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The Journal of Immunology, 2006, 176: 6862-6872.
Copyright © 2006 by The American Association of Immunologists

Precursor B Cell Receptor Signaling Activity Can Be Uncoupled from Surface Expression1

F. Betul Guloglu* and Christopher A. J. Roman2,*,{dagger}

* School of Graduate Studies, Program in Molecular and Cellular Biology and {dagger} Department of Microbiology and Immunology and Morse Institute for Molecular Genetics, State University of New York–Downstate Medical Center at Brooklyn, NY


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Signals from the precursor BCR (preBCR) cause proliferation and differentiation of progenitor (pro-) B cells into pre-B cells. Given the very low amounts of surface preBCRs and the demonstrated cell autonomy of preBCR signaling, we examined the possible occurrence of preBCR signal propagation from intracellular membranes such as the endoplasmic reticulum (ER) and the trans-Golgi network (TGN) in transformed and primary pro-B cells. PreBCRs composed of normal Ig µ or truncated Dµ heavy chains (HCs) were redirected to intracellular sites via localization sequences appended to the HC cytoplasmic tail. PreBCR complexes retained in the TGN or shunted from the TGN to lysosomes were as or 50% as active as the corresponding wild-type preBCRs in directing preBCR-dependent events, including CD2 and CD22 expression and proliferation in primary pro-B cells. This occurred despite their low to undetectable surface expression in transformed cells, which otherwise allowed significant surface accumulation of wild-type preBCRs. In contrast, ER-retained preBCRs were inactive. These results suggest that preBCR signaling is remarkably tolerant of dramatic changes in its subcellular distribution within post-ER compartments and support the possibility that the preBCR can activate signaling pathways in the TGN as well as the plasma membrane.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In early B cell development, the functionality of an Ig H chain (HC)3 is tested by its ability to form a precursor BCR (preBCR) in association with an IgLC-like complex known as the surrogate L chain (SLC) and the Ig{alpha}beta signal transducers. The preBCR signals for survival, proliferation, and differentiation of progenitor (pro-) B cells into precursor B (pre-B) cells. Given the structural similarities of the SLC complex, composed of the invariant VpreB and {lambda}5 chains, with conventional, polymorphic Ig L chains (LCs), pairing with the SLC is usually predictive of the Ig HC’s ability to pair with an IgLC to form a mature BCR. In general, pro-B cells that make no HC or synthesize IgHCs that cannot form a preBCR with the SLC do not develop (1, 2). Rare exceptions are pro-B cells that produce HC clonotypes which do not require the SLC to form receptors and signal; in humans, such cells are deleted (3).

The preBCR is different from the BCR by virtue of the SLC complex. A consequence is that what would be the conventional Ag binding cleft is obstructed by non-Ig "unique regions" (URs) of VpreB and {lambda}5 (4, 5). As such, the mechanisms of how preBCR signaling is initiated and regulated in vivo are controversial. Interaction of the preBCR with galectin-1 (humans) and heparan sulfate (mice) present on the surface of stromal cells primarily via the UR of {lambda}5 (6, 7) support the model of ligand-dependent initiation of a preBCR signal in a manner analogous to Ag for the mature BCR. In contrast, biochemical evidence of cell-autonomous preBCR activity and the ability of preBCR+ pro/pre-B cells to proliferate and differentiate in vitro in the absence of stromal cells support the model of ligand-independent initiation of preBCR signaling (8, 9, 10). However, the biological importance of putative ligand interactions in early B cell development and immunity is not yet known. Furthermore, preBCR properties consistent with cell-autonomous signaling do not exclude the possibility of preBCR-specific ligands expressed by pre-B cells.

In these models, preBCR surface expression is a common and important aspect to the mechanism of activation of preBCR-dependent signal transduction and its regulation. For example, if extracellular preBCR ligands are necessary, then preBCRs must reach the plasma membrane to engage them. Alternatively, a model not contingent on ligand involvement is that engagement of critical signaling molecules can only occur at the plasma membrane (PM). Counterintuitively, at most, only 2% of preBCRs are on the cell surface, and the remainder is contained in the endoplasmic reticulum (ER) of freshly isolated primary pre-B cells (11). Normal HCs require association with SLCs to be transported from the ER; however, the SLC is a poor escort for surface transport of HCs compared with LCs, mainly due to the UR of {lambda}5 (8, 12). Conversely, the UR of {lambda}5 plays an important role in cell-autonomous signaling of the preBCR (8), which in turn causes internalization of preBCRs and presumably targets them to lysosomes. Thus, the low surface levels of the preBCR could be explained by the equilibrium between the slow export of newly synthesized preBCRs out of the ER and then rapid internalization and degradation after signaling.

Nevertheless, the relationship of cell surface preBCR expression to signaling activity remains correlative. Studies do suggest that preBCR complexes must leave the ER to become signaling competent. Biochemical evidence of preBCR activity was not detected in transformed pre-B cells expressing an IgHC that could not exit the ER because it could not associate with the SLC (11). Similarly, preTCR complexes retained in the ER via an ER retention sequence did not signal in vivo (13). However, whether the preBCR signal can be triggered by SLC-assembled preBCRs in the ER or from other intracellular compartments, such as the trans-Golgi network (TGN) or endosomes, is not known.

