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International Immunology Advance Access originally published online on November 7, 2006
International Immunology 2007 19(1):19-30; doi:10.1093/intimm/dxl118
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Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes: evidence for a role in internalization of the B cell receptor

Cathlin M. Mutch1, Ratna Sanyal1, Tammy L. Unruh1, Lana Grigoriou1, Minghua Zhu2, Weiguo Zhang2 and Julie P. Deans1

1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Health Sciences Center, 3330 Hospital Drive North West, Calgary, Alberta, T2N 4N1, Canada
2 Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA

Correspondence to: J. P. Deans; E-mail: jdeans{at}ucalgary.ca


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) are raft-associated transmembrane adaptor proteins with distinct functions in immediate/early phases of receptor signaling pathways. Heterogeneous rafts are thought to compartmentalize membrane-associated signaling events. In order to investigate the subcellular localization of LAB/NTAL and PAG/Cbp, they were expressed as fluorescent chimeric fusion proteins in a human B cell line and their distribution was examined, along with the corresponding endogenous proteins, before and after B cell receptor (BCR) stimulation. Both adaptors were distributed predominantly at the plasma membrane in resting cells and co-clustered with other raft-associated proteins; however, they distributed differently in buoyant membranes isolated by either detergent resistance or non-detergent methods, indicating that they might localize to distinct rafts. After activation, LAB/NTAL was internalized and co-localized with the BCR while PAG/Cbp remained on the cell surface. BCR internalization was reduced in LAB/NTAL-deficient murine B cells, suggesting a regulatory role for LAB/NTAL in activation-induced internalization of the BCR. The cytoplasmic domain of LAB/NTAL, and not the transmembrane/juxtamembrane region, was found to be essential for its internalization.

Keywords: adaptor proteins, B cell receptor, B lymphocytes, Cbp, endocytosis, heterogeneity, LAB, microdomains, NTAL, PAG, rafts


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) hereafter referred to as LAB and PAG, belong to a family of transmembrane adaptor proteins (TRAPs) that have very short extracellular sequences, single transmembrane spans and cytoplasmic domains containing multiple sites for tyrosine phosphorylation (1, 2). PAG is a broadly expressed TRAP that functions as a negative regulator of receptor signaling (3, 4). Phosphorylation of PAG, mediated by the Src family kinase Fyn (5, 6), recruits Csk to down-regulate Src family kinase activity. In contrast, expression of LAB is restricted to B lymphocytes and cells of the myeloid lineage, is rapidly tyrosine phosphorylated by Syk following receptor signaling, and associates with Sos1, Gab1, Grb2 and c-Cbl (7, 8). Unlike the apparent T cell counterpart LAT, LAB does not recruit phospholipase C{gamma} (PLC{gamma}) or affect intracellular calcium mobilization; however, sequestration of Grb2 by LAB enhanced calcium influx upon B cell receptor (BCR) stimulation in DT40 cells (9). Recent evidence from LAB-deficient mice indicates a prominent role for LAB as both a positive and negative regulator of receptor signaling in mast cells (10, 11). Effects in the B cell compartment are relatively minor and the function of LAB in B cells is still not entirely clear (12).

LAB and PAG, like the transmembrane adaptors LAT and LIME, possess a membrane-proximal palmitoylation motif (CXXC) and are constitutively associated with membrane rafts. Other known TRAP family members, LAX, SIT and TRIM, are not palmitoylated and appear to be excluded from rafts (1, 2). Rafts are implicated in intracellular protein trafficking and in compartmentalized signal transduction from the plasma membrane (13–18). In B lymphocytes, many of the signaling effectors within the BCR signaling cascade are associated with rafts (19, 20). The BCR itself localizes predominantly to non-raft domains in resting mature B cells but translocates to lipid rafts following antigen engagement. The dually acylated Src family kinase Lyn is constitutively associated with rafts and initiates signaling within these domains. Lyn and the tyrosine kinase Syk activate a cascade of phosphorylation events involving the Ig{alpha}–Igß subunits of the BCR and various other substrates, including LAB and PAG (5–8, 21). Following activation of signaling pathways from the plasma membrane, engaged BCRs are endocytosed to down-regulate receptor signaling and deliver antigen for processing and presentation on MHC (22). BCR internalization is predominantly an actin- and clathrin-dependent process (23–26), with a role for lipid rafts suggested by some (25–28), but not all (29), studies. Signaling requirements for BCR internalization are not yet clearly defined, presumably in part because of the complexity of multiple mechanisms underlying this important process. Activation of Src family kinases (Lyn) is required (28, 30, 31), but evidence for involvement of Syk has been inconsistent (30, 32). The small cytoplasmic adaptor protein, Bam32, upon phosphorylation by Lyn, links BCR signaling to actin polymerization via rac1, and BCR internalization is reduced in Bam32–/– DT40 cells (31, 33). BCR internalization occurs normally in PLC{gamma}-deficient DT40 cells (31), and does not require calcium mobilization (34). At least one study has contraindicated a role for protein kinase C (PKC) (34), but a recent report suggested involvement of the calcium-independent delta isoform of PKC in internalization of the BCR in both naive and anergic B cells (28).

