Cell adherence to fibronectin and the aggregation of the high affinity immunoglobulin E receptor synergistically regulate tyrosine phosphorylation of 105-115-kDa proteins.

Adherence of cells to extracellular matrix components modulates cellular responses. Here we compared the array of tyrosine phosphorylated proteins induced by the aggregation of the high affinity receptor for IgE (Fc epsilon RI) in fibronectin-adherent and in nonadherent rat basophilic leukemia (RBL-2H3) cells. Adherence to fibronectin in the absence of Fc epsilon RI aggregation induced tyrosine phosphorylation of 105-115-kDa proteins. This phosphorylation was reversed by EDTA and by a synthetic peptide containing the sequence Arg-Gly-Asp, demonstrating a requirement for fibronectin-integrin interaction. Aggregation of Fc epsilon RI in fibronectin-adherent cells markedly enhanced the tyrosine phosphorylation of the same 105-115-kDa proteins. There were minimal differences in tyrosine phosphorylation of other proteins induced by the aggregation of Fc epsilon RI in nonadherent and in fibronectin-adherent cells. Direct activation of protein kinase C and/or increase in calcium influx induced the phosphorylation of the 105-115-kDa proteins only in fibronectin-adherent cells. The magnitude of the phosphorylation of the 105-115-kDa proteins induced by the aggregation of Fc epsilon RI in fibronectin-adherent cells was substantially greater than the sum of that due to adherence to fibronectin and the aggregation of Fc epsilon RI in nonadherent cells. Therefore, cell adherence and the aggregation of Fc epsilon RI synergistically regulate tyrosine phosphorylation of the 105-115 kDa proteins.

Adherence of cells to extracellular matrix components modulates cellular responses. Here we compared the array of tyrosine phosphorylated proteins induced by the aggregation of the high affinity receptor for IgE (FccRI) in fibronectin-adherent and in nonadherent rat basophilic leukemia (RBL-2H3) cells. Adherence to fibronectin in the absence of FccRI aggregation induced tyrosine phosphorylation of 105-1 15-kDa proteins. This phosphorylation was reversed by EDTA and by a synthetic peptide containing the sequence Arg-Gly-Asp, demonstrating a requirement for fibronectinintegrin interaction. Aggregation of FccRI in fibronectin-adherent cells markedly enhanced the tyrosine phosphorylation of the same 105-1 15-kDa proteins. There were minimal differences in tyrosine phosphorylation of other proteins induced by the aggregation of FccRI in nonadherent and in fibronectin-adherent cells. Direct activation of protein kinase C and/or increase in calcium influx induced the phosphorylation of the 105-115-kDa proteins only in fibronectin-adherent cells. The magnitude of the phosphorylation of the 105-115-kDa proteins induced by the aggregation of FccRI in fibronectin-adherent cells was substantially greater than the sum of that due to adherence to fibronectin and the aggregation of FccRI in nonadherent cells. Therefore, cell adherence and the aggregation of FccRI synergistically regulate tyrosine phosphorylation of the 105-115 kDa proteins.
Integrins are the major cell surface receptors by which cells adhere to other cells or to the extracellular matrix (1)(2)(3)(4)(5). These interactions are important for many biological processes, including cell differentiation and proliferation, tumor metastasis, inflammation, and the immune response. Integrins are heterodimers of LY and /3 subunits, each of which is a transmembrane protein. The extracellular domain of the (Y subunit has three or four tandem repeats of a putative Ca2+ binding motif. Many integrins bind the tripeptide Arg-Gly-Asp sequence present on various extracellular matrix glycoproteins including fibronectin, laminin, and collagen (6).
