©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Adapter Molecule Grb2 by the FcR1 in the Mast Cell Line RBL2H3 (*)

Helen Turner (§) , Karin Reif (¶) , Juan Rivera (1), Doreen A. Cantrell (**)

From the (1) Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, P. O. Box 123, London WC2A 3PX, United Kingdom and the NIASD, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Antigenic cross-linking of the high affinity IgE receptor (FcR1) 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 FcR1-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 FcR1-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 FcR1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for FcR1-activated PTKs and binds to Grb2 SH2 domains in FcR1 activated but not quiescent cells. The subunit of the FcR1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2-binding protein described in the present report is not the FcR1 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 FcR1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to FcR1 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 FcR1 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.


INTRODUCTION

Antigenic cross-linking of the high affinity immunoglobulin E (IgE) receptor (FcR1) on mast cells and basophils results in expression of the production of cytokines and exocytotic secre-tion of allergic mediators (1) . The stimulation of PTKs() by the activated FcR1 is an immediate membrane proximal event crucial for FcR1 signal transduction and hence mast cell end function (2, 3, 4) . The FcR1 complex is composed of three subunits, the 45-kDa and 30-kDa chains and a homodimer of two disulfide-linked 10-kDa chains (5) . The intracellular tails of the and subunits contain a common motif, termed the tyrosine-based activation motif (TAM) of the general sequence EXYXL/IXYXL/I, (6) which is thought to couple the FcR1 to intracellular PTKs. TAMs are found also in the invariant subunits of the B cell and T cell antigen receptors (BCR and TCR) and thus appear to be an evolutionarily conserved sequence motif essential for the activation of lymphocytes (7, 8, 9, 10, 11) .

Members of the TAM-based receptor family are typically coupled to two subfamilies of PTKs. The FcR1 associates with the src-family tyrosine kinase p56lyn and a 72-kDa PTK, Syk (3, 12) . The BCR is similarily associated with p56lyn (13) and Syk whereas the TCR is predominantly associated with the src-family p59fyn and a kinase homologous to Syk, ZAP70 (14, 15) . It has also been reported that FcR1 cross-linking induces tyrosine phosphorylation and activation of the atypical src-like Bruton tyrosine kinase (16) suggesting that a third category of PTKs may contribute to FcR1 signaling. One common response to triggering of the FcR1, TCR, and BCR is tyrosine phosphorylation and activation of phospholipase C-1 (2, 17, 18) . This permits these receptors to control inositol polyphosphate and diacylglycerol production which in turn modulate intracellular calcium concentration and the activation of PKC, respectively. Calcium flux and PKC play a critical role in TCR signal transduction (19, 20) and are important signals for mast cell activation and the secretion of granule components such as histamine and 5-hydroxytryptamine (21, 22) .

A second, essential, PTK-controlled signaling pathway to originate from the TCR involves the guanine nucleotide-binding proteins p21ras (23) . The mechanism of TCR coupling to p21ras was proposed to involve the guanine nucleotide exchange protein Sos, the homologue of the Drosophila ``son of sevenless'' gene product (24, 25) . Sos is known to complex with the adapter protein Grb2/Sem 5 which is composed of one SH2 domain and two SH3 domains (26, 27) . In many cell systems, the SH3 domains of Grb2 bind to Sos whereas the interaction between the Grb2 SH2 domain and tyrosine-phosphorylated molecules such as the epidermal growth factor (EGF) receptor or Shc may regulate the function and cell localization of Sos (27, 28, 29, 30) .

Studies in T lymphocytes have identifed a number of tyrosine-phosphorylated proteins which can potentially bind to Grb2. These include Shc, a membrane-localized tyrosine phosphoprotein of 36-kDa and 75- and 116-kDa molecules (25, 31, 32, 33, 34) . The 75- and 116-kDa molecules are constitutively associated with the SH3 domains of Grb2 analogous to the Grb2/Sos association and are substrates for TCR-activated PTKs (32, 33) . The p75 is apparently a hematopoietic cell-specific protein. Its structure and protein sequence are not known, but on the basis of its association with the Grb2 SH3 domains, p75 is a candidate for a second Grb2 downstream effector molecule. Shc and p36 when tyrosine phosphorylated bind to the SH2 domain of Grb2. In TCR-activated T cells, both Shc and p36 are tyrosine phosphorylated and could potentially form a complex with Grb2 SH2 domains. However, the predominant complexes that can be detected are composed of p36-Grb2-Sos (25, 35) . Studies in B cells have generated evidence for regulation of Grb2 complexes by the BCR. In this system both Shc tyrosine phosphorylation and the formation of Shc-Grb2-Sos complexes are observed in response to BCR ligation (36, 37) The pattern of PTK activation in response to BCR or FcR1 triggering is similar, particularly in the stimulation of the 72-kDa PTK Syk (12) . However, there has been no analysis of the regulation of adapter molecules such as Grb2 and Shc in FcR1-stimulated cells. Accordingly, the object of the present studies was to examine whether Grb2 and associated molecules are regulated by the FcR1.

