Molecular analyses of the association of CD4 with two members of the transmembrane 4 superfamily, CD81 and CD82.
Previously, we have shown that CD81 and CD82, two members of the transmembrane 4 superfamily, form multimolecular membrane complexes by associating with each other and with CD4 or CD8 in T cells. In the present study, we further analyzed the molecular basis of the CD4 association with CD81 and CD82 by co-precipitation experiments. First, we examined the regions of CD4 involved in the association with CD81 and CD82 by employing chimeric proteins generated from CD4 and CD2. It was confirmed that CD4, but not CD2, was capable of binding with CD81 and CD82 in transfected cells. We found that the cytoplasmic region of CD4 was sufficient for the chimeric proteins to co-precipitate CD81, while both the cytoplasmic and extracellular regions of CD4 were required for them to efficiently co-precipitate CD82. We next found, by using truncated CD4 lacking the C-terminal 31 amino acids or mutated CD4 with the cysteine residues at 394 and 397 replaced by serine, that the p56lck binding site or the covalent modification with palmitic acid was not necessary for CD4 to associate with CD81 and CD82. Finally, we found that the binding of p56lck to CD4 strongly inhibited its association with CD81 and CD82. It is, therefore, suggested that CD4 exists at least in two physical states, one associated with p56lck and another associated with CD81 and CD82 in the absence or uncoupling of p56lck.
Imai
T
T
Shionogi Institute for Medical Science, Osaka, Japan.
Kakizaki
M
M
Nishimura
M
M
Yoshie
O
O
D000215:Acylation; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism*; D001402:B-Lymphocytes; D001483:Base Sequence; D001665:Binding Sites; D002051:Burkitt Lymphoma; D018801:CD2 Antigens / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D006801:Humans; D051861:Kangai-1 Protein; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D005810:Multigene Family; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D060225:Tetraspanin 28; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D018801:CD2 Antigens; D015704:CD4 Antigens; C554798:CD81 protein, human; C497688:CD82 protein, human; D051861:Kangai-1 Protein; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010169:Palmitic Acids; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D060225:Tetraspanin 28; D019308:Palmitic Acid; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)