Integrins do not only establish a physical link between cells and the extracellular matrix, but they also transduce signals into the cell. For example, the activation of integrins induces * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby protein tyrosine phosphorylation, increases in cytoplasmic pH, changes in intracellular Ca2+, or CAMP levels and initiates gene expression (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Many effects of integrins are due to their capacity to modify cellular responses to various stimuli; for instance, in T cells the activation of integrins facilitates the CD3-mediated interleukin production and cell proliferation (17)(18)(19)(20)(21). Thus, adherence by integrins can activate several intracellular signaling pathways. Similarly, the function of integrins is modulated by intracellular signals induced by other receptors (22)(23)(24)(25)(26); hence, the activation of T cells through the CD3 receptor complex results in an increase in the integrin-mediated adhesion of these cells to specific substrates (22,23). Likewise, phorbol myristate acetate (PMA)' stimulates protein kinase C and results in an increase in cell adhesion (22,24). These data suggest interactions between the intracellular signals transduced by integrins and other receptors.
There are high affinity receptors for IgE (FccRI) on the surface of mast cells, basophils, and related cultured cell lines such as the rat basophilic leukemia (RBL-2H3) cells (27,28). Aggregation of the FccRI by antigen activates these cells to release inflammatory mediators and cytokines. The activation of these cells results in many intracellular biochemical reactions, including protein tyrosine phosphorylation (29-35), phospholipase C activation (36), stimulation of phospholipases A2 (37) and D (38), and an increase in Ca2+ influx (39).
Recently, we reported that the mast cell analogue, RBL-2H3 cells, adhere strongly to fibronectin but weakly to other extracellular matrix components (40). The binding to fibronectin is Ca2+-dependent and can be inhibited by a synthetic peptide containing the sequence Arg-Gly-Asp, and as shown here is due to integrins. RBL-2H3 cells are widely used as a model for studying FccRI-induced signal transduction (41).
In the present studies, RBL-2H3 cells were used to examine the effect of adherence to fibronectin on protein tyrosine phosphorylation induced by the aggregation of FccRI. We show that the aggregation of FccRI induced the strong tyrosine phosphorylation of 105-115-kDa proteins only in fibronectin-adherent cells.
Antibodies-Mouse monoclonal anti-trinitrophenyl IgE (TNP-142) and mAb BD6 have been characterized previously (42). mAb BGD6 (IgG) and mAb BB3 (IgM) bind to RBL-2H3 cells, do not inhibit IgE binding, and do not induce histamine release.' Rabbit polyclonal antibody to chicken p1 integrins (43) that cross-reacts with & integrins of several species was a generous gift of Dr. Kenneth M.
Yamada (National Institute of Dental Research, National Institutes of Health, Bethesda, MD). Normal Rabbit IgG and goat anti-rabbit IgG were from Jackson Immunoresearch Laboratories, Inc. (Bar Harbor, ME). Horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY-20 was from ICN Immunobiologicals (Lisle, IL).
Cells and Cell Culture-The RBL-2H3 cloned subline of rat basophilic leukemia cells was maintained in complete medium (Eagle's minimum essential medium with Earle's balanced salt solution supplemented with 15% heat-inactivated fetal calf serum, 4 mM Lglutamine and antibiotic/antimycotic mixture) at 37 "C in 5% CO'. Cells were dissociated with 0.05% trypsin, 0.53 mM EDTA and subcultured three times a week.
Immunoprecipitation and Immunoblotting of Integrins-RBL-2H3 cells were 1'61-surface-labeled and then solubilized as described previously (44,45). Briefly, the labeled cells (5 X lO'/ml) were incubated for 30 min at 0 "C with 100 mM octyl glucoside in Buffer A (50 mM Tris-HC1, pH 8.0, 150 mM NaCl, 1 mM CaCl', 1 mM MgC12, 1 mM phenylmethylsulfonyl fluoride) and the lysates cleared by centrifugation at 44,000 X g for 30 min at 4 "C. For immunoprecipitation, 100 pg/ml of normal rabbit IgG or 100 pg/ml of rabbit anti-& integrins were preincubated with goat anti-rabbit IgG coupled to Sepharose 4B beads, whereas for affinity purification the BSA, fibronectin and the 120-kDa a-chymotrypsin fragment of fibronectin were directly coupled to the Sepharose 4B as recommended by the manufacturer. Precipitation of proteins from the lysates was for at least 16 h at 4 "C. The beads were washed extensively with Buffer A containing 25 mM octyl glucoside. The proteins then were eluted by boiling in sample buffer (75 mM Tris-HC1, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol) and analyzed by SDS, 10% polyacrylamide gel (PAGE) under nonreducing conditions. For immunoblotting, proteins from unlabeled RBL-2H3 cells were precipitated as above, separated by SDS-PAGE (lo%), and then transferred to nitrocellulose (42).