The data presented here show that the SH3 domain of Grb2 binds Sos and 75-, 120-, and 140-kDa tyrosine phosphoproteins whereas the major Grb2 SH2 domain-binding proteins are Shc and an unknown 33-kDa protein. Two of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to FcR1 ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain. Shc is apparently not a substrate for FcR1-activated PTKs and hence its association with Grb2 is not controlled by cell activation. The present report thus demonstrates that there are multiple Grb2-containing protein complexes in mast cells of which a subset are FcR1-regulated.


MATERIALS AND METHODS

Antibodies

The anti-phosphotyrosine monoclonal antibody 4G10 was purchased from Upstate Biotechnology Inc (Lake Placid, NY). The anti-phosphotyrosine antibody FB2 (W. Fantl, University of California) was purified from hybridoma supernatant. Polyclonal anti-Shc antibody was purchased from TCS Biologicals. Monoclonal anti-Dinitrophenyl (mouse IgE isotype) was purchased from Sigma. Anti-mouse Sos1 and anti-Grb2 were purchased from Upstate Biotechnology Inc. and Affiniti (Nottingham, United Kingdom), respectively. The monoclonal antibody JRK1, with specificity for the chain of FcR1 (38) , was a generous gift of Dr. Juan Rivera (NIAMS, Bethesda, MD).

Peptide Reagents and Fusion Proteins

The EGFR-Y1068 peptide sequence is PVPEpYINQS. The Trk-Y490 peptide sequence is IENPQpYFSDA. For use in affinity purification these peptides were coupled to Affi-Gel 10 beads (Bio-Rad). The following glutathione S-transferase (GST) fusions were used and have been previously described (28, 32) . GST-alone, GST-Grb2 (full-length Grb2), GST-µSH3 49L/203R double SH3 mutant, GST-Grb2NSH3 isolated amino-terminal SH3 domain (amino acids 1-58), GST-Grb2 CSH3 isolated carboxyl-terminal SH3 domain (amino acids 159-217) and GST-Sos (mSos1 carboxyl-terminal residues 1135-1336). The fusion proteins were coupled to glutathione-agarose beads (Sigma) for affinity purification experiments.

Cell Culture

The rat basophilic leukemia cell line RBL2H3 was a gift of Dr. Roberto Solari, Glaxo Research and Development, U.K. They were maintained in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated (56 °C for 30 min) fetal bovine serum and 2 m M glutamine. Only the adherent fraction of the cell cultures was passaged or used in experiments.

Cell Stimulation and Lysis

RBL2H3 monolayer cells were detached from the culture flask using a cell scraper, washed once, and primed in suspension with 1 µg/ml IgE anti-dinitrophenol (IgE anti-DNP) in RPMI 1640, 10% fetal bovine serum for 1 h at 37 °C. Receptor cross-linking was effected using 10 µg/ml keyhole limpet hemocyanin (KLH)-DNP conjugate (Calbiochem) at 37 °C. After stimulation all cells were placed on ice and pelleted immediately in a microfuge. The cells were lysed in a buffer containing 50 m M HEPES (pH 7.4), 1% (w/v) Nonidet P-40, 150 m M NaCl, 20 m M NaF, 20 m M iodoacetamide, 1 m M phenylmethylsulfonyl fluoride, 1 µg/ml protease inhibitors (leupeptin, pepstatin A, and chymotrypsin), and 1 m M NaVO.

Affinity Purifications and Western Blotting

Lysates (1.5 10RBL2H3) were precleared with Protein A-insoluble suspension (Sigma) for 15 min at 4 °C. Lysates for fusion protein affinity purifications were then precleared with glutathione-agarose bead suspension (Sigma). Lysates for peptide precipitations (Trk-Y490 and EGFR-Y1068) were cleared with Affi-Gel 10 (Pharmacia). Affinity purifications with specific reagents were carried out for 2 h at 4 °C with constant rotation. The beads were washed three times in 1 ml of lysis buffer and boiled in reducing SDS sample buffer for 10 min. Samples were resolved on 11% SDS-PAGE.

The resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) which was blocked in 5% non-fat milk for 1 h. The membrane was probed with a specific antibody followed by an appropriate second stage horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Amersham). Reactive bands were detected using the enhanced chemiluminescence system (Amersham).


RESULTS

Tyrosine Phosphorylation of Multiple Grb2 Binding Proteins Is an Immediate Consequence of FcR1 Cross-linking

The data in Fig. 1 show a Western blot analysis with anti-phosphotyrosine antibodies of total cell lysates from quiescent and FcR1-stimulated RBL2H3 cells. The experiment demonstrates that there is prominent FcR1-induced tyrosine phosphorylation of proteins at 80, 70, and 55 kDa. This phosphorylation is rapid, detected within 2 min and sustained for a period of 30 min. In control experiments no increase in tyrosine phosphorylation of any proteins in RBL2H3 cells which have been incubated with monomeric IgE but not exposed to cross-linking multivalent antigen was observed. Binding experiments with a GST-fusion protein of wild type Grb2 were carried out in order to establish whether FcR1-induced tyrosine phosphoproteins include a subset of Grb2-binding proteins. The data from this experiment are shown in Fig. 2. In precipitates purified from RBL2H3 cell lysates with GST-Grb2, there are multiple tyrosine phosphoproteins in both quiescent and activated cells. Many proteins are observed which are basally tyrosine phosphorylated in unstimulated cells. However, there is an FcR1-induced increase in tyrosine phosphorylation of proteins migrating at 120, 110, 75, 65, and 30-33 kDa.

The experiment in Fig. 2explores also the SH domain specificity of FcR1-induced tyrosine-phosphorylated Grb2-binding proteins. Affinity binding experiments were performed with truncated or mutated Grb2 fusion proteins comprising the isolated Grb2 NH-terminal and COOH-terminal SH3 domains, called GST-Grb2NSH3 and GST-Grb2CSH3, respectively. In addition, GST-Grb2 with mutations in both SH3 domains which leave the SH2 domain intact (GST-Grb2 µSH3) was used. Fig. 2shows a Western blot analysis with anti-phosphotyrosine antibodies on proteins affinity purified from RBL2H3 lysates using the panel of GST-Grb2 variants in comparison with wild-type GST-Grb2. The data show that a prominent FcR1-induced tyrosine phosphoprotein of 75 kDa was detected in the precipitates isolated with wild type GST-Grb2, GST-Grb2NSH3, or GST-Grb2CSH3 fusion proteins. Both GST-Grb2 SH3 domain fusion proteins also purified tyrosine-phosphorylated proteins of 38, 52-55, 120, and 140 kDa. Cross-linking of the FcR1 induced tyrosine phosphorylation of 33- and 65-kDa proteins which bound to the wild type GST-Grb2 fusion protein. These species did not bind the isolated Grb2 SH3 domains. In addition to the isolated SH3 domains, further characterization of domain specificities was possible using the GST-Grb2 µSH3 molecule. This is a full-length Grb2 construct which has point mutations introduced into each of the SH3 domains but an intact SH2 domain. The FcR1-induced 75-kDa tyrosine phosphoprotein did not bind to the GSTGrb2 µSH3 fusion protein, nor did the 110-kDa tyrosine phosphoprotein. However, FcR1-induced tyrosine phosphoproteins of 33 and 65 kDa bound efficiently to the GST-Grb2 µSH3 protein. As well, a 55-kDa tyrosine phosphoprotein and a doublet of high molecular mass tyrosine-phosphorylated proteins at 120-140 kDa were detected binding to GST-Grb2 µSH3. The latter species were inducibly tyrosine phosphorylated in FcR1-activated RBL2H3 to varying degrees between experiments.


