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

Cell Signal. 2005 Jul;17(7):817-25. doi: 10.1016/j.cellsig.2004.11.003. Epub 2004 Dec 10.


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.

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • GTP-Binding Protein beta Subunits / metabolism
  • GTPase-Activating Proteins / genetics
  • Genes, Reporter
  • Humans
  • Luciferases / genetics
  • Microscopy, Fluorescence
  • Pheromones / pharmacology
  • Protein Structure, Tertiary
  • RGS Proteins / antagonists & inhibitors
  • RGS Proteins / genetics
  • RGS Proteins / metabolism*
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics


  • GTP-Binding Protein beta Subunits
  • GTPase-Activating Proteins
  • Pheromones
  • RGS Proteins
  • Recombinant Fusion Proteins
  • SST2 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Luciferases