Trends in Biotechnology
Volume 28, Issue 8, August 2010, Pages 407-415
Journal home page for Trends in Biotechnology

Review
Lighting up multiprotein complexes: lessons from GPCR oligomerization

https://doi.org/10.1016/j.tibtech.2010.05.002Get rights and content

Spatiotemporal characterization of protein–protein interactions (PPIs) is essential in determining the molecular mechanisms of intracellular signaling processes. In this review, we discuss how new methodological strategies derived from non-invasive fluorescence- and luminescence-based approaches (FRET, BRET, BiFC and BiLC), when applied to the study of G protein-coupled receptor (GPCR) oligomerization, can be used to detect specific PPIs in live cells. These technologies alone or in concert with complementary methods (SRET, BRET or BiFC, and SNAP-tag or TR-FRET) can be extremely powerful approaches for PPI visualization, even between more than two proteins. Here we provide a comprehensive update on all the biotechnological aspects, including the strengths and weaknesses, of new fluorescence- and luminescence-based methodologies, with a specific focus on their application for studying PPIs.

Section snippets

Background

Biological processes proceed through a sequence of specific protein–protein interactions (PPIs) along intracellular signaling cascades. Characterization of these interactions is thus essential to the understanding of cellular mechanisms. Using genetic approaches (e.g. yeast two-hybrid screens) 1, 2, it is possible to reveal new PPIs, which are subsequently confirmed and validated by additional biochemical approaches, such as immobilized PPI assays (e.g. co-immunoprecipitation and pull-down

Fluorescence RET (FRET)

Classical RET techniques, including fluorescence RET (FRET) and bioluminescence RET (BRET), use the nonradiative transfer of energy (Box 1) between donor and acceptor fluorescent molecules as a measure of their proximity. For example, cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP) can be used for FRET in live cells. When CFP is excited and FRET occurs, CFP emission decreases and YFP emission increases (Figure 1a). The presence of specific receptor homo- or

PPIs at the surface of a living cell

The difficulties encountered with classical RET techniques for analysis of GPCR homo- and heterocomplex assembly at the cell surface can be circumvented using the SNAP-tag technology (Table 1) [33]. The SNAP-tag method is based on irreversible and specific reaction of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives, which can be selectively labeled with a variety of chemical fluorophores (Figure 1c). The first remarkable characteristic of

Protein-fragment complementation assays toward the study of protein oligomerization

Protein-fragment complementation assays (PCAs) facilitate direct detection of PPIs and the study of their dynamic events in living cells 39, 40. In brief, PCAs consist of the structural and functional reconstitution of an active protein, typically an enzyme or FP, from two inactive halves that are genetically fused to the interacting proteins of interest. When FP or LP fragments are used for complementation, these assays are known as bimolecular fluorescence or luminescence complementation

Detection of higher-order protein complexes

The RET techniques described above are well-suited for detection of interactions between two proteins that form homo- or heterodimer complexes; however, a given protein can also be part of multiprotein complexes involving numerous interactions with different receptor partners. The formation of highly organized protein structures can be extremely specific, especially if they dictate the final functional output of a specific receptor oligomer. Thus, the existence of high-order oligomer complexes

Concluding remarks

Fluorescence- and luminescence-based assays are useful tools for the visualization and characterization of noncovalent PPIs in many cell types and organisms. In recent years, new optical techniques based on RET and protein-fragment complementation have enabled researchers to detect specific PPIs in diverse biological fields. Thus, as the use of these approaches increases dramatically, it is timely to revise their significant strengths and weaknesses to encourage reliable biotechnological

Acknowledgements

This work was supported by grants SAF2008-01462 and Consolider-Ingenio CSD2008-00005 from Ministerio de Ciencia e Innovación (to F.C.) and a start-up package from the Department of Pharmacology and Chemical Biology, University of Pittsburgh and by the National Institutes of Health grants DK087688 (J.-P.V.). F.C. and V.F.D. belong to the Neuropharmacology and Pain accredited research group (Generalitat de Catalunya, 2009 SGR 232). We thank Benjamín Torrejón and Esther Castaño from the Scientific

References (87)

  • J.F. Mercier

    Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer

    J. Biol. Chem.

    (2002)
  • A. Keppler

    Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro

    Methods

    (2004)
  • A. Gautier

    An engineered protein tag for multiprotein labeling in living cells

    Chem. Biol.

