Structural determinants of allosteric agonism and modulation at the M4 muscarinic acetylcholine receptor: identification of ligand-specific and global activation mechanisms

Abstract

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M(4) mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M(1)/M(4) mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M(4) mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile(93) and Lys(95) as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe(186) in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp(432) in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr(439) and Tyr(443), were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.

FIGURE 1.

Mutations and ligands investigated in the current study. A, snake diagram of the M₄ mAChR highlighting mutated residues. B–E, structures of the endogenous agonist, ACh (B), the M₁/M₄-preferring agonist, xanomeline (C), the functionally selective bitopic agonist, McN-A-343 (D), and the allosteric ligand, LY2033298 (E).

FIGURE 2.

Agonist affinity estimates are differentially modified by M₄ mAChR mutations. Bars represent the difference in pK_B of each agonist, derived using either a competitive or allosteric ternary complex model (see “Experimental Procedures”), relative to the wild-type receptor value for that agonist. Data represent the mean ± S.E. of at least three experiments performed in triplicates. n.d. indicates that there was no detectable binding; *, significantly different to wild-type, p < 0.05, one-way analysis of variance, Dunnett's post-test.

FIGURE 3.

Identification of residues that govern LY2033298 affinity and cooperativity with ACh at the M₄ mAChR. The competition between 100 pm [³H]QNB and a fixed concentration of ACh was determined in the absence (bars) or presence (circles) of increasing concentrations of LY2033298 at the indicated mAChR constructs. The fixed concentration of ACh was 10 μm for all experiments except for F186A (30 μm) and Y439A (1 mm). The curves drawn through the points represent the best global fit of an allosteric ternary complex model (Equation 2) to each pair of datasets, with the cooperativity between LY2033298 and [³H]QNB (α′) fixed to a value of 1. Data points represent the mean ± S.E. of at least three experiments performed in triplicates.

FIGURE 4.

[³H]NMS dissociation kinetic studies confirm that Phe¹⁸⁶ likely contributes to the allosteric binding site for LY2033298 at the M₄ mAChR. Concentration-effect relationships for LY2033298 on the dissociation rate of [³H]NMS at the wild type or F186A M₄ mAChR stably expressed in membranes from Chinese hamster ovary-FlpIn cells. Data represent mean ± S.E. of three experiments performed in triplicates.

FIGURE 5.

Identification of residues that govern orthosteric and allosteric agonist signaling efficacy at the M₄ mAChR. Peak levels of pERK1/2 were assessed as described under “Experimental Procedures” and normalized to the response elicited by 10% FBS. The curves drawn through the points at each receptor construct represent the best global fit of an operational model of agonism (Equation 1) to each family of datasets, with the affinity of each agonist fixed to the pK_B value determined in separate binding assays (Table 1). Data points represent the mean ± S.E. of at least three experiments performed in triplicates.

FIGURE 6.

Agonist signaling efficacy estimates are differentially modified by M₄ mAChR mutations. Bars represent the difference in log τ of each agonist, derived from an operational model of agonism (see “Experimental Procedures”), relative to the wild-type receptor value for that agonist. Data represent the mean ± S.E. of at least three experiments performed in triplicate. n.d. indicates that there was no detectable response; *, significantly different to wild type, p < 0.05, one-way analysis of variance, Dunnett's post-test.