We found that adenosine stimulation
itself elicits activation of calcium response and PI3K-signaling related molecules such CT99021 solubility dmso as Gab2 in mast cells. In addition, prolonged culture of mast cells with monomeric IgE resulted in enhancement of Gab2 phosphorylation upon adenosine loading. However, in the absence of FcεRI-signaling, adenosine itself failed to induce degranulation response in mast cells even when cells were cultured with IgE for 48 h. Our results of the present study clearly indicate that FcεRI-signaling through the FcRβ-ITAM is indispensable for amplified PI3K-signaling and degranulation response in mast cells stimulated with low-dose antigen and adenosine. With regard to the molecular mechanisms for amplification of PI3K-signaling pathway, Bohnacker et al. recently
reported that activation of class 1B PI3K p110γ:p84 complex via adenosine receptors is crucial 9. Unlike adenosine receptors, FcεRI stimulation triggers activation of class 1A PI3K including p110δ:p85 complex 37. It is thus unclear how two different Obeticholic Acid manufacturer PI3K isoforms cooperate to generate synergistic activation. In this study we demonstrated that tyrosine phosphorylation of FcRβ was synergistically amplified upon costimulation of FcεRI and adenosine receptors (Fig. 8B). The finding suggests the possibility that adenosine stimulation augments the FcεRI-mediated activation of class 1A PI3K. Since (i) adenosine increased FcεRI-induced tyrosine phosphorylation of Gab2 at Tyr452 residue, which is a potential binding site of p85 subunit of class 1A PI3K 38 and (ii) Gab2 deficiency generally results in severe impairment of PI3K-signaling and degranulation in mast cells 27–29, 39, 40, we consider that the enhanced Gab2 phosphorylation may result in amplification
of activation of class 1A PI3K. In the synergism of Gab2 phosphorylation, we observed that adenosine itself elicits tyrosine phosphorylation of Gab2 in mast cells in an FcRβ-ITAM-dependent Digestive enzyme manner. We currently consider that sensitization of mast cells with IgE largely contributes to FcRβ-ITAM-dependent tyrosine phosphorylation of Gab2 in mast cells upon adenosine loading. Because (i) the tyrosine phosphorylation of Gab2 did not occur in FcεRI-negative BMMC derived from FcRβ−/− mice (Fig. 6B), (ii) prolonged-culture of mast cells with IgE (SPE-7 or IgE-3) resulted in enhancement of Gab2 tyrosine phosphorylation following adenosine stimulation (Fig. 5), and (iii) SPE-7 was more helpful IgE clone for the Gab2 phosphorylation than IgE-3 although both IgE increased levels of FcRβ protein (data not shown) and FcεRI expression on the cell surface (Fig. 4A).