The 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 on B lymphocytes. They are linked to other proteins to form B cell antigen receptor (BCR) complexes which transduce the signal triggered by the binding of antigen. In order to study the components that interact with BCR complexes in the cell it is essential that they are accessible to biochemical studies. Therefore, we have developed a simple and rapid method that allows the purification and labelling of B lymphocyte plasma membranes. For this, B cells are attached to polyacrylamide beads. Upon disruption of the cells, bead-bound membranes are obtained which expose the cytoplasmic side into the medium. The membrane proteins can then be radioiodinated and eluted with detergents. The combination of the improved methods for the preparation of bead-attached membrane patches and radiolabelling of the proteins has allowed for the first time an investigation into the cytoplasmic side of the BCR complex. All the subunits that had been previously described could be detected in 2D autoradiographs. Furthermore, it could be shown that the protein Ig-beta, which is part of an Ig-associated heterodimer, is predominantly labelled at the extracellular domain. The second component, Ig-alpha, is labelled to a higher degree at its intracellular domain. In addition, further proteins could be detected exclusively at the cytoplasmic side of the membrane. Results from 2D autoradiographs show that they may form heterodimers. These proteins are candidates for the interaction of BCR complexes with further members of the signalling cascade, such as protein tyrosine kinases and/or G proteins.
Persin
C
C
Paul-Ehrlich-Institut, Langen, Germany.
Korn
M
M
Vogel
L
L
Haustein
D
D
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D003593:Cytoplasm; D003902:Detergents; D006579:Heterozygote; D046911:Macromolecular Substances; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry*; D012995:Solubility
D003902:Detergents; D046911:Macromolecular Substances; D011947:Receptors, Antigen, B-Cell