©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Functions of the FcR1 and Subunits in the Control of FcR1-mediated Tyrosine Kinase Activation and Signaling Responses in RBL-2H3 Mast Cells (*)

(Received for publication, September 8, 1994; and in revised form, November 14, 1994)

Bridget S. Wilson Nicholas Kapp (§) Rebecca J. Lee Janet R. Pfeiffer A. Marina Martinez Yehudit Platt Francois Letourneur (1) Janet M. Oliver (¶)

From the Department of Pathology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 Basel Institute for Immunology, CH-4058 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In RBL-2H3 rat tumor mast cells, cross-linking the high affinity IgE receptor, FcR1, activates the protein-tyrosine kinases Lyn and Syk and initiates a series of responses including protein-tyrosine phosphorylation, inositol 1,4,5-trisphosphate synthesis, Ca mobilization, secretion, membrane ruffling, and actin plaque assembly. The development of chimeric receptors containing cytoplasmic domains of individual subunits of the heterotrimeric (alphabeta(2)) FcR1 has simplified analyses of early signaling events in RBL-2H3 cells. Here, RBL-2H3 cells were transfected with cDNAs encoding the extracellular and transmembrane domains of the interleukin-2 receptor alpha subunit (the Tac antigen) joined to the C-terminal cytoplasmic domains of the FcR1 and beta subunits (TT and TTbeta). Both sequences contain tyrosine activation motifs implicated in antigen receptor signal transduction. TT and TTbeta are expressed independently of the native FcR1, as demonstrated by the ability of Tac cross-linking agents to trigger the clustering and internalization through coated pits of both chimeric receptors without co-clustering the FcR1. A full range of signaling activities is induced by TT cross-linking; the TT-induced responses are slower and, except for Lyn activation, smaller than the FcR1-induced responses. In striking contrast, TTbeta cross-linking elicits no tyrosine phosphorylation or signaling responses, it impairs basal activities measured in secretion and anti-PY (anti-phosphotyrosine antibody) immune complex kinase assays, and it antagonizes FcR1-induced Lyn and Syk activation, protein-tyrosine phosphorylation, and signaling responses. We hypothesize that the isolated beta subunit binds a specific kinase or coupling protein(s) required for signaling activity, sequestering it from the signal-transducing subunit. Binding the same kinase or coupling protein to the beta subunit of the intact FcR1 may serve instead to present it to the adjacent subunit, resulting in enhanced kinase activation and signaling responses.


INTRODUCTION

The high affinity IgE receptor, FcR1, (^1)of mast cells and basophils belongs to the family of multichain immune system receptors, that includes the T cell receptor (TCR), the mIg receptor of B cells, and several members of the Fc receptor family. These receptors lack intrinsic enzyme activity. Instead, they recruit and activate cytoplasmic protein-tyrosine kinases that phosphorylate tyrosine residues in characteristic receptor subunit cytoplasmic sequences called variously tyrosine activation motifs (TAMs), antigen receptor homology 1 motifs, and antigen recognition and response motifs(1, 2, 3) . TAMs are 20-25 amino acid sequences containing two YXXL/I motifs separated by approximately ten residues(4, 5) . They are found in the and chains of the multisubunit TCR, in the Ig-alpha and Ig-beta chains of the mIg receptor, and in the beta and chains of the heterotrimeric (alphabeta(2)) FcR1. The TAM-containing FcR1 subunit is additionally found in association with at least two other Fc receptors, FcRI and FcRIIIA(6, 7, 8, 9) , and in the subset of T cells, where it replaces the TCR subunit(10) . Current studies suggest that the first response of immune system cells to antigen receptor cross-linking is TAM phosphorylation mediated by members of the Src protein-tyrosine kinase family(11) . This primary phosphorylation provides sites for the binding and activation of members of the Syk family of protein-tyrosine kinases and for the binding of a range of SH2 domain-containing protein-tyrosine kinase substrates (reviewed in (3) ). Once phosphorylated by receptor-associated kinases, these proteins initiate the downstream responses of signal transduction.

Recently chimeric receptors containing irrelevant extracellular and transmembrane domains and specific cytoplasmic domains have been used to dissect the signaling activities of individual subunits of the multichain immune system receptors. In particular, it was found that chimeric receptors expressing the TAM-containing cytoplasmic tails of the TCR and chains can both stimulate tyrosine phosphorylation and signaling responses when transfected into T cell lines and cross-linked by antibodies to the extracellular domain(12, 13, 14, 15) . T cells transfected with chimeric receptors consisting of a membrane-anchored form of Syk in T cells also have tyrosine phosphorylating and signaling activity following cross-linking(16) . These studies suggest there is redundancy in the pathways that initiate TCR signaling and that Syk activation is the critical event leading to downstream responses. Similar studies in transfected RBL-2H3 cells have yielded less clear-cut results. Thus, cross-linking transfected receptors consisting of the extracellular and transmembrane domains of the Tac antigen coupled to the cytoplasmic domains of the FcR1 subunit (TT) was shown to induce tyrosine phosphorylation and secretory responses varying from 50% of FcR1-mediated responses (17) to 10% of FcR1-mediated responses (12) to no response at all (18) . In addition, no tyrosine phosphorylating or secretory activity was found in association with cross-linked chimeric receptors consisting of the extracellular and transmembrane domains of Tac coupled to the TAM-containing C-terminal cytoplasmic domain of the FcR1 beta subunit (TTbeta; (17) ).

Here, we report further studies on the distribution, associated kinases, and signaling activities of TT and TTbeta expressed at high density on RBL-2H3 cells. Our results with cells expressing TT provide evidence that cross-linking alone is sufficient to activate both Syk and Lyn and to initiate a full range of downstream responses. In contrast, TTbeta cross-linking not only fails to activate tyrosine kinases and signaling responses but it impairs tyrosine kinase activation and signal transduction in response to FcR1 cross-linking.


EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

RBL-2H3 cells were cultured in minimal essential medium (MEM; Life Technologies, Inc.) with 15% fetal bovine serum (FBS) on either tissue culture or suspension grade culture dishes. The expression vector for the chimeric receptor, TT, was described by Letourneur and Klausner(12) . The TTbeta expression vector was identical except that the extracellular and transmembrane portions of human Tac were joined to the cytoplasmic C-terminal portion of the rat FcR1 beta subunit(19) . To generate stable transfectants, cells (1.2 times 10^7 RBL-2H3/ml in 0.8 ml of MEM, 20% FBS) were supplemented with 20 µg of linearized vector DNA and 1 µg of the linearized neomycin resistance plasmid, pFneo, electroporated in a Bio-Rad Gene Pulser at 500 µF and 250 volts, held for 10 min on ice, then transferred to a flask containing 30 ml of fresh MEM, 20% FBS. After 24 h, the medium was replaced with fresh MEM, 20% FBS containing 1.2 mg/ml G418 (Geneticin; Life Technologies, Inc.); the G418 concentration was reduced to 0.12 mg/ml after 72 h. A clone of TT-transfected cells demonstrating high binding activity toward fluorescein isothiocyanate (FITC)-conjugated anti-Tac antibodies (4 µg/ml; Amac, Inc., Westbrook, ME) and a strong secretory response to cross-linking the endogenous FcR1 was chosen for further study. Stably transfected TTbeta cells were selected by labeling cell populations with FITC-anti-Tac and sorting the 5% of cells with the highest surface expression of TTbeta. Surface expression of both TT and TTbeta chimeric receptors was routinely monitored by flow cytometry using FITC-conjugated anti-Tac monoclonal antibodies and the Simply Cellular (San Juan, Puerto Rico) bead standards.

