title: Characterization of α(2B)-adrenoceptor ligand binding in the presence of muscarinic toxin α and delineation of structural features of receptor binding selectivity.
abstract: Muscarinic toxin α (MTα), a peptide isolated from the venom of the African black mamba, was recently found to selectively antagonize the human α(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 α(2B)-adrenoceptor in the presence of MTα. In equilibrium binding experiments, MTα decreased the binding of the orthosteric ligands, but failed to completely displace these. This effect of MTα was due to noncompetitive inhibition of B(max) without change in radioligand affinity. On the contrary, cellular signaling via the α(2B)-adrenoceptor could be titrated to zero despite the incomplete receptor blockade. To locate binding sites for MTα on the receptor protein, we generated chimeric receptors of α(2B)- and α(2A)- or α(2C)-adrenoceptors. Data based on these constructs revealed the extracellular loop two (ECL2) as the structural entity that enables MTα binding. Cumulative exchange of parts of ECL2 of α(2B) for α(2A)-adrenoceptor sequence resulted in a gradual decrease in the affinity for MTα, indicating that MTα binds to the α(2B)-adrenoceptor through multiple sites dispersed over the whole ECL2. Together the results suggest that binding of MTα to the α(2B)-adrenoceptor occludes orthosteric ligand access to the binding pocket. Putative homomeric receptor complexes as factors underlying the apparent noncompetitivity are also discussed.
authors.affiliation: Department of Biosciences, Åbo Akademi University, Tykistökatu 6, FIN-20520 Turku, Finland.
authors.forename: Katja
authors.initials: K
authors.lastname: Näreoja
authors.forename: Johnny
authors.initials: J
authors.lastname: Näsman
mesh_terms: D058647:Adrenergic alpha-2 Receptor Agonists / Q000378:metabolism / Q000494:pharmacology; D058669:Adrenergic alpha-2 Receptor Antagonists / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000495:Allosteric Site; D000818:Animals; D000068438:Brimonidine Tartrate; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D004546:Elapid Venoms / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D050506:Mutant Chimeric Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D054730:Protein Interaction Domains and Motifs; D011807:Quinolizines / Q000378:metabolism / Q000494:pharmacology; D011810:Quinoxalines / Q000378:metabolism / Q000494:pharmacology; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D030162:Reptilian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D018411:Spodoptera
chemical_list: C547768:ADRA2A protein, human; C547771:ADRA2B protein, human; C547774:ADRA2C protein, human; D058647:Adrenergic alpha-2 Receptor Agonists; D058669:Adrenergic alpha-2 Receptor Antagonists; D004546:Elapid Venoms; D008024:Ligands; D050506:Mutant Chimeric Proteins; D009498:Neurotoxins; D010455:Peptides; D011807:Quinolizines; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D030162:Reptilian Proteins; C574795:muscarinic toxin alpha, Dendroaspis polylepis; C054290:L 657743; D000068438:Brimonidine Tartrate