COMPOSITION OF THE INTRACELLULAR DOMAINS OF THE B-CELL ANTIGEN RECEPTOR COMPLEX STUDIED FROM THE CYTOPLASMIC SIDE OF THE MEMBRANE

Immunoglobulins of the classes M and D function as antigen receptors o n B lymphocytes. They are linked to other proteins to form B cell anti gen receptor (BCR) complexes which transduce the signal triggered by t he binding of antigen. In order to study the components that interact with BCR complexes in the cell it is essential that they are accessibl e to biochemical studies. Therefore, we have developed a simple and ra pid method that allows the purification and labelling of B lymphocyte plasma membranes. For this, B cells are attached to polyacrylamide bea ds. Upon disruption of the cells, bead-bound membranes are obtained wh ich expose the cytoplasmic side into the medium. The membrane proteins can then be radioiodinated and eluted with detergents. The combinatio n of the improved methods for the preparation of bead-attached membran e patches and radiolabelling of the proteins has allowed for the first time an investigation into the cytoplasmic side of the BCR complex. A ll the subunits that had been previously described could be detected i n 2D autoradiographs. Furthermore, it could be shown that the protein Ig-beta, which is part of an Ig-associated heterodimer, is predominant ly labelled at the extracellular domain. The second component, Ig-alph a, is labelled to a higher degree at its intracellular domain. In addi tion, further proteins could be detected exclusively at the cytoplasmi c side of the membrane. Results from 2D autoradiographs show that they may form heterodimers. These proteins are candidates for the interact ion of BCR complexes with further members of the signalling cascade, s uch as protein tyrosine kinases and/or G proteins.