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107 Direct Physical Association between Purified CCR5 and CD4
S. Phogat*, X. Xiao , L. Wilson, A. Phogat, and D. S. Dimitrov
NCI at Frederick, NIH, MD, USA
Background: CD4 and CCR5 are required for the initiation of signal transduction and serve as major HIV receptors. It has been previously demonstrated the cell surface-associated molecules can be coimmunoprecipitated and that 2-domain (but not 4-domain) soluble CD4 (sCD4) can partially displace MIP-1a bound to cell surface associated CCR5. However, it was not clear whether the CD4-CCR5 interaction is direct or mediated through other molecules. Evidence for direct physical association between purified molecules not only would resolve this issue but could also provide new possibilities for detailed study of the CCR5-CD4 interaction.
Methods: Detergent solubilized CCR5 (dsCCR5) was produced and purified by a methodology developed in J. Sodroski’s laboratory based on cells engineered to express very high levels of CCR5 tagged with a nanopeptide recognized by the mAb 1D4. Binding to sCD4 was measured by using an ELISA-type of assay.
Results: Purified dsCCR5 bound specifically and with high affinity to 2-domain and 4-domain sCD4. Antibodies to CD4 inhibited the binding in a dose-dependent manner that correlated with their binding to CD4. The equilibrium dissociation constant of the dsCCR5-sCD4 association at 4(C was in the nanomolar range and similar for the 2- and 4-domain sCD4.
Conclusions: The finding that CCR5 directly associates with CD4 could have implications for possible cross-talk between receptors belonging to 2 unrelated families, further understanding of the mechanisms of HIV-1 entry and development of candidate vaccines based on CD4-CCR5 complexes with HIV-1 envelope glycoproteins.
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