Cross-linking of the IgE-loaded high-affinity IgE receptor (FcεR1) by multivalent Ags results in mast cell activation and subsequent release of multiple proinflammatory mediators. The dose-response curve for FcεR1-mediated degranulation is bell-shaped, regardless of whether the IgE or the Ag concentration is varied. Although overall calcium influx follows this bell-shaped curve, intracellular calcium release continues to increase at supraoptimal IgE or Ag concentrations. As well, overall calcium mobilization adopts more transient kinetics when stimulations are conducted with supraoptimal instead of optimal Ag concentrations. Moreover, certain early signaling events continue to increase whereas degranulation drops under supraoptimal conditions. We identified SHIP, possibly in association with the FcεR1 β-chain, as a critical negative regulator acting within the inhibitory (supraoptimal) region of the dose-response curve that shifts the kinetics of calcium mobilization from a sustained to a transient response. Consistent with this, we found that degranulation of SHIP-deficient murine bone marrow-derived mast cells was not significantly reduced at supraoptimal Ag levels. A potential mediator of SHIP action, Bruton’s tyrosine kinase, did not seem to play a role within the supraoptimal suppression of degranulation. Interestingly, SHIP was found to colocalize with the actin cytoskeleton (which has been shown previously to mediate the inhibition of degranulation at supraoptimal Ag doses). These results suggest that SHIP, together with other negative regulators, restrains bone marrow-derived mast cell activation at supraoptimal IgE or Ag concentrations in concert with the actin cytoskeleton.

Mast cell degranulation is induced by multivalent allergens which cross-link immunoglobulin E (IgE) molecules that are bound to high-affinity IgE receptors (FcεR1) at the cell surface (3). Receptor aggregation leads subsequently to the activation of phosphatidylinositol 3-kinase (PI3K), generating phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. PIP3 then attracts various intracellular proteins with pleckstrin homology (PH) domains that play critical roles in triggering degranulation. These include phospholipase C-␥ (PLC-␥) (12) and the tyrosine kinase Btk (44). PLC-␥ hydrolyzes PI-4,5-biophosphate, thereby generating diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (the latter triggering the release of intracellular calcium from the endoplasmic reticulum). The tyrosine kinase Btk (44) phosphorylates and activates PLC-␥, thereby sustaining calcium release from the endoplasmic reticulum and promoting the influx of extracellular calcium through I CRAC channels in the plasma membrane (45). The Src homology 2 (SH2) domaincontaining inositol-5Ј-phosphatase (SHIP) acts as a gatekeeper of antigen-induced degranulation by hydrolyzing PIP3 (18), and there is considerable interest in identifying molecules that interact with SHIP in the hope that some of these molecules might modulate its activity and, in turn, regulate mast cell degranulation. , that become phosphorylated and targets of SH2-containing proteins upon cell stimulation; and a C-terminal SH2 domain. SHIP appears to interact with Shc via different intermolecular mechanisms, depending on the cell type involved. In myeloid cells, for example, SHIP appears to interact with Shc in a bidentate manner, with SHIP's SH2 domain binding to one of the three phosphorylated tyrosines within Shc's CH domain while SHIP's two phosphorylated NPXY motifs bind to Shc's PTB domain (34). In B lymphocytes, on the other hand, the adapter protein Grb2 appears to be required for an efficient association between Shc and SHIP, and a ternary complex of SHIP, Shc, and Grb2 is formed (16). Furthermore, studies with T cells suggest that Shc interacts solely via its PTB domain with one of SHIP's two phosphorylated NPXY motifs (29). Common to all three models, however, the SH2 domain of Shc is not involved in the Shc-SHIP interaction and therefore might be available to recruit one or more regulatory proteins into the Shc-SHIP complex.To date, very few proteins have been shown to bind to the SH2 domain of Shc. These include the signal-transducing subunits of the B-cell receptor (2) and the ␤-chain of FcεR1 (23), following receptor activation. Interestingly, unlike the B-cell receptor system, where both signaling components, Ig-␣ and Ig-␤, interact with Shc, only the ␤-subunit of FcεR1 appears to associate with this adapter protein. In addition, the Shc SH2 domain has been shown to bind to the adapter protein Gab2 (4) and mPAL, a protein whose expression is restricted to tissues containing actively dividing cells (47).

B cell antigen receptors (BCRs) are multimeric transmembrane protein complexes comprising membrane-bound immunoglobulins (mIgs) and Ig-␣͞Ig-␤ heterodimers. In most cases, transport of mIgs from the endoplasmic reticulum (ER) to the cell surface requires assembly with the Ig-␣͞Ig-␤ subunits. In addition to Ig-␣͞Ig-␤, mIg molecules also bind two ER-resident membrane proteins, BAP29 and BAP31, and the chaperone heavy chain binding protein (BiP). In this article, we show that neither Ig-␣͞Ig-␤ nor BAP29͞BAP31 nor BiP bind simultaneously to the same mIgD molecule. Blue native PAGE revealed that only a minor fraction of intracellular mIgD is associated with high-molecular-weight BAP29͞BAP31 complexes. BAP-binding to mIgs was found to correlate with ER retention of chimeric mIgD molecules. On high-level expression in Drosophila melanogaster S2 cells, mIgD molecules were detected on the cell surface in the absence of Ig-␣͞Ig-␤. This aberrant transport was prevented by coexpression of BAP29 and BAP31. Thus, BAP complexes contribute to ER retention of mIg complexes that are not bound to Ig-␣͞Ig-␤. Furthermore, the mechanism of ER retention of both BAP31 and mIgD is not through retrieval from a post-ER compartment, but true ER retention. In conclusion, BAP29 and BAP31 might be the long sought after retention proteins and͞or chaperones that act on transmembrane regions of various proteins.