Davis Christopher N, Bradley Stefania Risso, Schiffer Hans H, Friberg Mikael, Koch Kristian, Tolf Bo-Ragnar, Bonhaus Douglas W, Lameh Jelveh
ACADIA Pharmaceuticals Inc, 3911 Sorrento Valley Blvd, San Diego, CA 92121, USA.
BMC Pharmacol. 2009 Dec 2;9:14. doi: 10.1186/1471-2210-9-14.
Activation of muscarinic M1 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 M1 receptors activated by allosteric agonists undergo the same regulatory fate as M1 receptors activated by orthosteric agonists.
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 M1 receptors and stimulated interaction of the receptors with beta-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 M1 receptors with AC-260584 or xanomeline resulted in persistent activation of M1 receptors, suggesting that continual receptor signaling might impede or delay receptor endocytosis into intracellular vesicles.
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.
毒蕈碱型M1受体的激活是通过正构激动剂与乙酰胆碱结合位点的相互作用，或通过变构激动剂与受体上不同位点的相互作用介导的。本研究的重点是确定变构激动剂激活的M1受体是否经历与正构激动剂激活的M1受体相同的调节命运。
将正构激动剂卡巴胆碱、氧化震颤素-M和毛果芸香碱与变构激动剂AC-42、AC-260584、N-去甲基氯氮平和占诺美林进行了比较。所有配体均激活M1受体并刺激受体与β-抑制蛋白-1的相互作用。所有配体均降低细胞表面结合并导致总受体结合丧失。用高渗蔗糖处理可阻断受体内化，表明所有配体均诱导网格蛋白包被小泡的形成。然而，去除正构激动剂后，内化的受体可循环至细胞表面，但变构激动剂则不能。尽管所有配体均导致细胞表面受体结合丧失，但用AC-260584或占诺美林处理后未观察到细胞内小泡。用AC-260584或占诺美林短暂刺激M1受体导致M1受体持续激活，这表明持续的受体信号传导可能会阻碍或延迟受体内吞入细胞内小泡。
这些结果表明，变构激动剂在诱导不同调节途径的能力上与正构配体不同，且彼此之间也不同。因此，不同变构配体诱导的信号传导和调节途径具有配体特异性。
