CD4 and CCR5 constitutively interact at the plasma membrane of living cells: a confocal fluorescence resonance energy transfer-based approach.
Human immunodeficiency virus entry into target cells requires sequential interactions of the viral glycoprotein envelope gp120 with CD4 and chemokine receptors CCR5 or CXCR4. CD4 interaction with the chemokine receptor is suggested to play a critical role in this process but to what extent such a mechanism takes place at the surface of target cells remains elusive. To address this issue, we used a confocal microspectrofluorimetric approach to monitor fluorescence resonance energy transfer at the cell plasma membrane between enhanced blue and green fluorescent proteins fused to CD4 and CCR5 receptors. We developed an efficient fluorescence resonance energy transfer analysis from experiments carried out on individual cells, revealing that receptors constitutively interact at the plasma membrane. Binding of R5-tropic HIV gp120 stabilizes these associations thus highlighting that ternary complexes between CD4, gp120, and CCR5 occur before the fusion process starts. Furthermore, the ability of CD4 truncated mutants and CCR5 ligands to prevent association of CD4 with CCR5 reveals that this interaction notably engages extracellular parts of receptors. Finally, we provide evidence that this interaction takes place outside raft domains of the plasma membrane.
Gaibelet
Gérald
G
IPBS/CNRS, 205 Route de Narbonne, 31062 Toulouse cedex, France.
Planchenault
Thierry
T
Mazères
Serge
S
Dumas
Fabrice
F
Arenzana-Seisdedos
Fernando
F
Lopez
André
A
Lagane
Bernard
B
Bachelerie
Françoise
F
D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology* / Q000378:metabolism / Q000821:virology; D031541:Fluorescence Resonance Energy Transfer; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D015497:HIV-1 / Q000472:pathogenicity / Q000502:physiology; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D011485:Protein Binding; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism*; D015728:Receptors, HIV / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D008164:Luminescent Proteins; D019713:Receptors, CCR5; D015728:Receptors, HIV; D011993:Recombinant Fusion Proteins