Cell Activation

For studies with the endogenous FcR1, cells were incubated overnight with a saturating amount (1 µg/ml) of monoclonal anti-dinitrophenol IgE (anti-DNP IgE; (20) ), followed by washing with modified Hanks' buffered saline (21) containing 0.1% bovine serum albumin (Hanks'-BSA) and activation at 37 °C by the addition of 0.1 µg/ml of DNP-conjugated BSA (DNP-BSA; Molecular Probes, Eugene, OR) or 1 µg/ml rabbit anti-IgE(22) . For studies with the chimeric receptors, cells were incubated for 10 min at room temperature with 2 µg/ml biotinylated anti-Tac (Amac, Inc.), then washed and activated at 37 °C by the addition of 25 µg/ml avidin (Calbiochem). Transfected receptors were also primed with unmodified anti-Tac (Amac, Inc.) followed by cross-linking with 25 µg/ml unmodified or lissamine rhodamine sulfonyl chloride (LRSC)-conjugated goat anti-mouse IgG (Jackson Immunochemicals, West Grove, PA).

Microscopy

Receptor clustering and internalization were observed by fluorescence microscopy of monolayers of IgE- and anti-Tac-primed cells on glass coverslips. Cells were incubated at 37 °C with DNP-BSA and/or LRSC-conjugated goat anti-mouse IgG, then fixed and permeabilized with 2% paraformaldehyde plus 0.02% saponin for 30 min at room temperature. Fixed IgE-receptor complexes were labeled with rabbit anti-IgE (22) followed by FITC-conjugated anti-rabbit IgG (Jackson Immunochemicals). Receptor distributions were observed using a Zeiss Photomicroscope III. Chimeric receptors were also primed in suspension with biotinyl-anti-Tac antibody, followed by cross-linking with streptavidin conjugated to 15 nm colloidal gold particles (Amersham Corp.) and processing for transmission electron microscopy(23) .

To observe cross-linker-induced membrane ruffling and spreading, monolayers of primed cells were incubated for 10 min at 37 °C with cross-linker (DNP-BSA or avidin), then fixed with 2% glutaraldehyde and processed for scanning electron microscopy(24) . To observe actin plaque assembly, monolayers of activated cells were fixed with paraformaldehyde and labeled with rhodamine-phalloidin(25) .

Secretion Assays

Cells were plated into 24-well tissue culture dishes at a density of 2 times 10^5 cells/well and incubated for 16-24 h with 0.4 µCi/ml [^3H]serotonin (5-hydroxytryptamine, DuPont NEN). Anti-DNP IgE (1 µg/ml) was added to designated wells. The dishes were washed with 1 ml of Hanks'-BSA medium, held at room temperature for 10 min while designated wells were primed with biotinylated anti-Tac antibody, washed again, and a total of 0.2 ml of warmed Hanks'-BSA plus cross-linking reagents (DNP-BSA and/or avidin) was added to each well. After incubation at 37 °C for 20 min, 0.3 ml of ice-cold phosphate-buffered saline (PBS) was added to each well and [^3H]serotonin release was measured by liquid scintillation counting of 0.3-ml aliquots of the supernatants from each well. Percent serotonin release was calculated based upon wells lysed with 1% Triton X-100 and corrected for blanks taken prior to incubation. Each measurement was performed in duplicate.

Flow Cytometric Assays for Cross-linker-induced Protein-Tyrosine Phosphorylation

IgE and biotinyl-anti-Tac-primed RBL-2H3 cells (1 times 10^6/ml; 0.5-ml aliquots) were activated in suspension at 37 °C with DNP-BSA or avidin, reactions were terminated by the addition of fixative (4% paraformaldehyde, 0.04% saponin in PBS, pH 7.4), and washed cells were incubated sequentially for 30 min at room temperature in PBS with 1% BSA (PBS-BSA) containing 1 µg/ml affinity-purified rabbit polyclonal anti-phosphotyrosine antibody (anti-PY; prepared by J. Potter and G. Deanin as described in (26) ) and with FITC-conjugated goat anti-rabbit IgG antibody (1:35 in PBS-BSA; Cappell, Durham, NC). Washed cells were suspended in 1 ml of PBS and mean channel fluorescence determined for at least 10,000 cells using a Coulter Epics Elite flow cytometer.

Biochemical Analysis of Cross-linker-induced Protein-Tyrosine Phosphorylation

Primed, adherent cell cultures (6 times 10^6 cells/100-mm plate) were activated at 37 °C with DNP-BSA and/or avidin and reactions halted by the addition of 5 ml of ice-cold PBS. Supernatants were aspirated and the cells lysed by scraping with 1.0 ml of ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM NaV0(4), 0.1 µg/ml each of leupeptin and antipain, 1% Brij 96, pH 7.2). Lysates were centrifuged for 4 min at 13,000 times g in a refrigerated microcentrifuge, the supernatant protein content determined, and 100 µg of protein was separated by SDS-PAGE on 10% linear or 7.5-12% gradient gels and transferred to nitrocellulose. Membranes were blocked with 1% immunoglobulin-free BSA (Sigma) for 1 h at room temperature, incubated sequentially for 1 h at 4 °C with 1 µg/ml affinity-purified rabbit anti-PY and with S-protein A (0.2 µCi/ml; Amersham), then dried and analyzed by autoradiography or by use of a Molecular Dynamics PhosphorImager with ImageQuant software.

1,4,5-IP(3) Assays

1,4,5-IP(3) levels in antigen- or avidin-activated cell suspensions were determined using a radioreceptor assay modified from Challiss et al.(27) as described(28) .

