Critical Role for the Second Extracellular Loop in the Binding of Both Orthosteric and Allosteric G Protein-coupled Receptor Ligands

The second extracellular (E2) loop of G protein-coupled receptors (GPCRs) plays an essential but poorly understood role in the binding of non-peptidic small molecules. We have utilized both orthosteric ligands and allosteric modulators of the M2 muscarinic acetylcholine receptor, a prototypical Fami...

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Bibliographic Details
Published in:The Journal of biological chemistry 2007-08, Vol.282 (35), p.25677-25686
Main Authors: Avlani, Vimesh A., Gregory, Karen J., Morton, Craig J., Parker, Michael W., Sexton, Patrick M., Christopoulos, Arthur
Format: Article
Language:English
Subjects:
Allosteric Regulation - physiology
Allosteric Site - physiology
Amino Acid Substitution
Animals
Cattle
Cysteine - chemistry
Cysteine - genetics
Humans
Ligands
Models, Molecular
Protein Binding - physiology
Protein Structure, Tertiary - genetics
Receptor, Muscarinic M2 - chemistry
Receptor, Muscarinic M2 - genetics
Rhodopsin - chemistry
Rhodopsin - genetics
Structural Homology, Protein
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Summary:The second extracellular (E2) loop of G protein-coupled receptors (GPCRs) plays an essential but poorly understood role in the binding of non-peptidic small molecules. We have utilized both orthosteric ligands and allosteric modulators of the M2 muscarinic acetylcholine receptor, a prototypical Family A GPCR, to probe possible E2 loop binding dynamics. We developed a homology model based on the crystal structure of bovine rhodopsin and predicted novel cysteine substitutions that should dramatically reduce E2 loop flexibility via disulfide bond formation and significantly inhibit the binding of both types of ligands. This prediction was validated experimentally using radioligand binding, dissociation kinetics, and cell-based functional assays. The results argue for a flexible “gatekeeper” role of the E2 loop in the binding of both allosteric and orthosteric GPCR ligands.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M702311200