Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipypirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Timeresolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligandreceptor complexes toward a high affinity dimeric state. G protein-coupled receptors (GPCRs)3 trigger a wide palette of signaling pathways (1, 2), including G protein-independent responses (3). These receptors display multiple conformational and functional states, dependent on the cellular context, differentially selected and stabilized by ligands, and discriminated by downstream protein partners (4 -9). The occurrence of distinct receptor conformational species is supported by structural arguments provided by metal ion site engineering (10) or in situ disulfide cross-linking (11) and by direct monitoring of receptor intramolecular rearrangements through fluorescencebased methods (for reviews see Refs. 8,9,12).Few studies focused on the initial ligand binding step, its kinetic description, and its relationship with functionally relevant receptor conformational states. These aspects were addressed by monitoring intermolecular fluorescence resonance energy transfer (FRET) between a GFP-tagged receptor (donor) and a fluorescent ligand (acceptor). Both neurokinin A binding to class A tachykinin NK2 receptors (4, 13) and parathyroid hormone binding to class B parathyroid hormone receptors (14) proceeded in two steps, featuring two kinetically distinguishable conformational states. Whether such biphasic binding reactions are a general feature of GPCRs, independent on the pharmacological nature of the ligand, and whether they reflect different receptor functional states or sequential binding steps remain important questions to be elucidated.Muscarinic cholinergic receptors (15) disp...
Two fluorescent derivatives of the M1 muscarinic selective agonist AC-42 were synthesized by coupling the lissamine rhodamine B fluorophore (in ortho and para positions) to AC42-NH(2). This precursor, prepared according to an original seven-step procedure, was included in the study together with the LRB fluorophore (alone or linked to an alkyl chain). All these compounds are antagonists, but examination of their ability to inhibit or modulate orthosteric [(3)H]NMS binding revealed that para-LRB-AC42 shared several properties with AC-42. Carefully designed experiments allowed para-LRB-AC42 to be used as a FRET tracer on EGFP-fused M1 receptors. Under equilibrium binding conditions, orthosteric ligands, AC-42, and the allosteric modulator gallamine behaved as competitors of para-LRB-AC42 binding whereas other allosteric compounds such as WIN 51,708 and N-desmethylclozapine were noncompetitive inhibitors. Finally, molecular modeling studies focused on putative orthosteric/allosteric bitopic poses for AC-42 and para-LRB-AC42 in a 3D model of the human M1 receptor.
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