Ligand-Receptor-G-Protein Molecular Assemblies on Beads for Mechanistic Studies and Screening by Flow Cytometry (original) (raw)
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BioTechniques
G protein-coupled receptors (GPCR) and cellular signaling elements are prime targets for drug discovery. Sensitive realtime methods that expand the analytical capabilities for these elements can play significant roles in basic research and drug discovery. Here, we describe novel approaches for the real-time fluorescence analysis of GPCRs. Using the G proteincoupled N-formyl peptide receptor (FPR) as a model system in concert with a fluorescent ligand, we showed the quantitative solubilization of his-tagged FPRs in 1% dodecyl maltoside. Solublized receptors reconstitute in dodecyl maltoside with a mixture of bovine brain G i /G o showing an apparent K d of 100 nM. Solubilized receptors were also bound to Ni 2+ -silica particles and were detected in a flow cytometer by the binding of fluorescent ligand. The efficiency of receptor uptake by the particles was in excess of 80% with an apparent affinity for the bead in the nM range. The receptors had largely homogeneous dissociation characteristics, an appropriate K d for the ligand in the low nM range and a high site number, with several million receptor molecules per particle. However, the G protein reconstitution was not detected on the beads, apparently for steric reasons. These approaches for displaying receptors could prove useful in drug discovery and in the analysis of the molecular assemblies in signal transduction.
Techniques: GPCR assembly, pharmacology and screening by flow cytometry
Trends in Pharmacological Sciences, 2004
Flow cytometers are well known for their ability to analyze and sort cells at high rates based on physiological responses and expression of protein markers. The potential for flow cytometry in G-protein-coupled receptor (GPCR) research, however, is less well appreciated. Potential applications include: (i) the homogenous discrimination of free and bound ligands or proteins in both cellular and microsphere-based assays; and (ii) multiplexed (‘suspension array’) analysis of cell responses and protein–protein interactions. Innovative sample-handling systems also provide sub-second resolution of interaction kinetics and 1 second per well throughput of microliter-sized samples from multiwell plates. Flow cytometric methods using microspheres for analysis of GPCRs that interact with intracellular and extracellular binding partners such as ligands, G proteins and kinases have been established. These analyses can produce quantitative pharmacological data analogous to radioligand assays, and, in some cases, the probes can be integrated into the assembly as fluorescent fusion proteins.