Identification of orthosteric and allosteric site mutations in M2 muscarinic acetylcholine receptors that contribute to ligand-selective signaling bias.

Muscarinic acetylcholine receptors contain at least one allosteric site that is topographically distinct from the acetylcholine, orthosteric binding site. Although studies have investigated the basis of allosteric modulation at these receptors, less is known about putative allosteric ligands that activate the receptor in their own right. We generated M(2) muscarinic acetylcholine receptor mutations in either the orthosteric site in transmembrane helices 3 and 6 (TM3 and -6) or part of an allosteric site involving the top of TM2, the second extracellular (E2) loop, and the top of TM7 and investigated their effects on the binding and function of the novel selective (putative allosteric) agonists (AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl), 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)-3,3-dihydro-2(1H)-quinolinone), and N-desmethylclozapine) as well as the bitopic orthosteric/allosteric ligand, McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium). Four classes of agonists were identified, depending on their response to the mutations, suggesting multiple, distinct modes of agonist-receptor interaction. Interestingly, with the exception of 77-LH-28-1, allosteric site mutations had no effect on the affinity of any of the agonists tested, but some mutations in the E2 loop influenced the efficacy of both orthosteric and novel selective agonists, highlighting a role for this region of the receptor in modulating activation status. Two point mutations (Y104(3.33)A (Ballesteros and Weinstein numbers in superscript) in the orthosteric and Y177A in the allosteric site) unmasked ligand-selective and signaling pathway-selective effects, providing evidence for the existence of pathway-specific receptor conformations. Molecular modeling of 77-LH-28-1 and N-desmethylclozapine yielded novel binding poses consistent with the possibility that the functional selectivity of such agents may arise from a bitopic mechanism.