Abstract
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.
Highlights
(NHMRC) of Australia Program Grant 519461. □S The on-line version of this article contains supplemental Table 1 and Figs. 1 and 2. 1 Recipient of an National Health and Medical Research Council (NHMRC) Biomedical Postgraduate Research Scholarship, a
Rationale for the Choices of Orthosteric and Allosteric Site Mutations—Fig. 1 shows a snake diagram of the secondary structure of the M2 muscarinic acetylcholine receptors (mAChRs), indicating residues mutated in the current study
Our results at the M2 mAChR are in general agreement with recent findings made for some of the novel selective
Summary
(NHMRC) of Australia Program Grant 519461. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1 and 2. 1 Recipient of an NHMRC Biomedical Postgraduate Research Scholarship, a. The interaction between the two sites with such ligands can conceivably occur concomitantly, as recently demonstrated for the bitopic orthosteric/allosteric ligand McN-A-343 [35] and novel hybrid oxotremorine-bis(ammonio)alkane compounds [36], or via interchange between two distinct binding modes (orthosteric versus allosteric), as may be the case for small molecule modulators of the ghrelin receptor [37]. Given these diverse modes of allosteric agonist-receptor interaction, the structurefunction relationships that underlie them and/or their functional consequences are likely to vary compared with those of classic orthosteric agonists. Of particular interest is whether or not allosteric agonists have a higher propensity than orthosteric agonists to engender functional selectivity (i.e. to promote unique conformational states of the receptor that activate specific subsets of the total complement of pathways available to that receptor in a given cell type) [38]
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