Figure 2: Anti-phosphotyrosine Western blot showing SH domain specificity of tyrosyl proteins binding to Grb2 in RBL2H3 cells. 2 10RBL2H3 cells/point were primed as described and stimulated with 10 µg/ml KLH-DNP for the time indicated in minutes at 37 °C. Lysates were affinity purified for 2 h with the Grb2 fusion protein indicated. The panel of GST fusion proteins used were the wild type GST-Grb2, the single SH3 domain truncations GST-Grb2NSH3 and GST-Grb2CSH3, and the double SH3 mutant GST-Grb2 µSH3. GST alone coupled to glutathione-agarose beads was included as a negative control. Samples were resolved on 11% SDS-PAGE under reducing conditions before transfer to PVDF membrane, blocking with non-fat milk, and Western blot analysis with 4G10.



From these binding studies it can be concluded that there are FcR1-induced tyrosine phosphoproteins of 33 and 65 kDa which can bind to Grb2 SH2 domains. In addition, there is an FcR1-induced tyrosine phosphoprotein of 75 kDa which binds to Grb2 SH3 domains. Other tyrosine phosphoproteins that associate with Grb2 SH3 domains and SH2 domains have molecular masses of 110-120 and 120-140 kDa, respectively. The binding experiments using truncated and mutated Grb2 fusion proteins show that the proteins migrating in the 110-120 kDa range may in fact be resolved into both basally and FcR1-induced tyrosine-phosphorylated proteins. Similarly, there are multiple tyrosine-phosphorylated proteins at 52-55 kDa binding to the intact GST-Grb2 fusion protein that resolve into both basally phosphorylated proteins and FcR1-induced tyrosine phosphoproteins upon analysis with the truncated and mutant Grb2 fusion proteins.

Tyrosine Phosphoproteins That Associate with the SH3 Domains of Endogenous Grb2 in RBL2H3 Cells

Initial experiments showed that there is a complicated pattern of FcR1-induced tyrosine phosphoproteins which are capable of binding to Grb2 fusion proteins. Therefore, to establish whether there are FcR1-induced complexes between tyrosine-phosphorylated proteins and endogenous Grb2, we used affinity purification protocols to isolate Grb2 and associated proteins from RBL2H3 cell lysates. This technique has been described in detail previously (32) and has been used effectively to examine Grb2 complexes in TCR-activated T-lymphocytes. Briefly, a synthetic tyrosine phosphopeptide corresponding to the autophosphorylation site Tyr-1068 in the carboxyl-terminal tail of the epidermal growth factor receptor (EGFR-Y1068) was used to precipitate Grb2 from RBL2H3 cells. The EGFR-Y1068 peptide binds to the Grb2 SH2 domain with nanomolar affinity and thus competitively blocks associations between cellular proteins and the SH2 domains of Grb2. Use of this peptide allows the copurification of Grb2 and proteins complexed with Grb2 SH3 domains. Grb2 was also purified from RBL2H3 cells using a GST fusion protein of a proline-rich fragment from the carboxyl terminus of murine Sos (GST-Sos). The GST-Sos fusion protein used in these experiments binds the Grb2 SH3 domain and therefore isolates Grb2 and proteins associated with the Grb2 SH2 domain. GST-Sos competitively prevents the copurification of Grb2 SH3-binding proteins. The data in Fig. 3 show an anti-Grb2 Western blot of the cellular proteins purified with GST-Sos or EGFR-Y1068 and indicate that these two reagents isolate equivalent levels of endogenous Grb2. Moreover, a comparison of the levels of Grb2 purified with EGFR-Y1068 or GST-Sos with Grb2 levels in total cell lysates (Fig. 3 A) shows that these reagents are capable of affinity purifying the majority of endogenous Grb2 from RBL2H3 cells. The data in Fig. 3 B show that it is possible to copurify Sos with Grb2 when the EGFR-Y1068 motif but not GST-Sos is used as an affinity matrix.


Figure 3: The EGFR-Y1068 peptide and GST-Sos fusion protein affinity purify Grb2 from RBL2H3 cell lysate. RBL2H3 cell lysates containing 2 10cell equivalents were precleared as described and affinity purified with either EGFR-Y1068 coupled to Affi-Gel-10 or GST-Sos fusion protein coupled to glutathione-agarose beads. Post-nuclear proteins were acetone precipitated from 2 10RBL2H3 (total lysate). The samples were resolved on 11% SDS-PAGE under reducing conditions before transfer to PVDF and blocking of the membrane. A, the membrane was then probed with 0.5 µg/ml anti-Grb2 for 1 h and developed with anti-mouse Ig-horseradish peroxidase second stage antibody. B, the PVDF membrane was blocked and probed with 1 µg/ml anti-mSos1 for 2 h before development with anti-rabbit Ig-horseradish peroxidase second stage antibody.



