Trends in Pharmacological Sciences
Techniques: GPCR assembly, pharmacology and screening by flow cytometry
Section snippets
Flow cytometric technology
Flow cytometry began in the 1960s primarily as a means of analyzing and sorting populations of cells. Its development paralleled and was driven, in part, by the introduction of monoclonal antibodies to study the intricacies of the immune system. Fluorescent probes compatible with the detection of other ligands, the physiology of cell response, the subcellular analysis of DNA and chromosomes, and non-cellular microsphere-based analysis were soon introduced. The flow cytometric analysis of
Receptor solublization and display of ligand–receptor and protein–protein interactions
A detergent-solubilized GPCR provides simultaneous access to the external ligand binding site and the internal G-protein binding site. Solubilization of GPCRs in 1% dodecyl maltoside (DOM), particularly rhodopsin, dates back to at least 1985. There are now several reports that indicate that some solubilized GPCRs retain their binding activity for both ligands and G proteins 16, 17. These GPCRs include wild-type and mutant N-formyl peptide receptor (FPR), FPR fused to the green fluorescent
Assaying signal transduction mechanisms of molecular assemblies
Fusion proteins have aided the exploration of mechanisms for ternary complex signal transduction 31, 32. A flow cytometric analysis of ternary complex disassembly following addition of GTPγS [21], long believed to initiate signal transduction through separation of the α-subunit from the βγ-subunits of the G protein, has been performed [21]. These ternary LRG complexes were constructed with one component fluorescently labeled, denoted by F, and the disassembly of the complex was monitored by the
Microspheres are evaluated flow cytometrically as sensors and in multiplex arrays
Microspheres are now used widely to detect solution components. Microspheres are particularly useful in cell lysates for detecting the presence of phosphorylated signaling components using phosphospecific antibodies [Upstate Biotechnology (http://www.upstate.com); Biorad (http://www.bio-rad.com)]. As such, these microspheres act as sensors for the components that are present in the solution. However, it is also possible to quantitatively examine the affinity of an assembly in solution even when
Assemblies of signaling molecules
Although the focus of this article has been on the important problem of soluble transmembrane proteins, generic ligand–receptor and protein–protein interactions are also accessible for quantitative analysis, multiplexing, sub-second analysis and, as described later, high-throughput screening. The assembly of synthetic biotinylated and phosphorylated GPCR tail peptides with wild-type and mutant arrestins has been investigated [38]. In this study, a set of affinities and the minimal GPCR tail
High-throughput flow cytometry
High-throughput in flow cytometry has been achieved by analysis and sorting rates of ≥10 000 particles per second, and by multiplexing. Flow cytometry has however been limited by the time required to deliver individual samples from multiwell plates. Commercial systems are limited typically to two samples per minute, each sample handled individually and the data from each sample treated as a single file. Several sample-handling systems have been described that enhance throughput by delivering
Concluding remarks
Flow cytometry, long regarded primarily as a clinical tool, has entered the arena of GPCR research and discovery. Flow cytometry offers the potential for high content, sub-second kinetic resolution, homogeneous assembly analysis, discrimination of multiple particle populations and high throughput. The versatility of the platform, the ease of alternating between cellular and molecular analyses or the ability to perform both simultaneously should prove attractive.
Acknowledgements
This work was supported by NIH Grant EB00264.
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