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Analysis of chimeric RGS proteins in yeast for the functional evaluation of protein domains and their potential use in drug target validation
Journal article   Peer reviewed

Analysis of chimeric RGS proteins in yeast for the functional evaluation of protein domains and their potential use in drug target validation

Seena K Ajit and Kathleen H Young
Cellular signalling, v 17(7), pp 817-825
Jul 2005
DOI: https://doi.org/10.1016/j.cellsig.2004.11.003
PMID: 15763424
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

Animals Cercopithecus aethiops COS Cells Genes, Reporter GTP-Binding Protein beta Subunits - metabolism GTPase-Activating Proteins - genetics Humans Luciferases - genetics Microscopy, Fluorescence Pheromones - pharmacology Protein Structure, Tertiary Rats Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism RGS Proteins - antagonists & inhibitors RGS Proteins - genetics RGS Proteins - metabolism Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - metabolism Saccharomyces cerevisiae Proteins - genetics
For the identification of regulators of G-protein signaling (RGS) modulators, previously, we developed a luciferase based yeast pheromone response (YPhR) assay to functionally investigate RGS4 (K.H. Young, Y. Wang, C. Bender, S. Ajit, F. Ramirez, A. Gilbert, B.W. Nieuwenhuijsen, in: D.P. Siderovski (Ed.), Meth. Enzymol. 389 Regulators of G_protein Signaling, Part A, 2004.). To extend the diversity of this assay, additional RGS proteins were evaluated for functional complementation in a RGS (sst2Delta) knockout yeast strain. For RGS proteins that did not function in their native form, a series of chimeric constructs were generated with the N terminus of RGS4 fused in frame with the partial or full-length RGS cDNA of interest. RGS4 N terminus fused to either full-length or the C terminus of RGS7 successfully complemented sst2Delta. On the contrary, the RGS7N/RGS4C chimera (N terminus of RGS7 in frame with RGS domain of RGS4) was not effective, showing that N terminus of RGS4 helps in targeting. RGS10 exists as two splice variants, differing only by 8 amino acids (aa) in the N terminus, being either 168 aa (RGS10S), or 174 aa (RGS10). While RGS10 was functional in yeast, RGS10S required the presence of the N terminus of RGS4 for its activity. Although the same RGS4 N terminus domain was present in chimeras generated, the GTPase accelerating protein (GAP) function observed was not similar, suggesting differences in the RGS domain function. In conclusion, the use of RGS4 N terminus chimeric constructs enabled us to develop a selectivity assay for different RGS proteins.

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Web of Science research areas
Cell Biology
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