MOLECULAR ANALYSES OF THE ASSOCIATION OF CD4 WITH 2 MEMBERS OF THE TRANSMEMBRANE-4 SUPERFAMILY, CD81 AND CD82

Previously, we have shown that CD81 and CD82, two members oi the trans membrane 4 superfamily;, form multimolecular membrane complexes by ass ociating with each other and with CD4 or CD8 in T cells. In the presen t study, we further analyzed the molecular basis of the CD4 associatio n with CD81 and CD82 by co-precipitation experiments. First, we examin ed the regions of CD4 involved in the association with CD81 and CD82 b y employing chimeric proteins generated from CD4 and CD2. It was confi rmed 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 bot h the cytoplasmic and extracellular regions of CD4 were required for t hem to efficiently co-precipitate CD82. We next found, by using trunca ted CD4 lacking the C-terminal 31 amino acids or mutated CD4 with the cysteine residues at 394 and 397 replaced by serine, that the p56(lck) binding site or the covalent modification with palmitic acid was not necessary for CD4 to associate with CD81 and CD82. Finally, we found t hat the binding of p56(lck) to CD4 strongly inhibited its association with CD81 and CD82. It is, therefore, suggested that CD4 exists al lea st in two physical stales, one associated with p56(lck) and another as sociated with CD81 and CD82 in the absence or uncoupling of p56(lck).