To address these issues, we have created a panel of preBCR complexes that were redirected within the secretory/endosomal system and tested their signaling activity in primary and transformed pro-B cells. Our results show that preBCR signaling activity can be relatively indifferent to dramatic changes in surface levels. They are consistent with the model that cell-autonomous signaling can be initiated from both intracellular, post-ER membranes and the cell surface.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Expression constructs

The creation of cDNAs encoding mouse µ HC 17.2.25, Dµ HC, human µHC TG.SA, and {lambda}1 has been described previously (12). The cDNAs were subcloned into MiG (14), a murine retroviral construct that contains the marker gene GFP linked to the cDNA of interest via an internal ribosome entry site. cDNAs encoding mouse µ and Dµ IgHCs with ER, TGN, or lysosome localization signals fused to the cytoplasmic tail (Table I) were created using standard overlap PCR mutagenesis and were verified by sequencing (Genewiz; oligonucleotides and strategies available upon request).


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  		Table I. Amino acid sequence of cytoplasmic domains of chimeric µHC clones with ER, TGN- and lysosomal localization signal sequences attached to the cytoplasmic region of WT µa

 
Cells and in vitro cell culture

The v-abl-transformed Rag1–/–{lambda}5–/– and Rag1–/– pro-B cell lines were described previously (15). Short-term primary IL-7-dependent pro-B cell cultures were established by harvesting and plating total bone marrow of 4- to 6-wk-old Rag1–/– (16) and Rag1–/–{lambda}5–/– mice (17) in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies), antibiotics (1% penicillin-streptomycin, L-Glutamine), 5 x 10–5 M 2-ME, and rIL-7 (100 U/ml = 5 ng/ml; Cell Science). Cells were seeded at a density of 0.5–2 x 106 cell/ml and maintained in culture for 2–3 days before retroviral infections (18, 19).

Retroviral infections

Retroviruses were produced by cotransfection of HEK293 cells (by calcium phosphate) with the retroviral plasmids plus p{psi}ECO-encoding ecotropic helper functions (20). V-abl-transformed and primary cells were spin-infected with recovered supernatants (14). Briefly, viral supernatants and polybrene (4–8 µg/ml) were added to 0.5–2 x 106 bone marrow cells per well in 12- or 24-well plates, followed by centrifugation at 2500 rpm at 25°C for 1.5 h. Supernatants were then replaced with fresh medium supplemented with 100 U/ml IL-7 after infection. For double infections, the spin infection was repeated twice with 1 day between infections. Double infections of primary IL-7-dependent pro-B cells were done by first infecting cells with the {lambda}1LC-MiG virus, then splitting the infected cells into separate wells for infection with HC viruses that did not contain a marker gene. Cells were analyzed 2–4 days after infection by flow cytometry and Western blot as described in the figures.

Analysis of surface marker expression and cell growth

The following Abs (directed against mouse Ags except where noted) were used to stain cells for flow cytometry by standard protocols: anti-CD19-TRI and anti-IgM-PE from Caltag Laboratories; anti-preBCR-biotin (SL156), anti-{lambda}5-biotin (LM34), anti-CD2-PE, anti-CD22-PE, and streptavidin-PE from BD Pharmingen; anti-human IgM-PE from Southern Biotechnology Associates. Analyses of CD2, CD22, and proliferation were performed as described elsewhere (21). Briefly, the values shown for CD2 and CD22 induction are the percentage of CD19+GFP+ cells that were also CD2+ and CD22+, 4 and 2 days after infection, respectively.

Calculation of relative growth. Relative growth was defined as the fold change in the percentage of CD19+GFP+ cells in cultures after 24 or 48 h divided by the fold change in the percentage of CD19+GFP– cells in the same culture over the same time period (see Figs. 4, A and B, and 6C). The fold change in CD19+GFP– cells in each sample was defined as 1 (no change in relative growth rate) in the bar graphs.


Figure 4
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  		FIGURE 4. Activity of normal and redirected preBCR complexes in primary pro-B cells. Bar graphs plot the relative activity of normal µ and Dµ preBCRs and their redirected counterpart preBCRs in Rag1–/–{lambda}5+ (A) and Rag1–/–{lambda}5–/– cells (B) with respect to relative growth (left), CD2 induction (middle), and CD22 induction (right). In all graphs, the bar showing the activity of the normal µ (µWT) preBCRs is black; and the dotted line represents the activity of the normal Dµ-preBCR. Cells were infected with the indicated control (–) or HC-containing retroviruses; the relative growth of infected (GFP+) and uninfected (GFP–) CD19+ cells in each culture was measured as in Materials and Methods (Rag–/–{lambda}5+ n = 14 for empty vector, Dµ, µWT, µER4, µTGN4; n > 3 for the rest, Rag–/–{lambda}5–/– n ≥3 for all HCs; SE bars are shown). The values shown in the middle and right graphs represent the percentage of CD19+GFP+ cells that were CD2+ or CD22+. A, Rag–/–{lambda}5+ cells, n = 17 (CD2) and n = 21 (CD22) for empty vector, Dµ, µWT, µER4, µTGN4; n > 3; B, Rag–/–{lambda}5–/– cells, n ≥ 3 (CD2 and CD22) for all HCs; SE bars are shown. C, Relative abilities of normal and redirected preBCRs to sustain growth under limiting IL-7 in 5 days. The numbers plotted are the relative growth of the CD19+GFP+ population at the indicated concentration of IL-7 divided by the relative growth of the CD19+GFP+ population in 100 U/ml IL-7 cultures.

