гладких мышцах трахей морских свинок, сокращен ных под воздействием метахолина [44].…”

“…Длительность расслабления глад кой мускулатуры трахеи под воздействием кармо терола была более длительной по сравнению с формотеролом и салметеролом [44]. Кроме того, в экспериментальных условиях in vivo и in vitro про демонстрированы быстрое начало и длительность действия препарата [45].…”