Identification and characterization of the CXCR4 chemokine receptor in human T cell lines: ligand binding, biological activity, and HIV-1 infectivity.
The CXCR4 chemokine receptor has been shown to respond to the C-X-C chemokine stromal-derived factor (SDF-1) and has recently been shown to be an important coreceptor for HIV-1 infection. In the present paper we have tested a number of human lymphocyte cell lines, including Jurkat, HUT78, CEM, and Sup-T1 for the presence of CXCR4 receptors. We found that these T cell lines bind SDF-1alpha and SDF-1beta with high affinity. The CXCR4 Ab 12G5 inhibited both SDF-1 binding and HIV-1LAI-mediated fusion of CEM. Scatchard analysis revealed the presence of approximately 150,000 SDF-1alpha-binding sites per cell with a Kd between 5 and 10 nM. Cross-competition experiments using unlabeled SDF-1alpha and SDF-1beta revealed that both chemokines are equally capable of displacing their radiolabeled counterparts. Internalization studies with [125]I-SDF-1alpha revealed that Jurkat cells internalized greater than 90% of the ligand by 2 h at 37 degrees C. SDF-1alpha was also chemotactic for Jurkat cells and caused an increase in the rate of extracellular acidification that was half-maximal at 18 nM SDF-1alpha and could be inhibited by pretreatment with the SDF-1 proteins, pertussis toxin, or the Ab 12G5. Finally, SDF-1alpha also caused an increase in the cytosolic Ca2+ concentration in Sup-T1 cells that was abolished by preincubating the cells with pertussis toxin or PMA and inhibited by the Ab 12G5. This molecular characterization of CXCR4 receptors should prove useful in clarifying receptor interaction with SDF-1 proteins and with HIV-1 glycoprotein, with the ultimate aim of targeting the viral interaction for therapeutic intervention.
Hesselgesser
J
J
Department of Immunology, Berlex BioSciences, Richmond, CA 94804, USA.
Liang
M
M
Hoxie
J
J
Greenberg
M
M
Brass
L F
LF
Orsini
M J
MJ
Taub
D
D
Horuk
R
R
D001665:Binding Sites; D002459:Cell Fusion / Q000276:immunology; D002460:Cell Line; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D015658:HIV Infections / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008024:Ligands; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011991:Receptors, Virus / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism* / Q000821:virology; D014407:Tumor Cells, Cultured
C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D016207:Cytokines; D008024:Ligands; D019718:Receptors, CXCR4; D011991:Receptors, Virus; D019204:GTP-Binding Proteins