TY - JOUR T1 - The M1 Muscarinic Receptor Allosteric Agonists, AC-42 and TBPB Bind to a Unique Site Distinct from the Acetylcholine Orthosteric Site JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.110.065771 SP - mol.110.065771 AU - Marlene A Jacobson AU - Constantine Kreatsoulas AU - Danette Pascarella AU - Julie O'Brien AU - Cyrille Sur Y1 - 2010/07/21 UR - http://molpharm.aspetjournals.org/content/early/2010/07/21/mol.110.065771.abstract N2 - Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), TBPB (1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one) and the clozapine metabolite, N-desmethylclozapine. Previously, Spalding et al (Mol Pharmacol (2002) 61:1297-1302) defined a minimal epitope for AC-42 comprising the first 45 amino acids, the third extracellular loop and seventh transmembrane domain. Using chimeric M1 and M3 receptor constructs, the AC42 minimal epitope has been extended to also include transmembrane II. Phe 77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-desmethlyclozapine did not require Phe 77. To further map the binding site of AC-42, TBPB and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into a M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of tryptophan 101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists, AC-42, TBPB and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains. ER -