Protein Tyrosine Phosphorylation-Flat bottom Immulon-2 enzyme-linked immunosorbent assay wells (Dynatech Laboratories, Inc., Chantilly, VA) were coated with 30 pg/ml of fibronectin by incubating wells for 2 h at 37 "C as described previously (40). Control wells received phosphate-buffered saline only. Wells were washed once with blocking buffer (phosphate-buffered saline supplemented with 20 mg/ml BSA) and then unoccupied sites were blocked by incubating 200 pl of this buffer in each well for 2 h at 37 "C. The wells were then washed three times with PIPES buffer (25 mM PIPES, 110 mM NaCl, 5 mM KCl, 5.6 mM glucose, 1 mM CaCl', and 0.1% BSA, pH 7.4). RBL-2H3 cells (2 X lo6 cells/ml) were suspended in PIPES buffer, and 40 pl was added to the wells for 20 min at 37 "C in an incubator. The cells were then solubilized by adding 40 p1 of an ice-cold solubilizing buffer (phosphate buffer containing 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaC1, 50 mM NaF, 1 mM Na3V04, 50 pg/ml leupeptin, 0.5 unit/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride; final concentrations) (29). The lysates were boiled in sample buffer and proteins separated by SDS-PAGE (10%). The proteins were then transferred to nitrocellulose paper and immunoblotted with horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY-20 and then visualized as described previously (33). In some experiments, the cells were allowed to adhere to fibronectin for 20 min at 37 "C and then incubated with 1 mM of the peptide Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro for 30 min at 37 "C. In another set of experiments, cells were allowed to adhere to fibronectin for 20 min at 37 "C and then excess EDTA  To examine the effect of adhering to fibronectin on FcrRI-induced tyrosine phosphorylation, RBL-2H3 cells (lo6 cells/ml) were sensitized with a 1:10,000 dilution of ascitic fluid containing the anti-TNP mouse monoclonal IgE (TNP-142) in complete medium (Eagle's minimum essential medium with Earle's balanced salt solution supplemented with 15% heat-inactivated fetal calf serum, 4 mM Lglutamine, and antibiotic/antimycotic mixture). After 60 min at room temperature, cells were washed three times with PIPES buffer and then resuspended in this buffer at 2 X lo6 cells/ml. The cells were then added to the wells and were incubated for 20 min at 37 "C. Then, 20 p1 of the antigen DNPwHSA (50 ng/ml final concentration) were added to the wells. This antigen concentration induced optimal histamine release and protein tyrosine phosphorylation (29, 31, 40). After a 25-min incubation at 37 "C, histamine content in the supernatants of one set of wells was determined by automated fluorometric analysis (46). Spontaneous histamine release during the incubation period was less than 5% in all experiments. To an identical set of wells, 20 pl of ice-cold lysing buffer was added, and the lysates were examined for tyrosine phosphorylation as described above. In other experiments, the cells were stimulated with the optimal concentrations of PMA (40 nM final concentration), Ca'+ ionophore A23187 (final concentration 0.5 p~) , or the combination of both for 25 min at 37 "C.
