Sequential Requirements of the N-Terminal Palmitoylation Site and SH2 Domain of Src Family Kinases in the Initiation and Progression of Fc?RI Signaling
Journal - Molecular and Cellular Biology
Initial biochemical signaling originating from high-affinity immunoglobulin E receptor (Fc?RI) has been ascribed to Src family kinases. To understand the mechanisms by which individual kinases drive the signaling, we conducted reconstitution experiments: Fc?RI signaling in RBL2H3 cells was first suppressed by a membrane-anchored, gain-of-function C-terminal Src kinase and then reconstructed with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-myc epitope. Those constructs derived from Lyn and Fyn, which are associated with detergent-resistant membranes (DRMs), physically interacted with resting Fc?RI and reconstructed clustering-induced signaling that leads to calcium mobilization and ERK1 and -2 activation. c-Src-derived construct, which was excluded from DRMs, failed to interact with Fc?RI and to restore the signaling, whereas creation of palmitoylatable Cys3 enabled it to interact with DRMs and with Fc?RI and to restore the signaling. Deletion of Src homology 3 (SH3) domain from the Lyn-derived construct did not alter its ability to transduce the series of signaling. Deletion of SH2 domain did not affect its association with DRMs and with Fc?RI nor clustering-induced tyrosine phosphorylation of Fc?RI ß and ? subunits, but it almost abrogated the next step of tyrosine phosphorylation of Syk and its recruitment to Fc?RI. These findings suggest that Lyn and Fyn could, but c-Src could not, drive Fc?RI signaling and that N-terminal palmitoylation and SH2 domain are required in sequence for the initial interaction with Fc?RI and for the signal progression to the molecular assembly.
TCR isoform containing the Fc receptor chain exhibits structural and functional differences from isoform containing CD3
Journal - International Immunology
The structure and function of the TCR-CD3 complex containinga homodimer of the gamma chain of the high affinity receptorfor IgE (FcR) (FcR+ TCR) was investigated by transfecting theFcR gene into a CD3–, CD3–, FcR– T cell line.Introduction of FcR, as well as CD3, induced a high expressionof the TCR-CD3 complex on the cell surface. Transfected FCRformed a homodimer and associated firmly with the TCRßdimer but only weakly with the CD3. Stimulation of both FcRand CD3 transfectants by antibodies against TCR or CD3 inducedaccumulation of inositol phosphates, the Ca2+ response, IL-2production, and growth inhibition. On the other hand, antigenstimulation of transfectants expressing FcR as well as CD3 inducedIL-2 production, but only the latter exhibited the antigen-inducedgrowth inhibition. In vitro kinase assay suggested that theCD3 dimer but not the FcR dimer associates with the Fyn kinase.These results indicate that the FcR homodlmer Is able to forma functional TCR complex but that the mode of assembly and thesignaling function of FcR+ TCR, including its association withtyrosine klnase(s), may differ from the TCR-CD3 complex containingCD3 homodimers (+ TCR). This provides an example which illustratesthat different TCR isoforms mediate distinct signals and functions.
|Keywords : ||assembly of TCR-CD3 complexes • protein tyrosine kinase • signal transduction • TCR isoforms • transfection|