Distinct Structural Changes in a G Protein-coupled Receptor Caused by Different Classes of Agonist Ligands

Abstract

The activity of G protein-coupled receptors can be modulated by different classes of ligands, including agonists that promote receptor signaling and inverse agonists that reduce basal receptor activity. The conformational changes in receptor structure induced by different agonist ligands are not well understood at present. In this study, we employed an in situ disulfide crosslinking strategy to monitor ligand-induced conformational changes in a series of cysteine-substituted mutant M 3 muscarinic acetylcholine receptors. The observed disulfide cross-linking patterns indicated that muscarinic agonists trigger a separation of the N-terminal segment of the cytoplasmic tail (helix 8) from the cytoplasmic end of transmembrane domain I. In contrast, inverse muscarinic agonists were found to increase the proximity between these two receptor regions. These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most G protein-coupled receptors, our findings should be of broad general relevance.

Heparin Induces Rat Aorta Relaxation via Integrin-Dependent Activation of Muscarinic M 3 Receptors

“…Heparinbinding motifs were described in proteins with sequences such as XBBBXXBX and XBBXBX (where B and X are basic and hydropathic residues, respectively). 42 M 3 receptors do not have heparin-binding sites on the extracellular loops, 43 which is in accordance with the absence of heparin binding to muscarinic receptors ( Figure 4C and 4D). In addition, other possibilities to explain heparin-induced relaxation were evaluated.…”

A Fluorescence Resonance Energy Transfer-based M2 Muscarinic Receptor Sensor Reveals Rapid Kinetics of Allosteric Modulation

“…Allosteric ligands are defined by the fact that in the absence of an orthosteric ligand, they do not affect the activity of a receptor (7) but that they alter the affinities of orthosteric ligands, presumably because of conformational changes in the receptor (8 -10). Recent experimental evidence supports the hypothesis that allosteric ligands can induce specific receptor conformations by direct binding to an allosteric ligand site and that this may result in changes of the agonist-induced activity of the receptor (11).…”

Helix 8 of the M1Muscarinic Acetylcholine Receptor: Scanning Mutagenesis Delineates a G Protein Recognition Site

“…That agonist binding induces a conformational change involving H8 was suggested by a cysteine substitution and induced disulfide cross-linking study on the M 3 mAChR (Li et al, 2007). Increased spatial separation or conformational mobility between the N-terminal segment of H8 and TM1 is a favored interpretation.…”

“…H8, in particular, a dileucine motif toward the C-terminal end, also has been implicated in control of receptor expression (Sawyer et al, 2010) and is suggested to be part of a dimerization interface involving TM1 (Fung et al, 2009). In the M 3 mAChR, cysteine substitution and disulfide cross-linking studies indicated that the N-terminal part of H8 moves away from TM1 during agonist activation (Li et al, 2007) and that residues in H8 can undergo both agonistindependent and -dependent cross-linking to G q ␣ (Hu et al, 2010).…”