 

Figure 6
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  		FIGURE 6. Properties of BCRs containing redirected HCs. A, Flow cytometry to detect surface expression of normal and redirected µ{lambda}1 BCRs expressed in Rag1–/–{lambda}5–/– primary cells. Cells were infected with an unmarked HC retrovirus and {lambda}1 linked to GFP and stained with an anti-{lambda}1 Ab. The relative fluorescence intensity of the mean and the percentage of cells considered positive for {lambda}1 surface expression are shown in the upper right of each dot plot. No staining was detected in {lambda}1-only-expressing cells (data not shown). Similar results were obtained with an anti-Igµ Ab (data not shown). B, Western blot for µ HC expression in infected cells from A. As in previous figures, the lower band is the immature species, and the upper band is the mature species. C, Induction of CD2 (left) and CD22 (right) by different BCR complexes (n = 4, SE is shown). Bar graphs were derived as in Fig. 4.

 
Comparison of the relative abilities of preBCRs to support growth over a concentration gradient of IL-7. Two days after infection, each sample was equally divided into six separate cultures, and each was subcultured in different concentrations of IL-7 in 10-fold dilutions from 100 U/ml (5 ng/ml) to 0.01 U/ml (0.5 pg/ml) (see Fig. 4C). After 5 days, the growth of CD19+GFP+ cells relative to the growth of CD19+GFP– cells in each culture was calculated as above. The numbers plotted in the line graph were calculated by dividing the relative fold change in CD19+GFP+ cells at the given IL-7 concentration to the relative fold change in CD19+GFP+ cells in the 100 U/ml IL-7 cultures.

Western blotting and immunoprecipitations

Western blots were performed as described previously (12). The following Abs were used for Western blot and immunoprecipitation: rabbit and goat anti-mouse IgM, µ HC-specific, hamster {gamma}-globulin from the Jackson ImmunoResearch Laboratories and anti-mouse CD79a and CD79b from BD Pharmingen. All AP- and HRP-conjugated secondary Abs were from the Jackson Immunoresearch Laboratories and Caltag Laboratories. Coimmunoprecipitation analysis of Ig{alpha}/Igbeta with Igµ was performed according to Stevens et al. (22) with minor changes. Briefly, cells were lysed in 1% digitonin lysis buffer at 4°C for 10 min, cell lysates were precleared at 4°C for 1 h, followed by incubation with protein G beads for 30 min. Supernatant was incubated with 3 µl of hamster anti-mouse Igbeta (or anti-Igµ) or hamster {gamma}-globulin (isotype control) overnight. Ab was immunoprecipitated with 20 µl of protein G beads for 1 h, washed three times with digitonin lysis buffer, and eluted by boiling in reducing 1x SDS sample buffer. Eluates were resolved on 8–10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Membranes were blotted with anti-Igbeta or anti-Igµ Abs. Protein deglycosylation with endoglycosidase H (Endo H) or PNGase F (New England Biolabs) was performed according to the manufacturer’s protocols.

Detection of tyrosine-phosphorylated proteins

Stimulation of v-abl-transformed cells was performed according to Mielinz et al. (11). Briefly, cells (5 x 106/ml) were incubated in complete serum-free RPMI 1640 medium for 2 h at 37°C. Cells were washed and stimulated with 350 µM H2O2 and 100 µM sodium orthovanadate (in 1xPBS) for 2–3 min. Stimulation was terminated with cold 1xPBS. Cells were lysed in digitonin lysis buffer (1% digitonin, water soluble; Sigma-Aldrich), 150 mM NaCl, 10 mM triethanolamine, 1 mM EDTA (pH 7.5), 1x protease inhibitor mixture (complete tablet; Roche) at 4°C for 15 min. Supernatant was incubated with 2 µl of anti-Igbeta (or anti-Igµ) Ab overnight. Abs were immunoprecipitated with 20 µl of protein G beads for 1 h, washed three times with digitonin lysis buffer, and eluted by boiling in reducing 1xSDS sample buffer. Eluates were resolved on 12% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Membranes were blotted with anti-phosphotyrosine (4G10-HRP; Upstate Biotechnology), anti-Igbeta, or anti-Igµ Abs.

Ab uptake experiment

Following Natarajan and Lindstadt (23), HC-expressing and control cells were incubated with RPE-conjugated anti-mouse IgM Ab at 37°C (experimental) or on ice (control starting amount). After 30 min, cells incubated at 37°C were treated with 5 volumes of ice-cold acid buffer (PBS and HCl, pH 2.0) for 45 s, then acidic buffer was neutralized by the addition of 0.2 volumes of 1 M HEPES (pH 7.6), followed by fixation with the addition of 0.5 volumes of 4% paraformaldehyde. The relative amount of Ab internalized at that time point relative to the starting amount (defined as 100%) was determined by flow cytometry.

Drug treatment

To block degradative pathways, v-abl-transformed and primary cells were treated with 40 mM NH4Cl, 0.1 mg/ml leupeptin (Roche), or vehicle only (DMSO) for 5–6 h at 37°C. To block endocytosis of surface receptors, primary and v-abl-transformed cells were incubated with 10–25 µM cytochalasin D (Sigma-Aldrich) for 1–6 h.

