Development of RGS Modulator Screening Platform Based on Fluorescently Labeled and Chimeric Proteins
Date Issued
2011
Date
2011
Author(s)
Lin, Chang-Jia
Abstract
Heterotrimeric G-protein system composed by Gα and Gβγ subunits is the most prevalent cellular signaling molecule, mediating the delivering of extracellular signal from G-protein coupled receptor (GPCR) to intracellular effectors. The ligand bound GPCR activates G-proteins and switches on the signaling cascade via the GTP bound form Gα subunit. The signaling event terminates till the GTP hydrolysis catalyzed by the intrinsic GTPase activity of Gα, rendering G-protein system return to ground state and ready for the next signal.
The regulator of G-protein signaling (RGS) belongs to GTPase accelerating protein (GAP) family, which regulates signaling events downstream GPCR by promoting the GTP hydrolysis of Gα, therefore, reducing the duration and amplitude of activated signaling. The critical role on G-protein signaling implies RGS as a potential candidate for drug targeting, which attracts increasing professional and academic attention in last decade. However, the difficulty on analyzing RGS activity limits the progress of RGS physiologic research. There is no clinical RGS drug until today.
In this study, we focused on development of RGS4 and RGS9 drug screening platform based on highly expressible chimeric and fluorescently labeled proteins. In RGS9 part, we used a fluorescently modified chimeric Gα to probe the interaction with RGS9. By using 96-well plate and fluorescent ELISA, high throughput, highly sensitive, real-time monitoring of Gαt-RGS9 interaction could be achieved. Also, we developed RGS4- Gαi1 interaction assay via fluorescently labeled RGS4. In this part, through analysis of cysteines in RGS4 sequence, we successfully identified the background labeling came from Cys148 which also played a role in allosteric regulation of RGS4. Furthermore, Cys95 would be an essential residue for protein stability. Based on these results, we introduced few labeling sites on RGS4- Gαi1 interface to construct mutants for signaling optimization.
Subjects
G-protein system
regulator of G-protein signaling (RGS)
RGS modulator
RGS drug screening
fluorescent labeling
chimeric G-alpha
protein-protein interaction
RGS allosteric regulation
Type
thesis
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