Measuring Intracellular Calcium Concentration

[Ca](i) was measured in individual, fura-2/AM-loaded RBL-2H3 cells using fluorescence ratio imaging microscopy as described. (^2)Briefly, cells were seeded into a Leiden coverslip dish/CO(2) microincubator (Medical Systems Corp., Greenvale, NY) mounted on a Zeiss IM35 inverted fluorescence microscope equipped with a 100-watt mercury arc lamp and computer-controlled filter wheels and shutters (CRG Precision Electronics, Houston, TX). The microscope was interfaced to a Photometrics (Tucson, AZ) Series 200 CCD array camera (29) that acquired fluorescence images during experiments at approximately 2-s intervals. A Microvax II computer controlled both filter wheel movement and image acquisition.

Untreated or IgE-primed cells were loaded with 2 µM fura-2/AM (Molecular Probes) for 30 min at room temperature. Cells were primed with 2 µg/ml biotinyl-anti-Tac IgG for the last 10 min of loading with fura-2 and activated by exchanging the room temperature medium with fresh medium containing DNP-BSA or avidin at 35 °C. Cells (3-10 per field in each experiment) were observed using 360 and 385 nm excitation filters and a 510 nm emission filter. At the end of each experiment, average ratio values were calculated for a user-defined area within each cell in background-subtracted, ratioed images. The time resolution of ratioed images was 2-5 s. Average ratio values were converted to [Ca](i) as described in Grynkiewicz et al.(30) using a calculated K(d) for fura-2/Ca of 155 nM.

Immune Complex Kinase Assays

Suspensions of primed, transfected RBL-2H3 cells (6 times 10^6 cells/ml; 0.5-ml aliquots) were activated with DNP-BSA or avidin, lysed as described above, and immune complexes were generated by incubating supernatants with rabbit polyclonal antibodies directed against phosphotyrosine (anti-PY; described above), against a peptide corresponding to the C-terminal 28 amino acids of porcine Syk reported in Taniguchi et al.(31) (anti-Syk; provided by Dr. R. Geahlen, Purdue University), and against Lyn (Santa Cruz Biotechnology, Santa Cruz, CA); the antibodies were preabsorbed to protein A-Sepharose beads (Pharmacia, Uppsala, Sweden) as described(37) . Kinase activity was measured by adding 40 µl of kinase buffer (25 mM Hepes, 10 mM MnCl(2), pH 7.5) containing 10-25 µCi of [-P]ATP (Amersham, 3000 Ci/mmol) to the washed immune complexes and transfer to a 30 °C heat block for 1-3 min. Precipitates were washed three times with 1 ml of ice-cold kinase buffer, followed by the addition of 40 µl of Laemmli sample buffer containing 5% 2-mercaptoethanol and protein separation by SDS-PAGE. P-Proteins were visualized by autoradiography or PhosphorImager analysis of the dried gels.


RESULTS

Expression of the Chimeric Receptors

Transfected cells were selected for study based on their high binding of FITC-anti-Tac IgG to TT and TTbeta, respectively (illustrated for TT cells in Fig. 1, A-C) and their strong secretory response (approximately 50% [^3H]serotonin release in 20 min) to cross-linking the native FcR1 (Fig. 2, A and C, column 2). Fluorescent bead standards were used to convert mean fluorescence intensity of FITC-anti-Tac-labeled cells measured by flow cytometry to molecules of FITC-anti-Tac bound per cell. Assuming that each anti-Tac antibody occupies two chimeric receptors, it was calculated that the transfectants express approximately 400,000 TT and 200,000 TTbeta molecules/cell. This is comparable with FcR1 expression levels in RBL-2H3 cells (200,000-300,000/cell; (32) ).


Figure 1: The distribution of chimeric and native receptors on transfected RBL-2H3 cells. Monolayers of transfected cells on glass coverslips were incubated with anti-DNP-IgE plus FITC-conjugated or biotinylated anti-Tac IgG. In A and B, TT receptors were labeled with FITC-anti-Tac IgG for 10 min, respectively, at room temperature (A) or 37 °C (B). In C and D, TT receptors were labeled with anti-Tac IgG for 10 min at room temperature, then cross-linked by 10-min incubation at 37 °C with LRSC-conjugated goat anti-mouse IgG (C); the cells were fixed and the IgE-primed FcR1 labeled with rabbit anti-IgE IgG and FITC-anti-rabbit IgG (D). Cells in A-D were observed by fluorescence microscopy. In E and F, TT (E) and TTbeta (F) cells were incubated for 10 min at room temperature with biotinyl-anti-Tac IgG, cross-linked for 10 min at 37 °C with avidin-conjugated 15-nm colloidal gold particles, then fixed and processed for transmission electron microscopy. Arrowheads point to gold particles in coated pits; arrows indicate gold particles in uncoated intracytoplasmic vesicles. In A-D, magnification: times 800. In E and F, bar = 10 µm.




Figure 2: Secretory responses induced by receptor cross-linking. [^3H]Serotonin-loaded transfected cells were primed with anti-DNP-IgE and biotinyl-anti-Tac IgG and activated for 20 min (A, C) or for the times indicated (B, D) with cross-linking agents. Cross-linker-induced [^3H]serotonin release was measured as described under ``Experimental Procedures.'' Results in B and D are corrected for spontaneous [^3H]serotonin release. In A and C, columns 1-3, TT and TTbeta transfectants, respectively, were incubated for 20 min with no addition, DNP-BSA, and avidin. In addition, TT cells were incubated with anti-mouse IgG (A, column 4), and TTbeta cells were incubated with avidin plus DNP-BSA following a 5-min preincubation with avidin alone (C, column 4). In B and D, TT and TTbeta cells were incubated with DNP-BSA (B and D, solid circles), avidin (B, solid triangles) or with DNP-BSA plus avidin following a 5-min preincubation in avidin alone (D, open triangles). Results are representative of three separate experiments, each performed in duplicate.



Chimeric Receptors Are Expressed Independently of the FcR1

To establish that the transfected receptors are expressed independently of the native FcR1, one receptor type was cross-linked, and the distributions of both species were examined by fluorescence microscopy. Noncross-linked native receptors are distributed uniformly over the whole cell surface (illustrated in Refs. 22 and 24). The results in Fig. 1show that chimeric TT receptors also maintain a uniform distribution when incubated with FITC-anti-Tac antibody for 10 min at 4 °C (not illustrated) or at room temperature (Fig. 1A). A small amount of the FITC-anti-Tac-receptor complex is internalized when incubation is for 10 min at 37 °C (visible as small intracytoplasmic vesicles in Fig. 1B). Identical results were obtained with TTbeta cells (not illustrated). To avoid this loss of receptor during priming, all functional studies reported here were performed using transfected cells that were primed by 10-min incubation at room temperature with anti-Tac antibody.

The addition of LRSC-conjugated goat anti-mouse IgG to cross-link anti-Tac-receptor complexes on TT cells causes the rapid clustering and internalization of chimeric receptors (Fig. 1C). Fig. 1D shows that the noncross-linked FcR1 retains a uniform cell surface distribution during the avidin-induced cross-linking, clustering, and internalization of transfected receptors. Again, experiments with TTbeta transfectants (not illustrated) yielded identical results.

