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.
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.
1 We have studied the di erence in receptor binding activity between partial and full b 2 -adrenoceptor agonists and the abilities of the agonists to interact with Ser 204 and Ser 207 in the ®fth transmembrane region of the b 2 -adrenoceptor, amino acid residues that are important for activation of the b 2 -adrenoceptor. (7)-isoprenaline) and b 2 -(83% and 74% of (7)-isoprenaline) adrenoceptors. 4 The a nities of full agonists, TA-2005 and (7)-isoprenaline, were markedly decreased by substitution of Ala for Ser 204 (S204A) of the b 2 -adrenoceptor, whereas this substitution slightly reduced the a nities of partial agonists, (+)-salbutamol and (+)-salmeterol. Although the a nities of full agonists for the S207A-b 2 -adrenoceptor were decreased, those of partial agonists for the S207A-b 2 -adrenoceptor were essentially the same as for the wild type receptor. 5 The constitutively active mutant (L266S, L272A) of the b 2 -adrenoceptor had an increased a nity for all four agonists. The a nities of full agonists were decreased by substitution of Ser 204 of the constitutively active mutant, whereas the degree of decrease was smaller than that caused by the substitution of the wild type receptor. Although the a nities of (+)-salbutamol and (+)-salmeterol for the S207A-b 2 -adrenoceptor were essentially the same as those for the wild type b 2 -adrenoceptor, the a nities of (+)-salbutamol and (+)-salmeterol for the constitutively active b 2 -adrenoceptor were decreased by substitution of Ser 207 . 6 These results suggest that Ser 204 and Ser 207 of the wild type and constitutively active b 2 -adrenoceptors di erentially interacted with b 2 -selective agonists.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.