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Cell Signal

Title:		Analysis of chimeric RGS proteins in yeast for the functional evaluation of protein domains and their potential use in drug target validation
Author(s):		Ajit SK; Young KH;
Address:		"Wyeth Research, Neuroscience Discovery Research, CN 8000, Princeton NJ 08543, USA"
Journal Title:		Cell Signal
Year:		2005
Volume:		20041210
Issue:		7
Page Number:		817 - 825
DOI:		10.1016/j.cellsig.2004.11.003
ISSN/ISBN:		0898-6568 (Print) 0898-6568 (Linking)
Abstract:		"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"
Keywords:		"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/a;"
Notes:		"MedlineAjit, Seena K Young, Kathleen H eng England 2005/03/15 Cell Signal. 2005 Jul; 17(7):817-25. doi: 10.1016/j.cellsig.2004.11.003. Epub 2004 Dec 10"