Heterogeneity in raft composition potentially compartmentalizes the functions of constituent membrane proteins (35), although there are few reports of distinct rafts in lymphocytes (27, 36–38). The adaptor proteins LAB and PAG have similar structural organization and are both associated with membrane rafts via presumptive palmitate modification of membrane-proximal cysteine residues; however, they have distinct functions in receptor signaling and might segregate into distinct rafts. Here, we report studies that revealed differential distribution of LAB and PAG in microdomains in resting B lymphocytes, selective internalization of LAB after receptor stimulation and a role for LAB in regulating internalization of the BCR.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Generation of green fluorescent protein fusion constructs
Cloning of full-length human LAB cDNA into pEGFPN1 (BD Biosciences Clonetech, Palo Alto, CA, USA) was previously described (39). The entire human PAG sequence (including the 5' untranslated region and excluding the stop codon) was cloned from BJAB cDNA using the forward primer 5'-AGTCGGTACCTACTGGACAAACATTTCCTCC-3' and reverse primer 5'-GACTGGATCCAGCCTGGTAATATCTCTGCC-3'. The forward primer incorporated a KpnI restriction site and the reverse primer incorporated a BamHI restriction site that allowed the amplified sequence to be inserted into the multiple cloning site (MCS) of the pEGFPN1 vector.

Truncated LAB-green fluorescent protein (GFP) incorporated amino acid residues 1–35 of human LAB in the pEGFPN1 vector. These residues encompass the extracellular, transmembrane and juxtamembrane domains of human LAB (including the CXXC motif for palmitoylation). Truncated LAB was cloned from BJAB cDNA using the forward primer 5'-AGTCAGATCTTCACCAGGCCACGCATCACAAG-3' and reverse primer 5'-GACTGGATCCGACCTCTTTGCACCTGGGCG-3'. The forward primer incorporated a BglII restriction site and the reverse primer incorporated a BamHI restriction site that allowed the amplified sequence to be inserted into the MCS of pEGFPN1. A LAB-PAG-GFP construct was also created; the cytoplasmic domain of PAG (residues 46–432) was cloned from BJAB cDNA using the forward primer 5'-AGTCGGATCCGCCGCGACAGCATAGTGGG-3' and reverse primer 5'-GACTACCGGTAGCCTGGTAATATCTCTGCC-3'. The forward primer incorporated a BamHI restriction site and the reverse primer incorporated an AgeI restriction site that allowed the amplified sequence to be inserted into the truncated LAB-GFP vector.

A PAG-LAB-GFP fusion protein comprising the extracellular, transmembrane and juxtamembrane domains of human PAG (residues 1–47) and the cytoplasmic domain of human LAB (residues 36–243) was also expressed in the pEGFPN1 vector. Residues 1–47 of PAG were cloned from BJAB cDNA using the forward primer 5'-AGTCGCTAGCTACTGGACAAACATTTCCTCC-3' and reverse primer 5'-GACTAAGCTTTCGCGGCTTCTTTTCCCTGTC-3'. The forward primer incorporated an NheI restriction site and the reverse primer incorporated a HindIII restriction site that allowed the amplified sequence to be inserted into the MCS of pEGFPN1. The cytoplasmic domain of LAB was cloned from BJAB cDNA using the forward primer 5'-AGTCAAGCTTTCAGAGAAAATCTACCAGCAG-3' and reverse primer 5'-GACTACCGGTGGGGCTTCTGTGGCTGCCACCTC-3'. The forward primer incorporated a HindIII restriction site and the reverse primer incorporated an AgeI restriction site that allowed the amplified sequence to be inserted into the truncated PAG-GFP vector.