Two-dimensional Electrophoresis-This was as described previously with some modification (47). RBL-2H3 cells were added to wells coated with only BSA or fibronectin as described above. After 20 min at 37 "C, some wells received either PIPES buffer (control) or antigen for 25 min at 37 "C. Lysates were prepared as above, transferred to urea buffer (9.5 M urea, 8% Nonidet P-40), boiled for 15 min, centrifuged, and separated by isoelectrofocusing (pH 3-10), followed by SDS-PAGE (4-20%). The proteins were then transferred to nitrocellulose and analyzed for tyrosine phosphorylation as above.

RESULTS
Immunoprecipitation and Immunoblotting of Integrins in RBL-2H3 Cells-Previously we reported that RBL-2H3 cells adhere to fibronectin with characteristics that suggest a role for integrins, i.e. the binding was Caz+-dependent and was inhibited by the peptide Arg-Gly-Asp (40). In the present report we further defined the surface receptors on the cells involved in binding to fibronectin. By immunofluorescence, a rabbit polyclonal antibody to chicken p1 integrins (43) that cross-reacts with PI integrins of several species bound to permeabilized RBL-2H3 cells. The antibody to p1 integrins precipitated a 125-kDa glycoprotein from 'z51-surface-labeled cells (Fig. 1A). Proteins of similar molecular mass were also precipitated by both the 120-kDa fragment of fibronectin and by intact fibronectin (Fig. lA). The 130-kDa surface-labeled proteins precipitated with both the 120-kDa fragment of fibronectin and by fibronectin is probably the a component of integrins. Additional unidentified proteins were also precipitated by intact fibronectin. Unlabeled proteins that were precipitated by fibronectin were immunoblotted with anti-pl integrins antibody (Fig. 1B). Again a 125-kDa protein was. observed in the immunoblots. These data indicate that RBL-2H3 cells bound to fibronectin through p1 integrins.
Adherence to Fibronectin Induces Tyrosine Phosphorylation of 105-115-kDa Proteins-As shown in Fig. 2, the adherence of RBL-2H3 cells to fibronectin induced the phosphorylation on tyrosine of proteins, the most prominent being in the range of 105-115 kDa (pp105-115). In time course experiments, the phosphorylation of the pp105-115 induced by interaction with fibronectin was first apparent within 5 min, reached a peak by 20 min and decreased slightly at 60 min (Fig. 3). It was also detectable in adherent cells that had attached to plastic plates by culture overnight in the fetal calf serum-containing medium (data not shown). Thus, tyrosine phosphorylation of pp105-115 continues as long as the cells are adherent. Previously we observed that the binding of RBL-2H3 cells to fibronectin was Ca2+-dependent and was inhibited by a pep-  tide containing the Arg-Gly-Asp sequence (40). We therefore examined the effect of the peptide Arg-Gly-Asp on protein tyrosine phosphorylation in fibronectin-adherent cells. Adherent cells were incubated with 1 mM of a synthetic peptide containing the Arg-Gly-Asp sequence or Arg-Gly-Glu. The addition of a synthetic peptide containing the Arg-Gly-Asp sequence, but not Arg-Gly-Glu, reversed the phosphorylation of pp105-115 (Fig. 4A). Similarly, chelating Ca2' in the medium by adding EDTA rapidly reversed the tyrosine phosphorylation of pp105-115 (Fig. 4R). The action of EDTA is most likely to disrupt cell binding to fibronectin, because the 1-min incubation is too short to deplete intracellular Ca". The rapid reversal of the phosphorylation of pp105-115 suggests the presence of active tyrosine phosphatase(s) in the cells. Taken together, these results indicate that tyrosine phosphorylation of pp105-115 is tightly coupled to adherence mediated by integrins.
To determine if cell adherence and spreading on other biological surfaces induces tyrosine phosphorylation of pp105-115, RBL-2H3 cells were plated on surfaces coated with 30 pg/ml of antibodies to different RBL-2H3 surface molecules. Although this resulted in cell adherence and spreading (Fig. 5), there was no increase in the tyrosine phosphorylation of pp105-115 (Fig. 6). Therefore, tyrosine phosphorylation of pp105-115 resulted from the integrinmediated adherence of cells to fibronectin.
