Abstract
G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.
Highlights
G protein-coupled receptors (GPCRs)4 have outstanding importance as targets for drug action [1, 2]
This amino acid forms a junction between E2 and TM7 that appears to be essentially involved in GPCR activation
Receptor activation leads to G protein stimulation, which was measured using the GTP analogue [35S]GTP␥S that binds to an activated G protein but is not hydrolyzed [23]
Summary
G protein-coupled receptors (GPCRs) have outstanding importance as targets for drug action [1, 2]. Conformational changes include extracellular receptor regions, and a critical role of the second extracellular loop (E2) for GPCR activation and ligand binding has emerged [5,6,7,8,9]. Our strategy is based on the hypothesis that activation-related three-dimensional changes involve the whole receptor protein and include allosteric sites. Allosteric ligands should be useful to probe activation-related spatial rearrangement of the receptor protein outside the orthosteric binding area. Applying allosteric small molecules as probes, we provide evidence that the full agonist acetylcholine and a partial agonist induce divergent conformational changes in the extracellular receptor region. We identified an amino acid, M2Trp422, that plays a key role for allosteric/orthosteric binding and activation cooperativity This amino acid forms a junction between E2 and TM7 that appears to be essentially involved in GPCR activation
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