THE ANTIGANGLIOSIDE MONOCLONAL-ANTIBODY AA4 INDUCES PROTEIN-TYROSINE PHOSPHORYLATIONS, BUT NOT DEGRANULATION, IN RAT BASOPHILIC LEUKEMIA-CELLS

The monoclonal antibody AA4 (mAb AA4) recognizes novel alpha-galactosy l derivatives of G(D1b) on rat basophilic leukemia (RBL-2H3) cells. Th e binding of mAb AA4 induced protein tyrosine phosphorylations without histamine release. Several of the same proteins including Fc epsilon RI beta, Fc epsilon RI gamma, p72(syk), and phospholipase C-gamma 1 we re tyrosine-phosphorylated by mAb AA4 binding and by the activation of the high affinity IgE receptor, Fc epsilon RI. There was also activat ion of the p53/56(lym) and p72(syk) protein-tyrosine kinases, but comp ared to direct Fc epsilon RI activation, mAb AA4 did not result in inc reased tyrosine phosphorylation of pp105-115 or pp125(FAK), and the re ceptor subunits (Fc epsilon RI beta and Fc epsilon RI gamma) were more heavily phosphorylated. Furthermore, the time course of the phosphory lations with mAb AA4 was slower than that induced by Fc epsilon RI agg regation. By immunofluorescence, the tyrosine-phosphorylated proteins after mAb AA4 stimulation were localized in patches at the cell membra ne and in areas of cell-cell contact, whereas after Fc epsilon RI acti vation, there was a reticular cytoplasmic pattern. There were no prote in tyrosine phosphorylations either when Fc epsilon RI was saturated w ith IgE or when F(ab')(2) fragments of mAb AA4 were used, although the F(ab')(2) fragments still induced morphological changes. There was al so coprecipitation of the beta and gamma subunits of Fc epsilon RI wit h the anti-ganglioside antibody. These data strongly suggest the invol vement of Fc epsilon RI in the antiganglioside-induced protein tyrosin e phosphorylations. Moreover, phosphorylations of these proteins inclu ding the beta and gamma chains of Fc epsilon RI and activation of p53/ 56(lyn) and p72(syk) did not result in histamine release.