In transformed chicken fibroblasts the PI integrins are phosphorylated on tyrosine (48)(49)(50). However, our experiments suggest that pp105-115 is not the P1 integrins. RBL-2H3 cell lysates from cells that had been adherent on fibronectin were affinity-purified with anti-phosphotyrosine or with anti-P1 integrins adsorbents. The precipitated phosphorylated proteins (eluted with phenyl phosphate) or integrins were transferred to nitrocellulose paper and probed with either anti-phosphotyrosine or anti-P1 integrin antibodies.
The P1 integrins was not precipitated by the anti-phosphotyrosine affinity column, although the 105-115-kDa phosphorylated proteins could be detected in these immunoblots (data not shown). Similarly, the antibodies to PI integrin did not precipitate tyrosine-phosphorylated pp105-115 (data not shown). These results, together with the difference in the apparent molecular mass between and pp105-115, demonstrate that pp105-115 is not PI integrin.

FctRIAggregation Induces Strong Tyrosine Phosphorylation of pp105-115 in Fibronectin-adherent but Not in Nonadherent
RBL-2H3 Cells-Recently, we and others (29-35) have reported the rapid tyrosine phosphorylation of several proteins coupled to FccRI-mediated signal transduction in RBL-2H3 cells. Here, we compared the array of tyrosine phosphorylated proteins induced by the aggregation of the FccRI in fibronectin-adherent to those in nonadherent RBL-2H3 cells. FccRI aggregation of adherent and nonadherent RBL-2H3 cells led  to tyrosine phosphorylation of 38-, 72-, 80-, 97-, 105-115-, and 140-kDa proteins (Fig. 7, lanes 3 and 4 ) . The extent of the phosphorylation of the 38-, 72-, 80-, 97-, and 140-kDa proteins was slightly increased in adherent cells when compared with nonadherent cells. For example, by densitometry there was a 24% increase in tyrosine phosphorylation of the 72-kDa protein in adherent cells. However, the intensity of phosphorylation of the proteins in the molecular mass 105-115 kDa range was markedly enhanced in cells attached to fibronectin. The extent of the phosphorylation of the 105-115-kDa proteins induced by FccRI in fibronectin-adherent cells was substantially greater than that induced by both adherence to fibronectin and by the aggregation of FccRI in nonadherent cells (Fig. 7, lanes 2 and 3 versus lane 4 ) . Aggregating FccRI on cells adherent to antibody-coated surfaces strongly phosphorylated the 38-, 72-, 80-, 97-, and 140-kDa proteins (Fig. 8, lane 1 versus lanes 4-6); however, the phosphorylation of the 105-115-kDa proteins was strikingly less than that induced by FccRI aggregation on cells adherent to fibronectin (Fig. 8, lane 3 versus lanes 4-6). Hence, adherence to fibronectin by integrins is essential for optimal tyrosine phosphorylation of the 105-115-kDa proteins induced by the aggregation of FccRI. Two-dimensional electrophoresis was used to confirm that in fibronectin-adherent cells the 105-115-kDa proteins phosphorylated by adherence and by FccRI aggregation are identical (Fig. 9). Adherence to fibronectin resulted in the tyrosine phosphorylation of several proteins that correspond to the pp105-115 seen in the previous exper- iments (Fig. 9, A versus B ) . Tyrosine phosphorylation of the same pp105-115 was enhanced in adherent cells in which FccRI were aggregated (Fig. 9, B versus C); no additional proteins of this size range were phosphorylated on tyrosine. Taken together, the data indicate that adherence to fibronectin by integrin and the aggregation of FccRI synergistically regulated tyrosine phosphorylation of pp105-115.

