Fluorescent protein complementation assays: new tools to study G protein-coupled receptor oligomerization and GPCR-mediated signaling

Abstract

G protein-coupled receptor (GPCR) signaling is mediated by protein-protein interactions at multiple levels. The characterization of the corresponding protein complexes is therefore paramount to the basic understanding of GPCR-mediated signal transduction. The number of documented interactions involving GPCRs is rapidly growing, and appreciating the functional significance of these complexes is clearly the next challenge. New experimental approaches including protein complementation assays (PCAs) have recently been used to examine the composition, plasma membrane targeting, and desensitization of protein complexes involved in GPCR signaling. These methods also hold promise for better understanding of drug-induced effects on GPCR interactions. This review focuses on the application of fluorescent PCAs for the study of GPCR signaling. Potential applications of PCAs in high-content screens are also presented. Non-fluorescent PCA techniques as well as combined assays for the detection of ternary and quaternary protein complexes are briefly discussed.

Figure 1

BiFC and multicolor BiFC to visualize GPCR oligomerization. Principle of BiFC (A) and multicolor BiFC (B). N- or C- terminal fragments (n or c) of fluorescence proteins (e.g. Venus, V and Cerulean, C) are fused to the carboxy-termini of the receptors (X, Y, and Z). Receptor dimerization leads to the complementation of functional FPs. (C) Multicolor BiFC for the simultaneous detection of A_2AR homomers and A_2AR/D₂R heteromers in neuronal cells. Prolonged treatment with quinpirole, a D₂R agonist, leads to decreased Venus (Vn/Cc) over Cerulean (Cn/Cc) fluorescence, interpreted as an alteration in GPCR homo-/heteromer abundance (Adapted from Vidi et al. 2008a). (D) Drug-induced changes in the composition and sub-cellular localization of CB₁R homomers and CB₁R/D₂R heteromers following persistent activation of CB₁R with CP55,940 (Adapted from (Przybyla and Watts 2010)).

Figure 2

Applications of combined fluorescence techniques to study receptor trafficking. (A) The expression of a dominant negative COPII Rab GTPase, Sar1 H79G, can be used as a tool to prevent GPCR export from the endoplasmic reticulum (ER). A_2AR fused to the Venus fluorescent protein was expressed in the CAD neuronal cell model. Plasma membrane expression of A_2AR was inhibited upon coexpression of Sar1 H79G (arrows). The N-terminal fragment of GAP43 fused to mCherry was used to label the plasma membrane (mCherry-MEM). Scale bar, 5 μm. (B) Fluorescence resonance energy transfer (FRET) measured with fluorescence lifetime imaging in cells expressing A_2AR-Cerulean (A_2AR-(C) and A_2AR-Venus (A_2AR-V), or A_2AR-Vn/A_2AR-Vc BiFC fusions, as well as Sar1 H79G. Cells were selected for analysis that displayed intracellular signals (gray bars) or plasma membrane signals (black bar). FRET evidenced by decreased Cerulean lifetime was measured in cells expressing A_2AR-C and A_2AR-V and at the plasma membrane from cells expressing A_2AR-C and A_2AR-Vn/A_2AR-Vc (‘trimer’). In contrast, no BiFC-FRET signal was detected in intracellular compartments. The data suggest that GPCR dimers may be trafficked from the ER and assemble into higher-order oligomers at the plasma membrane.

Figure 3

Possible application of BiFC in high-content screening for novel AC interacting proteins. The ‘sensitization interactome’ of AC will be examined using viral-mediated BiFC. Stable cell lines coexpressing GPCRs and AC-Vn will be infected with a Vc-tagged retroviral cDNA library. Complementation in the absence of GPCR stimulation represents pre-assembled complexes, constitutive interactions, or potential false positives. In contrast, cells revealing drug-induced complementation suggest the presence of a new interaction that occurs as a result of receptor activation and could represent novel AC-protein interactions relevant to the ‘sensitization interactome’.