Signaling and protein associations of a cell permeable CD40 complex in B cells.
Signaling through the CD40 receptor activates diverse molecular pathways in a variety of immune cell types. To study CD40 signaling complexes in B cells, we produced soluble CD40 cytoplasmic domain multimers that translocate across cell membranes and engage intracellular CD40 signaling pathways. As visualized by fluorescence microscopy, rapid transduction of recombinant Antennapedia-isoleucine zipper (Izip)-CD40 cytoplasmic domain fusion protein (Antp-CD40) occurred in both the DND39 B cell line and human tonsillar B cells. Upon cellular entry, Antp-CD40 activated NF-kappaB-dependent transcription, induced proteolytic processing of p100 to the p52/NF-kappaB2 subunit, and increased expression of CD80 and CD54 on the surface of B cells. Antp-CD40 transduction of B cells did not, however, activate detectable levels of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase and did not up-regulate CD95 expression. Analysis of Antp-CD40 complexes recovered from transduced B cells revealed that Antp-CD40 associated with endogenous TRAF3 and Ku proteins. Multimerization of Antp-CD40, or extensive clustering of transmembrane CD40, diminished the disruptive effect of the T254A mutation in the TRAF2/3 binding site of the CD40 cytoplasmic domain. Taken together, these results indicate that Antp-CD40 mimics some of the natural CD40 signaling pathways in B cells by assembling partially functional signaling intermediates that do not require plasma membrane localization. We present a novel approach for delivering pre-activated, soluble receptor cytoplasmic domains into cells and recovering intact signaling complexes for molecular analysis.
Zoog
Stephen J
SJ
Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT 06877, USA.
Papov
Vladimir V
VV
Pullen
Steven S
SS
Jakes
Scott
S
Kehry
Marilyn R
MR
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D019013:CD40 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2; D048008:TNF Receptor-Associated Factor 3; D014162:Transfection
D019013:CD40 Antigens; D046911:Macromolecular Substances; D016328:NF-kappa B; D011506:Proteins; D011993:Recombinant Fusion Proteins; D047992:TNF Receptor-Associated Factor 2; D048008:TNF Receptor-Associated Factor 3