epidermal-growth-factor and Lymphoma--B-Cell

A novel member of the EGF-TM7 family, mEMR4, was identified and characterized. The full-length mouse EMR4 cDNA encodes a predicted 689-amino acid protein containing two epidermal growth factor (EGF)-like modules, a mucin-like spacer domain, and a seven-transmembrane domain with a cytoplasmic tail. Genetic mapping established that mEMR4 is localized in the distal region of mouse chromosome 17 in close proximity to another EGF-TM7 gene, F4/80 (Emr1). Similar to F4/80, mEMR4 is predominantly expressed on resident macrophages. However, a much lower expression level was also detected in thioglycollate-elicited peritoneal neutrophils and bone marrow-derived dendritic cells. The expression of mEMR4 is up-regulated following macrophage activation in Biogel and thioglycollate-elicited peritoneal macrophages. Similarly, mEMR4 is over-expressed in TNF-alpha-treated resident peritoneal macrophages, whereas interleukin-4 and -10 dramatically reduce the expression. mEMR4 was found to undergo proteolytic processing within the extracellular stalk region resulting in two protein subunits associated noncovalently as a heterodimer. The proteolytic cleavage site was identified by N-terminal amino acid sequencing and located at the conserved GPCR (G protein-coupled receptor) proteolytic site in the extracellular region. Using multivalent biotinylated mEMR4-mFc fusion proteins as a probe, a putative cell surface ligand was identified on a B lymphoma cell line, A20, in a cell-binding assay. The mEMR4-ligand interaction is Ca2+-independent and is mediated predominantly by the second EGF-like module. mEMR4 is the first EGF-TM7 receptor known to mediate the cellular interaction between myeloid cells and B cells.

Topics: Amino Acid Sequence; Amino Acids; Animals; Binding Sites; Biotinylation; Blotting, Western; Cells, Cultured; Chromosome Mapping; Cloning, Molecular; Cytoplasm; Dendritic Cells; DNA, Complementary; Epidermal Growth Factor; Flow Cytometry; Genetic Linkage; Genetic Vectors; Ligands; Lymphoma, B-Cell; Macrophage Activation; Macrophages; Mice; Models, Biological; Molecular Sequence Data; Myeloid Cells; Neutrophils; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sequence Homology, Amino Acid; Signal Transduction; Tissue Distribution; Up-Regulation

The 120 kD product of the c-Cbl oncogene is a prominent substrate of protein tyrosine kinases that lacks a known catalytic activity but possesses an array of binding sites for cytoplasmic signalling proteins. An oncogenic form of Cbl was recently identified in the 70Z/3 pre-B cell lymphoma which has a small deletion at the N-terminus of the Ring finger domain. This form of Cbl, termed 70Z-Cbl, exhibits an enhanced level of tyrosine phosphorylation compared with c-Cbl. Here we demonstrate that the expression of 70Z-Cbl induces a tenfold enhancement in the kinase activity of the EGF receptor in serum-starved and EGF-stimulated cells. In serum-starved cells this results in EGF receptor autophosphorylation and the recruitment of Grb2, Shc and Sos1 but does not induce a corresponding increase in MAP kinase activity. Furthermore the expression of 70Z-Cbl greatly enhances EGF-induced tyrosine phosphorylation of the protein tyrosine phosphatase SHP-2. We also show that the Cbl/EGF receptor complex is predominantly associated with CrkII and is distinct to the Grb2/Shc/Sos1 complex that associates with the EGF receptor. These findings therefore demonstrate a biochemical effect of an oncogenic Cbl protein and support predictions from C. elegans that Cbl functions as regulator of receptor tyrosine kinases.

Topics: 3T3 Cells; Adaptor Proteins, Signal Transducing; Animals; Cell Line, Transformed; Culture Media, Serum-Free; Cyclin-Dependent Kinases; Epidermal Growth Factor; ErbB Receptors; GRB2 Adaptor Protein; Guanine Nucleotide Exchange Factors; Humans; Lymphoma, B-Cell; Mice; Oncogene Protein v-cbl; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Retroviridae Proteins, Oncogenic; Tumor Cells, Cultured; Tyrosine; Ubiquitin-Protein Ligases

The CD53 antigen is a prototype member of the transmembrane-4 superfamily which includes several tumor antigens. In this report we have studied the changes in the cellular binding of phorbol esters after stimulation with monoclonal antibody (mAb) MRC OX-44 (anti-CD53) and epidermal growth factor (EGF) using a fluorochrome-phorbol ester binding assay. Incubation of a rat B cell lymphoma cell line with this mAb or EGF induces the appearance of high- and low-affinity phorbol ester binding sites and changes the total number of binding sites. Four binding sites with different characteristics have been detected. The binding data suggest that two structurally different receptors, CD53 antigen and EGF receptor, induce a similar change in the functional protein kinase C expressed in the cell which might be implicated in the responses elicited after cell stimulation.

Topics: Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Binding Sites; Epidermal Growth Factor; Lymphoma, B-Cell; Phorbol Esters; Rats; Signal Transduction; Tetraspanin 25; Tumor Cells, Cultured