Multicolor BiFC analysis of competition among G protein β and γ subunit interactions

Abstract

We have applied multicolor BiFC to study the association preferences of G protein β and γ subunits in living cells. Cells co-express multiple isoforms of β and γ subunits, most of which can form complexes. Although many βγ complexes exhibit similar properties when assayed in reconstituted systems, knockout experiments in vivo suggest that individual isoforms have unique functions. BiFC makes it possible to correlate βγ complex formation with functionality in intact cells by comparing the amounts of fluorescent βγ complexes with their abilities to modulate effector proteins. The relative predominance of specific βγ complexes in vivo is not known. To address this issue, multicolor BiFC can determine the association preferences of β and γ subunits by simultaneously visualizing the two fluorescent complexes formed when β or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete to interact with limiting amounts of a common γ or β subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). Multicolor BiFC also makes it possible to determine the roles of interacting proteins in the subcellular targeting of complexes, study the formation of protein complexes that are unstable under isolation conditions, determine the roles of co-expressed proteins in regulating the association preferences of interacting proteins, and visualize dynamic events affecting multiple protein complexes. These approaches can be applied to studying the assembly and functions of a wide variety of protein complexes in the context of a living cell.

A novel orange-colored bimolecular fluorescence complementation (BiFC) assay using monomeric Kusabira-Orange protein

“…Although the KON154 and KOC155 pair showed bright fluorescence, it is possible that frequent self-assembly of fragments results in a higher background signal. To address this, the S/N ratio of mKO2-based BiFC was estimated by a BiFC competition assay [3,8,17,18]. In this assay, a competitor-induced decrease in BiFC fluorescence reflects the positive signal derived from specific proteinprotein interactions and the remaining fluorescence reflects the background signal derived from the self-assembly of BiFC fragments [3].…”

Bimolecular Fluorescence Complementation (BiFC): A 5-year Update and Future Perspectives

“…The competitive nature of bJun interactions was also demonstrated using the multicolor BiFC assay to analyze interactions among bJun, bFos, and bATF2 (15). BiFC competition analysis has been successfully used in other BiFC experiments as well (17,19,62). However, it is important to note that the competitor must be co-expressed with the two fusion proteins, if not before the expression of the two fusions, since the BiFC complex is essentially irreversible once formed (8,33,43,4851).…”

Sensing of biomolecular interactions using fluorescence complementing systems in living cells

“…Although many β and γ complexes exhibit similar properties when assayed in reconstituted systems, in vivo knockout experiments suggest that individual isoforms have unique functions. Application of BiFC enables researchers to correlate β and γ complex formation with functionality in intact cells by comparing the amounts of fluorescent β and γ complexes with their abilities to modulate effector proteins (Hynes et al, 2008). There is growing awareness of the complexity in the organization of 7TM receptor signaling pathways, and in the ability of their ligands to modulate and direct this signaling, the simplicity of BiFC measurements allows for high-content and advanced imaging applications (Rose et al, 2010).…”