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.
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References
Malbon C. C. (2004) Frizzleds:new members of the superfamily of G-proteincoupled receptors. Front. Biosci. 9, 1048–1058.
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.
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.
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.
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.
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.
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.
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© 2006 Humana Press Inc.
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Wang, Zx., Broach, J.R., Peiper, S.C. (2006). Functional Expression of CXCR4 in Saccharomyces cerevisiae in the Development of Powerful Tools for the Pharmacological Characterization of CXCR4. In: Ali, H., Haribabu, B. (eds) Transmembrane Signaling Protocols. Methods in Molecular Biology™, vol 332. Humana Press. https://doi.org/10.1385/1-59745-048-0:115
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DOI: https://doi.org/10.1385/1-59745-048-0:115
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