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

Rebois, R V; Robitaille, Mélanie; Pétrin, Darlaine; Zylbergold, Peter; Trieu, Phan; Hébert, Terence E.

doi:10.1016/j.ymeth.2008.06.006

Cited by 46 publications

(34 citation statements)

References 33 publications

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“…In cells expressing ACII-RLuc and G␤ 1 /␥ 2 fusions to YFP fragments, activation of cotransfected ␤ 2 AR resulted in stronger BRET signals, possibly reflecting conformational changes within the complex (Rebois et al, 2006). The association between a GPCR, a G protein subunit, and an effector was also revealed in BiFC-BRET assays using cells coexpressing ACII-RLuc with FPfragment fusions to ␤ 2 AR and G␥ 2 (Rebois et al, 2008). These experiments highlight the use of BiFC-BRET as a novel method to study and examine the existence of GPCR signaling complexes.…”

Section: Combining Techniques To Monitor Interactions Between Multiplmentioning

confidence: 87%

“…2D) was given by Rebois et al (Rebois et al, 2008): the ␤ 2 AR was fused to N-or C-terminal fragments from Venus or GLuc, and BRET signals were detected in cells expressing the fourreceptor fusion constructs (Rebois et al, 2008). A similar approach was used to demonstrate D 2 homo-oligomeric complexes with four protomers (Guo et al, 2008), a finding supported by modeling of the two interaction interfaces and confirmed by cross-linking experiments (Guo et al, 2008).…”

Section: Combining Techniques To Monitor Interactions Between Multiplmentioning

confidence: 87%

“…The assembly of trimeric G proteins was shown in cells expressing G␣ i1 -RLuc and G␤ 1 /G␥ 2 fusions to YFP fragments . Likewise, the association of the type II adenylyl cyclase (ACII) or the inward-rectifier potassium channel Kir 3.1 with G␤ 1 /␥ 2 was demonstrated in cells expressing ACII-RLuc or Kir 3.1 -RLuc and G␤ 1 /␥ 2 fusions to YFP fragments (Rebois et al, 2006;Rebois et al, 2008). In cells expressing ACII-RLuc and G␤ 1 /␥ 2 fusions to YFP fragments, activation of cotransfected ␤ 2 AR resulted in stronger BRET signals, possibly reflecting conformational changes within the complex (Rebois et al, 2006).…”

Section: Combining Techniques To Monitor Interactions Between Multiplmentioning

confidence: 99%

See 2 more Smart Citations

Fluorescent and Bioluminescent Protein-Fragment Complementation Assays in the Study of G Protein-Coupled Receptor Oligomerization and Signaling

Vidi

Watts²

2009

Mol Pharmacol

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Most cellular functions, including signaling by G protein-coupled receptors (GPCRs), are mediated by protein-protein interactions, making the identification and localization of protein complexes key to the understanding of cellular processes. In complement to traditional biochemical techniques, noninvasive resonance energy transfer (RET) and protein-fragment complementation assays (PCAs) now allow protein interactions to be detected in the context of living cells. In this review, fluorescent and bioluminescent PCAs are discussed and their application illustrated with studies on GPCR signaling. Newly developed techniques combining PCA and RET assays for the detection of ternary and quaternary protein complexes are also presented.The identification and localization of protein-protein interactions is central to the understanding of biological processes such as extracellular signal integration, gene expression regulation, as well as most cellular metabolic pathways. Immunoprecipitation (IP) and pull-down assays have been used extensively to demonstrate protein-protein interactions. Although these techniques remain very useful, they necessitate the disruption of biological samples and the solubilization of membrane proteins, and they generally provide little information on the subcellular localization of the protein complexes (Milligan and Bouvier, 2005). Fluorescence and bioluminescence resonance energy transfer (FRET and BRET), as well as reporter complementation assays, are noninvasive approaches that overcome some limitations of classic biochemical techniques. They allow the examination of proteinprotein interactions in their native context: in living cells or even in living animals. Moreover, information on the subcellular localization of the interaction can be gained with microscopic FRET or bimolecular fluorescence complementation (BiFC) analysis.Multiple protein-protein interactions play essential roles in signaling events mediated by GPCRs. Oligomerization between GPCRs is now recognized to modulate the pharmacological characteristics of the receptors and influence their coupling to G proteins (Fuxe et al., 2007;Milligan, 2007;Pin et al., 2007). The interaction between GPCRs and G proteins is also well documented. GPCR activation upon ligand binding induces conformational changes in the receptor structure that promotes a GDP-to-GTP nucleotide exchange to the ␣ subunit of the interacting trimeric G protein. GTP-bound G␣ subunits then dissociate from (or change conformation relative to) the receptor and the ␤/␥ subunits, allowing interactions between G␣ as well as G␤/␥ with effector proteins (Lambert, 2008). In contrast to early models in which individual components of the system were envisioned as free-

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Section: Combining Techniques To Monitor Interactions Between Multiplmentioning

confidence: 87%

Section: Combining Techniques To Monitor Interactions Between Multiplmentioning

confidence: 87%

Section: Combining Techniques To Monitor Interactions Between Multiplmentioning

confidence: 99%

See 1 more Smart Citation

Fluorescent and Bioluminescent Protein-Fragment Complementation Assays in the Study of G Protein-Coupled Receptor Oligomerization and Signaling

Vidi

Watts²

2009

Mol Pharmacol

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“…Alternatively, cell surface-expressed snap-tagged GPCRs can be covalently labeled with similar membrane-impermeant donor and acceptor fluorophores [45]. Recently, protein fragment complementation (PFC) techniques have also been applied to detect GPCR dimerization and oligomerization when used in combination with FRET/BRET measurements [46][47][48][49]. To this end, a reporter protein (e.g., green fluorescent protein or luciferase variants) is split into two nonfunctional fragments, which are genetically fused to the Cterminus of the GPCRs of interest.…”

Section: Detection Of Gpcr Dimerizationmentioning

confidence: 99%

Targeting Chemokine Receptor Dimers: Are There Two (or More) to Tango?

Vischer¹,

Nijmeijer²,

Parmentier

2010

Methods and Principles in Medicinal Chemistry

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“…Young-Hwa Song and Matthias Wilmanns describe the use of BiFC analysis in combination with high-resolution structural biology methods for determination of the architectures of multi-protein complexes [5]. Terence Hébert and colleagues report the detection of ternary protein complexes using a combination of complementation and resonance energy transfer assays [6]. Cathy Berlot and colleagues describe the use of multicolor BiFC analysis for quantitative analysis of the relative efficiencies of complex formation among different G protein subunits [7].…”

Section: Several Approaches Have Been Developed For Visualization Of mentioning

confidence: 99%

Overcoming uncertainty through advances in fluorescence imaging of molecular processes in cells

Kerppola¹

2008

Methods

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

Cited by 46 publications

References 33 publications

Fluorescent and Bioluminescent Protein-Fragment Complementation Assays in the Study of G Protein-Coupled Receptor Oligomerization and Signaling

Fluorescent and Bioluminescent Protein-Fragment Complementation Assays in the Study of G Protein-Coupled Receptor Oligomerization and Signaling

Targeting Chemokine Receptor Dimers: Are There Two (or More) to Tango?

Overcoming uncertainty through advances in fluorescence imaging of molecular processes in cells

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