To determine whether any of the tyrosine phosphoproteins observed in GST-Grb2 affinity purifications bind to endogenous Grb2 via its SH3 domains, we performed Western blot analysis with phosphotyrosine antibodies on Grb2 complexes isolated with EGFR-Y1068 peptide. As shown in Fig. 4 A, the EGFR-Y1068 peptide precipitates a 75-kDa tyrosine phosphoprotein from FcR1-activated but not quiescent RBL2H3 cells. The EGFR-Y1068 peptide also purified two tyrosine phosphoproteins of 110 and 120 kDa which were apparently equally phosphorylated in quiescent and FcR1-activated cells. These tyrosine phosphoproteins appear to bind to endogenous Grb2 via its SH3 domains. They do not coprecipitate with Grb2 which has been isolated using the GST-Sos fusion protein to compete out Grb2 SH3-binding proteins (Fig. 4 B). They also comigrate with the 75-, 110-, and 120-kDa tyrosine phosphoproteins detected in the binding experiments using GST-Grb2 single SH3 domain fusion proteins (Fig. 2). Affinity purification with the GST-Grb2 fusion protein produces the characteristic pattern of tyrosine phosphoproteins observed in previous experiments. It should be noted that there was a variable nonspecific binding of a 50-52-kDa doublet of tyrosine phosphoproteins to control affinity purifications comprising GST alone or Affi-Gel beads alone.


Figure 4: A, the EGFR-Y1068 phosphopeptide purifies tyrosine phosphoproteins from stimulated RBL2H3. RBL2H3 were primed with IgE anti-DNP and stimulated with KLH-DNP for 15 min as described. Lysates were made, and affinity purifications were carried out on 2 10cell equivalents/lane with the following reagents: Affi-Gel-10 beads alone, EGFR-Y1068 coupled to Affi-Gel-10, GST-Grb2 fusion protein, and GST alone coupled to glutathione-agarose beads. The samples were resolved under reducing conditions on 11% SDS-PAGE before transfer and blocking as described above. The membranes were then probed with 1 µg/ml 4G10 for 1 h and developed with anti-mouse Ig-horseradish peroxidase. B, binding of 55- and 33-kDa tyrosine phosphoproteins to endogenous Grb2 in RBL2H3. RBL2H3 cells were stimulated and lysed as described previously. Lysates from 2 10cells/point were precleared and affinity purified for 2 h with either GST-Grb2, GST-Grb2 µSH3, or GST-Sos. After 11% SDS-PAGE and transfer to PVDF membrane, Western blot analysis was carried out with 1 µg/ml 4G10. The membrane was then stripped and reprobed with 1 µg/ml anti-Shc overnight at 4 °C and developed with anti-rabbit Ig-horseradish peroxidase.



Tyrosine Phosphoproteins of 33 and 55 kDa Associate with Endogenous Grb2 SH2 Domains

The data in Fig. 4 B show anti-phosphotyrosine Western blots of the endogenous Grb2 complexes isolated from RBL2H3 cells with GST-Sos. These data indicate that the 120-, 140-, and 65-kDa tyrosine phosphoproteins observed binding to the normal and the double SH3 mutant GST-Grb2 fusion proteins do not coprecipitate with endogenous Grb2. However, an FcR1-induced tyrosine phosphoprotein of 33 kDa was detected in the endogenous Grb2 complexes purified by GST-Sos. In addition, a tyrosine phosphoprotein of 55 kDa was observed to bind to endogenous Grb2 in both quiescent and FcR1-stimulated RBL2H3. These p33 and p55 molecules appeared to bind to Grb2 SH2 domains based on their pattern of binding to the panel of Grb2 fusion proteins. As well, they did not coprecipitate with the Grb2 complexes purified with the EGFR-Y1068 peptide, and their interaction with endogenous Grb2 was competed by the EGFR-Y1068 peptide that binds to the Grb2 SH2 domain (data not shown).

