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© The Rockefeller University Press, 0021-9525/1997//651 $5.00
The Journal of Cell Biology, Volume 139, Number 3, , 1997 651-664

Article

Phorbol Esters and SDF-1 Induce Rapid Endocytosis and Down Modulation of the Chemokine Receptor CXCR4



Natalie Signoret*, Joanne Oldridge*, Annegret Pelchen-Matthews*, Per J. Klasse*, Thanh Tran{ddagger}, Lawrence F. Brass{ddagger}, Mette M. Rosenkilde§, Thue W. Schwartz§, William Holmes||, Walt Dallas||, Michael A. Luther||, Timothy N.C. Wells, James A. Hoxie{ddagger}, and Mark Marsh*

* Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom; {ddagger} Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; § Laboratory for Molecular Pharmacology, Rigshospitalet 6321, DK-2100 Copenhagen, Denmark; || Division of Molecular Sciences, Glaxo Research Institute, Research Triangle Park, North Carolina 27709; and Geneva Biomedical Research Institute, GlaxoWellcome Research and Development SA, 1228-Plan-Les-Ouabes, Geneva, Switzerland

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time ~5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms.

SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1–mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.

Abbreviations used in this paper: HA, hemagglutinin; HIV, human immunodeficiency virus; MIP, macrophage inflammatory peptide; PDB, phorbol dibutyrate; SDF, stromal cell–derived factor.

Address all correspondence to Mark Marsh, Medical Research Council, Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, UK. Tel.: (44) 171-380-7807. Fax: (44) 171-380-7805. E-mail: m.marsh{at}ucl.ac.uk

J. Oldridge, A. Pelchen-Matthews, P.J. Klasse, and M. Marsh were supported by grants from the UK Medical Research Council. N. Signoret was supported by a European Union Training and Mobility of Researchers programme grant number FMRX-CT96-0058. J.A. Hoxie was supported by grants from the National Institutes of Health AI40880 and AI38225 and L.F. Brass by National Institutes of Health grant number HL 40387. M. Marsh and J.A. Hoxie were supported by a North American Treaty Organization collaboration grant.


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