Characterization of 伪_2B-adrenoceptor ligand binding in the presence of Muscarinic toxin 伪 and delineation of structural features of receptor binding selectivity

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• 作者：Katja Nä ; reoja ; Johnny Nä ; sman ; ^{jonasman@abo.fi}
• 关键词：伪2-Adrenoceptor ; Chimeric receptor ; Extracellular loop ; Ligand binding ; Mamba snake ; Muscarinic toxin
• 刊名：European Journal of Pharmacology
• 出版年：2012
• 期刊代码：24_00142999
• 类别：neu
• 出版时间：15 May, 2012
• 卷：683
• 期：1-3
• 页码：63-70
• 文件大小：948 K

摘要

Muscarinic toxin 伪 (MT伪), a peptide isolated from the venom of the African black mamba, was recently found to selectively antagonize the human 伪_2B-adrenoceptor. To gain more information about the binding of this peptide toxin, we studied the properties of the [³H]UK14,304 agonist and the [³H]MK-912 antagonist binding to the 伪_2B-adrenoceptor in the presence of MT伪. In equilibrium binding experiments, MT伪 decreased the binding of the orthosteric ligands, but failed to completely displace these. This effect of MT伪 was due to noncompetitive inhibition of B_max without change in radioligand affinity. On the contrary, cellular signaling via the 伪_2B-adrenoceptor could be titrated to zero despite the incomplete receptor blockade. To locate binding sites for MT伪 on the receptor protein, we generated chimeric receptors of 伪_2B- and 伪_2A- or 伪_2C-adrenoceptors. Data based on these constructs revealed the extracellular loop two (ECL2) as the structural entity that enables MT伪 binding. Cumulative exchange of parts of ECL2 of 伪_2B for 伪_2A-adrenoceptor sequence resulted in a gradual decrease in the affinity for MT伪, indicating that MT伪 binds to the 伪_2B-adrenoceptor through multiple sites dispersed over the whole ECL2. Together the results suggest that binding of MT伪 to the 伪_2B-adrenoceptor occludes orthosteric ligand access to the binding pocket. Putative homomeric receptor complexes as factors underlying the apparent noncompetitivity are also discussed.