In many cells the adapter molecule Shc is tyrosine phosphorylated in response to receptor stimulation and forms a complex with Grb2 SH2 domains. Western blot analysis with Shc antisera has shown that RBL2H3 cells express two isoforms of Shc. The 55-kDa isoform is predominant and the 46-kDa Shc protein is a minor component of the Shc population in these cells. The data in Fig. 4 B show that a tyrosine phosphoprotein of 55 kDa is coprecipitated with endogenous Grb2 by the GST-Sos fusion protein from both quiescent and FcR1-stimulated RBL2H3 cells. This protein had a slightly lower mobility than the nonspecific 50-52-kDa proteins described earlier and could thus possibly represent tyrosine phosphorylated 55-kDa Shc isoform. Accordingly, the blots were reprobed with a Shc antiserum. Fig. 4 B shows that the 55-kDa Shc isoform binds to the wild type and SH3-mutated Grb2 fusion proteins and also can be copurified with endogenous Grb2 by GST-Sos. The binding of Shc to endogenous Grb2 was not influenced by the activation state of the RBL2H3.

To further analyze the tyrosine phosphorylation of Shc in RBL2H3 cells, we used a tyrosine phosphopeptide Trk-Y490, which corresponds to a high affinity binding site for Shc SH2 domains in the Trk subunit of the nerve growth factor receptor. This was used as a reagent to affinity purify Shc from RBL2H3 cells. The data in Fig. 5 A show an anti-Shc Western blot of proteins affinity purified with the Trk-Y490 peptide from RBL2H3 cells. The 55- and 46-kDa Shc isoforms bind to the Trk-Y490 peptide but not to the EGFR-Y1068 peptide used here as a specificity control. The data in Fig. 5 B show an anti-phosphotyrosine Western blot of Trk-Y490 peptide precipitates isolated from quiescent and FcR1-triggered RBL2H3 cells. The 55-kDa Shc isoform is tyrosine phosphorylated in both quiescent and stimulated cells. These data demonstrate also that the 55-kDa tyrosine phosphoprotein binding to GST-Grb2 and GST-Sos comigrates with the tyrosine-phosphorylated 55-kDa Shc protein purified with Trk-Y490. The nonspecific 50-52-kDa protein doublet can once again be observed binding to control Affi-Gel beads alone but with a different mobilty to either of the Shc isoforms.

The 33-kDa Grb2 SH2 Domain-binding Tyrosine Phosphoprotein Is Not the Subunit of the FcR1

The accumulated evidence from the experiments in Figs. 1-5 show that FcR1 triggering results in tyrosine phosphorylation of a 33-kDa protein which binds to endogenous Grb2 in an SH2 domain-mediated association. The subunit of the FcR1 is 30-33 kDa and is known to be tyrosine phosphorylated in response to receptor triggering. Accordingly, we examined whether the p33 Grb2-binding protein was the subunit of the FcR1 (Fig. 6). Western blot analysis with the FcR1 chain antibody JRK1 showed that the chain can be immunoprecipitated with the anti-phosphotyrosine antibody FB2 from FcR1-stimulated but not quiescent cells (Fig. 6). Furthermore, it was possible to observe a hyperphosphorylated population of JRK1-immunoreactive chain molecules in total lysates from FcR1-activated RBL2H3. This latter population comigrated on SDS-PAGE with the population selected for by the FB2 anti-phosphotyrosine immunoprecipitation. However, the 33-kDa Grb2-binding tyrosine phosphoprotein detected by anti-phosphotyrosine Western blot analysis of GST-Grb2 affinity purifications from FceR1-stimulated RBL2H3 was not immunoreactive with the JRK1 antibody and is thus not the subunit of the FcR1. It is apparent, moreover, that the SDS-PAGE mobility of p33 is different from the tyrosine-phosphorylated chain.


Figure 6: The p33 tyrosine phosphoprotein binding the Grb2 SH2 domain in RBL2H3 cells is not the FcR1 chain. Affinity purifications of lysates from 2 10quiescent or FcR1-stimulated RBL2H3 were carried out using GST-Grb2. Post-nuclear protein from 2 10stimulated or unstimulated RBL2H3 cells was acetone-precipitated. Immunoprecipitations using 2 µg/point of the anti-phosphotyrosine antibody FB2 were carried out on lysates from 2 10quiescent or FcR1-stimulated RBL2H3. The samples were resolved and transferred as described. The membrane was probed with the mouse monoclonal antibody JRK1, which is reactive for the FcR1 chain, overnight at 4 °C. The Western blot was then developed with an anti-mouse Ig-horseradish peroxidase second stage antibody. An arrow marks the position of antibody light chain in the anti-phosphotyrosine immunoprecipitations.