A LAX-LAB-GFP fusion protein comprising the extracellular, transmembrane and juxtamembrane domains of LAX (residues 1–68) and the cytoplasmic domain of LAB (residues 36–243) was also expressed in the pEGFPN1 vector. Truncated LAX was cloned from BJAB cDNA using the forward primer AGTC 5'-GCTAGCCACGAGGCCTCTGTGCCCCTC-3' and reverse primer 5'-GACTAAGCTTTTGTCGCTTCTTCCGTTTATT-3'. The forward primer incorporated an NheI restriction site and the reverse primer incorporated a HindIII restriction site that allowed the amplified sequence to be inserted into the MCS of pEGFPN1. The cytoplasmic domain of LAB was cloned as above and inserted into the truncated LAXTR-GFP vector. The sequence of all constructs was confirmed.

Cells and transfections
BJAB cells were maintained in RPMI 1640 supplemented with 7.5% fetal bovine serum (FBS). BJAB cells were transfected with pEGFPN1 vectors using electroporation: 1 x 107 BJAB cells were incubated with 20 µg DNA for 30 min at 37°C prior to electroporation at 340 V and 950 µF (Gene Pulser II, Bio-Rad Laboratories, Hercules, CA, USA). Transiently transfected cells were maintained in 10% FBS/RPMI and used for microscopy 24 h after transfection. Stable transfected cell lines were obtained by sorting for GFP-positive cells (BD FACSVantage Sort Enhanced System, BD Biosciences Immunocytometry Systems, Franklin Lakes, NJ, USA) and were maintained in 10% FBS/RPMI supplemented with 1 mg ml–1 geneticin (Invitrogen Corporation, Carlsbad, CA, USA). Primary human B cells were isolated from human tonsils obtained from the Alberta Children's Hospital. B cells were enriched to >95% by T cell rosetting using sheep RBCs pre-treated with 2-aminoethylisothiouronium bromide (Sigma, St Louis, MO, USA). Murine lymphocytes were isolated from the spleens of LAB–/– or wild-type C57Bl/6 mice. Following mechanical disruption of whole spleens, the RBCs were removed by hypotonic lysis.

Antibodies
Anti-GFP (rabbit serum) was purchased from Eusera (University of Alberta, Edmonton, Alberta, Canada). NAP-07, an IgG1 mouse mAb against human LAB, and MEM-255, an IgG2a mouse mAb against human PAG, were purchased from Abcam Inc. (Cambridge, MA, USA). Rabbit anti-PAG was a gift from André Veillette (IRCM, Montréal, Quebec, Canada). Anti-CD45 (mIgG1), anti-CD20 B1 (mIgG2a) and anti-CD59 MEM-43/5 (mIgG2b) were purchased from BD Biosciences PharMingen (San Diego, CA, USA), Coulter (Miami, FL, USA) and Monosan (Uden, Netherlands), respectively. Biotinylated anti-phosphotyrosine 4G10 (Upstate, Waltham, MA, USA) was generated using the EZ-Link-Sulfo-NHS-Biotin Kit from Pierce Biotechnology Inc. (Rockford, IL, USA). Unconjugated, Cy3-conjugated and FITC-conjugated F(ab')2 goat anti-human IgM Fc5µ specific, biotin-conjugated F(ab')2 goat anti-mouse IgM, Cy3-conjugated F(ab')2 goat anti-mouse-IgG and HRP-conjugated rabbit anti-mouse IgG were purchased from Jackson Immunoresearch Laboratories Inc. (West Grove, PA, USA). Alexa488-conjugated rabbit anti-mouse IgG was purchased from Molecular Probes Inc. (Eugene, OR, USA).

Isolation of detergent-resistant membranes
Detergent-resistant membranes (DRMs) were isolated from 108 cells as previously described (27), using 1% Triton X-100. After sucrose density gradient centrifugation, eight fractions (1.5 ml each) were collected from the top to the bottom of each gradient. An aliquot of each fraction and the pellet were dissolved in SDS sample buffer. Equal cell equivalents from each gradient fraction were subjected to SDS-PAGE and transferred to Immobilon P (Millipore Corporation, Bedford, MA, USA) for western blotting. For DRM imaging, 108 cells were stained prior to lysis with 50 µg primary antibody for 15 min followed by 50 µg Cy3-conjugated goat anti-mouse IgG for 15 min. DRMs were isolated and transferred to a fresh 14 x 89-mm ultra-clear centrifuge tube, mixed with 12 ml MBS [25 mM 2-(N-morpholino)-ethanesulphonic acid and 150 mM NaCl] to dilute the sucrose, and concentrated by centrifugation at 37 000 r.p.m for 1 h in a SW41 Ti rotor in a Beckman XL-70 ultracentrifuge (Palo Alto, CA, USA). The pelleted DRMs were suspended in 200 µl Tris buffer (10 mM Tris and 150 mM NaCl) and a sample was loaded on a poly-lysine-coated slide for imaging.

Non-detergent isolation of light membranes
A non-detergent lipid raft isolation method was used exactly as described (40), except that a step gradient rather than a linear gradient was used. Briefly, cells were washed and re-suspended in buffer containing 20 mM Tris–HCl pH 7.8, 250 mM sucrose, 1 mM CaCl2 and 1 mM MgCl2. Cell membranes were disrupted on ice by repeated passage through a 22-gauge needle. Lysates were centrifuged at 1000 x g for 10 min at 4°C and supernatants transferred to a chilled ultracentrifuge tube. The pellets were re-suspended in buffer and subjected to another round of shearing and centrifugation. The combined supernatants were mixed with an equal volume of 50% iodixanol (Optiprep, Axis-Shield PoCAs, Oslo, Norway), overlayered with a 20%/5% iodixanol step gradient and centrifuged at 17 000 r.p.m. for 3 h in a SW41 Ti rotor in a Beckman XL-70 ultracentrifuge. To prepare for imaging, the membranous material at the upper interface was treated as for DRMs.

Immunoprecipitations
Cells (1 x 107) were stimulated with 10 µg F(ab')2 goat anti-human IgM at 37°C followed by lysis in buffer containing 1% maltoside (Sigma–Aldrich Corporation, St Louis, MI, USA), 10 mM Tris, 150 mM NaCl, 1 µg ml–1 leupeptin, 1 µg ml–1 aprotinin, 1 mM phenylmethylsulphonylfluoride, 1 mM EDTA, 1 mM Na2MoO4 and 1 mM Na3VO4. LAB and PAG were immunoprecipitated using mouse anti-LAB, rabbit anti-PAG or rabbit anti-GFP and Protein A-Sepharose (Repligen Corporation, Waltham, MA, USA). Immunoprecipitates were subjected to SDS–PAGE and transferred to Immobilon P membranes.

Western blotting
Membranes were blocked with 5% BSA. Phosphotyrosine blots were performed using biotinylated 4G10 followed by HRP-conjugated neutralite avidin (Southern Biotechnology Associates, Birmingham, AL, USA). All other blots were performed using primary antibodies followed by HRP-conjugated Protein A (Bio-Rad Laboratories) for rabbit primary antibodies or HRP-conjugated rabbit anti-mouse IgG for mouse primary antibodies. Western blots were developed using SuperSignal chemiluminescent substrate (Pierce Biotechnology Inc.) and visualized using the Fluor-S MAX MultiImager (Bio-Rad Laboratories).

Immunofluorescence microscopy
For surface staining, cells were fixed with 1% PFA (Electron Microscopy Sciences, Fort Washington, PA, USA) in PBS for 5 min at room temperature either before or after cell-surface labeling as indicated. For intracellular staining, cells were fixed in 2% PFA (BJAB cells) or 4% PFA (tonsil B cells) for 10 min at room temperature, permeabilized with 0.1% Triton for 5 min at room temperature and incubated with antibodies at room temperature in the presence of 1% BSA. The cells were visualized using a DeltaVision Restoration Microscopy System (Applied Precision, Issaquah, WA, USA) with an Olympus IX70 microscope, a mercury arc lamp and a series 300 CCD camera system cooled to –40°C (Photometrics, Muchen, Germany). Stacks of optical sections at 0.2-µm intervals were acquired using an Olympus 60X/1.40 NA oil immersion objective. GFP, FITC and Alexa488 were visualized using the standard FITC filter set (Ex 490/20; Em 528/38) and Cy3 was visualized using the standard RD filter set (Ex 555/28; Em 617/73). Single-labeled control samples were imaged separately to confirm negligible bleed-through of fluorophores in filter sets used for other fluorophore imaging channels (data not shown). Digital deconvolution was performed on the stacks of fluorescent optical sections using SoftWoRX constrained iterative deconvolution (Applied Precision).

For image display and co-localization analysis, the intensities for each wavelength were scaled using SoftWoRX image analysis software. The range of intensities for each fluorophore in a given image was scaled from minimum to maximum detected signal. Specifically, each image exhibited a bimodal distribution of pixel intensity with the first (very minor) distribution corresponding to background fluorescence and the second distribution corresponding to specific fluorescence. The minimum image fluorescence was set at a level higher than the background fluorescence node. This was done for all images except those of endogenous PAG where the minimum image fluorescence was set at a level higher than isotype control fluorescence. The maximum image fluorescence was set as the maximal pixel intensity detected. Scaled images were imported into CoLocalizer Pro to determine the extent of co-localization (Colocalization Research Software, Boise, ID, USA). The software assigned a color to every pixel and the percentage of green pixels co-localized with red pixels ([no. of yellow pixels / (no. of green pixels + no. of yellow pixels)] x 100) was calculated and reported as percent co-localization.

Flow cytometric measurement of BCR internalization
Splenocytes (2.4 x 107) were washed in RPMI and re-suspended in ice-cold PBS. Cells were incubated on ice with 24 µg of biotin-conjugated F(ab')2 goat anti-mouse IgM for 30 min, washed twice with ice-cold PBS to remove unbound antibody, re-suspended at 3 x 106 cells per 500 µl in ice-cold PBS and aliquoted into 3 x 106 cell samples. Each sample was incubated at 37°C for defined periods, at the end of which 500 µl of ice-cold PBS was added and the sample placed on ice. Remaining cell-surface BCR was stained on ice for 30 min with PE-conjugated streptavidin (Becton Dickinson, Mississauga, Ontario, Canada). The cells were washed with ice-cold PBS followed by fixation in 1% PFA and analysis by flow cytometry (FACScan, Becton Dickinson). The mean fluorescence intensity (MFI) of BCR staining at time 0 (i.e. after cell labeling on ice) was set at 100%. The percent BCR remaining on the cell surface at later time points was calculated using the equation [(MFI 37°C / MFI 4°C) x 100]. Results were expressed as mean ± standard error and analyzed using an unpaired Student's t-test. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
GFP fusion constructs
Full-length LAB and PAG were conjugated to GFP as illustrated in Fig. 1(A), and expressed in the BJAB mature human B cell line. Both constructs localized to the plasma membrane, as shown in later figures. The raft localization of LABFL-GFP and PAGFL-GFP was verified by sucrose density gradient centrifugation of 1% Triton lysates, in which both proteins re-distributed to buoyant fraction 4 (Fig. 1B), the same as the endogenous proteins (data not shown). As an additional measure of appropriate localization and functional integrity of the fusion proteins, we determined whether LABFL-GFP and PAGFL-GFP were phosphorylated upon BCR stimulation. Endogenous LAB and PAG immunoprecipitated from parental BJAB cells were rapidly and transiently phosphorylated following BCR stimulation, as expected (7, 8, 21; Fig. 1C). LABFL-GFP and PAGFL-GFP were immunoprecipitated from stably transfected BJAB cells using anti-GFP and were phosphorylated following BCR stimulation with kinetics similar to those of the endogenous proteins (Fig. 1C).


