Fc γ receptor type IIb induced recruitment of inositol and protein phosphatases to the signal transductory complex of human B-cell

Abstract

Co-clustering of Fc γRIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of Fc γRIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human Fc γRIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human Fc γRIIb, synthetic peptides were designed to cover almost the entire intracellular Fc γRIIb domain, including Fc γRIIb1 and Fc γRIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53–56 kDa being the most prominent one. A fraction of the 53–56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating Fc γRIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both Fc γRIIb1 and Fc γRIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to Fc γRIIb1 and Fc γRIIb2 specific sequences. Synthetic peptide representing Fc γRIIb1 and Fc γRIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50–56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to Fc γRIIb1 or Fc γRIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human Fc γRIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SHIP and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the Fc γRIIb co-clustered BCR are responsible for the Fc γRIIb mediated negative regulation of human B-cell activation.