Molecular associations of class II MHC antigens in mitogen-stimulated B cells.
Previously, we showed that murine B cell membrane proteins undergo rearrangements in the plasma membrane to form new molecular associations in response to mitogenic stimulation. These complexes were covalently stabilized by photoreactive cross-linking agents and were analyzed by SDS PAGE. We have now identified certain complexes that involve class II MHC products, the Ia antigens. Upon stimulation of B cells with LPS, Ia surface molecules (as identified by radioimmunoprecipitation with polyclonal anti-Ia antiserum) enter into a molecular complex with a 95-kd membrane-associated protein (p95) to form a 200-kd complex that may be stabilized by the cross-linking agent dithiobisphenylazide (DTPA). This molecular association is not observed upon stimulation with mitogenic anti-Ig reagents, nor with the polyclonal B cell activator 8-bromoguanosine. p95 is not a disulfide-linked molecule itself, and by separate immunoprecipitation experiments we have established that it is not a component of surface Ig, transferrin receptor, the B cell Fc receptor, or CR1, the receptor for complement component C3b. Further analysis of the association of Ia antigens with surface proteins, with the use of monoclonal antibodies directed against I-A or I-E, has demonstrated that each subregion gene product forms a unique molecular association. Precipitation of radiolabeled lysates from LPS-activated B cells with anti-I-A reveals the aforementioned association with p95. In contrast, the I-E antigen apparently forms complexes with a multimer of a 15-kd protein to give complexes of 45, 60, 75, and 90 kd. When analyzed by two-dimensional diagonal gels (nonreducing/reducing), only the I-E bands are revealed by autoradiography, indicating that the putative p15 that associates with I-E may not be accessible to surface labeling. The disparate molecular associations for I-A and I-E suggest that the formation of these distinct protein complexes may be functionally related to a different role in the process of cellular activation for each of these Ia subregion gene products.
D000818:Animals; D001386:Azides; D001402:B-Lymphocytes / Q000276:immunology* / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D006649:Histocompatibility Antigens; D000949:Histocompatibility Antigens Class II; D007075:Immunoglobulin M; D008070:Lipopolysaccharides / Q000276:immunology; D046911:Macromolecular Substances; D008285:Major Histocompatibility Complex; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D011947:Receptors, Antigen, B-Cell
D001386:Azides; D003432:Cross-Linking Reagents; D006649:Histocompatibility Antigens; D000949:Histocompatibility Antigens Class II; D007075:Immunoglobulin M; D008070:Lipopolysaccharides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011947:Receptors, Antigen, B-Cell; C025369:4,4'-dithiobis(phenylazide)