Previously, we used colloidal gold labeling techniques to show that clustering of cross-linked FcR1 is followed by their internalization through coated pits (illustrated in (23) and (24) ). The results in Fig. 1, E and F, show that gold-labeled cross-linked TT and TTbeta are also internalized through coated pits and are later located in uncoated intracytoplasmic vesicles.

Signaling Activities of the Transfected Receptors

In RBL-2H3 cells, FcR1 cross-linking with multivalent antigen elicits functional responses including secretion, ruffling, spreading, and actin plaque assembly(25, 33) . These responses are associated with biochemical changes, including protein-tyrosine phosphorylation, 1,4,5-IP(3) generation, Ca stores release, and Ca influx(34, 35) . The signaling activities of the transfected TT and TTbeta receptors were characterized by comparing the responses of biotinyl-anti-Tac-primed chimeric receptors cross-linked with avidin with the responses of IgE-primed native FcR1 cross-linked with DNP-BSA.

Secretion

The secretory responses of RBL-2H3 cells to cross-linking the chimeric and native receptors are illustrated in Fig. 2. Optimum conditions for priming and cross-linking chimeric receptors were first established using TT transfectants, reported in previous studies to mediate secretion in response to cross-linking agents(12, 17) . [^3H]Serotonin-loaded cells were primed with biotinyl-anti-Tac antibody (0.5-4 µg/ml) for 10 min at room temperature, washed with Hanks'-BSA medium, and activated with avidin (10-50 µg/ml) for 20 min at 37 °C. Maximum [^3H]serotonin release occurred in cells that were primed with 2 µg/ml biotinylated anti-Tac antibody and activated with 25 µg/ml avidin (Fig. 2A, column 3). This maximum response was typically 40-60% of that observed when the endogenous receptor was primed with 1 µg/ml anti-DNP IgE and activated with 0.1 µg/ml DNP-BSA (Fig. 2A, column 2), previously shown to be optimal conditions for FcR1-mediated secretion(22) . A somewhat lower maximum secretory response was observed when anti-Tac primed TT receptors were cross-linked with anti-mouse IgG (Fig. 2A, column 4). The reduced secretory response of RBL-2H3 cells to TT in comparison with FcR1 cross-linking was maintained over the entire time course of secretion (Fig. 2B). Piceatannol, a Syk-selective tyrosine kinase inhibitor(36, 37) , abolished secretion mediated through both the TT and native receptors (not illustrated).

Cells expressing TTbeta showed a small but consistent increase in the rate of basal or spontaneous secretion in comparison with TT-transfected cells (compare Fig. 2A, column 1, with Fig. 2C, column 1). Although this may simply reflect variability in basal activity between different subpopulations of RBL-2H3 cells, it is striking that the TTbeta cells selected by Jouvin et al.(17) showed the same property.

TTbeta cross-linking agents caused a decrease in basal secretion in TTbeta cells (compare Fig. 2C, columns 1 and 3). TTbeta cross-linking also resulted in a 30-50% reduction in FcR1-mediated secretion measured over 20 min in TTbeta-transfected cells (compare Fig. 2C, columns 2 and 4). The reduction in FcR1-mediated secretion due to TTbeta cross-linking is apparent throughout the time course of FcR1-induced secretion (Fig. 2D). Control experiments established that the binding of biotinyl-anti-Tac IgG to TTbeta receptors, without subsequent TTbeta cross-linking, does not alter either basal or FcR1-mediated secretion. The addition of avidin to TTbeta transfectants without prior priming with biotinyl-anti-Tac IgG also has no effect on basal or FcR1-mediated secretion. These results provided the first indication that chimeric receptors can specifically affect the signaling activity of the native receptor.

Membrane and Cytoskeletal Responses

Fig. 3and Fig. 4illustrate the ruffling, spreading and F-actin redistribution responses of activated RBL-2H3 cells. Resting cells show a characteristically rounded or moderately spread cell body with a microvillous cell surface (Fig. 3A) and a fairly uniform submembranous distribution of F-actin (Fig. 4A). Cross-linking the native FcR1 on either TT or TTbeta transfectants causes a dramatic cell spreading response accompanied by a transformation of the cell surface to a lamellar architecture (Fig. 3B) and the redistribution of F-actin into plaques at the ventral (adherent) cell surface (Fig. 4B). Like FcR1 cross-linking, TT cross-linking activates cell spreading, ruffling, and actin plaque assembly (Fig. 3C and Fig. 4C). In contrast, TTbeta cross-linking causes no obvious membrane or cytoskeletal responses (Fig. 3D and Fig. 4D).


Figure 3: Cross-linker-induced ruffling and spreading in transfected RBL-2H3 cells. Transfected cells cultured on glass coverslips were primed with anti-DNP-IgE and biotinyl-anti-Tac IgG, rinsed in Hanks'-BSA medium, and incubated for 10 min at 37 °C in medium alone (A), in DNP-BSA (B), or in avidin (C, D). The cells were fixed with 2% glutaraldehyde and processed for scanning electron microscopy as described under ``Experimental Procedures.'' Cells in A-C are TT transfectants. Cells in D are TTbeta transfectants. Bar = 10 µm.




Figure 4: Cross-linker-induced actin plaque assembly in transfected cells. The experiment is the same as described in Fig. 3except that cells were fixed after activation in 2% paraformaldehyde, 0.02% saponin, and F-actin was fluorescence labeled using rhodamine-phalloidin. The fluorescence photomicrographs illustrate F-actin distribution at the ventral cell surface. Cells are TT transfectants incubated in medium alone (A), in DNP-BSA (B), or in avidin (C) and TTbeta transfectants incubated in avidin (D). Magnification: times 800.



Protein-Tyrosine Phosphorylation

In RBL-2H3 cells, FcR1 cross-linking activates at least two receptor-associated protein-tyrosine kinases, Lyn and Syk(38) , and causes the tyrosine phosphorylation of multiple substrates(34) . Here, flow cytometry of anti-PY labeled cells was used to define the effects of cross-linking the transfected receptors on the total levels of protein-tyrosine phosphorylation in RBL-2H3 cells (Fig. 5), and anti-PY immunoblotting was used to determine if the same or different proteins are phosphorylated in response to cross-linking the different receptors (Fig. 6).


Figure 5: Cross-linker-induced protein-tyrosine phosphorylation measured by flow cytometry. IgE and biotinyl-anti-Tac-primed TT (A) and TTbeta (B) cells were incubated in suspension with DNP-BSA and/or avidin for various times, then fixed and labeled with rabbit anti-PY antibody followed by FITC-anti-rabbit IgG. The mean fluorescence intensity per cell was measured for 10,000 cells/sample by flow cytometry as described under ``Experimental Procedures.'' Data are corrected for mean fluorescence intensity in parallel unstimulated samples and are representative of three similar experiments. Activation was with DNP-BSA (A and B, open circles), avidin (A and B, open triangles), anti-mouse IgG (A, closed triangles) and DNP-BSA plus avidin following 5-min preincubation with avidin alone (B, solid circles).




