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. 2012 Oct 26;287(44):37066-77.
doi: 10.1074/jbc.M112.408534. Epub 2012 Sep 10.

Stimulus bias provides evidence for conformational constraints in the structure of a G protein-coupled receptor

Karen J Gregory  1 , Patrick M Sexton, Andrew B Tobin, Arthur Christopoulos
Affiliations

Stimulus bias provides evidence for conformational constraints in the structure of a G protein-coupled receptor

Karen J Gregory et al. J Biol Chem. .

Abstract

A key characteristic of G protein-coupled receptors (GPCRs) is that they activate a plethora of signaling pathways. It is now clear that a GPCR coupling to these pathways can be regulated selectively by ligands that differentially drive signaling down one pathway in preference to another. This concept, termed stimulus bias, is revolutionizing receptor biology and drug discovery by providing a means of selectively targeting receptor signaling pathways that have therapeutic impact. Herein, we utilized a novel quantitative method that determines stimulus bias of synthetic GPCR ligands in a manner that nullifies the impact of both the cellular background and the "natural bias" of the endogenous ligand. By applying this method to the M(2) muscarinic acetylcholine receptor, a prototypical GPCR, we found that mutation of key residues (Tyr-80(2.61) and Trp-99(3.28)) in an allosteric binding pocket introduces stimulus bias in response to the atypical ligands AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl) and 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)- 3,3-dihydro-2(1H)-quinolinone). By comparing stimulus bias factors among receptor internalization, G protein activation, extracellular-regulated protein kinase 1/2 (ERK1/2) signaling, and receptor phosphorylation, we provide evidence that Tyr-80(2.61) and Trp-99(3.28) act either as molecular switches or as gatekeeper residues that introduce constraints limiting the active conformation of the M(2) muscarinic acetylcholine receptor and thereby regulate stimulus bias. Furthermore, we provide evidence that downstream signaling pathways previously considered to be related to each other (i.e. receptor phosphorylation, internalization, and activation of ERK1/2) can act independently.

