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 cross-linking 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.
Highlights
The superfamily of G protein-coupled receptors (GPCRs)2 represents the largest group of cell surface receptors found in nature [1,2,3,4,5]
In conjunction with a three-dimensional model of the M3 muscarinic receptor, this study provides the first piece of direct structural information as to how the receptor conformations induced by GPCR agonists and inverse GPCR agonists differ from each other at the molecular level
During the past few years, we have applied an in situ disulfide cross-linking strategy to gain insight into agonist-induced conformational changes in the M3 muscarinic receptor, a prototypical class I GPCR
Summary
Materials—Carbamylcholine chloride (carbachol), acetylcholine bromide, atropine sulfate, N-methylscopolamine bromide, cupric sulfate (CuSO4), 1,10-phenanthroline, N-ethylmaleimide, and mammalian protease inhibitor mixture were purchased from Sigma. [3H]NMS (82.0 Ci/mmol) and myo[3H]inositol (20 Ci/mmol) were obtained from PerkinElmer Life Sciences. Membrane homogenates prepared from transfected COS-7 cells (ϳ10 –20 g of membrane protein per tube) were incubated with the muscarinic antagonist/inverse agonist, [3H]NMS, for 2 h (22 °C) in 1 ml of buffer A. Receptor-containing membranes prepared from one 100-mm dish (ϳ1 mg of protein) were suspended in 1 ml of buffer A containing 25 M Cu(II)-phenanthroline, either in the presence or in the absence of different muscarinic ligands. Receptor-containing membranes were incubated on ice for 30 min either in the presence or in the absence of 5 M urea and used for disulfide cross-linking studies, exactly as described by Ward et al [23]. To quantitate the intensities of immunoreactive bands, we used scanning densitometry employing the program NIH ImageJ
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