Advertisement

Functional Expression of CXCR4 in Saccharomyces cerevisiae in the Development of Powerful Tools for the Pharmacological Characterization of CXCR4

  • Zi-xuan Wang
  • James R. Broach
  • Stephen C. Peiper
Part of the Methods in Molecular Biology™ book series (MIMB, volume 332)

Summary

CXCR4, the receptor for stromal cell-derived factor (SDF)-1, was expressed in Saccharomyces cerevisiae coupled to the pheromone response pathway via a chimeric Gα 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 pheromoneresponsive. 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.

Key Words

CXCR4 Saccharomyces cerevisiae GPCR constitutively active mutant (CAM) antagonists FUS1-HIS3, FUS1-lacZ 

References

  1. 1.
    Malbon C. C. (2004) Frizzleds:new members of the superfamily of G-proteincoupled receptors. Front. Biosci. 9, 1048–1058.PubMedCrossRefGoogle Scholar
  2. 2.
    Bardwell L., Cook J. G., Inouye C. J., and Thorner J. (1994) Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae. Dev Biol 166, 363–379.PubMedCrossRefGoogle Scholar
  3. 3.
    Ozcan S., Dover J., and Johnston M. (1998) Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae. EMBO J. 17, 2566–2573.PubMedCrossRefGoogle Scholar
  4. 4.
    Zhang W. B., Navenot J. M., Haribabu B., et al. (2002) A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists. J. Biol. Chem 277, 24,515–24,521.PubMedCrossRefGoogle Scholar
  5. 5.
    Klein C., Paul J. I., Sauve K., et al. (1998) Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast. Nat. Biotechnol. 16, 1334–1337.PubMedCrossRefGoogle Scholar
  6. 6.
    Zhang W. B., Wang Z. X., Murray J. L., Fujii N., Broach J., and Peiper S. C. (2004) Functional expression of CXCR4 in S. cerevisiae: development of tools for mechanistic and pharmacologic studies. Ernst. Schering. Res. Found Workshop 125–152.Google Scholar
  7. 7.
    Fujii N., Oishi S., Hiramatsu K., et al. (2003) Molecular-size reduction of a potent CXCR4-chemokine antagonist using orthogonal combination of conformation-and sequence-based libraries. Angew. Chem. Int. Ed. Engl. 42, 3251–3253.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Zi-xuan Wang
    • 1
  • James R. Broach
    • 2
  • Stephen C. Peiper
    • 3
  1. 1.Department of PathologyMedical College of GeorgiaAugusta
  2. 2.Department of Molecular BiologyPrinceton UniversityPrinceton
  3. 3.Department of PathologyMedical College of GeorgiaAugustaGA

Personalised recommendations