DISCUSSION

The data presented here show that in RBL2H3 mast cells, as in many cell systems, the guanine nucleotide exchange factor for p21ras, Sos, is complexed to the SH3 domains of Grb2. The data show also that there are FcR1-induced complexes between tyrosine phosphoproteins and the adapter molecule Grb2. These include two basally tyrosine-phosphorylated 110- and 120-kDa molecules and a 75-kDa protein substrate for FcR1-activated PTKs which binds to the Grb2 SH3 domain. Two protein substrates for FcR1-activated tyrosine kinases were observed to bind GST fusion proteins of Grb2 but more importantly were also found in association with endogenous Grb2. A 75-kDa protein which was tyrosine phosphorylated in activated but not quiescent RBL2H3 cells was associated with the SH3 domains of Grb2. We also detected a 33-kDa tyrosine phosphoprotein complexed to Grb2 SH2 domains in FcR1 stimulated but not quiescent cells. Previous studies of Grb2-binding proteins in T cells have illustrated how experiments that identify Grb2-binding proteins solely on the basis of affinity purifications with GST-Grb2 fusion proteins can be misleading. This point is made again by the present study. Hence wild type GST-Grb2 fusion protein experiments suggested that in RBL2H3 lysates there are tyrosine phosphoproteins of 33, 55, 65, 120, and 140 kDa which are capable of binding to Grb2 SH2 domains. The 33- and 65-kDa proteins in particular are substrates for FcR1-activated PTKs as evidenced by their inducible tyrosine phosphorylation. However, only the constitutively tyrosine-phosphorylated 55-kDa and the FcR1-induced 33-kDa tyrosine phosphoproteins were detected in association with endogenous Grb2 SH2 domains. Similarly, only a subset of the tyrosine-phosphorylated proteins identified as Grb2 SH3-binding proteins by the in vitro binding experiments were present in the in vivo Grb2 complexes isolated from RBL2H3 cells: a 75-kDa FcR1-induced tyrosine phosphoprotein and two proteins of 110 and 120 kDa, respectively, which were equivalently tyrosine phosphorylated in quiescent and activated cells and appeared to associate constitutively with Grb2 SH3 domains.

It has been described in many cell systems that tyrosine-phosphorylated Shc forms a complex with Grb2 (28, 39) . In the present study, Shc was observed to be tyrosine phosphorylated in quiescent RBL2H3 cells, and no increases in Shc tyrosine phosphorylation were detected in response to FcR1 triggering. This suggests that Shc is not a major substrate for FcR1-activated tyrosine kinases in RBL2H3 cells. In accordance with the lack of FcR1 control of Shc tyrosine phosphorylation, no FcR1-induced changes in Shc-Grb2 complex formation were observed in RBL2H3 cells although Shc was constitutively associated with Grb2 in an SH2 domain-mediated association. It has been previously noted that there is no significant formation of Shc-Grb2 complexes in response to TCR ligation in T cells (25, 35) . In contrast, in B cells ligation of the BCR induces tyrosine phosphorylation of Shc and induces a Shc-Grb2-Sos complex that is proposed to link the BCR to p21ras (36, 37) . Interestingly, Shc is a reasonably abundant protein in both T cells and mast cells. Moreover, in both cell types cytokines such as interleukin-2 and interleukin-3 can stimulate high levels of Shc tyrosine phosphorylation and induce Shc-Grb2 complexes indicating that there are no technical problems in detecting such complexes in these cells (40, 41, 42) .

In the absence of inducible tyrosine phosphorylation of Shc, a possible candidate for linking signals transduced by the FcR1 to the Grb2 signaling pathway is a 33-kDa tyrosine phosphoprotein. This p33 is tyrosine phosphorylated in response to FcR1 stimulation and binds Grb2 SH2 domains. The subunit of the FcR1 has a similar molecular weight to p33 and is tyrosine phosphorylated in response to receptor ligation (17) . We have shown, however, that the p33 tyrosine phosphoprotein observed to bind the SH2 domain of endogenous Grb2 is not the chain of the FcR1. This does not exclude an indirect interaction between Grb2 and the FcR1. The FcR1-induced p33 molecule appears functionally analogous to the TCR-induced p36 phosphoprotein observed as the major tyrosine-phosphorylated species to bind Grb2 SH2 domains in activated T cells (25, 35) . It remains to be determined, however, whether p36 and p33 share any structural homology. In this context, studies in a variety of cell systems have identified many tyrosine kinase substrates with apparently diverse functions which can bind to Grb2 SH2 domains. These include adapters such as Shc, tyrosine phosphatases such as R-PTP and Syp, and growth factor receptors and receptor subunits such as the EGF receptor and IRS-1 (27, 43, 44) . Accordingly, it appears that multiple mechanisms have evolved to couple cellular PTKs to Grb2, of which the p36 and now p33 molecules may be one type.

