carbocyanines and telenzepine

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins in the human genome. Events in the GPCR signaling cascade have been well characterized, but the receptor composition and its membrane distribution are still generally unknown. Although there is evidence that some members of the GPCR superfamily exist as constitutive dimers or higher oligomers, interpretation of the results has been disputed, and recent studies indicate that monomeric GPCRs may also be functional. Because there is controversy within the field, to address the issue we have used total internal reflection fluorescence microscopy (TIRFM) in living cells to visualize thousands of individual molecules of a model GPCR, the M(1) muscarinic acetylcholine receptor. By tracking the position of individual receptors over time, their mobility, clustering, and dimerization kinetics could be directly determined with a resolution of approximately 30 ms and approximately 20 nm. In isolated CHO cells, receptors are randomly distributed over the plasma membrane. At any given time, approximately 30% of the receptor molecules exist as dimers, and we found no evidence for higher oligomers. Two-color TIRFM established the dynamic nature of dimer formation with M(1) receptors undergoing interconversion between monomers and dimers on the timescale of seconds.

Topics: Animals; Benzenesulfonates; Binding, Competitive; Carbocyanines; Cell Membrane; CHO Cells; Cricetinae; Cricetulus; Fluorescent Dyes; Humans; Kinetics; Magnetic Resonance Spectroscopy; Microscopy, Fluorescence; Molecular Dynamics Simulation; Molecular Structure; Muscarinic Antagonists; Pirenzepine; Protein Multimerization; Radioligand Assay; Receptor, Muscarinic M1; Time Factors; Transfection

The measurement of ligand receptor binding parameters for G-protein-coupled receptors is indispensable in the drug discovery process. Traditional ligand receptor binding assays require scale-up of cells and membrane preparations, which is an expensive and time-consuming process. In this report, the authors describe the development of a homogeneous live-cell binding assay for GPCRs using a fluorophore-labeled nonpeptide ligand. The model assay used Cy3B-labeled telenzepine and Chinese hamster ovary cells expressing M1 muscarinic acetylcholine receptors. This homogeneous live-cell fluorescence binding assay format is superior to the traditional binding methods because it measures binding of a ligand to intact receptors on living cells. The assay requires no washing or separation steps, thereby allowing a real-time kinetic readout for the determination of ligand association and dissociation from the intact receptors. The results also suggest that miniaturization is feasible without compromising the data quality.

Topics: Animals; Atropine; Benzenesulfonates; Biological Assay; Carbocyanines; CHO Cells; Cricetinae; Cricetulus; Fluorescent Dyes; Ligands; Miniaturization; Muscarinic Antagonists; Parasympatholytics; Pirenzepine; Quinuclidinyl Benzilate; Receptor, Muscarinic M1; Receptors, G-Protein-Coupled

Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands. Receptor expression levels varied from approximately 1 to 10 pmols receptor per mg protein, and ligand concentrations were in the 0.5- to 1.0-nM range. The FP assays were successfully miniaturized to 1536-well formats using Cy3B-labeled ligands, significantly reducing reagent consumption, particularly the receptor source, without compromising assay reliability. Z' factor values determined for the FP receptor assays in both 384- and 1536-well formats were found to be > 0.5, indicating the assays to be robust, reliable, and suitable for HTS purposes.

Topics: Animals; Atropine; Binding, Competitive; Carbocyanines; CHO Cells; Cricetinae; Fluorescence Polarization; Fluorescent Dyes; Humans; Kinetics; Miniaturization; Neurokinin A; Peptides; Pirenzepine; Receptors, Cell Surface; Receptors, Muscarinic; Receptors, Neurokinin-1; Sensitivity and Specificity; Substance P