Confocal imaging

WT and "redirected" µHCs were visualized in HC-infected primary and v-abl-transformed cells via staining with anti-IgM-Cy5 and one of the following compartmental markers: Lysotracker, to probe late endosomes/lysosomes; wheat gluten (WGA), to detect Golgi, early endosomes, and plasma membrane; transferrin, to stain early endosomes; and anti-GRP78, to detect the ER. For Lysotracker and transferrin staining, cells were incubated in fresh medium with 5 µM Lysotracker Red DND-99 (Molecular Probes and Invitrogen Life Technologies) for 15 min or with 10–20 µg/ml rhodamine-conjugated mouse transferrin (Rockland) for 15–30 min at 37°C according to the manufacturer’s instructions. Cells were washed twice with 1xPBS and fixed in 2–4% paraformaldehyde for 15 min at room temperature. Following washing with 1xPBS, cells were incubated first in permeabilization buffer (0.1% saponin (Sigma-Aldrich), 0.1% sodium azide, and 5% FBS in 1xPBS) for 15 min and an additional 15–45 min in permeabilization buffer with 10–75 µg/ml Cy5-conjugated anti-mouse IgM (The Jackson Laboratory) at room temperature. For ER and Golgi stains, 1–10 µg/ml tetramethylrhodamine isothiocyanate-conjugated WGA (Molecular Probes) or 0.2 µg/ml rabbit anti-GRP78 (1/1000, H-129; Santa Cruz Biotechnology) was added along with anti-mouse IgM at this step. Cells labeled with anti-GRP78 were visualized using Rhodamine Red-X-conjugated F(ab')2 anti-rabbit IgG (The Jackson Laboratory). Following washing several times with 1xPBS, cells were resuspended in mounting medium (Prolong Gold antifade reagent; Molecular Probes) and 20 µl was placed on slides (VWR Superfrost Plus). Images were analyzed with a confocal imaging system (Bio-Rad MRC-1024 krypton/argon laser and Olympus IX70 inverted microscope).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Creation and localization of redirected preBCR complexes

Fusion of known ER, TGN, and lysosomal targeting/retention sequences to the cytoplasmic tail of IgHCs was used as a strategy to redirect preBCR complexes to different subcellular membranes (Table I). Fusions were made to a representative normal SLC-dependent µ HC that forms functional preBCRs and supports B cell development in vivo and in vitro (21, 24), and Dµ (25), a truncated HC that makes signaling-impaired preBCR complexes that do not progress through the secretory pathway as efficiently as µ preBCRs (12, 26). The efficacy of intracellular retention of the redirected µ HCs and the corresponding preBCR complexes was first tested in Abelson lines, which, unlike primary pre-B cells, accumulate readily detectable amounts of surface preBCRs (Refs. 8 and 21 ; Figs. 1 and 2). As such, the efficacy of the retention/localization sequences was clearly evident in these cells.


Figure 1
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  		FIGURE 1. Localization of redirected preBCR complexes. A and B, Flow cytometry of the Rag1–/– Abelson cell line 1-2 infected with the indicated full-length (µ) and Dµ HC retroviruses to detect surface expression of the preBCR epitope SL156 (A) or Igµ HCs (B). The staining profile of the control MiG vector was indistinguishable from DµLamp and is not shown. C, Western blot of extracts from infected Ableson-transformed cells as in A and B for HC expression. The upper bands are the mature, Golgi-modified species that is Endo Hf resistant (see text; data not shown); the lower bands are the immature, ER species. Note that in µER4 (lane 5) and DµER1 and DµLamp (lanes 11 and 14), only the immature species is observed. D, Representative confocal images of individual Rag1–/– Abelson-transformed cells infected with the indicated HC retrovirus and stained with either WGA (top row; PM and TGN) or calnexin (bottom row; ER) both in red, plus an anti-IgHC µ Ab in green. Areas of overlap appear yellow/orange.

 

Figure 2
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  		FIGURE 2. Incubation of Abelson-transformed and primary Rag1–/– cells with lysosomal or trafficking inhibitors promotes the accumulation of mature HCs without changing surface expression. A, Western blot of Abl-transformed (top row) and primary (bottom row) Rag1–/– cells infected with the indicated HC left untreated (control) or treated with either NH4CL/leupeptin or cytochalasin D. B, Surface expression of preBCR complexes as visualized with the Igµ Ab from untreated (control) and treated cells in A. Staining with the SL156 anti-preBCR mAb showed the same result (data not shown).

 
µ HCs appended with different ER-retention sequences showed variable degrees of ER localization and retention. PreBCR complexes with µER1, µER2, and µER3 HCs still reached the cell surface, although at lower levels than preBCRs with wild-type µ (µWT; Fig. 1, A and B, and data not shown; Table II). In contrast, little, if any, µER4 was detected on the cell surface. Western blot analysis showed that µER4 was expressed as well as µWT, but no mature (TGN-modified, Endo-Hf-resistant) species was detected (Fig. 1C, lane 5). Confocal microscopy revealed that little to no µER4 colocalized with WGA, which stains the PM and Golgi, whereas discreet areas of overlap were evident with µWT (Fig. 1D, cf. WGA second and third panels from the left). Conversely, coincident staining of µER4 with calnexin and GRP78 (BiP) showed extensive overlap and confirmed ER localization (Fig. 1D, lower panels, and data not shown). Mutation of KK to AA in the µER4 retention sequence (µER4mut) allowed WT levels of the resultant preBCR surface expression and mature HC species (Fig. 1, A and B), suggesting that preBCR assembly was not perturbed by the additional cytoplasmic sequences. Thus, only ER4 efficiently retained µ-preBCR complexes in the ER.


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  		Table II. Comparison of metabolism and signaling competency of WT and redirected preBCRs with ER, TGN, and lysosomal targeting signalsa

 
Among the four TGN localization sequences tested, only TGN4 enforced low levels of µ and preBCR surface expression (Fig. 1, A and B, and data not shown; Table II). Western blot showed this was not due to reduced protein expression (Fig. 1C). µHCs/preBCRs with mutations of critical amino acid residues in the TGN4 localization sequence (µTGN4mut) exhibited WT levels of surface expression (Fig. 1, A–C). Although µTGN4 was expressed dramatically less on the surface than µWT and µTGN4mut, all three proteins showed similar amounts of mature Golgi-modified forms (Fig. 1C). Similarly, confocal imaging showed that µTGN4 was found nearly exclusively in WGA-positive, calnexin-negative intracellular compartments (Golgi), but not in the PM (Fig. 1D).

These retention sequences also retained Dµ complexes. However, Dµ preBCRs are ordinarily not transported out of the ER with the same efficiency as µ preBCRs (12, 27), and attachment of the ER1 localization signal was sufficient to abolish the already low surface expression of Dµ (Fig. 1, A and B). This corresponded to the lack of mature Dµ species by Western blot (Fig. 1C). The effects of the ER1 localization sequence were reversed by mutation (DµER1mut). Similarly, the TGN4 localization signal also abolished Dµ surface expression (Fig. 1, A and B, bottom row), but unlike ER1, the proportion of the mature, Golgi-modified form of DµTGN4 was the same as Dµ (Fig. 1C, lanes 10–14). Confocal imaging showed extensive overlap of intracellular DµTGN4 and WGA, supporting the idea that it is primarily localized in the Golgi (Fig. 1D).

µ and Dµ HCs were also linked to a lysosomal targeting motif of lysosome-associated membrane protein 1 (Lamp-1), an integral lysosomal membrane protein (28). Most of Lamp-1 was shown to be directly routed to lysosomes from the TGN rather than indirectly by endocytosis from the plasma membrane (29, 30, 31). IgM and preBCR staining of transformed cells infected with DµLamp or µLamp showed no detectable HC on the surface (Fig. 1, A and B). Unlike the TGN4 fusions, Western blot showed that there were very low levels of Golgi-modified mature forms of µLamp and DµLamp compared with the WT counterparts (Fig. 1C).

To test whether mature TGN forms of µLamp HCs were being made but rapidly shunted to lysosomes for degradation, transformed and primary Rag1–/–{lambda}5+ cells infected with WT or redirected µHCs were treated with a combination of ammonium chloride (NH4Cl) and leupeptin. NH4Cl affects trafficking of endosomal vesicles and increases the pH of acidic endosomes, and leupeptin inhibits lysosomal and proteosomal enzymes. Under these conditions, the mature forms of µWT, µTGN4, and µLamp HCs accumulated in both cell types (Fig. 2A, lanes 2, 4, 11, and 13). Cytochalasin D treatment, which inhibits actin filament formation and thus endosomal trafficking (32), also increased levels of mature µWT, µTGN4, and µLamp proteins, most dramatically in primary cells (Fig. 2A). However, none of these treatments led to any change in corresponding preBCR surface expression by either cell type (Fig. 2B). These treatments did not affect µER4, which remained immature in all cases. In contrast to µ, the mature forms of Dµ, DµTGN4, DµER1, or DµLamp did not accumulate with these treatments (data not shown), suggesting Dµ complexes are processed differently than µ within the endosomal system. Measurement of surface biotinylated preBCRs confirmed the flow cytometry results (data not shown). These data suggested that the µLamp proteins were primarily localized to lysosomes and confirmed that the redirection sequences behaved similarly in primary and Abelson-transformed cells.

An anti-Igµ Ab uptake assay was used to further characterize any surface expression of redirected preBCRs (23). In this assay, the relative total amount of a conjugated anti-Igµ Ab internalized over a given time period is measured. In the Abelson-transformed Rag1–/–{lambda}5+ cells, the amount of anti-Igµ Ab internalized by cells expressing µTGN4 preBCR complexes was only slightly higher than preBCR-negative cells and cells expressing µER4, Dµ, or DµTGN4-preBCRs, whereas cells expressing µWT and µTGN4mut internalized severalfold higher amounts of Ab (Fig. 3A). No internalized Ab was detected in Abelson cells expressing µLamp and DµLamp preBCRs. Also, no uptake was detected in primary cells with any HCs, consistent with the undetectable surface expression by flow cytometry (Fig. 3B). Both Ab uptake and internalization rate assays (8, 23) determined that µWT, µTGN4mut, and µER4mut behaved similarly, demonstrating these activities were not affected by the extra cytoplasmic sequences (Fig. 3 and data not shown). This result also suggested that low to no detectable surface expression of µTGN4 and DµTGN4 was probably not due to faster internalization of these HCs from the cell surface.


Figure 3
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  		FIGURE 3. Measurement of surface preBCR expression by uptake of anti-Igµ Abs. Control (–) or HC-infected Rag1–/– Abelson-transformed (A) or primary cells (B) were incubated with R-PE labeled anti-Ig µ Ab and the relative amount of this Ab internalized by cells during a 0.5- to 1-h period was measured as described in Materials and Methods. Note that in A, the µ-{lambda}1 sample was from Rag1–/–{lambda}5–/– cells infected with both the µ HC and a {lambda}1 LC virus; all other samples were infected with the HC only. The scale of the bars in B is ~2-fold greater than those depicted in A. n = 6 and n = 3 for transformed and primary cells, respectively, and SEs are shown.

 
Signaling competency of redirected preBCR complexes

The signaling competency of redirected preBCR complexes was tested in the Rag1–/– Abelson line and in primary IL-7-dependent pro-B cells from Rag1–/– mice. In these systems, preBCRs are composed of retrovirally expressed HCs that assemble with endogenous SLC and Ig{alpha}beta components (21). µWT-preBCR expression in primary cells results in a {lambda}5-dependent induction of CD2 and CD22 (Fig. 4, A (Rag1–/–{lambda}5+ cells) and B (Rag1–/–{lambda}5–/– cells), middle and right bar graphs), increased relative growth (Fig. 4, A and B, left graphs), and increased survival in low concentrations of IL-7 compared with preBCR-negative (i.e., uninfected and control-infected) pro-B cells (Fig. 4C and Ref. 21). In Abelson lines incubated with phosphatase inhibitors, relative levels of total phosphotyrosine increase in cells expressing functional µWT preBCRs compared with preBCR-negative cells (Fig. 5A and Ref. 8).


Figure 5
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  		FIGURE 5. PreBCR-dependent induction of tyrosine phosphorylation and assembly of redirected preBCR complexes. A, Western blot to detect tyrosine-phosphorylated proteins in Rag1–/– v-abl-transformed cells that were infected either with the indicated control (–) or µ HC retroviruses. Cells were serum starved, incubated with pervanadate/H2O2 to inhibit intrinsic phosphatase activity, extracts prepared, and extracted proteins subjected to immunoprecipitation with an anti-Igbeta mAb (Materials and Methods). Immunoprecipitates were resolved on SDS-PAGE, blotted to nitrocellulose, and then probed with either the 4G10 mAb, which detects tyrosine-phosphorylated proteins (top panel) or with an anti-Igbeta Ab (middle panel). Total extracts from the same cells were also probed directly by Western blot with an anti-µ HC Ab to confirm that equivalent levels of the corresponding µ HC were expressed in each cell population used for the assay. B, Normal and redirected µ HCs associate with Igbeta. Extracts from v-abl-transformed Rag1–/– cells infected with the indicated HC retrovirus were subjected to immunoprecipitation with an anti-Igbeta or control (C) Ab and then material precipitated were resolved by SDS-PAGE and probed by Western blot for µ HCs.

 
In these assays, µER4 preBCRs were either inactive or profoundly impaired, promoting little, if any, proliferation, induction of CD2 or CD22 surface expression (Fig. 4A), or survival (Fig. 4C) in primary Rag1–/–{lambda}5+ cells. Total phosphotyrosine levels in µER4-expressing Abelson cells were much lower than those in µWT, but were still above control preBCR-negative cells (Fig. 5A). The lower levels were not due to impaired preBCR assembly, because µER4 could be coimmunoprecipitated with Igbeta as well as, if not better than, the other HC alleles (Fig. 5B) and the SL156 preBCR epitope, which requires {lambda}5 and µHC, was detected intracellularly in µER4-expressing cells (data not shown). Interestingly, despite lower overall tyrosine phosphorylation of higher m.w. proteins compared with µWT, phosphorylation of 32- to 40-kDa polypeptides that corresponded to Igbeta/Ig{alpha} dramatically increased in µER4-expressing cells. This suggests that preBCR-dependent ITAM phosphorylation can occur in the ER with functional HCs that can associate with {lambda}5. However, this did not correlate with productive signaling events in primary cells. Consistent with their inability to completely retain preBCR complexes in the ER, the signaling activity of µER3 was comparable to WTµ, and µER1 and µER2 were ~50% less active (Table II).

In contrast to ER4, preBCR retention mediated by the TGN4 sequence had little effect on preBCR signaling activity. µTGN4 and DµTGN4-preBCRs were nearly as active as µWT and Dµ-preBCRs, respectively, for proliferation and CD2/CD22 induction in primary cells (Fig. 4A; the relative signaling efficiency of other HCs is shown in Table II). µTGN4 was also nearly as active at conferring a growth advantage to pro-B cells at lower IL-7 concentrations as µWT (Fig. 4C). The nearly WT levels of preBCR activity was also evident in Rag1–/– Abelson lines expressing the µTGN4 preBCR, in which preBCR-dependent increases in total phosphotyrosine levels of high m.w. proteins was similar to that of µWT-expressing cells (Fig. 5A). The activity of µTGN4 was indistinguishable from µTGN4mut in primary cells (Fig. 4A), even though the latter was expressed on the surface of the Abelson line at WT levels (Fig. 1A). Similarly, µLamp-preBCRs were active, exhibiting an intermediate activity that was comparable to µER1 and Dµ (Fig. 4A), despite there being no detectable surface expression (Fig. 1A). Interestingly, the profile of µLamp-dependent phosphotyrosine exhibited both the prominent 32-kDa phosphorylation characteristic of µER4 but also the higher m.w. proteins common with µWT and µTGN4 (Fig. 5A).

However, signaling by Dµ preBCRs was intolerant to the other redirection sequences. In contrast to µ, DµER1 and DµLamp did not mediate any preBCR-dependent signaling (Fig. 4A and data not shown). This corresponded to the absence of mature forms of these Dµ HCs by Western blot (Fig. 1C, lanes 11 and 14) even in the presence of endosomal/lysosomal inhibitors (data not shown).

In general, any signaling mediated by the redirected HCs was, like the parental HCs, dependent on the SLC complex. Lamp and ER HCs did not show any activity in primary Rag1–/–{lambda}5–/– cells (Fig. 4B). A low level of activity, substantially lower than in {lambda}5+ cells, was detected in primary Rag1–/–{lambda}5–/– cells infected with µTGN4, µTGN4mut, and µER1mut HCs (Fig. 4B). This corresponded to a minor fraction of mature HC species detected by Western blot analysis of Rag1–/–{lambda}5–/– transformed cells that expressed these HCs (data not shown). However, no HCs could be detected on the surface of these cells by flow cytometry (data not shown). This suggested that the appended cytoplasmic sequences of these particular HCs may have actually promoted some low level of {lambda}5/SLC-independent HC export from the ER to the TGN, in turn causing a low level of signaling in {lambda}5’s absence. Thus, the low level of {lambda}5-independent signaling is consistent with SLC-independent enhanced ER export driven by the redirection sequences. For ER1, this was evident when the retention functions were inactivated (ER1mut).

Activity of redirected BCR complexes

Targeting sequences also affected the intracellular trafficking of BCR complexes. Previously, we showed that Rag1–/–{lambda}5–/– transformed and primary pro-B cells coexpressing HCs and the {lambda}1LC expressed high levels of surface receptors primarily because HCs can leave the ER more efficiently in association with {lambda}1 than with the SLC (12, 21). In the Rag1–/–{lambda}5–/– cells, µTGN4-{lambda}1 and µER4-{lambda}1 complexes could be detected on the surface, though in qualitatively different ways from each other and at much lower levels than µWT (Fig. 6A). In contrast, µLamp-{lambda}1 was virtually undetectable on the surface (Fig. 6A). Western blot showed that comparable amounts of each HC was expressed, but the highest amounts of mature Golgi-modified forms were from µWT and µTGN4 HCs; in contrast, µLamp had intermediate levels and those of µER4 were barely detectable (Fig. 6B). These results indicate that the ER4 and Lamp-1 sequences can most effectively prevent surface expression of the resultant HC-{lambda}1 receptor complexes. However, as suggested for the µLamp-preBCR, and unlike µER4-{lambda}1, the µLamp1-{lambda}1 complexes pass through the TGN without reaching the cell surface to an appreciable degree.

The signaling activities of the HC-{lambda}1 receptors, here measured by their relative abilities to induce CD2 and CD22, most directly correlated with relative levels of the mature HC species detected by Western blot, not relative surface expression levels (Fig. 6C). For example, µWT and µTGN4 exhibited comparable activity, even though the surface staining intensity of µTGN4-{lambda}1 complexes was ~10-fold less than those of µWT (Fig. 6 and data not shown). Similarly, µER4 was virtually inactive, and µLamp exhibited an intermediate activity despite surface staining intensity nearly 100-fold less than WT. Therefore, the relative tolerance of signaling activity with respect to subcellular redistribution is not unique to preBCR but also a property of the BCR.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Surface expression of preBCR complexes has been tightly linked to their signaling capability. IgHCs that cannot associate with the SLC, typically obligatory for receptor transport to the surface, do not contribute to B cell repertoire (33, 34). It is thought that preBCR expression in the plasma membrane may be critical to engage signal transduction pathways and extracellular ligands. To address the importance of surface expression and putative receptor-ligand interactions in signaling, we have created a panel of preBCRs with functional full-length HCs that were either not detectable on the surface (µER4, µLamp) or were present over 10-fold less (µER1, µTGN4) than WT on the surface of Abelson lines, which are permissive for WT preBCR surface accumulation. A combination of biochemical and immunofluorescent approaches showed that µER4 was retained in the ER, whereas µLamp and µTGN4 resided primarily in the Golgi and endosomes. That µER4 was inactive supports the model that preBCR signaling cannot be propagated in the ER, at least not to induce proliferation and differentiation, and with normal HCs that can assemble into preBCR complexes. Surprisingly, µTGN4 preBCRs were as active as µWT complexes. Similarly, µLamp preBCR retained ~50% WT activity, and DµTGN4 was as active as Dµ. Thus, in contrast to the existing paradigm, relative levels of preBCR signaling by these HCs were disproportionate to their relative levels of surface expression.

One interpretation of these data is that preBCR signaling is tolerant of dramatic decreases in steady-state surface expression. However, in vivo studies have suggested that a linear relationship exists between preBCR signal strength and levels of IgHC and SLC expression, which are indirectly reflected in surface expression levels. For example, transgenically expressed IgHCs were more effective at enforcing allelic exclusion when expressed in homozygosity, and in the absence of VpreB-1, efficiency of pre-B development was proportional to VpreB-2 gene and protein dose (35, 36, 37). Similarly, in our system we have observed that substantially lowering µWT expression resulted in proportionally lower signaling activity and surface levels (F. B. Guloglu, unpublished observations). In contrast, relative levels of surface expression of the redirected preBCRs did not always predict their preBCR signaling activity. Rather, a better correlation did exist between redirected preBCR signaling activity and the relative amounts of constituent mature, TGN-modified HCs. For µLamp, these mature forms were revealed after inhibition of the lysosomal system. Taken together, these data support the model that surface expression per se may not be critical for signaling, and that signaling may be propagated from intracellular, post-ER membranes such as the TGN.