One well-documented role of Grb2 is to couple tyrosine kinases to the guanine nucleotide exchange protein Sos and hence to p21ras signaling pathways (45) . We have observed that triggering of the FcR1 in RBL2H3 cells results in activation of p21ras and downstream effector molecules such as Raf-1 and the MAP kinase ERK2.() The current data demonstrate that Sos is constitutively associated with Grb2 SH3 domains in RBL2H3 cells. Accordingly, the FcR1-induced 33-kDa tyrosine phosphoprotein that binds to Grb2 SH2 domain may be an adapter that couples the FcR1 to Grb2-Sos complexes thereby permitting FcR1 stimulation of p21ras. However, it has been recognized increasingly that Grb2 may link PTKs to molecules other than Sos. For example, a second p21ras guanine nucleotide exchange protein C3G binds to Grb2 SH3 domains in PC12 cells (46) . As well, the GTPase dynamin can bind to and be activated by Grb2 SH3 domains in vitro and in vivo (47) . In the present report, we have shown that in RBL2H3 cells Grb2 SH3 domains do not only bind the p21ras guanine nucleotide exchange protein Sos. There are also tyrosine-phosphorylated proteins of 75, 110, and 120 kDa which associate with endogenous Grb2 SH3 domains. By analogy with Sos, C3G, and dynamin, the p75, p110, and p120 molecules are candidates for alternate Grb2 effector molecules. The p75 molecule is of particular interest in this context because it is a substrate for FcR1-activated PTKs. One of the major substrates for FcR1-activated PTKs has been recently cloned and is a 75-kDa protein termed SPY or HS1, which has an SH3 domain and also contains proline-rich sequences reminiscent of SH3-binding domains characterized in molecules such as Sos (48) . Western blot analysis of endogenous Grb2 complexes in both T cells and RBL2H3 cells demonstrated that HS1 is not the Grb2-associated p75 described in this study.() Associations between Grb2 and p75 have not been detected in fibroblasts suggesting that this molecule has a limited tissue distribution and hence function (32) . In preliminary analysis the FcR1-induced p75 appears identical to a 75-kDa protein that is a substrate for TCR-activated PTKs and is constitutively associated with Grb2 SH3 domains in T cells (32) . We propose that p75 may be a common signaling element for the TAM-based receptor family that includes the TCR and the FcR1. Recently, p120, which is a substrate for TCR-activated PTKs, was shown to bind to the NH-terminal SH3 domain of Grb2 fusion proteins (34) . The constitutively tyrosine-phosphorylated 120 kDa protein described herein to associate with endogenous Grb2 complexes was not p120although, p120 was detected in the Grb2 fusion protein complexes isolated from RBL2H3 cells and corresponded to the 120-kDa FcR1-induced tyrosine phosphoprotein seen in these complexes (data not shown).

The role of the FcR1 is to regulate the exocytosis of mast cell granules and to modulate cytokine gene expression. The present data have shown that the FcR1 induces tyrosine phosphorylation of multiple Grb2-binding proteins, strongly suggesting that this adapter protein is involved in coupling the FcR1 to one or more signal transduction pathways. It has been demonstrated that calcium and PKC have an important role in FcR1-regulated cellular responses (21) . We would propose that Grb2-coupled signaling pathways may also be important for FcR1-induced mast cell activation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Glaxo Research and Development, United Kingdom.

Supported by Boehringer Ingelheim Fonds.

**
To whom correspondence should be addressed. Tel.: 071-269-3307; Fax: 071-269-3417.

The abbreviations used are: PTKs, protein tyrosine kinases; TAM, tyrosine-based activation motif; BCR, B cell antigen receptor; TCR, T cell antigen receptor; EGF, epidermal growth factor; GST, glutathione S-transferase; DNP, dinitrophenol; KLH, keyhole-limpet-hemocyanin; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.

H. Turner and D. Cantrell, unpublished observations.

H. Turner, K. Reif, and S. Ley, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Narin Osman and Dr. Larry Samelson for valuable advice and discussions and Nicola O'Reilly and Elisabeth Li for peptide syntheses.


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