Techniques: GPCR assembly, pharmacology and screening by flow cytometry

https://doi.org/10.1016/j.tips.2004.10.009Get rights and content

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.

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.

References (52)

  • T.A. Key

    N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity

    J. Biol. Chem.

    (2003)
  • U. Gether

    Fluorescent labeling of purified beta 2 adrenergic receptor. Evidence for ligand-specific conformational changes

    J. Biol. Chem.

    (1995)
  • R. Seifert

    GPCR-Galpha fusion proteins: molecular analysis of receptor-G-protein coupling

    Trends Pharmacol. Sci.

    (1999)
  • R.M. Potter

    Arrestin variants display differential binding characteristics for the phosphorylated N-formyl peptide receptor carboxyl terminus

    J. Biol. Chem.

    (2002)
  • F. Goubaeva

    Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides

    J. Biol. Chem.

    (2003)
  • A. Chigaev

    FRET detection of cellular alpha 4-integrin conformational activation

    Biophys. J.

    (2003)
  • J. Szollosi

    Applications of fluorescence resonance energy transfer for mapping biological membranes

    J. Biotechnol.

    (2002)
  • S.M. Young

    High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening

    J. Biomol. Screen.

    (2004)
  • B.S. Edwards

    Flow cytometry for high-throughput, high-content screening

    Curr. Opin. Chem. Biol.

    (2004)
  • R.J. Lefkowitz

    Dancing with different partners: protein kinase a phosphorylation of seven membrane-spanning receptors regulates their G protein-coupling specificity

    Mol. Pharmacol.

    (2002)
  • L.A. Sklar

    Real-time spectroscopic analysis of ligand-receptor dynamics

    Annu. Rev. Biophys. Biophys. Chem.

    (1987)
  • J.P. Nolan et al.

    The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions

    Nat. Biotechnol.

    (1998)
  • L.A. Sklar

    Flow cytometric analysis of ligand-receptor interactions and molecular assemblies

    Annu. Rev. Biophys. Biomol. Struct.

    (2002)
  • J.P. Nolan

    Cytometric approaches to the study of receptors

  • Sklar, L.A. Flow Cytometry in Biotechnology, Oxford University Press (in...
  • T.C. George

    Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer

    Cytometry

    (2004)
  • Cited by (26)

    • Application of antibiotic-derived fluorescent probes to bacterial studies

      2022, Methods in Enzymology
      Citation Excerpt :

      The primary focus was on the measurement and/or quantification of different biomarkers of immune response, such as TNF-α or interleukins (Ding et al., 2018). Another focus was on detection of interactions with G-protein-coupled receptors (GPCRs) (Waller et al., 2004). In recent years, flow cytometry has gained increasing attention for infectious disease research (Steen, 2000).

    • Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors

      2014, Biochimica et Biophysica Acta - Biomembranes
      Citation Excerpt :

      The results are summarized in Fig. 2. This approach was based on the establishment of flow cytometric methods for monitoring ligand binding by Sklar and co-workers [58,80], and on the application of flow cytometry to genetic screening of large mutational libraries in yeast developed primarily by Wittrup and co-workers [139,140]. It was also based on the discovery of binding-dependent fluorescence changes in the intensity and wavelength of fluorescence emission of the ligand [K7(NBD),Nle12] α-factor and other related α-factor analogs [62,64,100].

    • Advances in Multiple Analyte Profiling

      2008, Advances in Clinical Chemistry
      Citation Excerpt :

      Modern flow cytometry is presently amenable to assay miniaturization down to 8 μl with the introduction of platforms such as HyperCyt®. In recent studies from our laboratory, we routinely analyzed up to 3000 cells from each sample well with an average sampling time of ∼1.5 s. HyperCyt® has been successfully adapted to cell-based end point assays [66], and studies investigating cell–cell adhesion [48, 67], and fluorescent ligand binding to cellular receptors [68, 69]. Fluorescence polarization utilizes linearly polarized light for excitation, and the emission is detected to infer changes in molecular orientation and mobility.

    View all citing articles on Scopus
    View full text