    (2008)
  • C.D. Hu

    Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation

    Mol. Cell

    (2002)
  • S. Zhang

    Combinatorial marking of cells and organelles with reconstituted fluorescent proteins

    Cell

    (2004)
  • T.F. Massoud

    Reporter gene imaging of protein–protein interactions in living subjects

    Curr. Opin. Biotechnol.

    (2007)
  • T.R. Hynes

    Visualization of G protein βγ dimers using bimolecular fluorescence complementation demonstrates roles for both β and γ in subcellular targeting

    J. Biol. Chem.

    (2004)
  • M. Heroux

    Functional calcitonin gene-related peptide receptors are formed by the asymmetric assembly of a calcitonin receptor-like receptor homo-oligomer and a monomer of receptor activity-modifying protein-1

    J. Biol. Chem.

    (2007)
  • F. Philip

    Signaling through a G protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model

    J. Biol. Chem.

    (2007)
  • Y. Liang

    Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes

    J. Biol. Chem.

    (2003)
  • D. Fotiadis

    Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors

    Curr. Opin. Struct. Biol.

    (2006)
  • B. Jastrzebska

    Functional and structural characterization of rhodopsin oligomers

    J. Biol. Chem.

    (2006)
  • C. Brock

    Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement

    J. Biol. Chem.

    (2007)
  • L.F. Agnati

    Receptor–receptor interactions: a novel concept in brain integration

    Prog. Neurobiol.

    (2010)
  • S. Ferre

    Functional relevance of neurotransmitter receptor heteromers in the central nervous system

    Trends Neurosci.

    (2007)
  • J. Gandia

    Detection of higher-order G protein-coupled receptor oligomers by a combined BRET-BiFC technique

    FEBS Lett.

    (2008)
  • P.A. Vidi

    Adenosine A2A receptors assemble into higher-order oligomers at the plasma membrane

    FEBS Lett.

    (2008)
  • R.V. Rebois

    Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

    Methods

    (2008)
  • R.A. Cardullo

    Theoretical principles and practical considerations for fluorescence resonance energy transfer microscopy

    Methods Cell Biol.

    (2007)
  • S.J. Briddon

    Plasma membrane diffusion of G protein-coupled receptor oligomers

    Biochim. Biophys. Acta

    (2008)
  • J. Miller et al.

    Using the yeast two-hybrid system to identify interacting proteins

    Methods Mol. Biol.

    (2004)
  • M. Selbach et al.

    Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK)

    Nat. Methods

    (2006)
  • J. Gandia

    Light resonance energy transfer-based methods in the study of G protein-coupled receptor oligomerization

    Bioessays

    (2008)
  • C.F. Becker

    C-Terminal fluorescence labeling of proteins for interaction studies on the single-molecule level

    ChemBiochem.

    (2006)
  • K. Palczewski

    G protein-coupled receptor rhodopsin

    Annu. Rev. Biochem.

    (2006)
  • V.P. Jaakola

    The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist

    Science

    (2008)
  • J.P. Vilardaga

    Measurement of the millisecond activation switch of G protein-coupled receptors in living cells

    Nat. Biotechnol.

    (2003)
  • P.S. Park

    Oligomerization of G protein-coupled receptors: past, present, and future

    Biochemistry

    (2004)
  • R. Maggio

    G protein-coupled receptor oligomerization provides the framework for signal discrimination

    J. Neurochem.

    (2007)
  • G. Milligan

    G protein-coupled receptor hetero-dimerization: contribution to pharmacology and function

    Br. J. Pharmacol.

    (2009)
  • J.P. Pin

    International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the recognition and nomenclature of G protein-coupled receptor heteromultimers

    Pharmacol. Rev.

    (2007)
  • J.P. Vilardaga

    Conformational cross-talk between alpha2A-adrenergic and mu-opioid receptors controls cell signaling

    Nat. Chem. Biol.

    (2008)
  • G. Valentin

    Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments

    Nat. Methods

    (2005)
  • Cited by (0)

    View full text