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Figures

FIGURE 1.
FIGURE 1.
Agonist-mediated internalization of wild type M2 mAChRs. CHO-FlpIn cells stably expressing the V5-tagged wild type M2 mAChR (A and B) were exposed to 100 μm ACh (■), 100 μm CCh (●), 100 μm arecoline (▴),1 μm pilocarpine (⧫), 30 μm 77-LH-28-1 (□), 30 μm AC-42 (△), 30 μm NDMC (◊), or 300 μm McN-A-343 (○) for up to 30 min prior to quantification of cell surface receptors with [3H]NMS. Concentration-response curves were constructed for the indicated agonists at the V5-tagged wild type M2 mAChR (C) following 30 min of agonist stimulation. Data represent the mean ± S.E. of n = 3–5 experiments performed in triplicate; error bars not shown lie within the dimensions of the symbol.
FIGURE 2.
FIGURE 2.
Time course for agonist-mediated internalization of cell surface mutant M2 mAChRs. A and B, CHO-FlpIn cells stably expressing the Y802.61A mutant M2 mAChR were exposed to 100 μm ACh (■), 1 mm CCh (●), 300 μm arecoline (▴), 300 μm pilocarpine (⧫), 1 μm 77-LH-28-1 (□), 10 μm AC-42 (△), 10 μm NDMC (◊), or 300 μm McN-A-343 (○) for the indicated times prior to quantification of cell surface receptors with [3H]NMS. C and D, CHO-FlpIn cells stably expressing the W993.28A mutant M2 mAChR were exposed to 300 μm ACh (■), 1 mm CCh (●), 100 μm arecoline (▴), 1 μm pilocarpine (⧫), 30 nm 77-LH-28-1 (□), 1 μm AC-42 (△), 30 μm NDMC (◊), or 300 μm McN-A-343 (○) for up to 30 min prior to quantification of cell surface receptors with [3H]NMS. E and F, CHO-FlpIn cells stably expressing the Y177A mutant M2 mAChR were exposed to 100 μm ACh (■), 1 mm CCh (●), 300 μm arecoline (▴), 300 μm pilocarpine (⧫), 10 μm 77-LH-28-1 (□), 30 μm AC-42 (△), 10 μm NDMC (◊), or 300 μm McN-A-343 (○) for up to 30 min prior to quantification of cell surface receptors with [3H]NMS. Data represent the mean ± S.E. for n = 3–4 experiments conducted in triplicate. Error bars not shown lie within the dimensions of the symbol.
FIGURE 3.
FIGURE 3.
Time course for agonist-mediated reduction in [3H]QNB-accessible binding sites at mutant M2 mAChRs. A and B, CHO-FlpIn cells stably expressing the Y1043.33A mutant M2 mAChR were exposed to 10 mm ACh (■), 10 mm CCh (●), 10 mm arecoline (▴), 10 mm pilocarpine (⧫), 30 mm 77-LH-28-1 (□), 30 mm AC-42 (△), 30 mm NDMC (◊), or 300 mm McN-A-343 (○) for up to 30 min prior to measurement of [3H]QNB-accessible receptors. C and D, CHO-FlpIn-cells stably expressing the Y4036.51A mutant M2 mAChR were exposed to 10 mm ACh (■), 10 mm CCh (●), 10 mm arecoline (▴), 10 mm pilocarpine (⧫), 30 mm 77-LH-28-1 (□), 30 mm AC-42 (△), 30 mm NDMC (◊), or 300 mm McN-A-343 (○) for up to 30 min prior to measurement of [3H]QNB-accessible receptors. Data represent the mean ± S.E. for n = 3–4 experiments conducted in triplicate. Error bars not shown lie within the dimensions of the symbol.
FIGURE 4.
FIGURE 4.
Agonist concentration-response curves for internalization of mutant M2 mAChRs. A–C, after agonist stimulation for 30 min, concentration-response curves were constructed as follows before determination of cell surface expression with [3H]NMS: A, ACh (■), CCh (●), arecoline (▴), 77-LH-28-1 (□), and AC-42 (△) at the Y802.61A; B, ACh (■), CCh (●), arecoline (▴), 77-LH-28-1 (□), AC-42 (△), and NDMC (◊) at the W993.28A; C, ACh (■), CCh (●), arecoline (▴), and AC-42 (△) at the Y177A. D and E, after agonist stimulation for 30 min, concentration-response curves were constructed as follows before determination of [3H]QNB binding levels: D, NDMC (◊) and AC-42 (△) at the Y1043.33A; E, NDMC (◊) and AC-42 (△) at the Y4036.51A. Data represent the mean ± S.E. for n = 3–4 experiments conducted in triplicate. Error bars not shown lie within the dimensions of the symbol.
FIGURE 5.
FIGURE 5.
Determination of bias factors quantifying agonist functional selectivity at wild type and mutant M2 mAChRs. Agonist concentration-response data derived from this study and from our earlier study (19) were fitted to Equation 2 to derive log(τ/KA) values for each agonist at each pathway. These values were normalized to that derived for ACh at each pathway, and the resulting normalized values (Δlog(τ/KA)) were analyzed using Equation 3 under “Experimental Procedures” to yield the final log(bias) factor (ΔΔlog(τ/KA)) for each agonist between the two pathways. *, indicates statistical significance.
FIGURE 6.
FIGURE 6.
Validation of 32P labeling and immunoprecipitation of M2 mAChRs. After 32P labeling, either non-transfected CHO-FlpIn cells (CHO-nt) or those stably expressing the wild type M2 mAChR were immunoprecipitated with either anti-M2 antibody (A) or anti-V5 antibody (B), resolved by SDS-PAGE, and transferred to nitrocellulose membranes before autoradiography was performed as described under “Experimental Procedures.” The nitrocellulose membrane corresponding to the autoradiograph in A was then reprobed with anti-V5 antibody (C). The nitrocellulose membrane corresponding to the autoradiograph in B was then reprobed with anti-M2 antibody (D).
FIGURE 7.
FIGURE 7.
Agonist-mediated phosphorylation of wild type and allosteric site-mutated M2 mAChRs. After 32P labeling, CHO-FlpIn cells stably expressing M2 mAChR constructs were exposed to agonists for 5 min and then immunoprecipitated with anti-V5, resolved by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography. The panels on the right are representative autoradiographs, and those on the left are grouped autoradiography data. A, phosphorylation of wild type M2 mAChR after treatment with 100 μm ACh, 100 μm pilocarpine, 30 μm NDMC, 1 mm McN-A-343, or 30 μm 77-LH-28-1. B, phosphorylation of Y802.61A receptors after treatment with 100 μm ACh, 300 μm pilocarpine, 30 μm NDMC, 1 mm McN-A-343, or 10 μm 77-LH-28-1. C, phosphorylation of Y177A receptors after treatment with 100 μm ACh, 300 μm pilocarpine, 30 μm NDMC, 1 mm McN-A-343, or 30 μm 77-LH-28-1. Data represent n = 3–4 experiments performed in duplicate and normalized to the percent of the ACh-stimulated response (itself designated as 100%). *, denotes a significant difference from basal, as determined by one-way ANOVA and Dunnett's post-test. In all instances, the ACh response was also significantly different from basal.
FIGURE 8.
FIGURE 8.
Agonist-mediated phosphorylation of orthosteric site-mutated M2 mAChRs. After 32P labeling, CHO-FlpIn cells stably expressing M2 mAChR constructs were exposed to agonists for 5 min and then immunoprecipitated with anti-V5 antibody, resolved by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography. The panels on the right are representative autoradiographs, and those on the left are grouped autoradiography data. A, phosphorylation of W993.28A receptors after treatment with 300 μm ACh, 300 μm pilocarpine, 30 μm NDMC, 1 mm McN-A-343, or 3 μm 77-LH-28-1. B, phosphorylation of Y1043.33A receptors after treatment with 10 mm ACh, 10 mm pilocarpine, 30 mm NDMC, 1 mm McN-A-343, or 100 mm 77-LH-28-1. C, phosphorylation of Y4036.51A receptors after treatment with 10 mm ACh, 10 mm pilocarpine, 30 μm NDMC, 1 mm McN-A-343, or 30 μm 77-LH-28-1. Data represent n = 3 experiments performed in duplicate and normalized to the percent of the ACh-stimulated response (itself designated as 100%). *, denotes a significant difference from basal, as determined by one-way ANOVA and Dunnett's post-test. In all instances, the ACh response was also significantly different from basal.
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