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

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.

FIGURE 1.

Agonist structures, and snake diagram of M₂ mAChR highlighting mutated residues. Residues previously reported to be involved in prototypical allosteric modulator binding are shown in squares, whereas residues implicated in orthosteric ligand binding are shown in heavy circles. Trp-99^3.28 is highlighted within a diamond, because this residue has been implicated in both orthosteric and allosteric ligand binding.

FIGURE 2.

Novel selective agonists display low efficacies at the wild type M₂ mAChR. A and B, peak levels of phosphorylated ERK1/2 (pERK1/2) were assessed as described under “Experimental Procedures,” corrected for basal level, and normalized to the response elicited by 10% FBS. Data represent the mean ± S.E. of 5–16 experiments performed in duplicate. C and D, agonists were pre-equilibrated with membranes from FlpInCHO cells stably expressing the wild type M₂ mAChR prior to exposure to [³⁵S]GTPγS, as indicated under “Experimental Procedures.” Data were corrected for basal level and normalized to the maximal response elicited by ACh. Data represent the mean ± S.E. of 3–5 experiments performed in duplicate. E, y ordinate response data for equivalent concentrations of each agonist at each pathway in A–D were plotted against one another. In this manner, the bias for a given agonist toward one pathway relative to the other can be visualized. The error bars not shown lie within the dimensions of the symbol.

FIGURE 3.

Functional affinity estimates from pERK1/2 assays of novel selective agonists display a different pattern of responses to receptor mutations relative to orthosteric agonists. The bars represent the difference in pK_A of each agonist, derived using an operational model of agonism (see “Experimental Procedures”), relative to the wild type receptor value for that agonist. The combined mutations, E172Q/D173N/E175Q and E172Q/D173N/E175Q/Y177A/T423^7.36A, are indicated by EDGE and EDGE+Y+T, respectively. Data represent the mean ± S.E. of 5–16 experiments performed in duplicate. no resp., there was no detectable agonism; *, significantly different from wild type, p < 0.05, one-way ANOVA, Dunnett's post-test. #, the agonist was capable of eliciting the maximal system response, such that its pK_A was constrained to equal that determined by radioligand binding. Note that no data are shown for Y104^3.33A, because this mutant did not mediate ERK1/2 phosphorylation in response to any agonist tested. Data are also not shown for AC-42, because this compound did not elicit any response at the wild type receptor (but see Table 3).

FIGURE 4.

Signaling efficacy estimates from pERK1/2 assays of novel selective agonists display a different pattern of responses to receptor mutations relative to orthosteric agonists. The bars represent the difference in log τ_C estimates (coupling efficiency corrected for receptor expression level) of each agonist derived from an operational model of agonism (see “Experimental Procedures”), relative to the wild type receptor value for that agonist. Data are not shown for AC-42, because this compound did not elicit any response at the wild type receptor (but see Table 4). All other details are as for Fig. 3.

FIGURE 5.

Novel selective agonists display a different pattern of responses for coupling to the ERK1/2 pathway at various receptor mutations relative to orthosteric agonists. Peak levels of phosphorylated ERK1/2 (pERK1/2) were assessed as described under “Experimental Procedures,” corrected for basal level, and normalized to the response elicited by 10% FBS. Data represent the mean ± S.E. of 3–16 experiments performed in duplicate. The error bars not shown lie within the dimensions of the symbol.

FIGURE 6.

Novel selective agonists display a different pattern of responses for promoting [³⁵S]GTPγS binding relative to orthosteric agonists. Agonists were pre-equilibrated with membranes from FlpInCHO cells stably expressing the indicated M₂ mAChR prior to exposure to [³⁵S]GTPγS, as indicated under “Experimental Procedures.” Data represent the mean ± S.E. of 4–16 experiments performed in duplicate. The error bars not shown lie within the dimensions of the symbol.

FIGURE 7.

Functional affinity estimates from [³⁵S]GTPγS binding assays of novel selective agonists display a different pattern of responses to receptor mutations relative to orthosteric agonists. Data represent the mean ± S.E. of 4–5 experiments performed in duplicate. n.d., not determined; *, significantly different from wild type, p < 0.05, one-way ANOVA, Dunnett's post-test. All other details are as for Fig. 3.

FIGURE 8.

Signaling efficacy estimates from [³⁵S]GTPγS binding assays of novel selective agonists display a different pattern of responses to receptor mutations relative to orthosteric agonists. Data represent the mean ± S.E. of 4–5 experiments performed in duplicate. n.d., not determined; *, significantly different from wild type, p < 0.05, one-way ANOVA, Dunnett's post-test. All other details are as for Fig. 4.

FIGURE 9.

Mutation of Y104^3.33A selectively abrogates agonist coupling to the ERK1/2 pathway. A, agonist concentration-response curves for ERK1/2 phosphorylation determined in FlpInCHO cells stably expressing the Y104^3.33A M₂ mAChR. Data represent the mean ± S.E. of 4–5 experiments performed in duplicate. All other details are as for Fig. 2. B and C, agonist concentration-response curves for [³⁵S]GTPγS binding at the same mutant. Data represent the mean ± S.E. of 4–5 experiments performed in duplicate. All other details are as for Fig. 2. D and E, agonist concentration-response curves for receptor-mediated Ca²⁺ mobilization at the wild type M₂ mAChR (D) or the Y104^3.33A M₂ mAChR (E). The peak Ca²⁺ response, determined as described under “Experimental Procedures,” was corrected for the background level of fluorescence and normalized to the response elicited by 2 μm ionomycin. Data represent the mean ± S.E. of 4–5 experiments performed in duplicate. Data represent the mean ± S.E. of 4–5 experiments performed in duplicate. The error bars not shown lie within the dimensions of the symbol.

FIGURE 10.

Molecular modeling suggests novel binding poses for 77-LH-28-1 and NDMC. Shown are views from the top of the extracellular space (A and B) and from the side of the TM bundle (C and D) of an M₂ mAChR docked with either 77-LH-28-1 (A and C) or NDMC (B and D). The key Asp-103^3.32 residue is shown in purple. Residues found to affect the binding affinity are shown in blue, whereas residues predominantly affecting signaling efficacy are shown in green. The numbering of TM regions is also indicated.