Abstract
Antigenic cross-linking of the high affinity IgE receptor (Fc epsilon R1) on mast cells results in protein tyrosine kinase activation. The object of the present study was to explore the regulation of the SH2 and SH3 domain containing adapter molecule Grb2 by Fc epsilon R1-stimulated PTK signal transduction pathways. Affinity purification of in vivo Grb2 complexes together with in vitro experiments with Grb2 glutathione S-transferase fusion proteins were used to analyze Grb2 complexes in the mast cell line RBL2H3. The data show that in RBL2H3 cells several different proteins are complexed to the SH3 domains of Grb2. These include the p21ras guanine nucleotide exchange factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for Fc epsilon R1-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins which suggests that Grb2 may be a pleiotropic adapter. Two Grb2 SH2-binding proteins were also characterized in RBL2H3 cells; the adapter Shc and a 33-kDa molecule. Shc is constitutively tyrosine phosphorylated in unstimulated cells and Fc epsilon R1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for Fc epsilon R1-activated PTKs and binds to Grb2 SH2 domains in Fc epsilon R1 activated but not quiescent cells. The beta subunit of the Fc epsilon R1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2-binding protein described in the present report is not the Fc epsilon R1 beta chain and its identity is unknown. The present report thus demonstrates that there are multiple Grb2 containing protein complexes in mast cells of which a subset are Fc epsilon R1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to Fc epsilon R1 ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain. We propose that protein complex formation by Grb2 is an important consequence of Fc epsilon R1 cross-linking and that this may be a signal transduction pathway which acts synergistically with calcium/PKC signals to bring about optimal mast cell end function.
Highlights
Antigenic cross-linking of the high affinity immunoglobulin E (IgE) receptor (FceRl) on mast cells and basophils results in expression of the production of cytokines and exocytotic secre
Th e da ta pr esen ted here show t hat in RBL2H 3 mast cells, as in man y cell systems, t he gu anine nucl eotid e excha nge factor for p21ras, Sos, is compl exed to the SH 3 domains of Grb 2
Two pr otein su bstrates for FceRI-acti va ted tyrosine kin ases were observed to bind gluta t hione S -t ransferase (GST) fus ion pr otein s of Grb2 bu t mor e imp ortantly were found in association with endogenous Grb2
Summary
Vol 270, No 16, Issue of April 21, pp. 9500-9506, 1995 Printed in U.S.A. Regulation of the Adapter Molecule Grb by the FceRl in the Mast Cell Line RBL2H3*. The data show that in RBL2H3 cells several different proteins are complexed to the SH3 domains of Grb2 These include the p21ras guanine nucleotide exchange factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for FceRl-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb effector proteins which suggests that Grb may be a pleiotropic adapter. Th ere has been no a nalys is of th e regul a tion of a da pter molecul es such as Gr b2 a nd Shc in FceR1-stimula te d cells Accord ingly, t he object of th e pr esen t st udies was to exa mine wh eth er Gr b2 an d associa te d molecules a re regul a ted by t he FceRl. Th e data pr esented here show t hat t he SH3 dom ain of Grb bind s Sos a nd 75-, 120-, a nd 140-kD a ty ros ine ph osphoproteins whe reas t he major Gr b2 SH2 domain-binding pr otein s a re Sh e a nd a n unknown 33-kDa pr otein. Th e pr esen t report th us dem onstra tes th a t the re are mu ltiple Gr b2-containing pr otein complexes in mast cells of which a subset a re FceR1-regul ated
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