Functional expression of CXCR4 in Saccharomyces cerevisiae in the development of powerful tools for the pharmacological characterization of CXCR4.
CXCR4, the receptor for stromal cell-derived factor (SDF)-1, was expressed in Saccharomyces cerevisiae, coupled to the pheromone response pathway via a chimeric Galpha subunit. Engagement of CXCR4 by SDF-1 resulted in expression of reporter genes, HIS3 or lacZ, under the transcriptional control of a FUS1 promoter, which is pheromone-responsive. CXCR4 mutants with constitutive signaling activity were generated by random mutagenesis of receptor coding sequences and selection for complementation of histidine auxotrophy in the yeast strain by autonomous expression of the FUS1-HIS3 reporter gene. Linkage of CXCR4 to the pheromone response pathway in yeast provides a system that lends itself to screening of receptor antagonists. The use of constitutively active mutants to screen for inhibitors of the weak partial agonist and inverse agonist pharmacologic types offers a sensitive, efficient approach that is independent of ligand.
Wang
Zi-xuan
ZX
Department of Pathology, Medical College of Georgia, Augusta, USA.
Broach
James R
JR
Peiper
Stephen C
SC
D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D006801:Humans; D011401:Promoter Regions, Genetic; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D019718:Receptors, CXCR4; D043562:Receptors, G-Protein-Coupled; D029701:Saccharomyces cerevisiae Proteins