Activation via the B cell antigen receptor (BCR) by antigen leads to B cell proliferation and immunoglobulin (Ig) secretion. However, engagement of the BCR simultaneously with the Ig receptor on B cells (FcgammaRIIB), delivers a potent inhibitory signal that prevents B cell proliferation and antibody secretion. Such co-ligation, which can be mediated by antigen-antibody complexes that can exist in later stages of the immune response, is thought to act as a """"""""feedback suppression"""""""" to limit excessive antibody production. Absence of this receptor results in enhanced antibody and anaphylactic responses in Fcgamma RIIB knock-out mice. In humans, certain rheumatoid factors, such as an IgM that reacts with the Fc portion of IgG, interfere with FcgammaRIIB-mediated regulation of antibody production. Thus, understanding the molecular mechanisms of this signaling process has important implications for B cell activation and regulation in normal physiology and pathology. FcgammaRIIB crosslinking with the BCR leads to inhibition of BCR-induced calcium entry into cells, diminution of Ras activation and attenuation of specific transcription factor activation, ultimately leading to inhibition of B cell proliferation. Recently, the inositol phosphatase, SHIP, has been identified as essential for mediating the inhibitory signaling via FcgammaRIIB. However, the mechanism(s) by which SHIP regulates these seemingly disparate pathways is completely unknown. This proposal will attempt to define the molecular mechanisms of inhibitory signaling via FcgammaRIIB and SHIP. Using the approaches of reconstitution of a cell line lacking SHIP expression and overexpression of mutant SHIP proteins, Aim I will address the molecular mechanism(s) by which SHIP regulates the calcium entry into cells. The region(s) of SHIP necessary for this regulation will also defined. The role of SHIP in regulating the two phosphoinositide substrates of SHIP, IP4 and IP3, and the implications in vivo will be rigorously tested.
Aim 2 address the role of SHIP in protecting B cells against FcgammaRIIB-induced apoptosis and attempts to identify the specific signaling molecules. The second part of this aim will attempt to """"""""rescue"""""""" the FcgammaRIIB-mediated inhibitory signaling. A detailed molecular understanding of FcgammaRIIB-mediated inhibitory signaling may yield important clues to B cell activation and desensitization, and point to possible future therapeutic interventions in certain autoimmune states.