Figure 6: Cross-linker-induced protein-tyrosine phosphorylation in TT transfectants. IgE plus biotinyl-anti-Tac-primed TT transfectants were incubated at 37 °C for 5 min with no addition (lane 1) or for the times indicated with DNP-BSA (lanes 2-4) or avidin (lanes 4 and 5), then lysed, centrifuged, and 100 µg of supernatant protein separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and tyrosine phosphorylated species were detected by incubation with rabbit anti-PY followed by S-protein A as described under ``Experimental Procedures.'' Radiolabeled proteins were detected by autoradiography. The migration positions of Syk, Lyn, and the FcR1 beta subunit are indicated; the beta subunit is further indicated by a small arrow.



Cross-linking IgE-primed FcR1 with 0.1 µg/ml DNP-BSA causes an increase in total anti-PY-reactive protein that reaches its highest levels by 2 min after cross-linking and declines by 10 min (Fig. 5, A and B). The addition of avidin or anti-mouse IgG to unprimed TT or TTbeta transfected cells caused no change in anti-PY reactivity (not illustrated). However, cross-linking biotinyl-anti-Tac-primed TT receptors with avidin or anti-mouse IgG increases total tyrosine phosphorylation (Fig. 5A). Like TT-induced secretion, the TT-induced protein-tyrosine phosphorylation response is typically slower and smaller than the response to FcR1 cross-linking.

In contrast with TT transfectants, there is no detectable increase in anti-PY-reactive proteins when avidin is added to biotinyl-anti-Tac-primed TTbeta transfected cells (Fig. 5B). Furthermore, the addition of TTbeta cross-linking agents 5 min before DNP-BSA substantially reduces the FcR1-mediated protein-tyrosine phosphorylation response (Fig. 5B).

The results of anti-PY immunoblotting showed that a very similar array of proteins is tyrosine phosphorylated in response to FcR1 and TT cross-linking. In particular, a major tyrosine-phosphorylated band at 72 kDa was absent from resting cell lysates (lane 1) but present under both cross-linking conditions (lanes 2-6). This protein was previously identified as the protein-tyrosine kinase, Syk(37, 38) . One protein, the FcR1 beta subunit, is tyrosine-phosphorylated only in response to FcR1 and not TT cross-linking. Consistent with previous results(17) , we were unable to detect a new anti-PY-reactive band corresponding to TT itself in avidin-activated cells.

Anti-PY-reactive Lyn is observed by immunoblotting in resting as well as activated cells (Fig. 6, lanes 1-6). It has been established that the activation and inactivation of Src kinases are both mediated by tyrosine phosphorylation, although on distinct tyrosine residues (reviewed in (39) ). Thus the anti-PY reactivity observed here in resting cells may reflect Lyn phosphorylation on an inhibitory tyrosine that is recognized on immunoblots by our polyclonal anti-PY antibody.

As expected from the flow cytometry results (Fig. 5), avidin-induced TTbeta cross-linking caused no detectable protein-tyrosine phosphorylation in anti-PY immunoblotting experiments; DNP-BSA-induced FcR1 cross-linking in TTbeta cells yielded a pattern of protein tyrosine phosphorylation that was indistinguishable from DNP-BSA-activated TT cells; and the FcR1-induced protein-tyrosine phosphorylation response was reduced substantially when TTbeta was cross-linked 5 min before the addition of DNP-BSA (not illustrated). The reduction in FcR1-induced phosphorylation due to concomitant TTbeta cross-linking resulted from a proportional decrease in the phosphorylation of all protein species, not a targeted decrease in the phosphorylation of specific proteins.

1,4,5-IP(3) Synthesis

We showed previously (28) that 1,4,5-IP(3) production is initiated rapidly by FcR1 cross-linking and is maintained over at least 10 min of activation. Here, we determined that TT cross-linking elicits an 1,4,5-IP(3) synthesis response that begins more slowly but is comparable with the FcR1-induced response by 5 min of activation (data not shown). The tyrosine kinase inhibitor, piceatannol, eliminated 1,4,5-IP(3) synthesis in response to both FcR1 and TT cross-linking, implicating the tyrosine kinase-activated RBL-2H3 cell enzyme, PLC1(34, 40) , in 1,4,5-IP(3) synthesis initiated through both the native and transfected receptors.

No 1,4,5-IP(3) was synthesized in response to TTbeta cross-linking. Furthermore, TTbeta cross-linking reduced FcR1-induced 1,4,5-IP(3) synthesis by more than 50% (data not shown).

Ca Mobilization

FcR1 cross-linking increases cytoplasmic Ca levels ([Ca](i)) through the 1,4,5-IP(3)-dependent release of Ca stores and through the activation of Ca influx(41, 42) . The results in Fig. 7show representative Ca responses in TT (A-C) and TTbeta (D-F) transfectants. Table 1documents the lag time from the addition of stimulus to the onset of response for the same cells.


Figure 7: Cross-linker-induced Ca responses in transfected cells. TT (A-C) and TTbeta (D-F) transfected RBL-2H3 cells were loaded with fura-2 and the [Ca] of individual cells was monitored by ratio imaging microscopy as described under ``Experimental Procedures.'' In each panel, [Ca]versus time is plotted for three cells representing typical responses for the indicated transfectant and experimental conditions. Table 1gives the total numbers of cells examined under each condition. Extracellular Ca was present throughout the experiments illustrated in A, B, D, E, F, and as indicated in C. Treatments were: A, IgE-primed TT transfected cells activated with 0.1 µg/ml DNP-BSA; B and C, biotinyl-anti-Tac-primed TT transfectants activated with 25 µg/ml avidin; D, IgE-primed TTbeta transfected cells stimulated with 0.1 µg/ml DNP-BSA; E, biotinyl-anti-Tac-primed TTbeta transfectants incubated with 25 µg/ml avidin; F, IgE- and biotinyl-anti-Tac-primed TTbeta transfectants treated with avidin 5 min before activation with 0.1 µg/ml DNP-BSA.





Fura-2-labeled TT transfectants incubated with 0.1 µg/ml DNP-BSA in the presence of extracellular Ca responded after a short lag (Table 1) with an abrupt rise in [Ca](i) followed by a plateau that decreased slowly over time in most cells (Fig. 7A). Thirty-two of 33 TT transfectants incubated with 25 µg/ml avidin in the presence of extracellular Ca also showed Ca responses. However, the average lag time before the onset of avidin-activated Ca responses was almost four times longer than than the lag time before DNP-BSA-induced responses (Table 1). In addition, the Ca responses to avidin were quite heterogeneous (Fig. 7B). Although in some cells TT cross-linking led to Ca responses that were indistinguishable from optimal antigen, in others the response was smaller and in some cases consisted of Ca oscillations superimposed on a small base line increase in [Ca](i). This pattern of increased lag times and smaller responses is typical of cells stimulated with suboptimal concentrations of antigen (around 1 ng/ml DNP-BSA).^2

The two phases of the Ca response, Ca stores release and Ca influx, can be observed separately by activating cells sequentially in the absence and presence of extracellular Ca. When activation was in Ca-free medium, the lag time from the addition of avidin to the onset of responses was again longer and more variable than the lag time for DNP-BSA-treated cells (Table 1). The traces in Fig. 7C show that both components of the Ca response were activated by TT cross- linking. One avidin-treated cell showed a single Ca spike due to Ca stores release during incubation in nominally Ca-free medium followed by a sustained elevation of [Ca](i) when complete medium was added. This resembles a typical response to optimal amounts of DNP-BSA.^2 Two other cells showed smaller responses to TT cross-linking characterized by two or three Ca oscillations when stimulation was in nominally Ca-free medium and a lower plateau when complete medium was added (Fig. 7C). This pattern is again typical of cells activated with suboptimal concentrations of antigen (approximately 1 ng/ml DNP-BSA).^2

The lag times (Table 1) and Ca responses of TTbeta transfectants to DNP-BSA (Fig. 7D) were not significantly different from those of TT transfectants (Fig. 7B) and nontransfected cells.^2 In 17 of 18 cells, TTbeta cross-linking caused no Ca responses at all (Fig. 7E). One cell showed a single Ca spike resembling the spontaneous oscillations that occur occasionally in unstimulated cells. TTbeta cross-linking 5 min prior to the addition of DNP-BSA increased the average lag time for the onset of the FcR1-induced Ca response from 35 to 51 s (Table 1), a difference that is significant at p < 0.05 using Student's t test. However, the magnitude of the Ca response in DNP-BSA and avidin cross-linked TTbeta cells was not reduced compared with TTbeta cells activated with DNP-BSA alone (Fig. 7F).

Kinase Activation through the Transfected Receptors

The effects of cross-linking native and chimeric receptors on the in vitro tyrosine phosphorylating activities of anti-Syk and anti-PY immune complexes are illustrated in Fig. 8, A and B.


Figure 8: Immune complex kinase assays. IgE plus anti-Tac-primed RBL-2H3 transfectants were activated, lysed, and their supernatants immunoprecipitated with anti-Syk (A) or anti-PY (B) antibodies. Kinase activities in the immunoprecipitates were measured as described under ``Experimental Procedures.'' Sources of immune complexes were: lane 1, unstimulated TT cells; lane 2, DNP-BSA-treated (1 min) TT cells; lane 3, avidin-treated (5 min) TT cells; lane 4, unstimulated TTbeta cells; lane 5, DNP-BSA-treated (1 min) TTbeta cells; lane 6, avidin-treated (5 min) TTbeta cells; lane 7, avidin (5 min) pretreated TTbeta cells stimulated with DNP-BSA plus avidin (1 min). In A, a large arrowhead identifies Syk and small arrowheads point to a set of unknown phosphoproteins. In B, an arrowhead again identifies Syk and dashes point to the 53- and 56-kDa isoforms of Lyn. The identity of Lyn in anti-PY assays is verified by its co-electrophoresis with autophosphorylated Lyn from an anti-Lyn immunoprecipitate (B, lane 8). Results are representative of three replicate assays.



Anti-Syk Immune Complex Kinase Activity

Syk activity was determined from the level of Syk autophosphorylation in anti-Syk immune complex kinase assays; previous studies (37, 38) established that Syk activity toward exogenous substrates increases in parallel with its autophosphorylating activity. The results in Fig. 8A, lanes 1 and 4, show that no Syk autophosphorylation occurs in immunoprecipitates from resting TT or TTbeta cells. FcR1 cross-linking causes a rapid activation of Syk, demonstrated by its strong autophosphorylation in TT and TTbeta cells (Fig. 8A, lanes 2 and 5). Syk autophosphorylation is also increased in response to TT cross-linking (Fig. 8A, lane 3). Quantitative analyses of PhosphorImager data showed that Syk phosphorylation following 5-min TT cross-linking was 14.3% of Syk phosphorylation induced by 1 min FcR1 cross-linking.

TTbeta cross-linking causes no increase in Syk activity measured in in vitro kinase assays (Fig. 8A, lane 6). TTbeta cross-linking 5 min prior to the addition of DNP-BSA has very little effect on FcR1-induced Syk activation (Fig. 8A, lane 7).

Although anti-Syk immune complexes from resting cells or from cells activated through the TTbeta receptor do not support Syk phosphorylation, they do phosphorylate an unidentified high molecular weight species and a series of unknown lower molecular weight proteins (arrowheads in Fig. 8A, lanes 1, 4, and 6). In contrast, anti-Syk immune complexes from cells activated through the FcR1 and TT receptors support Syk phosphorylation but the unknown phosphoproteins are significantly less prominent (Fig. 8A, lanes 2, 3, 5, and 7). The simplest interpretation is that Syk associates in resting cells with an uncharacterized active kinase and its substrates. These proteins either dissociate from Syk or the kinase is inhibited in response to TT and FcR1 cross-linking.

Anti-PY Immune Complex Kinase Activity

Kinase assays performed with anti-Lyn immunoprecipitates show high autophosphorylating activity regardless of receptor cross-linking(37) . Fig. 8B, lane 8, illustrates this observation and establishes that RBL-2H3 cells contain both the 53- and 56-kDa splice variants of Lyn. Therefore Lyn activation was inferred here from the results of anti-PY immune complex kinase assays (Fig. 8B, lanes 1-7). These assays also provided an independent measure of Syk activity in resting and activated cells.

Anti-PY immunoprecipitates from resting TT and TTbeta cells do not phosphorylate Syk but support a modest phosphorylation of the 53-kDa isoform of Lyn (Fig. 8B, lanes 1 and 4). FcR1 cross-linking causes a large increase in the phosphorylation of Syk and of the 53- and 56-kDa isoforms of Lyn in both TT and TTbeta cells (Fig. 8B, lanes 2 and 5). In addition, a series of co-precipitated kinase substrates are phosphorylated in anti-PY immune complexes prepared from DNP-BSA-treated cells.

The results in Fig. 8B, lane 3, confirm that TT cross-linking increases the Syk phosphorylating activity of anti-PY immune complexes. PhosphorImager analysis of this experiment showed that Syk phosphorylation following 5 min of TT cross-linking was 21% of Syk phosphorylation measured after 1 min of FcR1 cross-linking. TT cross-linking also increases the tyrosine phosphorylation of Lyn in anti-PY immune complex kinase assays (Fig. 8C, lane 3). In this case, PhosphorImager analysis showed that phosphorylation of the 53- and 56-kDa Lyn isoforms following 5 min of TT cross-linking was, respectively, 46 and 145% (average 117% calculated from the sum of pixels in both bands) of Lyn phosphorylation induced by 1 min of FcR1 cross-linking. An unidentified higher molecular weight protein species also shows increased phosphorylation in anti-PY immune complexes from avidin-activated TT cells.

Remarkably, TTbeta cross-linking causes a marked decrease in the basal phosphorylation of Syk, Lyn, and all other proteins in anti-PY in vitro kinase assays (Fig. 8B, lane 7). Furthermore, TTbeta cross-linking reduces the FcR1-stimulated phosphorylating activity of Lyn and Syk in anti-PY immune complexes by approximately 30% (illustrated in Fig. 8B, lane 6, and quantified by analyses of PhosphorImager data).


DISCUSSION

This work was initiated to explore the separate or synergistic functions of the individual subunits of the multichain FcR1. RBL-2H3 cells that express the native FcR1 at high density were transfected with chimeric receptors consisting of the extracellular and cytoplasmic domains of the Tac antigen joined to the cytoplasmic tails of the FcR1 beta and subunits. Because these cytoplasmic sequences both contain TAMs, the chimeric receptors were predicted to respond to cross-linking agents by tyrosine kinase activation and some or all of the signaling responses that normally follow FcR1 cross-linking. Indeed, Letourneur and Klausner (12) and Jouvin et al.(17) had previously demonstrated cross-linker-induced protein-tyrosine phosphorylation, secretion, and Ca mobilization responses to TT cross-linking in transfected RBL-2H3 cells. We determined that the transfected and native receptors are expressed at similar high densities on the cell surface and that they redistribute independently after cross-linking into clusters that are internalized through coated pits. These results established that there is no intermixing of chimeric and native receptor subunits in the transfectants used for these studies.

We found that TT cross-linking activates all the biochemical and functional responses that are associated with FcR1 cross-linking, including protein-tyrosine phosphorylation, 1,4,5-IP(3) synthesis, secretion, ruffling, spreading and actin plaque assembly. These responses to TT cross-linking show a slower onset than FcR1-induced responses and their final magnitude is only around 50% of the maximum responses to FcR1 cross-linking. TT cross-linking also stimulates Ca stores release and Ca influx responses resembling the responses of nontransfected cells to suboptimal concentrations of DNP-BSA.^2 Only FcR1 and not TT cross-linking causes FcR1 beta subunit tyrosine phosphorylation. With this exception, the major tyrosine-phosphorylated proteins detected by anti-PY immunoblotting are the same when cells are activated by TT and FcR1 cross-linking. Of course, it remains possible that minor substrates for protein-tyrosine phosphorylation differ when cells are activated through these different receptors. Indeed, preliminary experiments using the more sensitive method of [P]orthophosphate labeling and anti-phosphotyrosine immunoprecipitation indicate a simpler pattern of protein tyrosine phosphorylation in response to TT as compared with FcR1 cross-linking (work in progress). Surprisingly, no cross-linker-induced phosphorylation of TT protein itself was detected in this or a previous study(17) . These results confirm and substantially extend the analysis of TT receptor signaling activities begun by Letourneur and Klausner (12) and Jouvin et al.(17) . They contradict the report (18) that TT cross-linking does not activate transfected RBL-2H3 cells.

The TT-activated signaling pathway was explored further by in vitro immune complex kinase assays. Syk activation by TT cross-linking was demonstrated with both anti-Syk and anti-PY immune complexes. The extent of TT-induced Syk activation was around one fifth of the Syk activation response to FcR1 cross-linking in the same cells. Although cross-linking alone can activate Syk(16) , studies in T cells indicate that phosphotyrosine residues in the TAMs of cross-linked receptors enhance the activation of Syk family members by providing docking sites for their tandem SH2 domains(3) . Thus it is possible that the smaller activation of Syk by TT cross-linking than by FcR1 cross-linking is due in part to very low levels of avidin-induced TT receptor phosphorylation (below the limits of detection in our assays) in comparison with the strong DNP-BSA-induced phosphorylation of TAMs in the FcR1 beta and subunits demonstrated here and previously(34, 37, 43) . Complementary pharmacological studies showed that the Syk-selective tyrosine kinase inhibitor, piceatannol, abolishes signaling responses to both FcR1 (37) and TT cross-linking (this study), indicating that Syk activation is a critical event linking both the native and transfected receptors to downstream responses. An uncharacterized active kinase and several substrates co-precipitated with Syk from resting but not stimulated cells, raising the possibility that the inactive form of Syk exists in a complex with other proteins. Further study of these co-precipitated proteins may reveal mechanisms involved not only in receptor-mediated Syk activation but also in the modulation of basal Syk activity that occurs through the cell cycle(44) .

The extent of Lyn activation measured in anti-PY immune complex kinase assays was fairly similar between avidin-activated and DNP-BSA-activated TT cells. These results suggest that cross-linked TT is a more effective activator of Lyn than of Syk. Previously, Eiseman and Bolen (18) reported that TT cross-linking does not activate Lyn. However, the transfected cells used by these investigators had unusually small responses to FcR1 cross-linking, suggesting a general impairment of their signaling capacity. Substantial Lyn activation by TT cross-linking was also unexpected based on evidence (17) that Lyn protein associates specifically with TTbeta and does not co-precipitate with TT. To reconcile our results with Jouvin et al.(17) , we propose that Lyn may require only a transient or indirect interaction with cross-linked for activation.

If full, even though submaximal, signaling activity resides in the FcR1 subunit, what is the function of the FcR1 beta subunit? The FcR1 beta subunit C-terminal cytoplasmic domain sequence incorporates a TAM and Lyn has been observed previously to associate with the TTbeta construct(17) . In addition, it has been shown that deletion or mutation of the FcR1 beta subunit C-terminal cytoplasmic domain drastically reduces cross-linker-induced receptor phosphorylation and signaling responses(17, 23, 45) . Other recent studies have described specific mutations in the FcR1 beta subunit transmembrane domain that appear to be linked to atopy in humans (reviewed in (9) ). All of these reports point to an important role for the beta subunit in FcR1-mediated signaling. On the other hand, it has also been shown that a human FcR1 composed of just an alpha(2) complex is capable of signaling (45) and that a beta-less form of the FcR1 is commonly expressed on Langerhans cells (46) . Furthermore, our data indicate that cross-linking chimeric receptors containing the cytoplasmic domain of the isolated subunit can activate both Lyn and Syk and elicit all the signaling responses normally induced by FcR1 cross-linking. None of these results is consistent with an essential signaling function for the beta subunit.

