Spontaneous and ligand-induced trafficking of CXC-chemokine receptor 4.
A chimeric protein consisting of CXC-chemokine receptor 4 (CXCR4) and the green fluorescent protein (GFP) was used for studying receptor localization and trafficking in real time in stably transduced HeLa, U-937, CEM, and NIH/3T3 cells. CXCR4-GFP was fully active as a co-receptor in mediating human immunodeficiency virus (HIV) entry. Both CXCR4 and CXCR4-GFP were found to undergo significant spontaneous endocytosis. Only 51.5 +/- 7.8% of receptor molecules were found on the plasma membrane in CD4-positive cells, 43.9 +/- 8.5% were found in CD4-negative HeLa cells, 75.6 +/- 9.7% were found in U-937 cells, 72.5 +/- 7.9 were found in CEM cells, and almost none were found in in NIH/3T3 cells. Stromal cell-derived factor-1alpha induced rapid endocytosis of cell surface receptor molecules. A significant part of CXCR4 was targeted to lysosomes upon binding of the ligands, and recycling of internalized CXCR4 was not efficient. Only about 30% of receptor molecules recycled back to the cell surface in HeLa cells, 5% recycled in U937, and 10% recycled in CEM cells, suggesting that the protective effect of chemokines against HIV infection can be attributed not only to competition for binding but also to depletion of the co-receptor molecules from the cell surface. Envelope glycoprotein gp120 of syncytia-inducing/lymphocyte tropic HIV-1 strains induced rapid internalization of CXCR4 in both CD4-negative and CD4-positive cells, suggesting that gp120 is a high affinity ligand of CXCR4.
Tarasova
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NI
Molecular Aspects of Drug Design Section Applied Biosystems Laboratories Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA. Tarasova@ncifcrf.gov
Stauber
R H
RH
Michejda
C J
CJ
D016475:3T3 Cells; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000378:metabolism; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D015704:CD4 Antigens; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; C515256:Cxcl12 protein, mouse; D015699:HIV Envelope Protein gp120; D008024:Ligands; D008164:Luminescent Proteins; D019718:Receptors, CXCR4; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins