B cell antigen receptor assembly and Syk activation in the S2 cell reconstitution system.
Signal transduction from the B cell antigen receptor (BCR) involves a multitude of signaling molecules often organized in dynamic protein complexes. The molecular mechanisms operating during signaling are difficult to study solely by loss-of-function analysis. For a better understanding of the transient interaction of signaling molecules and their regulation by feedback loops, as well as their dynamic behavior in living cells, new techniques are required. We have developed a method allowing the reconstitution of the BCR complex and several of its key signaling elements in the evolutionary distant environment of the Drosophila S2 Schneider cell line. With this gain-of-function approach, we study here the assembly of the BCR complex and the control of its transport to the cell surface of S2 cells. We find that without binding to a light chain, the membrane-bound microm heavy chain (micromHC) homodimer, together with the Ig-alpha/Ig-beta heterodimer, can come to the cell surface where it is signaling competent. This finding could have implications for potential signaling functions of such a receptor molecule during pro-/pre-B cell development. We also studied the activation of the BCR-proximal kinase Syk. We found that a truncated Syk mutant lacking the first (N-terminal) SH2 domain and the linker regions, is still regulated by autoinhibition and can only become activated in the presence of the BCR. This indicates that the C-terminal SH2 domain of Syk is the dominant regulatory subunit of this kinase.
Wossning
Thomas
T
Department of Molecular Immunology, Biology III, University of Freiburg, Stübeweg 51, 79108 Freiburg, Germany.
Reth
Michael
M
D000818:Animals; D004331:Drosophila melanogaster / Q000276:immunology / Q000502:physiology; D004792:Enzyme Precursors / Q000276:immunology / Q000502:physiology*; D007075:Immunoglobulin M / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000502:physiology*; D011947:Receptors, Antigen, B-Cell / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000072377:Syk Kinase; D014162:Transfection
D004792:Enzyme Precursors; D007075:Immunoglobulin M; D047908:Intracellular Signaling Peptides and Proteins; D011947:Receptors, Antigen, B-Cell; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase