©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cooperativity and Segregation of Function within the Ig-/ Heterodimer of the B Cell Antigen Receptor Complex (*)

(Received for publication, October 23, 1995)

Phot Luisiri (1)(§) Young J. Lee (1)(§) Bartholomew J. Eisfelder (1) Marcus R. Clark (1) (2)(¶)

From the  (1)Departments of Medicine and (2)Pathology, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The B cell antigen receptor complex contains heterodimers of Ig-alpha and Ig-beta. The cytoplasmic tails of each of these chains contain two conserved tyrosines, phosphorylation of which initiates the signal transduction cascades activated by the receptor complex. Although the cytoplasmic domains of Ig-alpha and Ig-beta have been expressed individually and demonstrated to be competent signal transduction units, we postulated that within the context of a heterodimer, Ig-alpha and Ig-beta could have new, complementary or even synergistic functions. Therefore we developed a system to compare the signal transducing capacities of dimers of Ig-alpha/Ig-alpha, Ig-beta/Ig-beta, or Ig-alpha/Ig-beta. This was done by fusing the extracellular and transmembrane domains of either human platelet-derived growth factor receptor (PDGFR) alpha or beta to the cytoplasmic tail of either Ig-alpha or Ig-beta. Three cell lines expressing PDGFRbeta/Ig-alpha, PDGFRbeta/Ig-beta, or PDGFRalpha/Ig-beta together with PDGFRbeta/Ig-alpha were established in the murine B cell line A20 IIA1.6. While aggregation of each dimer by itself could induce the tyrosine phosphorylation of cellular substrates, only aggregation of the heterodimer induced the phosphorylation of substrates similar in range and intensity to that induced by the endogenous B cell antigen receptor complex. Interestingly, Ig-beta remarkably enhanced the rapidity (T(max) decreased from 5 to 1 min) and intensity (greater than 10-fold enhancement) of Ig-alpha phosphorylation. Conversely, the phosphorylation of Ig-beta was reduced to undetectable levels when co-aggregated with Ig-alpha. The enhancement of Ig-alpha phosphorylation by Ig-beta correlated with a lowering of the stimulation threshold for tyrosine kinase activation.


INTRODUCTION

A B cell's response to antigen, whether it be proliferation, differentiation, anergy, or deletion, is dependent upon recognition of that antigen by the B cell antigen receptor (BCR)(^1)(1, 2, 3) . The receptor is a multimeric complex consisting of the antigen-recognition substructure, membrane-bound immunoglobulin non-covalently associated with heterodimer(s) of Ig-alpha and Ig-beta(4, 5, 6) . Present evidence indicates that the cytoplasmic tails of Ig-alpha and beta (7) translate antigen engagement into cytoplasmic signaling events that initiate cellular responses(8, 9, 10, 11, 12) . Most proximally in the signaling cascade, one or more tyrosine kinases, including Syk and members of the Src family, are activated(13, 14, 15) . These in turn activate a variety of pathways whose constituents include Ras, phosphatidylinositol 3-kinase, and phospholipase C(1) . Embedded within the cytoplasmic tails of both Ig-alpha and beta is a sequence common to other multichain immune recognition receptor (MIRR) subunits including CD3, CD3, TCR, FcRIII, and FcRI, termed the immunoreceptor tyrosine-based activation motif (ITAM)(16, 17) . The motif contains two tyrosines, both of which are critical for initiating tyrosine kinase activation (10, 18) . Phosphorylation of these tyrosines facilitates the recruitment and activation of tyrosine kinases which contain SH2 domains, such as Syk and Fyn(19, 20, 21, 22) . Substrates for these kinases may also be recruited(22) . The presence of the ITAM in all MIRR chains involved in signal transduction has led some to suggest that apparently heterologous chains such as CD3 and TCR are functionally redundant and the presence of multiple ITAMs within each MIRR serve to increase the strength of signal which can be generated via the receptor. Evidence for this assertion has been obtained in studies of both the B and T cell antigen receptors(8, 12, 23, 24) . In contrast, we and others have provided evidence indicating that each heterologous ITAM containing chain has a distinct function(10, 11, 19, 25, 26) .

Many of the above studies utilized chimeras in which irrelevant extracellular and transmembrane domains were fused to the single cytoplasmic domain under study(18, 27, 28) . Although this approach has yielded considerable insight into ITAM-containing chains, it assumes that functions observed in the isolated circumstance of a single chimera are reflective of the function of that cytoplasmic domain within the intact receptor complex. This might not be true since most ITAM-containing chains, such as Ig-alpha and Ig-beta, are expressed on cell surfaces as heterodimers(29, 30, 31, 32) . Therefore, we postulated that within the context of a heterodimer, Ig-alpha and Ig-beta would have new, complementary or even synergistic functions, not predicted from studies of single chain chimeras.

As demonstrated in this report, Ig-alpha and beta have new and unpredicted functions in the context of a heterodimer. Using a novel chimera system, which allowed us to form either hetero- or homodimers of the cytoplasmic domains of Ig-alpha and Ig-beta, we observed that when Ig-beta is ligated independently it is able to activate tyrosine kinases. However, when co-aggregated with Ig-alpha, Ig-beta appears to remarkably enhance Ig-alpha phosphorylation. This in turn correlates with an increase in the range and intensity of cellular substrates phosphorylated by the heterodimer and a lowering of the stimulation threshold for tyrosine kinase activation.


MATERIALS AND METHODS

Construction of PDGFR/Ig-alpha/beta Chimeras

The construction and expression of the chimeras has been described in detail elsewhere. (^2)Briefly, cDNAs encoding the PDGFRalpha and beta chains (gift of A. Kazlauskas, National Jewish Center, Denver, CO) were mutated to introduce BamHI and EcoRI sites immediately after that portion of each cDNA which encodes the transmembrane domain. The introduction of these sites facilitated the insertion of cDNA fragments encoding the cytoplasmic domains of Ig-alpha and Ig-beta(25) . These fragments were assembled with an EcoRI/XhoI-flanked cDNA fragment containing multiple stop codons in pSK (Stratagene, La Jolla, CA), which were then subcloned into the expression vector pCB6/muTk (gift of H. Singh, University of Chicago, Chicago, IL) which contains a neomycin resistance gene, an IgM enhancer and a thymidine kinase promoter. The cDNAs encoding the chimeric constructs PDGFRbeta/Ig-alpha and PDGFRbeta/Ig-beta were transfected either separately or together into A20 IIA1.6 (33) by electroporation. Clones were derived by selection with G-418 and stained with anti-PDGFRalpha and anti-PDGFRbeta antibodies (Genzyme, Cambridge, MA) then FITC-conjugated anti-IgG(1) (Zymed, San Francisco, CA). They were analyzed by flow cytometry (FACScan, Becton Dickinson, Bedford, MA).

Reagents

Polyclonal anti-Ig-alpha and anti-Ig-beta antibodies were made by immunizing rabbits (HTI Bioproducts, Ramona, CA) with glutathione S-transferase fusion proteins containing the cytoplasmic domains of murine Ig-alpha or Ig-beta(25) . The serum of rabbits immunized with the Ig-alpha fusion protein was purified over a column (CNBr-activated Sepharose, Pharmacia Biotech Inc.) coupled to a peptide corresponding to the murine Ig-alpha cytoplasmic tail ITAM (amino acid residues 177-196)(34) , whereas the polyclonal anti-Ig-beta antibody was purified over a column containing the immunizing Ig-beta fusion protein. The anti-phosphotyrosine monoclonal antibodies FB2 and Ab2 were obtained from ATCC (Rockville, MD) and Oncogene Sciences (Uniondale, NY), respectively.

Cell Growth and Stimulation

For most experiments, cells were grown in Iscove's modified Dulbecco's medium (Sigma) supplemented with 10% fetal calf serum (HyClone, Logan UT), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 7.5% CO(2). For the experiments described in Fig. 6, cells were serum-starved (0.5% fetal calf serum) for 18 h before initiation of each experiment. For all the stimulation experiments described, aliquots of 10 times 10^6 cells were suspended in 300 µl of Iscove's modified Dulbecco's medium and incubated at 37 °C for 5 min. To stimulate cells via the endogenous BCR, cells were incubated with a rabbit polyclonal anti-IgG antibody (Jackson Immunoresearch, West Grove, PA) at 15 µg/ml for the times indicated in each experiment. To stimulate transfectants through the chimera, cells were incubated with PDGF-BB ligand (100 ng/ml, except where noted) (Sigma) for 5 min, followed by anti-PDGFRbeta antibody (5 µg/ml, except where noted) (Genzyme) for 3 min, then anti-IgG(1) antibody (5 µg/ml) (Jackson Immunoresearch).


Figure 6: Ig-alpha phosphorylation was enhanced when co-ligated with Ig-beta. 10 times 10^6 cells/sample of alpha/Ig-beta//beta/Ig-alpha were stimulated through chimeras with and without PDGF-BB. Cells were then lysed in 1% Nonidet P-40 at different time points after stimulation. The cell lysates were immunoprecipitated with a combination of anti-Ig-alpha and anti-Ig-beta antibodies. The immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred, and probed with Ab2. The immunoblot was then stripped and reprobed with a combination of anti-Ig-alpha and anti-Ig-beta antibodies.



Immunoprecipitation and Immunoblotting

Aliquots of stimulated or unstimulated cells were lysed on ice with an equal volume of 2% Nonidet P-40 lysis buffer(19) . After lysis, insoluble material was removed by centrifugation. Supernatants were incubated with FB2 (3 µg), anti-Ig-alpha antibody (3 µg), and/or anti-Ig-beta antibody and then with protein A-Sepharose beads (Pharmacia). Washed immunoprecipitates were boiled in SDS sample buffer, resolved by 7.5% or 10% SDS-PAGE, transferred to nylon membrane (Immobilon-P, Millipore, Bedford, MA), and finally immunoblotted with anti-Ig-alpha (1 µg/ml), anti-Ig-beta (1 µg/ml), or Ab2 (1 µg/ml) antibodies in 3% bovine serum albumin in 10 mM Tris (pH 8.0), 150 mM NaCl, 0.1% Triton X-100 (TBST). After washing in TBST, blots were incubated in either horseradish-conjugated goat anti-mouse IgG or goat anti-rabbit IgG antibodies (Amersham Corp.), washed in TBST, and then visualized by chemiluminescence (ECL, Amersham).


RESULTS

Construction, Expression, and Stimulation of PDGFR Chimeras

To examine if Ig-alpha and Ig-beta may function together to initiate pathways of cellular activation, we designed a system using the human PDGFRs, which allowed us to form either homo- or heterodimers of Ig-alpha and beta. Two forms of PDGFR exist, alpha and beta, each of which is recognized by specific monoclonal antibodies. Furthermore, although distinct, each has an equal affinity for the naturally occurring ligand, PDGF-BB(35) . Therefore, each chain can be expressed independently, yet made to form homodimers or predominantly heterodimers on singly or doubly transfected cells, respectively, by the addition of PDGF-BB. These dimers, which are representative of the resting complex (PDGF-BB does not induce tyrosine kinase activation; data not shown), can then be activated by specific antibodies (Fig. 1A).


Figure 1: A, stimulation of PDGFR chimeras. Homodimers or predominantly heterodimers were formed on singly (left) or doubly transfected (middle) cells, respectively, by first treating with PDGF-BB. Complexes were then aggregated with anti-PDGFRbeta antibodies, followed by goat anti-mouse antibodies. Omission of PDGF-BB before stimulating doubly transfected cells led to the aggregation of PDGFRbeta/Ig-alpha alone (right panel). B, schematic representation of PDGFR chimeras. cDNAs encoding the PDGFRalpha and beta chains were mutated to introduce BamHI and EcoRI sites immediately after that portion of each cDNA encoding the transmembrane domain (Tm). The introduction of these sites facilitated the insertion of cDNA fragments encoding the cytoplasmic domains of Ig-alpha and Ig-beta. Assembled cDNAs were cloned into the expression vector pCB6+/muTk, which contains a neomycin resistance gene, an IgM enhancer and a thymidine kinase promoter. C, flow cytometric analysis of A20 IIA1.6 cells expressing PDGFR chimeras. Expression of either surface IgG, PDGFRalpha or PDGFRbeta on wild type, beta/Ig-alpha, beta/Ig-beta, and alpha/Ig-beta//beta/Ig-alpha cells. To stain for surface IgG, 2 times 10^5 cells/sample from each cell line were incubated with FITC-conjugated anti-IgG at 4 °C. For chimera staining, 2 times 10^5 cells/sample from each cell line were incubated with anti-PDGFRalpha or anti-PDGFRbeta antibodies and subsequently FITC-conjugated anti-IgG1 at 4 °C. Also shown is staining of each cell line without primary antibody. As demonstrated by immunoprecipitation and immunoblotting, cells with equal staining intensity with anti-PDGFRalpha and anti-PDGFRbeta antibodies expressed approximately equal amounts of each protein (Fig. 3).




Figure 3: Only Ig-alpha was phosphorylated upon stimulation through the chimeras in alpha/Ig-beta//beta/Ig-alpha. 10 times 10^6 cells/sample of the wild type (WT) or alpha/Ig-beta//beta/Ig-alpha cells were either left unstimulated or were stimulated through the chimeras as in Fig. 2. Lysates from these cells were immunoprecipitated with FB2, anti-Ig-alpha, or anti-Ig-beta antibodies. Immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to nylon membrane, and then probed with Ab2, anti-Ig-alpha, or anti-Ig-beta antibodies.




Figure 2: The pattern of total protein phosphorylation in wild type (WT), beta/Ig-alpha, beta/Ig-beta, and alpha/Ig-beta//beta/Ig-alpha cells upon stimulation through endogenous receptor or through the chimeras. 10 times 10^6 cells/sample from each cell line were left unstimulated (us) or were stimulated through the endogenous receptor (Ig) or through the chimeras (Ch). Cells were then lysed in 1% Nonidet P-40 lysis buffer and cell lysates were immunoprecipitated with anti-phosphotyrosine antibodies (FB2). Immunoprecipitates were resolved by 10% SDS-PAGE, transferred to nylon membrane, and then probed with the anti-phosphotyrosine antibody Ab2.



We engineered cDNAs encoding for molecules in which either the cytoplasmic domains of Ig-alpha or Ig-beta were fused to the extracellular and transmembrane domains of either PDGFR alpha or beta (Fig. 1B). These cDNAs were expressed singly or in combination in A20 IIA1.6, a B cell lymphoma that lacks FcRII, to yield three cell lines expressing approximately equal levels of each chimera (beta/Ig-alpha (expressing PDGFRbeta/Ig-alpha), beta/Ig-beta and alpha/Ig-beta//beta/Ig-alpha) (Fig. 1C and Fig. 3).

The Cytoplasmic Domains of Both Ig-alpha and Ig-beta Are Needed to Induce the Efficient Tyrosine Phosphorylation of Cellular Proteins

We first asked if the cytoplasmic tails of Ig-alpha or Ig-beta alone, or the two chains together, were capable of inducing the tyrosine phosphorylation of cellular proteins in a manner similar to that induced via the endogenous BCR. Chimeras were stimulated by sequential incubation with PDGF-BB, followed by anti-PDGFRbeta antibody and rabbit anti-mouse IgG1. In parallel samples, the endogenous BCR on each transfectant was stimulated with polyclonal antibodies to IgG. After stimulation, cells were lysed and phosphotyrosine immunoprecipitates (with FB2) from each lysate were resolved by SDS-PAGE and analyzed by blotting with anti-phosphotyrosine antibodies (Ab2). As shown in Fig. 2, stimulation of the BCR on wild type and transfected cells induced a similar spectrum and intensity of tyrosine phosphorylation. In contrast, only in alpha/Ig-beta//beta/Ig-alpha cells did stimulation of chimeras induce tyrosine phosphorylation that was similar in distribution and intensity to that induced by the endogenous antigen receptor. In beta/Ig-alpha or beta/Ig-beta cells, stimulation of the chimeras could only induce the strong tyrosine phosphorylation of a subset of proteins. In related experiments, truncated co-expressed versions of each chimera which lacked cytoplasmic domains, PDGFRalpha/- and PDGFRbeta/-, were incapable of inducing any detectable tyrosine phosphorylation.^2 However, differences were observed in the induction via the chimeras in alpha/Ig-beta//beta/Ig-alpha cells and by the endogenous BCR. Stimulation of alpha/Ig-beta//beta/Ig-alpha failed to induce the tyrosine phosphorylation of proteins of 32 and 40 kDa. Subsequent immunoblotting revealed that the 32-kDa protein was Ig-alpha (data not shown). The 40-kDa protein appears to be a novel molecule which is associated with the endogenous Ig-alpha/beta heterodimer. (^3)These observations suggest that the chimeras do not utilize the BCR, or associated structures, to initiate tyrosine phosphorylation. Finally, at least one protein of 120-130 kDa was phosphorylated strongly in alpha/Ig-beta//beta/Ig-alpha, weakly in beta/Ig-alpha and beta/Ig-beta, and not at all by stimulation of the BCR. Since the chimeras are predicted to have molecular masses of approximately this size, we examined if this protein was a chimeric molecule.

Stimulation of Chimeric Heterodimers Induces the Tyrosine Phosphorylation of PDGFRbeta/Ig-alpha but Not PDGFRalpha/Ig-beta

The wild type A20 IIA1.6 and alpha/Ig-beta//beta/Ig-alpha cells were treated with PDGF-BB and then stimulated with anti-receptor antibodies as above. Anti-phosphotyrosine, anti-Ig-alpha, or anti-Ig-beta immunoprecipitates were resolved by SDS-PAGE and probed with antibodies of the same specificity in various combinations. Immunoblotting of the Ig-alpha and Ig-beta immunoprecipitates with the same antibodies confirmed that alpha/Ig-beta//beta/Ig-alpha cells but not wild type cells expressed PDGFRbeta/Ig-alpha (135 kDa) and PDGFRalpha/Ig-beta (125 kDa) (Fig. 3). Although both chimeras were expressed in readily detectable amounts, only PDGFRbeta/Ig-alpha was observed to be phosphorylated following stimulation. The phosphorylation of PDGFRbeta/Ig-alpha was detected in both Ig-alpha and Ig-beta immunoprecipitations, the latter presumably being a result of co-ligation during stimulation. The PDGFRbeta/Ig-alpha chimera co-migrated with a prominent tyrosine phosphoprotein precipitated from the lysates of stimulated cells. No tyrosine phosphoprotein corresponding to the PDGFRalpha/Ig-beta chimera was observed, even on overexposed gels (data not shown). From these results, it is readily apparent that PDGFRbeta/Ig-alpha, but not PDGFRalpha/Ig-beta, was inducibly phosphorylated in alpha/Ig-beta//beta/Ig-alpha cells. However, it is not clear if the differences in phosphorylation observed are due to differences intrinsic to each chain or due to interactions between the chains. To differentiate between these possibilities, we compared PDGFRbeta/Ig-alpha and PDGFR/Ig-beta phosphorylation in singly and doubly transfected cells.

The Phosphorylation of Ig-beta Was Extinguished in the Presence of Ig-alpha

We first examined the influence of Ig-alpha on Ig-beta phosphorylation by comparing PDGFR/Ig-beta phosphorylation in beta/Ig-beta and alpha/Ig-beta//beta/Ig-alpha. The chimeras were precipitated from the lysates of unstimulated cells (us) or cells stimulated with PDGF-BB ligand and anti-PDGFRbeta antibody followed by rabbit anti-mouse IgG(1) for 1, 2, or 5 min. A representative experiment is shown in Fig. 4(n = 4). In the beta/Ig-beta cell line, stimulation of the chimera induced its own phosphorylation, which was maximal at 2 min and transient (Fig. 4, upper panel). In contrast, phosphorylation of PDGFRalpha/Ig-beta was not detected in alpha/Ig-beta//beta/Ig-alpha cells, even though more PDGFRalpha/Ig-beta protein was immunoprecipitated (Fig. 4, lower panel). These results suggest that when expressed in isolation, Ig-beta can be phosphorylated. However, when ligated in the presence of Ig-alpha, its own phosphorylation is inhibited. These observations are in accordance with the minimal inducible tyrosine phosphorylation of Ig-beta observed following BCR ligation in A20 (Fig. 5C).


Figure 4: Ig-beta phosphorylation was extinguished to undetectable levels in alpha/Ig-beta//beta/Ig-alpha. 10 times 10^6 cells/sample of beta/Ig-alpha, beta/Ig-beta and alpha/Ig-beta//beta/Ig-alpha were stimulated through the chimeras as before and then lysed at different time points after stimulation. Cell lysates were immunoprecipitated with a combination of anti-Ig-alpha and anti-Ig-beta antibodies. Immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to nylon membrane, and blotted with Ab2 (upper panel). The immunoblot was then stripped and reprobed with a combination of anti-Ig-alpha and anti-Ig-beta antibodies (lower panel).




Figure 5: A, Ig-beta enhanced Ig-alpha phosphorylation. 10 times 10^6 cells/sample of beta/Ig-alpha and alpha/Ig-beta//beta/Ig-alpha cells were stimulated through their respective chimeras and then lysed at different time points after stimulation. Cell lysates were immunoprecipitated with combination of anti-Ig-alpha and anti-Ig-beta antibodies. Immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to nylon membrane, and probed with Ab2. The immunoblot was then stripped and reprobed with combination of anti-Ig-alpha and anti-Ig-beta antibodies. B, quantitation of the enhancement of Ig-alpha phosphorylation by Ig-beta. Immunoreactivities from the immunoblot in A were quantitated densiometrically. The specific tyrosine phosphorylation of Ig-alpha was calculated as: (immunoreactivity of sample to Ab2/immunoreactivity of sample to anti-Ig-alpha) times 100. This value was plotted as a function of time. C, the induction of Ig-alpha and Ig-beta tyrosine phosphorylation following BCR stimulation in wild type A20 IIA1.6. 10 times 10^6 cells/sample of wild type cells were stimulated with rabbit anti-mouse antibodies. Cells were then lysed, and the lysates were immunoprecipitated with FB2. The immunoprecipitates were resolved by 10% SDS-PAGE, transferred to nylon membrane, and probed with Ab2. The position of Ig-alpha and Ig-beta, which was determined by probing parallel samples with antibodies to these molecules (data not shown) is indicated.



Ig-beta Enhances the Phosphorylation of Ig-alpha

In Fig. 3and 4, it is apparent that only PDGFRbeta/Ig-alpha is tyrosine-phosphorylated following chimera stimulation and its phosphorylation is strong only when co-ligated with PDGFR/Ig-beta. To examine this further, we compared the inductive tyrosine phosphorylation of PDGFRbeta/Ig-alpha when expressed alone (beta/Ig-alpha) or with PDGFRalpha/Ig-beta (alpha/Ig-beta//beta/Ig-alpha). The cell lines beta/Ig-alpha and alpha/Ig-beta//beta/Ig-alpha were stimulated via the chimeras, lysed, and then immunoprecipitated with a combination of anti-Ig-alpha and anti-Ig-beta antibodies. Shown in Fig. 5are the results of a representative experiment (n = 3). Immunoprecipitates were resolved by SDS-PAGE, and after transfer to membrane, probed first with Ab2 (Fig. 5A, upper panel), then stripped and reprobed with a combination of anti-Ig-alpha and Ig-beta antibodies (lower panel). There were remarkable differences in both the degree and kinetics of PDGFRbeta/Ig-alpha phosphorylation with and without PDGFR/Ig-beta co-ligation. In the absence of PDGFR/Ig-beta, the tyrosine phosphorylation of PDGFRbeta/Ig-alpha was weak at 1 min, increased to a maximum at 5 min, and was essentially absent at 30 min. In contrast, in the presence of PDGFR/Ig-beta, PDGFRbeta/Ig-alpha phosphorylation was maximal at 1 min and thereafter decreased, being almost undetectable at 30 min. To analyze this data further, we densitometrically quantitated the immunoreactivity of each sample with Ab2 and anti-Ig-alpha antibodies and then plotted the ratio of these values as a function of time. As seen in Fig. 5B, co-ligation of PDGFR/Ig-beta enhanced the phosphorylation of PDGFRbeta/Ig-alpha approximately 12-fold at 1 min. This intensity and rapidity of tyrosine phosphorylation is similar to what is observed for endogenous Ig-alpha following ligation of the BCR (Fig. 5C).

It is possible that the observed differences in Ig-alpha phosphorylation with and without co-cross-linking Ig-beta were due to differences intrinsic to those cells in which both chains were expressed. To address this possibility, we examined if the degree of phosphorylation of PDGFRbeta/Ig-alpha was influenced by PDGFRalpha/Ig-beta co-cross-linking on the same cell line. Therefore, we studied alpha/Ig-beta//beta/Ig-alpha cells under two different stimulating conditions. First, following the stimulating protocol described above, which includes PDGF-BB, heterodimers were formed and then aggregated in alpha/Ig-beta//beta/Ig-alpha. In parallel, cells were stimulated without PDGF-BB, where only PDGFRbeta/Ig-alpha would have been aggregated. As predicted, the degree of PDGFRbeta/Ig-alpha phosphorylation was remarkably enhanced when heterodimers were first formed with PDGF-BB. The results from a representative experiment are shown in Fig. 6(n = 3).

Co-aggregation of PDGFRbeta/Ig-alpha and PDGFRalpha/Ig-beta Lowered the Threshold for Tyrosine Kinase Activation

Given the remarkable enhancement of Ig-alpha phosphorylation by Ig-beta, we postulated that the heterodimeric complex would be more efficient in its ability to activate tyrosine kinases than a homodimeric complex. Therefore, we compared the stimulation threshold for the induction of tyrosine phosphorylation in alpha/Ig-beta//beta/Ig-alpha and beta/Ig-alpha by titering out the primary stimulating antibody. Cells were otherwise stimulated and analyzed as in Fig. 2. As can be seen in Fig. 7, stimulating both transfectants with high concentrations of primary stimulating antibody induced the tyrosine phosphorylation of cellular proteins. However, the tyrosine phosphorylation of cellular proteins in beta/Ig-alpha diminished significantly after the first 4-fold dilution of anti-PDGFRbeta antibody. In contrast to alpha/Ig-beta//beta/Ig-alpha, the tyrosine phosphorylation was still detected after two dilutions of anti-PDGFRbeta antibody. Similar results were obtained when beta/Ig-beta was compared to alpha/Ig-beta//beta/Ig-alpha (data not shown). These results suggest that the heterodimeric structure of the BCR complex facilitates B cell responses to low doses of antigen.


Figure 7: The Ig-alpha/beta heterodimer had a lower threshold of stimulation than the Ig-alpha/alpha homodimer. 10 times 10^6 cells/sample of beta/Ig-alpha or alpha/Ig-beta//beta/Ig-alpha were stimulated through the chimeras as before except that the indicated decreasing concentrations of anti-PDGFRbeta antibody (serial 4-fold dilutions) were used. After stimulation cells were lysed in 1% Nonidet P-40 lysis buffer. Cell lysates were immunoprecipitated with FB2, and these were resolved by 10% SDS-PAGE, transferred, and probed with Ab2.




DISCUSSION

Herein we report that the ITAM-containing subunits within an immune recognition receptor complex can cooperate to efficiently initiate signal transduction cascades. Using a system that allowed us to compare the signal transduction capacities and physical properties of homo- and heterodimers of Ig-alpha and Ig-beta, we observed that the Ig-alpha/beta heterodimer induced the phosphorylation of a wider range of substrates at a lower threshold of stimulation than either homodimer. This synergy correlated with the ability of Ig-beta to enhance the tyrosine phosphorylation of Ig-alpha by more than 10-fold. Conversely, in the presence of Ig-alpha, Ig-beta phosphorylation was extinguished to undetectable levels. These data suggest that one of the major functions of Ig-beta is to enhance the phosphorylation and, therefore, the signal transducing capability of Ig-alpha. Furthermore, these data suggest that significant ``cross-talk'' or cross-modulation occurs between the subunits of the B cell antigen receptor.

Our results reveal a new level of complexity in the function of the BCR. Previous studies utilizing either fusion proteins, peptides or chimeras, have sought to characterize the functional capacities of individual cytoplasmic domains of the BCR complex(18, 19, 27, 28) . This reductionist approach assumes that each ITAM-containing domain is an isolated signaling unit whose capacities can simply be added to those of other domains to form an accurate picture of the whole receptor complex. Our data suggest that this assumption is not entirely valid. Rather, we would argue that while the study of each individual subunit reveals what it can do, it is only in the context of other receptor structures that one can elucidate what that subunit does do.

We have recently obtained data directly demonstrating that the coordinate activities of Ig-alpha and Ig-beta is of biological significance. We have established clones of WEHI 231, an immature B cell sensitive to apoptosis, expressing similar combinations of the chimeras described here. When the chimeras in these transfectants were stimulated, we observed that the induction of apoptosis required the cytoplasmic tails of both Ig-alpha and Ig-beta. In those experiments, as in the experiments described in this report, only the heterodimerized chimeras induced tyrosine kinase activation efficiently.^2

Phosphorylation of the ITAM tyrosines within the BCR cytoplasmic domains is a necessary and early event in the initiation of tyrosine kinase activation. Previously, we and others have demonstrated that members of the Src family of tyrosine kinases are constitutively associated with the resting receptor complex and that it is probably these kinases which mediate the phosphorylation of Ig-alpha and thereby initiate signaling by recruiting and activating SH2 domain containing secondary effectors(2, 13, 19, 36) . Which effectors are recruited by the receptor complex would be determined, in part, by which of the four tyrosine(s) (34, 37) in the cytoplasmic domain of Ig-alpha are phosphorylated upon receptor engagement(38) . From our data it is not clear if Ig-beta merely enhances the phosphorylation at previously modified tyrosines or directs the phosphorylation of new sites. This distinction is of potential significance because if Ig-beta directs the phosphorylation of Ig-alpha, the spectrum of substrates activated by the receptor complex would be altered.

One of the mechanisms by which Ig-beta could augment Ig-alpha phosphorylation would be to recruit novel kinases to the receptor complex. Previously, we found that phosphoproteins of 40 and 42 kDa bind in vitro to the cytoplasmic domain of Ig-beta via a phosphotyrosine-independent QTAT sequence embedded within the Ig-beta ITAM(19) . Recently, we have demonstrated that similar molecules are inducibly tyrosine-phosphorylated by BCR engagement and are associated with the native Ig-alpha/beta heterodimer.^3 Alternatively, it is possible that kinases are constitutively associated with Ig-beta because their SH2 domains bind a small subpopulation of phosphorylated Ig-beta tails. While we did not observe any phosphorylation of Ig-beta in our heterodimerized chimeras, there was a low level of Ig-beta phosphorylation in the native BCR, which minimally increased following receptor engagement (Fig. 5C). It is possible that such phosphorylation of PDGFRalpha/Ig-beta occurred at a level too low to detect.

Another possibility is that Ig-beta recruits SH2 domain-containing proteins, other than kinases, which bind to and protect Ig-alpha tyrosines from dephosphorylation. This is a plausible alternative since the tyrosine phosphatase CD45 is associated with the receptor complex and its function is necessary for BCR-mediated signal transduction.

Previously, models of antigen receptor-mediated signal transduction have assumed that each ITAM-containing chain within a receptor complex generates independent signals (Fig. 8, left). Some have postulated that the signals generated are redundant(12, 23, 24) , while others have demonstrated that some chains are functionally distinct and capable of preferentially coupling to selected secondary effectors(10, 11, 19, 25, 26) . However, our data support a model in which each chain within a heterodimer can cross-modulate the phosphorylative state and, by extension, the signal transducing capabilities of the other (Fig. 8, right). This leads to specialization of each chain's function within the multimeric whole. In the particular case of the BCR and tyrosine kinase activation, Ig-beta's function appears to be to facilitate the phosphorylation of Ig-alpha, which in turn allows the efficient activation of tyrosine kinases and possibly the recruitment of their substrates. The proof of this model will require an understanding of the mechanisms whereby Ig-beta enhances Ig-alpha phosphorylation.


Figure 8: Model of Ig-alpha/Ig-beta-mediated signal transduction. Previously, models of antigen receptor signal transduction have assumed that the cytoplasmic domains of each receptor chain was an independent signal transduction unit (left). In contrast, our data support a model in which cooperation between receptor components leads to the enhancement of signals initiated by particular chains (right).




FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM52736. 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.

§
These authors contributed equally to this work.

Investigator of the Arthritis Foundation. To whom correspondence should be addressed: Dept. of Medicine and Pathology, University of Chicago, 5841 S. Maryland, MC0930, Chicago, IL 60637. Tel.: 312-702-0202; Fax: 312-702-3467.

(^1)
The abbreviations used are: BCR, B cell antigen receptor; MIRR, multichain immune recognition receptor; ITAM, immunoreceptor tyrosine-based activation motif; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate.

(^2)
B. J. Eisfelder and M. R. Clark, submitted for publication.

(^3)
Y. J. Lee and M. R. Clark, manuscript in preparation.


ACKNOWLEDGEMENTS

We express our appreciation to Frank Fitch and Jean McGuire for their careful reading of this manuscript and helpful comments.


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