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. 2017 Apr 1:522:10-17.
doi: 10.1016/j.ab.2017.01.013. Epub 2017 Jan 20.

Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells

Christopher R Bodle  1 , Michael P Hayes  2 , Joseph B O'Brien  3 , David L Roman  4
Affiliations

Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells

Christopher R Bodle et al. Anal Biochem. .

Abstract

Cell based assessment tools and screening platforms are the preferred paradigm for small molecule identification and validation due to selectively identifying molecules with cellular activity and validation of compound activity against target proteins in their native environment. With respect to Regulator of G Protein Signaling (RGS) proteins, current cell based methodologies are either low throughput or monitor downstream signaling consequences. The increasing number of reports indicating RGS function in various disease pathogeneses highlights the need for a robust RGS inhibitor discovery and characterization paradigm. Promega's NanoBit Protein Complementation Assay utilizes NanoLuc, an engineered luciferase with enhanced luminescence characteristics which allow for both robust and kinetic assessment of protein interaction formation and disruption. Here we characterized 15 separate RGS: G protein interactions using this system. The binding profile of RGS: Gα interactions correlates to prior published biochemical binding profiles of these proteins. Additionally, we demonstrated this system is suitable for high throughput screening efforts via calculation of Z-factors for three of the interactions and demonstrated that a known small molecule inhibitor of RGS4 disrupts the RGS4: Gαi1 protein-protein interaction. In conclusion, the NanoBit Protein Complementation Assay holds promise as a robust platform for discovery and characterization of RGS inhibitors.

Keywords: Gα(i1); Gα(q); High throughput screening; NanoBit; RGS.

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Figures

Figure 1
Figure 1
Assembly of Protein Interaction Pairs. Four plasmids were constructed to produce fusion proteins of each possible large bit (LB) and small bit (SB) combination. Proteins depicted with a Nanobit piece on the left of the protein is fusion on the N-terminus of the RGS or G protein, and a Nanobit piece on the right of the protein represents fusion on the C-terminus of the RGS or G protein. Under each depiction is an example of how these fusion constructs are referred to in the text.
Figure 2
Figure 2
Optimization of Vector Pairs Using RGS4 and RGS17. RGS:Gαi1 complementation pairs were optimized using RGS4 and RGS17. A.) Representative kinetic read of interaction of RGS4: Gαi1. B.) Delineation of time range for area under the curve analysis, starting at AlF4 addition. 2C-D is a comparison of four vector pairs for the RGS4: Gαi1 interaction shown as increase in area under the curve following AlF4 addition (C) and fold increase in area under the curve (D) upon stimulation with AlF4. The same analysis was performed for the four optimal RGS17: Gαi1 vector pairs (E/F). Data are n=3 in at least duplicate, ± SD.
Figure 3
Figure 3
Characterization of 12 WT and 1 Mutant RGS Proteins Interaction with Gαi1 or Gαq. A.) Depicts the net increase in area under the curve in response to AlF4 stimulation for 13 RGS: Gαi1 protein pairs. B.) Normalized non- linear fit of luminescence over time to assess the rate of protein interaction. C.) Magnification of the boxed area in panel B. D.) RGS2 interaction with Gαi1 and Gαq in response to AlF4. E.) Non-linear fit of RGS2: Gαq as in panel B. Data are n=3 in at least duplicate, ± SD. Data for panel D are the background subtracted results of n=3 in at least duplicate, ± SEM.
Figure 4
Figure 4
Selective Disruption of RGS4: Gαi1 Interaction with 6383479. Small molecule inhibition of RGS4: Gαi1 interaction. A) n=3 raw luminescence trace of RGS4: Gαi1 in the presence or absence of RGS4 inhibitor 6383479. B) Quantification of 6383479 effect on RGS4: Gαi1 and control using AUC-compound treated/AUC-vehicle treated. Data are n=3 in at least duplicate, ± SD. P-value = 0.012
Figure 5
Figure 5
Characterization of RGS6L:Gβ5 Interaction. Interaction of RGS6L with Gβ5 was also detectable using this system. A.) Raw traces of RGS6Lα2: Gβ5 vector pairs. B.) Raw trace comparison of RGS6Lα2: Gβ5 interactions producing largest signal and the corresponding RGS6RH: Gβ5 pairing. Data are n=3 in at least duplicate, ± SD.
Figure 6
Figure 6
Amenability of RGS NanoBit™ Systems to High Throughput Screening. Suitability of NanoBit™ systems for high throughput campaigns. A/B.) The low magnitude interaction of RGS6RH: Gαi1 in a 96 well half area plate. The raw signal over time (A) along with Z-factor over time (B) are shown. C/D.) The high magnitude interaction of RGS8RH: Gαi1 was also tested in a similar manner. E/F.) Finally the RGS6L: Gβ5 interaction was assessed. Y-axis normalized to RGS8RH: Gαi1 signal to demonstrate varying magnitude of signal between protein pairs assessed.
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