The transmembranous domain of CD40 determines CD40 partitioning into lipid rafts.
Stimulation of CD40 has been previously shown to result in a release of ceramide in small sphingolipid-enriched rafts in the cell membrane [Grassmé et al., J. Immunol. 168 (2002) 298-307]. Those rafts fused to larger signaling platforms that served to cluster CD40. Here, we defined molecular mechanisms of CD40 clustering in membrane platforms. To this end, we replaced the transmembranous domain of CD40 with that of CD45, a molecule known to be excluded from lipid rafts. Murine T cells were stably transfected with wild-type CD40 or chimeric CD40/CD45. Flow cytometry confirmed normal binding properties of the mutant to CD40 ligand. Stimulation with CD40 ligand resulted in clustering of wild-type CD40, while the chimeric CD40/45 receptor failed to cluster. This correlated with a deficiency of the chimeric receptor to activate JNK, p38 MAP kinase and SAPK, known signaling molecules of the intracellular pathway initiated by CD40. Forced crosslinking of the CD40/45 chimeric receptor restored, at least partially, these signaling events. The results suggest that the transmembranous domain of CD40 is central for the recruitment to and clustering of CD40 in membrane platforms.
Bock
Jürgen
J
Department of Immunology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA.
Gulbins
Erich
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019013:CD40 Antigens / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D023201:CD40 Ligand / Q000378:metabolism / Q000494:pharmacology; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D048031:JNK Mitogen-Activated Protein Kinases; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism; D050356:Lipid Metabolism; D051379:Mice; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases
D019013:CD40 Antigens; D011994:Recombinant Proteins; D023201:CD40 Ligand; D048031:JNK Mitogen-Activated Protein Kinases; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D017493:Leukocyte Common Antigens