We approached FcR1 beta subunit function through detailed studies of the signaling activities of TTbeta transfectants. There is no Lyn activation in response to TTbeta cross-linking, establishing that any Lyn associated with the isolated beta subunit (17) is catalytically inactive. TTbeta transfectants also show no Syk activation, protein-tyrosine phosphorylation, or biochemical or functional responses to cross-linking agents. The absence of TTbeta-induced Syk activation, secretion, and protein-tyrosine phosphorylation was observed previously(17) . Based on spectrofluorimetric analyses in fura-2-labeled cell suspensions, Jouvin et al.(17) reported a small Ca mobilization response to TTbeta cross-linking. However, spectrofluorimetric studies are prone to mixing artifacts and problems due to dye leakage. Our single cell analyses clearly show that TTbeta cross-linking does not induce either Ca stores release or Ca influx responses. These results demonstrate that the presence of a TAM in the sequence of a transmembrane protein is not per se sufficient to couple receptor cross-linking to cell activation. Kim et al.(47) made a similar observation for the B cell mIg receptor, where both the Ig-alpha and Ig-beta chains of the receptor contain TAMs but Ig-alpha triggers tyrosine kinase activation much more efficiently that Ig-beta.

Further study indicated that TTbeta cross-linking not only fails to stimulate signaling responses, but actually reduces some of these responses to below basal levels. This was indicated by the reduction in basal secretion when TTbeta cross-linking agents were added to TTbeta-transfected cells (Fig. 2C) and by the marked reductions in both Lyn and Syk basal kinase activities in anti-PY immune complexes isolated after TTbeta cross-linking (Fig. 8C). Cross-linking TTbeta also has a negative effect on FcR1-mediated signaling, demonstrated by the substantial inhibition of FcR1-activated anti-PY immune complex kinase activity, protein-tyrosine phosphorylation, 1,4,5-IP(3) synthesis and secretion, and by the delayed onset of Ca responses, in cells co-stimulated with TTbeta cross-linkers.

The mechanism by which TTbeta cross-linking impairs basal and FcR1-mediated signal transduction is not known with certainty. One possibility is that TTbeta cross-linking activates a protein-tyrosine phosphatase that antagonizes basal and FcR1-activated protein-tyrosine phosphorylation, thus suppressing signaling responses. The loss of anti-PY-reactive proteins due to phosphatase activation would provide a direct explanation for the ability of TTbeta cross-linking agents to reduce the basal and FcR1-enhanced phosphorylation of Syk and Lyn in anti-PY immune complex kinase assays. However, RBL-2H3 cells have a high constitutive level of protein-tyrosine phosphatase activity as demonstrated by the ability of protein-tyrosine phosphatase inhibitors to mimic FcR1-induced cell activation (25, 28, 48) and by the rapid reversal of FcR1-mediated protein-tyrosine phosphorylation as soon as cross-linking is arrested (43) . This high background activity reduces the likelihood that a specific beta subunit-activated phosphatase controls signaling activity.

Alternatively, it is possible that the cross-linked TTbeta receptor inhibits signaling by binding and sequestering a kinase or a coupling protein that is required by the signal-transducing subunit. Since TTbeta cross-linking alone reduces signaling responses to below basal levels, this hypothesis implies that even basal secretory and other responses are controlled by receptor-dependent pathways that operate at a low level in the absence of receptor cross-linking. There is presently no evidence in RBL-2H3 cells for a non-kinase intermediate that couples the cross-linked FcR1 to kinase signaling by providing a docking platform for 5H2-domain proteins. Nevertheless, there is precedent for this hypothesis in the insulin receptor system where an accessory protein, IRS1, couples insulin binding to the amplification of a tyrosine kinase signaling cascade (reviewed in (49) ). Kinase sequestration is also possible. In particular, the association of Lyn with the isolated FcR1 beta subunit in TTbeta transfectants (17) may form a complex that has no catalytic activity of its own and reduces the amount of Lyn that can be activated by FcR1 cross-linking.

The hypothesis that the beta subunit binds a key signaling element suggests a means by which this subunit, that lacks independent signaling activity, may nevertheless modulate the signaling activity of the intact FcR1. We propose that the putative TTbeta-associated kinase or coupling protein associates with the beta subunit of the intact FcR1 as well as with the isolated beta subunit. Whereas binding of this protein to the isolated beta subunit sequesters it from the signal-transducing subunit and prevents cell activation, we hypothesize that binding the same protein to the beta subunit of the intact FcR1 may serve instead to present it to the adjacent subunit, enhancing kinase activation and signal transduction. A simple extension of this hypothesis can additionally explain the full, but submaximal, signaling activity of the TT receptor. We propose that the same kinase or coupling protein is activated by association with cross-linked TT, but is not topographically restricted in this case by binding to beta. The result is a less efficient signaling complex and slower and smaller signaling responses.


FOOTNOTES

*
This work was supported in part by Grant IM-68438 from the American Cancer Society and by National Institutes of Health Grant GM49814. 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 a postdoctoral fellowship from the University of New Mexico Cancer Research and Treatment Center.

To whom correspondence should be addressed: Cell Pathology Laboratory, Surge Bldg., University of New Mexico School of Medicine, Albuquerque, NM 87131. Tel.: 505-277-4364; Fax: 505-277-2999.

(^1)
The abbreviations used are: FcR1, the high affinity receptor for IgE; anti-PY, anti-phosphotyrosine antibody; DNP, dinitrophenol; BSA, bovine serum albumin; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; 1,4,5-IP(3), inositol 1,4,5-trisphosphate; LRSC, lissamine rhodamine sulfonyl chloride; PAGE, polyacrylamide gel electrophoresis; RBL-2H3, the 2H3 secreting subline of rat basophilic leukemia (RBL) cells; TAM, tyrosine activation motif; TCR, T cell receptor; MEM, minimal essential medium; PBS, phosphate-buffered saline.

(^2)
R. J. Lee and J. M. Oliver, submitted for publication.


ACKNOWLEDGEMENTS

This paper is dedicated to Dr. Grace G. Deanin, on the occasion of her retirement. Her passion for science, her commitment to students, and her generosity to colleagues are a legacy to cherish and perpetuate. We thank Dr. Richard Klausner, National Institutes of Health, for kindly providing access to cDNA constructs generated in his laboratory and Dr. Robert Geahlen, Purdue University, for his generous gifts of anti-Syk antibody and piceatannol. Larry Seamer from the University of New Mexico Cancer Research and Treatment Center's Cytometry Resource contributed importantly to the flow cytometric analyses of protein-tyrosine phosphorylation.


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