PMA and Cu2+ Ionophore Induce Tyrosine Phosphorylation of pplO5-115 Only in Fibromctin-adherent Cells-Ca2+ influx
and protein kinase C activation can induce protein tyrosine phosphorylation and are implicated in the signal transduction of various receptors (34, 51, 52). We therefore investigated whether Ca2+ influx and protein kinase C activation by themselves are sufficient to induce pp105-115 tyrosine phosphorylation. In nonadherent cells, treatment with the Ca2+ ionophore A23187 to increase intracellular Ca2+ and/or with PMA to activate protein kinase C did not induce tyrosine phosphorylation of the 105-115-kDaproteins (Fig. 10). In contrast, in fibronectin-adherent cells both the Ca2+ ionophore and PMA led to a significant increase in the tyrosine phosphoryl-  were prepared as above, transferred to urea buffer (9.5 M urea, 8% Nonidet P-40), boiled for 15 min, centrifuged, and the supernatant subjected to isoelectrofocusing (pH 3-10) followed by SDS-PAGE (4-20%). The proteins were then transferred to nitrocellulose and analyzed for tyrosine phosphorylation. Arrowheads mark the pp105-115. ation of the 105-115-kDa proteins (Fig. 10). Therefore, Ca2+ influx and protein kinase C activation by themselves are not sufficient to phosphorylate these proteins but require a second signal transduced by binding to fibronectin. These results again demonstrate the importance of integrins for tyrosine phosphorylation of pp105-115 induced by other stimuli.

DISCUSSION
Adherence of the RBL-2H3 cells to fibronectin-coated surfaces induced tyrosine phosphorylation of 105-115-kDa pro- teins. The experiments implicate integrins in playing an important role in signaling this protein tyrosine phosphorylation. First, cell adherence and spreading on biological surfaces other than fibronectin did not induce tyrosine phosphorylation of these proteins. Second, a synthetic peptide containing the Arg-Gly-Asp sequence reversed the adherence-induced protein tyrosine phosphorylation.
Third, protein tyrosine phosphorylation induced by adherence to fibronectin was dependent on the presence of extracellular Ca2+. These data strongly suggest that the pp105-115 phosphorylation is due to an interaction of PI integrins on RBL-2H3 cells with fibronectin. However, in the immunoprecipitation studies proteins other than the PI also bound to intact fibronectin ( Fig. 1). Thus, these other surface molecules could play a role in the tyrosine phosphorylation induced by adherence.
Although adherence of RBL-2H3 cells to fibronectin resulted in cell spreading, tyrosine phosphorylation of pp105-115 was not due to the morphological changes. The induction of tyrosine phosphorylation of pp105-115 by adherence to fibronectin occurred before there was significant cell spreading (within 5 min), reached a peak before the cells had completely spread (within 20 min), and was present long after the cells had fully spread. Furthermore, cell adherence and spreading on other biological surfaces did not induce the tyrosine phosphorylation of pp105-115. These data imply that tyrosine phosphorylation of pp105-115 induced by cell adherence to fibronectin is independent of cell spreading.
Protein tyrosine phosphorylation is a common signaling pathway for many different receptors (7-9, 29, 53-55). Integrins by activating protein tyrosine kinase(s) or inactivating a tyrosine phosphatase could result in enhanced tyrosine phosphorylation of pp105-115. The activation of integrins in other cells induced tyrosine phosphorylation of proteins with molecular mass similar to the ones reported here. For example, adherence of NIH 3T3 cells to fibronectin-coated surfaces induced tyrosine phosphorylation of 120-kDa proteins (7).
As we and others have reported previously (29-35), FctRI aggregation induced the tyrosine phosphorylation of several proteins. Some of these proteins, e.g. pp72, were phosphorylated only by the aggregation of FctRI. In contrast, either cell adherence or aggregation of the FctRI in nonadherent cells induced low level tyrosine phosphorylation of pp105-115 proteins. However, FctRI aggregation of fibronectin-adherent cells strongly phosphorylated pp105-115. Thus, although the stimulation of either integrins or FctRI alone induced some tyrosine phosphorylation of 105-115-kDa proteins, maximal phosphorylation was observed only after the aggregation of FccRI in fibronectin-adherent cells. The magnitude of phosphorylation of pp105-115 by FctRI aggregation in fibronectinadherent cells was not simply an additive effect of tyrosine phosphorylation induced by adherence to fibronectin and by the aggregation of FctRI in nonadherent cells. Therefore, integrins and FctRI are synergistically regulating the extent of the tyrosine phosphorylation of pp105-115.