Figure 1
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  		Fig. 1 LABFL-GFP and PAGFL-GFP chimeric proteins. The LABFL-GFP and PAGFL-GFP chimeric proteins comprise the extracellular, transmembrane and cytoplasmic domains of the adaptor proteins fused to the N-terminus of GFP (FL = full length). The configuration of the constructs is illustrated in (A), drawn approximately to scale. (B) The raft localization of LABFL-GFP and PAGFL–GFP was checked in BJAB cells stably expressing each construct. Cells were lysed in 1% Triton followed by sucrose density gradient centrifugation. Gradient fractions were subjected to western blotting using anti-GFP or anti-CD45 as indicated. CD45 identifies the detergent-soluble fractions at the bottom of the gradients. P represents the high-density insoluble pellet. (C) Parental BJAB cells or BJAB cells stably expressing LABFL-GFP and PAGFL-GFP were examined for LAB and PAG phosphorylation following BCR engagement. Endogenous LAB and PAG were immunoprecipitated from parental BJAB cells whereas LABFL-GFP and PAGFL-GFP were immunoprecipitated from stably transfected cell lines using anti-GFP. Tyrosine phosphorylation of the immunoprecipitated proteins was examined by western blotting. Kinetics of phosphorylation of LAB, PAG, LABFL-GFP and PAGFL-GFP are representative of at least three independent experiments each.

 
Organization of LAB and PAG at the plasma membrane
LABFL-GFP and PAGFL-GFP localized to the plasma membrane in transiently transfected BJAB B cells (Fig. 2). To examine their relative localization, transfected cells were permeabilized and stained with anti-PAG and anti-LAB. The upper limit of detection of complete co-localization was >90% (mean relative co-localization of four images each), as shown in cells expressing LAB-GFP or PAG-GFP stained with anti-LAB or anti-PAG, respectively (Fig. 2). Co-localization of PAGFL-GFP with endogenous LAB, and LABFL-GFP with endogenous PAG, was estimated at ~65% (mean relative co-localization of 10 images each), indicating predominant overlap but some separation of the fluorescent signals.


Figure 2
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  		Fig. 2 LAB-GFP and PAG-GFP co-localize at the cell surface and co-cluster with raft-associated proteins CD20 and CD59. (A) BJAB cells transfected with either LABFL-GFP or PAGFL-GFP were fixed, permeabilized and stained with anti-LAB or anti-PAG, as indicated, and anti-mouse IgG(Cy3). In (B–D), cells were incubated with murine mAbs specific for (B) CD20, (C) CD59 or (D) CD45 and goat anti-mouse IgG(Cy3), either after fixation (upper panels, labeled No XL) or before fixation (lower panels, labeled CDxx XL). In this and subsequent immunofluorescence figures, isotype controls were performed and the minimum image fluorescence was set at a level higher than isotype control fluorescence as described in Methods. Single-labeled control samples were imaged separately to confirm negligible bleed-through of fluorophores to the other channel. Results are representative of >90% of >100 cells observed in at least two independent experiments.

 
Separate clustering of different raft proteins has previously been used as an indication of their residency in distinct rafts (37, 41). LAB and PAG have only a few extracellular residues and there are no antibodies available that could be used to cluster either protein directly. We therefore examined the distribution of LABFL-GFP and PAGFL-GFP following aggregation of the raft-associated transmembrane protein CD20 (42) or glycophosphatidylinositol-linked CD59. In resting cells, CD20 showed a uniform punctate distribution around the circumference of the cell, but clustered into patches on the cell surface upon hyper-cross-linking (Fig. 2B); both LABFL-GFP and PAGFL-GFP co-clustered with CD20 (Fig. 2B). Like CD20, CD59 had a punctate distribution around the circumference of cells that were stained after fixation, but clustered on the cell surface upon hyper-cross-linking (Fig. 2C). In contrast to CD20, CD59 tended to cluster to one pole of the cell rather than into several large patches. LABFL-GFP and PAGFL-GFP both re-distributed to the same pole as clustered CD59 (Fig. 2C). Hyper-cross-linking the non-raft marker CD45 led to clustering of CD45 but the localization of LABFL-GFP and PAGFL-GFP appeared unchanged (Fig. 2D). These results suggested that LAB and PAG might reside in the same raft domains.

LAB internalizes with the BCR whereas PAG remains at the cell surface
Next, we examined the localization of LABFL-GFP and PAGFL-GFP relative to the BCR in resting and activated cells. When cells were fixed prior to staining with Cy3-conjugated F(ab')2 anti-IgM, the BCR showed a punctate distribution around the circumference of the cell with the brighter points of fluorescence largely coincident with those of LABFL-GFP and PAGFL-GFP (Fig. 3A, upper panels). The localization of LABFL-GFP and PAGFL-GFP following BCR stimulation was examined by incubating cells with Cy3-conjugated F(ab')2 anti-IgM at 37°C before fixation. After 10 min of stimulation, a large proportion of labeled BCR was internalized (Fig. 3A, lower panels), and we observed that LAB internalized following BCR engagement and co-localized with internalized BCR. In contrast, PAGFL-GFP did not internalize following BCR stimulation (Fig. 3A). We examined the localization of endogenous LAB and PAG in resting and activated BJAB cells, and obtained data that were similar to those obtained with the fluorescent chimeric proteins (Fig. 3B). All imaging results are representative of >90% of >100 cells observed in at least two independent experiments.


