23-kDa protein associated with CD40

CD40 is a 45-kDa glycoprotein member ofthe tumor necrosis factor receptor (TNFR) family expressed on B cells, thymic epithelial cells, dendritic cells, and some carcinoma cells. The unique capacity of CD40 to trigger immunoglobulin isotype switching is dependent on the activation of protein-tyrosine kinases, yet CD40 possesses no kinase domain and no known consensus sequences for binding to protein-tyrosine kinases. Recently, an intracellular protein (CD40bp/LAP-1/CRAF-1) which belongs to the family of TNFR-associated proteins was reported to associate with CD40. We describe a 23-kDa cell surface protein (p23) which is specifically associated with CD40 on B cells and on urinary bladder transitional carcinoma cells. Protein microsequencing revealed that p23 shows no homology to any known protein. A rabbit antibody raised against a peptide derived from p23 recognized a 23-kDa protein in CD40 immunoprecipitates. In contrast to CD40bp/LAP-1/CRAF-1, p23 was not associated with TNFR p80 (CD120b). These findings suggest that p23 is a novel member of the CD40 receptor complex. Interaction of the B-cell surface antigen CD40 with its ligand (CD40L) expressed on activated T cells plays a critical role in T-B cell collaboration, particularly in immunoglobulin isotype switching, in the induction of expression of B7 costimulatory molecules, in B-cell survival and in the formation of germinalcenter B cells (1-3). This is evidenced by the observations that mice with a disrupted CD40 gene fail to undergo isotype switching and to develop germinal centers in response to T-cell-dependent antigens (4, 5) and that B cells from CD40null mice fail to elicit an allogeneic response (G. Hollander, E. Castigli, and R.S.G., unpublished work). The unique capacity of CD40 to trigger immunoglobulin class switching in B cells suggests that CD40 ligation activates a unique signaling pathway(s). Our studies and those of others indicate that CD40 ligation activates protein-tyrosine kinases (PTKs), including Lyn and Syk and results in tyrosine phosphorylation of multiple substrates, including phosphatidylinositol 3-kinase, phospholipase C-,y2, and a 28-kDa phosphoprotein (6, 7). More importantly, PTK inhibitors and crosslinking of CD45 to CD40 inhibit CD40-mediated isotype switching (8, 9). However, CD40 has no kinase domain and no known consensus sequence for binding to PTK, suggesting that CD40 uses associated molecules for transducing intracellular signals. Recently, a member of the tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of proteins was found to bind to the cytoplasmic region of CD40 and to play a role in CD40-mediated induction of CD23 expression (1012). We have identified and biochemically characterized a 23-kDa cell surface protein (p23) associated with human CD40. p23 was not associated with CD95 (Fas) or with TNFR p80 (CD120b), suggesting that it could be important in CD40specific signaling. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. MATERIALS AND METHODS Cells. The human B-cell lines Raji, BJAB, and Ramos and the human bladder carcinoma cell line T24 were obtained from American Type Culture Collection. The human B-cell line Jijoye which expresses TNFR p80 (CD120b) was a kind gift from G. Mosialos and E. Kieff (Harvard Medical School). Human tonsil B cells were purified as described (13). Monoclonal Antibodies (mAbs) and Reagents. Mouse antihuman CD40 mAb 626.1 (IgGl) was a gift from S. M. Fu (University of Virginia, Richmond). Murine anti-major histocompatibility complex (MHC) class I mAb W6/32 (IgGl), and anti-HLA-DR mAb L243 (IgG2a) were obtained through American Type Culture Collection. Anti-MHC class II mAb 3B12 (IgGl) has been described (14). Anti-human Fas mAb ZB-4 (IgGl) was obtained from Kamiya Biomedical (Thousand Oaks, CA). Rat anti-human TNFR p80 mAb was purchased from Genzyme. Anti-CD81 mAb 5A6 (IgGl) was kindly provided by S. Levy (Stanford University). Tunicamycin was obtained from Boehringer Mannheim. Phosphatidylinositol-specific phospholipase C (PI-PLC) was purchased from Calbiochem. Anti-p23 peptide polyclonal antibody was raised in rabbits against a 17-aa peptide derived from p23 and conjugated to keyhole limpet hemocyanin (KLH) and was absorbed with KLH as described (15). Surface lodination and Immunoprecipitation. Surface iodination and immunoprecipitation were carried out with Bolton-Hunter reagent (16). In brief, 5 x 107 cells were washed with phosphate-buffered saline, and labeled with 2 mCi (74 MBq) of 1251 in the presence of Bolton-Hunter reagent for 30 min on ice. The cells were washed with PBS and lysed on ice for 30 min in 1% (vol/vol) Brij 96/150 mM NaCl/20 mM Hepes, pH 7.4/1 mM Na3VO4/50 mM NaF/1 mM phenylmethanesulfonyl fluoride containing leupeptin at 5 ,ug/ml and antipain, chymostatin, and pepstatin each at 1 ,ug/ml. The cell lysates were precleared with protein G-Sepharose beads (Pharmacia) precoupled with normal mouse immunoglobulin and then were immunoprecipitated with protein G beads preabsorbed with the indicated antibody. The immunoprecipitates were resolved by SDS/12% PAGE and the bands were visualized by autoradiography. 355 Metabolic Labeling. BJAB cells were first washed in minimal essential medium deficient in Met/Cys (GIBCO/ BRL) and incubated for 1 hr in the same medium supplemented with 5% fetal bovine serum that had been dialyzed against 150 mM NaCl/20 mM Hepes, pH 7.4. The cells were then incubated with [35S]methionine/[35S]cysteine (Tran35Slabel; ICN) for 5 hr, washed, and lysed in 1% Brij 96 lysis buffer. Immunoprecipitation was carried out as described above. Two-Dimensional Electrophoresis. Two-dimensional gel electrophoresis was carried out with a Bio-Rad kit in accord with the manufacturer's guidance. For first-dimension isoelectric focusing, radiolabeled immunoprecipitates were solubiAbbreviations: mAb, monoclonal antibody; MHC, major histocompatibility complex; NMS, normal mouse serum; PI-PLC, phosphatidylinositol-specific phospholipase C; TNFR, tumor necrosis factor receptor; TRAF, TNFR-associated factor. *To whom reprint requests should be addressed.