Abstract

We plan to monitor temporal and spatial arrangements of CD4 and chemokine receptors in living cells during the formation of HIV entry complexes. It is not clear if there are molecular interactions between CD4 and the chemokine receptors CXCR4 and CCR5 during HIV entry and if so if such interactions affect HIV entry and/or chemokine receptor signaling. We plan to address these issues by applying fluorescence resonance energy transfer (FRET) imaging to monitor the interactions of CD4 and the chemokine receptors in living cells. We have made progress in establishing cell lines for FRET imaging analyses. To visualize these two chemokine receptors and their interactions with CD4 in living cells, we have fused CD4, CXCR4 and CCR5 each with both cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at their C-termini and expressed them in HEK 293 cells. We have confirmed functionalities of tagged CXCR4 and CCR5 by monitoring signaling events upon receptor activation. We showed that CXCR4-CFP and CCR5-CFP trigger Ca2+ increases in response to their ligands.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000928-02
Application #
6987103
Study Section
(LIG)
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2004
Total Cost
Indirect Cost

  Institution

Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code

  Publications

Jin, Tian; Xu, Xuehua; Hereld, Dale (2008) Chemotaxis, chemokine receptors and human disease. Cytokine 44:1-8
Kramer, Jill M; Hanel, Walter; Shen, Fang et al. (2007) Cutting edge: identification of a pre-ligand assembly domain (PLAD) and ligand binding site in the IL-17 receptor. J Immunol 179:6379-83
Kramer, Jill M; Yi, Ling; Shen, Fang et al. (2006) Evidence for ligand-independent multimerization of the IL-17 receptor. J Immunol 176:711-5
Sohn, Hae Won; Tolar, Pavel; Jin, Tian et al. (2006) Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling. Proc Natl Acad Sci U S A 103:8143-8
Yi, Ling; Fang, Jun; Isik, Nilgun et al. (2006) HIV gp120-induced interaction between CD4 and CCR5 requires cholesterol-rich microenvironments revealed by live cell fluorescence resonance energy transfer imaging. J Biol Chem 281:35446-53
Jiao, Xuanmao; Zhang, Ning; Xu, Xuehua et al. (2005) Ligand-induced partitioning of human CXCR1 chemokine receptors with lipid raft microenvironments facilitates G-protein-dependent signaling. Mol Cell Biol 25:5752-62