title: Characterization of the human B cell RAG-associated gene, hBRAG, as a B cell receptor signal-enhancing glycoprotein dimer that associates with phosphorylated proteins in resting B cells.
abstract: Affinity-purified polyclonal antibodies against the hBRAG (human B cell RAG-associated gene) protein were generated to characterize hBRAG at the biochemical level. Immunoblotting and immunoprecipitation experiments with these antibody reagents demonstrate that this protein can be expressed in B cells as a membrane-integrated glycoprotein disulfide-linked dimer. However, both glycosylated and unglycosylated isoforms of hBRAG are detectable with these reagents. Additionally, their use in cell surface biotinylation and flow cytometry reveals subcellular hBRAG pools both at cell surface and intracellular locations. Co-immunoprecipitation experiments with hBRAG antisera detected the association of hBRAG with phosphorylated proteins in resting B cells, including the protein tyrosine kinase Hck, which is subsequently dephosphorylated upon B cell receptor (BCR) ligation. Consistent with its cell surface expression and possible link to BCR signaling, experiments in which alpha-hBRAG antibodies were used to generate early activation signals suggest a modest but specific element of tyrosine phosphorylation occurring through a putative hBRAG receptor. Additional experiments also suggest that hBRAG may be involved in positively enhancing BCR ligation-mediated early activation events. Collectively, these results are consistent with a function for hBRAG as a B cell surface signaling receptor molecule. Coupled with the earlier observation that hBRAG expression correlates with early and late B cell-specific RAG expression, we submit that hBRAG may mediate regulatory signals key to B cell development and/or regulation of B cell-specific RAG expression.
authors.affiliation: Department of Immunology, University of Toronto, Toronto M4N 3N5, Ontario, Canada.
authors.forename: L K
authors.initials: LK
authors.lastname: Verkoczy
authors.forename: B a
authors.initials: Ba
authors.lastname: Guinn
authors.forename: N L
authors.initials: NL
authors.lastname: Berinstein
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism*; D019921:Biotinylation; D002460:Cell Line; D019281:Dimerization; D005260:Female; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D020014:K562 Cells; D008214:Lymphocytes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009928:Organ Specificity; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011247:Pregnancy; D014176:Protein Biosynthesis; D011947:Receptors, Antigen, B-Cell / Q000502:physiology; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D015238:Sulfotransferases; D013601:T-Lymphocytes / Q000378:metabolism; D014158:Transcription, Genetic; D020298:U937 Cells
chemical_list: C411874:CHST15 protein, human; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D011947:Receptors, Antigen, B-Cell; D015238:Sulfotransferases