Differential regulation of muscarinic M(1 )receptors by orthosteric and allosteric ligands

BACKGROUND: Activation of muscarinic M(1 )receptors is mediated via interaction of orthosteric agonists with the acetylcholine binding site or via interaction of allosteric agonists with different site(s) on the receptor. The focus of the present study was to determine if M(1 )receptors activated by allosteric agonists undergo the same regulatory fate as M(1 )receptors activated by orthosteric agonists. RESULTS: The orthosteric agonists carbachol, oxotremorine-M and pilocarpine were compared to the allosteric agonists AC-42, AC-260584, N-desmethylclozapine and xanomeline. All ligands activated M(1 )receptors and stimulated interaction of the receptors with β-arrestin-1. All ligands reduced cell surface binding and induced the loss of total receptor binding. Receptor internalization was blocked by treatment with hypertonic sucrose indicating that all ligands induced formation of clathrin coated vesicles. However, internalized receptors recycled to the cell surface following removal of orthosteric, but not allosteric agonists. Whereas all ligands induced loss of cell surface receptor binding, no intracellular vesicles could be observed after treatment with AC-260584 or xanomeline. Brief stimulation of M(1 )receptors with AC-260584 or xanomeline resulted in persistent activation of M(1 )receptors, suggesting that continual receptor signaling might impede or delay receptor endocytosis into intracellular vesicles. CONCLUSION: These results indicate that allosteric agonists differ from orthosteric ligands and among each other in their ability to induce different regulatory pathways. Thus, signaling and regulatory pathways induced by different allosteric ligands are ligand specific.