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Review
. 2010 Aug;28(8):407-15.
doi: 10.1016/j.tibtech.2010.05.002. Epub 2010 Jun 9.

Lighting up multiprotein complexes: lessons from GPCR oligomerization

Francisco Ciruela  1 , Jean-Pierre Vilardaga, Víctor Fernández-Dueñas
Affiliations
Review

Lighting up multiprotein complexes: lessons from GPCR oligomerization

Francisco Ciruela et al. Trends Biotechnol. 2010 Aug.

Abstract

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.

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Figures

Figure 1
Figure 1
Fluorescence-based methods used in the study of protein–protein interactions. (a) Schematic representation of the basic FRET principle applied to the study of GPCR oligomerization. Two putative receptors, R1 and R2, bear a donor CFP (R1CFP) and an acceptor YFP fluorophore (R2YFP). If R1 and R2 interact, then the donor and acceptor fluorophores might be in close proximity (≤10 nm) and energy transfer between the two fluorophores can occur after donor excitation at 433 nm and emission at 475 nm and acceptor emission at 527 nm. The schematic GPCR and FP diagrams were prepared using the PyMOL molecular graphics system (DeLano Scientific), with the crystal structure of the sensory rhodopsin II and GFP from Aequorea victoria (PDB 1JGJ and 1EMA, respectively) as models. (b) Example of the DFRAP effect. Emission intensities of YFP (527 nm, yellow line) and CFP (480 nm, cyan line) recorded from cells coexpressing the μ-opioid and α2A adrenergic receptors tagged at the C-terminus with YFP and CFP, respectively. Emission intensities were recorded before and after YFP (the acceptor fluorophore) was photobleached via 5-min exposure to light at 500 nm. Adapted with permission from [18]. (c) Schematic representations of various technologies applied to the study of GPCR oligomerization. Two putative GPCRs, (i) bearing a Myc and a Flag epitope tag on their N-terminal domains or fused to (ii) O6-alkylguanine-DNA alkyltransferase (AGT; SNAP tag) or (iii) mutant O6-alkylguanine-DNA alkyltransferase (AGT*; CLIP tag), are labeled with specific antibodies against these tag sequences (i.e. Myc or Flag) or with specific substrates for either AGT or AGT*. The antibodies are conjugated to the acceptor (APC; or d2 molecule) and the donor (K, Eu3+-cryptate) FRET-pair fluorophore molecules. The SNAP-tag substrate O6-benzylguanine (BG) is conjugated to the donor (K or Cy3) and the CLIP-tag substrate O6-benzylcytosine (BC) to the acceptor (Cy5). The schematic AGT diagram was prepared using PyMOL (PDB 1EH6).
Figure 2
Figure 2
Schematic representation of the basic principle of BiFC and multicolor BiFC. (a) Two putative receptors, R1 and R2, each carry one half of YFP: R1 carries an N-terminal YFP (R1N-YFP); R2 carries a C-terminal YFP (R2C-YFP). The interaction of R1 and R2 generates a fluorescent complex formed by the two halves of YFP (N and C) and YFP fluorescence can therefore be directly visualized after excitation at 500 nm. Corresponding confocal microscopy images of HEK cells expressing the respective receptor constructs are shown below the schematic. Scale bar, 10 μm. Adapted with permission from [87]. GPCR and FP diagrams were prepared as described for Figure 1. (b) Application of multicolor BiFC to the simultaneous detection of receptor homodimers and heterodimers. Receptor R1 carries an N-terminal fragment of the Venus FP (R1VN), and receptor R2 carries either the N-terminus of the Cerulean FP (R2CN) or the C-terminus of the Cerulean FP (R3CC). The relative amount of heterodimer (R1/R2) versus homodimer (R2-R2) can be simultaneously visualized after excitation at 500 nm (Venus) or 433 nm (Cerulean). (c) Receptor oligomerization in response to drugs can be monitored using the approach in (b). For example, prolonged treatment with a receptor agonist resulted in a decrease in Venus fluorescence at 527 nm as measured against Cerulean fluorescence at 475 nm, thus indicating an agonist-induced change in the GPCR homo-/heterodimer ratio (R2/R2 vs R1/R2). Adapted with permission from [56], copyright © 2008 American Society of Pharmacology and Experimental Therapeutics.
Figure 3
Figure 3
Fluorescence-based methods used in the study of higher-order protein complexes. (a) Schematic representation of the basic SRET principle applied to the study of GPCR oligomerization. Three putative receptors, R1, R2 and R3 bearing a donor (Rluc), an acceptor and donor (GFP2) and acceptor (YFP) chromophores, are depicted as R1Rluc, R2GFP2 and R3YFP, respectively. When the three receptors are in close proximity, a sequential double energy transfer (BRET-FRET) might occur: Rluc emission at 395 nm excites GFP2; GFP2 emits at 510 nm, in turn exciting YFP, which then emits at 527 nm. The schematic GPCR diagrams were prepared as described in Figure 1. The crystal structure of luciferase from Renilla reniformis (PDB 2PSD) is shown. (b) The combination of PCA and RET techniques allows for the detection of tetrameric receptor complexes (R1-R2-R3-R4). BiLC generates a complementary luminescent protein (Rluc) that acts as a donor in a BRET process, with a complementary fluorescent acceptor protein (YFP) generated by BiFC. If these receptors are in close proximity, then donor–acceptor energy transfer can occur after Rluc substrate (coelenterazine H) oxidation. Excitation (475 nm) and emission (527 nm) wavelengths are indicated.
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