Characterization of α2B-adrenoceptor ligand binding in the presence of Muscarinic toxin a and delineation of structural features of receptor binding selectivity

Muscarinic toxin alpha (MT alpha), a peptide isolated from the venom of the African black mamba, was recently found to selectively antagonize the human alpha(2B)-adrenoceptor. To gain more information about the binding of this peptide toxin, we studied the properties of the [H-3]UK14,304 agonist and the [H-3]MK-912 antagonist binding to the alpha(2B)-adrenoceptor in the presence of MT alpha. In equilibrium binding experiments, MT alpha decreased the binding of the orthosteric ligands, but failed to completely displace these. This effect of MT alpha was due to noncompetitive inhibition of B-max without change in radioligand affinity. On the contrary, cellular signaling via the alpha(2B)-adrenoceptor could be titrated to zero despite the incomplete receptor blockade. To locate binding sites for MT alpha on the receptor protein, we generated chimeric receptors of alpha(2B)- and alpha(2A)- or alpha(2C)-adrenoceptors. Data based on these constructs revealed the extracellular loop two (ECL2) as the structural entity that enables MTa binding. Cumulative exchange of parts of ECL2 of alpha(2B) for alpha(2A)-adrenoceptor sequence resulted in a gradual decrease in the affinity for MT alpha, indicating that MT alpha binds to the alpha(2B)-adrenoceptor through multiple sites dispersed over the whole ECL2. Together the results suggest that binding of MT alpha to the alpha(2B)-adrenoceptor occludes orthosteric ligand access to the binding pocket. Putative homomeric receptor complexes as factors underlying the apparent noncompetitivity are also discussed. (C) 2012 Elsevier B. V. All rights reserved.