In previous studies the aggregation of FctRI in RBL-2H3 cell monolayers attached by culture in the presence of fetal calf serum induced the phosphorylation of a 110-kDa protein (34). This phosphorylation was Ca'+-dependent and was also induced by PMA and by Ca'+ ionophore. These similarities suggest that ppll0 reported previously may be one of the 105-115-kDa proteins. However, the ppll0 was characterized in cells grown as monolayers in the presence of fetal calf serum; these conditions were different from those in the present experiments. The similarities in these proteins could be fortuitous or a result of a true molecular relationship.
Recently, we have shown that adherence of RBL-2H3 cells to fibronectin-coated surfaces caused cell spreading, reorganization of the cytoskeleton, and redistribution of the secretory granules (40). As we have reported previously and confirm here, these changes are accompanied by a dramatic enhancement of histamine release induced by FctRI stimulation (Fig. 7). In the present experiments, adherence of the cells to surfaces coated with different antibodies also enhanced FctRI-mediated histamine release (Fig. 8); however, there was little increase in the phosphorylation of pp105-115. Thus, FctRI-mediated tyrosine phosphorylation of pp105-115 was greater in cells adherent to fibronectin compared with cells plated on antibody-coated surfaces, although both enhanced histamine release. These results suggest that marked increase in tyrosine phosphorylation of pp105-115 is not essential for enhanced histamine release.
The phosphorylation of pp105-115 in adherent cells was enhanced not only by the aggregation of FctRI, but also by stimulation with PMA and Ca2+ ionophore. In some experimental models, cell activation either induced or enhanced adherence to matrix proteins (22)(23)(24)(25)(26). Thus, cell stimulation could enhance the adherence of the RBL-2H3 cells that in turn may result in enhanced tyrosine phosphorylation of pp105-115. However, this probably does not explain the present observations. Stimulation of the cells by FctRI aggregation or with PMA did not increase the number of c.ells bound to fibronectin.' Furthermore, aggregating FctRI on cells adherent by culture for 24 h in fetal calf serum, i.e. all cells were attached to the tissue culture surface, still enhanced pp105-115 tyrosine phosphorylation (34). Another possible mechanism of the increased pp105-115 phosphorylation could be the redistribution of cellular proteins. Adherence of RBL-2H3 cells to fibronectin-coated surfaces induced massive reorganization of the cytoskeleton (40). Besides integrins, several tyrosine kinases and/or substrates, including talin, tensin, paxillin, and p~1 2 5~*~, accumulate at the sites of cellsubstratum contact (10, [48][49][50][56][57][58]. Several of these proteins are also hyperphosphorylated on tyrosine. Thus, in fibronectin-adherent cells, tyrosine kinases and/or substrates clustered at points of cell contact could be more accessible to signal transduction pathways. Similarly, FctRI aggregation, PMA, and Ca2+ ionophore induced reorganization of the cytoskeleton (59,60). Furthermore, PMA induced the serine/ threonine phosphorylation of cytoskeletal proteins and affects their interaction with integrins (61, 62). Thus, it is possible that in RBL-2H3 cells, FctRI aggregation, PMA, and Ca2+ ionophore affected the interaction of integrins with other proteins and as a result modulated the integrin-induced tyrosine phosphorylation of pp105-115.
It has been suggested that the signals transduced by integrins and those of other receptors may converge (4). Here we show that the signals induced by integrins and by FctRI synergistically regulated the extent of the phosphorylation of the same proteins. This suggests the convergence of the signals induced by integrins and by FctRI. Identifying the 105-115-kDa proteins and their cellular functions should shed light on their role in integrins and FctRI signal transduction pathways.