Studies in different systems have demonstrated that receptors or signaling intermediates can signal from intracellular membranes (38, 39, 40, 41, 42, 43, 44, 45). Many of these processes are known to require PM localization for ligand interaction and possibly to engage key signal transducers (46, 47). For example, the intensity of TCR stimulation regulates not only Ras isoform utilization but also the subcellular compartment from which Ras signals are propagated (48, 49). This may impact positive and negative selection during development or an immune response. However, mutated and oncogenic forms of receptor tyrosine kinases are no longer ligand dependent and can be active intracellularly (50, 51). In this way, the preBCR may be analogous to these constitutively active receptors, but unlike them can negatively regulate its own activity by driving its internalization and turnover and down-regulating SLC gene expression.

However, the data do not exclude the possibility that the PM has an important role in preBCR signaling. In one respect, this may be at the level of signaling output. It may be that preBCR expression in the PM is not important for CD2, CD22, and proliferation/survival, but is important for other preBCR-dependent phenomena not evaluated in this system. This includes allelic exclusion, a property which is lost both in v-abl-transformed cells and in our primary IL-7-dependent pro-B cultures with HCs expressed de novo via retroviruses (Ref. 52 and data not shown). Also not evaluated were hetero- and homotypic interactions that may promote later preBCR-dependent developmental changes (6, 7, 53, 54).

In another respect, it could be postulated that the Ig{alpha}beta heterodimer, rather than the intact preBCR, may reach the PM and signal. Several recent studies on the BCR have indicated that Ag cross-linking induces Ig dissociation from the Ig{alpha}beta heterodimer (55, 56). Whereas nearly all of the surface Ig is internalized, up to 30% of the Ig{alpha}beta heterodimers remain on the PM and continue to signal. Based on these observations, it is postulated that key steps in common for WT and TGN/Lamp-redirected preBCRs are 1) transport Ig{alpha}beta out of the ER and transit through the TGN and 2) "activation" of the Ig{alpha}beta molecules in such a way to render them competent to productively engage signal transduction pathways. After activation, Ig{alpha}beta may dissociate from the Ig components. HC-SLC components would be forwarded to lysosomes and activated Ig{alpha}beta would continue to the PM and signal. Whereas the first model postulated that the TGN is where signaling pathways are first engaged, this alternative model incorporates the idea that PM expression of Ig{alpha}beta may still be necessary for persistent and/or productive propagation of other subsequent preBCR-dependent developmental signals. A prediction from this and the first model is that preBCRs, by virtue of the {lambda}5UR, would be most efficient at inducing this hypothetical Ig{alpha}beta activation step, whereas {lambda}5UR-deleted preBCRs and BCRs would be less efficient. Activation may be a preBCR-intrinsic property; however, if ligand interaction facilitates this, both models imply this ligand is intravesicular. This alternative model is consistent with the HC-SLC-independent signaling activity of PM targeted Ig{alpha}beta fusion molecules (57, 58).

Nevertheless, these models do not exclude a role for an extracellular ligand in modulating preBCR signal transduction. Rather, several modes of preBCR signaling may coexist, which may depend on the individual properties of the preBCR clonotype. For example, ligand-dependent interactions may have a role in enhancing the intensity of preBCR-dependent signaling functions. This may be most important for certain HC clonotypes that may assemble with the SLC and transit through the secretory system but may not signal efficiently intracellularly via the cell-autonomous, possibly preBCR-intrinsic mechanism. A result may be that intact preBCRs that reach the surface are activated by SLC/preBCR ligand-dependent cross-linking (6).

The V(D)J recombination process often produces Ig genes that are nonproductive or that encode proteins that are structurally unsound. The "preBCR checkpoint" provides a functional screen for HC structure in the absence of any other information about future BCR properties. The primary and transformed pro-B cell culture systems here provide a rapid and effective means to systematically determine how the functionality of individual HC clonotypes with known biological properties is evaluated at this critical developmental stage. Expression of the redirected HCs in mice via transgenesis will help address the extent to which the resulting redirected preBCRs can support development in vivo or whether other interactions dependent on putative preBCR surface expression are necessary. In addition, the activity of redirected preBCRs in progenitor cells defective in particular signaling molecules will provide further insight into how the membrane dynamics of the preBCR control its interfacing with different signaling pathways. These types of approaches will ultimately determine how the IgHC structure influences these processes so as to control repertoire establishment and influence disease states.


   Acknowledgments
 
We gratefully thank Drs. S. Gottesman and W. Chirico (State University of New York–Downstate Medical Center) for critical reading of this manuscript and discussions. We are grateful to W. Oxberry (Department of Pathology Confocal Imaging Facility, State University of New York–Downstate Medical Center) for instruction and generosity and members of the Roman Laboratory for discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported in part by Research Project Grant 00-269-01-LBC from the American Cancer Society (to C.A.J.R.), by the New York City Council Speaker’s Fund of the New York Academy of Medicine (to C.A.J.R.), and by the State University of New York. Back

2 Address correspondence and reprint requests to Dr. Christopher Roman, Department of Microbiology and Immunology, State University of New York–Downstate Medical Center at Brooklyn, 450 Clarkson Avenue, Box 44, Brooklyn, NY 11203. E-mail address: Christopher.Roman{at}Downstate.edu Back

3 Abbreviations used in this paper: HC, H chain; preBCR, precursor BCR; LC, L chain; SLC, surrogate L chain; PM, plasma membrane; (S)LC, surrogate and/or conventional light chain; ER, endoplasmic reticulum; TGN, trans-Golgi network; Endo Hf, endoglycosidase; WT, wild type; WGA, wheat gluten; UR, unique region; Lamp-1, lysosome-associated membrane protein 1. Back

Received for publication January 26, 2006. Accepted for publication March 17, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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