Analysis of chimeric RGS proteins in yeast for the functional evaluation of protein domains and their potential use in drug target validation.
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.
Ajit
Seena K
SK
Wyeth Research, Neuroscience Discovery Research, CN 8000, Princeton NJ 08543, USA.
Young
Kathleen H
KH
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008856:Microscopy, Fluorescence; D010675:Pheromones / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
D044387:GTP-Binding Protein beta Subunits; D020690:GTPase-Activating Proteins; D010675:Pheromones; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; C094301:SST2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008156:Luciferases