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

Wen Bo Zhang, Jean Marc Navenot, Bodduluri Haribabu, Hirokazu Tamamuraz, Kenichi Hiramatu, Akane Omagari, Gang Pei, John P. Manfredi, Nobutaka Fujii, James R. Broach, Stephen C. Peiper

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206 Scopus citations

Abstract

CXCR4 is a G protein-coupled receptor for stromal-derived factor 1 (SDF-1) that plays a critical role in leukocyte trafficking, metastasis of mammary carcinoma, and human immunodeficiency virus type-1 infection. To elucidate the mechanism for CXCR4 activation, a constitutively active mutant (CAM) was derived by coupling the receptor to the pheromone response pathway in yeast. Conversion of Asn-119 to Ser or Ala, but not Asp or Lys, conferred autonomous CXCR4 signaling in yeast and mammalian cells. SDF-1 induced signaling in variants with substitution of Asn-119 to Ser, Ala, or Asp, but not Lys. These variants had similar cell surface expression and binding affinity for SDF-1. CXCR4-CAMs were constitutively phosphorylated and present in cytosolic inclusions. Analysis of antagonists revealed that exposure to AMD3100 or ALX40-4C induced G protein activation by CXCR4 wild type, which was greater in the CAM, whereas T140 decreased autonomous signaling. The affinity of AMD3100 and ALX40-4C binding to CAMs was less than to wild type, providing evidence of a conformational shift. These results illustrate the importance of transmembrane helix 3 in CXCR4 signaling. Insight into the mechanism for CXCR4 antagonists will allow for the development of a new generation of agents that lack partial agonist activity that may induce toxicities, as observed for AMD3100.

Original languageEnglish (US)
Pages (from-to)24515-24521
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number27
DOIs
StatePublished - Jul 5 2002

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Zhang, W. B., Navenot, J. M., Haribabu, B., Tamamuraz, H., Hiramatu, K., Omagari, A., Pei, G., Manfredi, J. P., Fujii, N., Broach, J. R., & Peiper, S. C. (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. Journal of Biological Chemistry, 277(27), 24515-24521. https://doi.org/10.1074/jbc.M200889200