Nuclear Localization in the Biology of the CD40 Receptor in Normal and Neoplastic Human B Lymphocytes.

CD40 was originally identified as 50-kD cell surface receptor in B lymphocytes, a tumor necrosis receptor (TNF) superfamily member containing Type I extracellular binding motif and other structural homologies to initiate signaling pathways. This receptor is constitutively expressed, and provides signaling for cellular proliferation and clonal expansion, as well as immunoglobulin production, isotype switching, and the development of humoral immunologic memory. CD40 signaling is primarily controlled through cognate ligand binding, predominantly involving its major physiologic ligand CD154 (CD40L), primarly from activated helpler (CD4+) T lymphocytes. Since CD40 plasma membrane receptor leads to a variety of immune and inflammatory responses, such as release of various cytokines and apoptotic mediators, expression of cellular adhesion molecules and matrix-degrading enzymes, this protein plays important roles in pathologic processes of chronic inflammatory diseases, various forms of autoimmunity, and other immuno-regulatory abnormalities. In these studies, we show that CD40 is one of the growing list of cell membrane growth factor receptors that are present in nucleus and cytoplasm of both normal and neoplastic B cells by immunohistochemistry. CD40 was also detected by western blotting analysis, immunofluorescence staining, as well as confocal microscopy. Transmission electron microscopy also confirmed the nuclear localization of CD40 in NHL-B cells. To examine the distribution of CD40 in normal peripheral blood B lymphocytes, CD40 proteins localize primarily in the cytoplasm and cell surface of G_o B cells, but not in the nucleus. However, after anti-IgM and CD154 activation of B cells in vitro, CD40 proteins are found not only in the cytoplasm, and cell surface, but also in the nucleus. Transfected recombinant CD40-GFP is shown to rapidly enter the nucleus by confocal microscopy. A putative NLS (KKVAKKPTNK) in the CD40 sequence is identified that when mutated from lysine to threonine, blocked entry of CD40-GFP into the nucleus. Co-immunoprecipitaion of CD40 with either importin-a or b suggests that the NLS of CD40 allows interaction with importin-a and b to form a protein complex that can target and bind to nuclear pore complex (NPC) to facilitate the transport CD40 protein from cytoplasm to nucleus. Newly synthesized ³⁵S-labeled CD40 experiments show that nascent CD40 protein molecules that are detected as monomers and dimers, shift from the plasma membrane of NHL-B cells to the cytoplasm and nucleus after 2.5 hour post chase, in vitro. Nuclear fractionation studies indicate that the CD40 protein is located in the nucleoplasm fraction, suggesting that in the nucleus, that CD40 may be involved with chromatin, as has been recently shown for other growth factor receptors (e.g. EGFR). Gal-4-reporter assays also demonstrate that pBIND-CD40C (CD40 fused to Gal4 DNA binding domain) increases luciferase acivity in a dose dependent manner reaching the maximal 4.4 fold induction. This data suggest that the CD40-intracellular domain has transactivation activity at the transcription level in the nucleus of B cells similar to EGFR of epithelial cells. Present studies indicate that the role of CD40 in neoplastic as well as normal B cells may involve nuclear activities in addition to its integral role as a plasma membrane growth factor receptor.