Figure 3
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  		Fig. 3 LAB-GFP co-caps and co-internalizes with the BCR whereas PAG-GFP remains at the cell surface. To image the BCR before and after stimulation, cells were incubated with fluorochrome-conjugated F(ab')2 anti-IgM after fixation (upper panels) or at 37°C for 10 min prior to fixation (lower panels). (A) BJAB cells transiently transfected with either LABFL-GFP or PAGFL-GFP were labeled/stimulated with F(ab')2 anti-IgM(Cy3). (B) Endogenous LAB and PAG in parental BJAB cells and (C) Endogenous LAB in primary human B cells were labeled with F(ab')2 anti-IgM(Cy3) (LAB-stained cells) or F(ab')2 anti-IgM(FITC) (PAG-stained cells). Cells were permeabilized and stained with anti-LAB and F(ab')2 anti-mouse IgG(Alexa488) or with anti-PAG and F(ab')2 anti-mouse IgG(Cy3). The BCR is false-colored red and the adaptor protein is false-colored green in both cases for the sake of clarity. Isotype control stained cells were imaged to confirm specific staining by antibodies (data not shown). Results are representative of at least 90% of >100 cells observed in each experiment. Imaging of LABFL-GFP and PAGFL-GFP was performed in at least six independent experiments, endogenous LAB and PAG in BJAB cells in two independent experiments and endogenous LAB in primary human B cells once. (D) Splenocytes were isolated from wild-type or LAB-deficient mice, labeled on ice with biotin-conjugated F(ab')2 anti-mouse IgM, washed with cold PBS and transferred to 37°C for the times indicated. BCR internalization was stopped with the addition of ice-cold PBS and remaining cell-surface IgM was detected with PE-conjugated streptavidin. Results are from four independent experiments conducted on four pairs of LAB-deficient and wild-type spleens. The MFI at time 0 was similar in all samples. Percent BCR remaining at the cell surface at each time point is plotted showing standard error.

 
LAB deficiency reduces BCR internalization
Co-internalization of endogenous LAB with the activated BCR was also observed in primary human B cells (Fig. 3C). The internalized pools of LAB were co-localized with internalized BCR, as seen also with endogenous LAB and LAB-GFP in BJAB cells. We therefore questioned whether LAB might have a role in internalization of the BCR. To test this, BCR internalization was measured in splenic B cells isolated from LAB-deficient and wild-type control mice. The BCR was labeled on ice with biotin-conjugated F(ab')2 anti-mouse IgM, and then internalization was initiated by raising the temperature to 37°C and tracked by flow cytometry using PE-conjugated streptavidin. In wild-type splenocytes, BCR internalization proceeded rapidly until only 20–25% remained on the cell surface (Fig. 3D). In LAB-deficient splenocytes, the kinetics of BCR internalization were similar but the amount of BCR remaining on the cell surface plateaued at ~40–45% (P < 0.05 at 20 min and later).

LAB and PAG distribute differently in isolated membrane microdomains
Differential internalization of LAB and PAG was difficult to reconcile with the data from Fig. 2 showing their co-localization and co-clustering with CD20 and CD59, which suggested that they might reside in the same raft domains. However, co-clustering could result from raft-independent molecular interactions and the limits of spatial resolution using wide-field immunofluorescence microscopy is in the order of 200 nm, substantially greater than the lowest estimates of the size of elemental rafts (43–45). We therefore investigated whether LABFL-GFP and PAGFL-GFP co-isolated with CD20 and CD59 in the same membrane fragments following lysis of the cells.

Raft-associated molecules labeled in live cells with fluorochrome-conjugated antibodies can be visualized in subsequently isolated DRMs by immunofluorescence microscopy (27). We used this technique to examine the distribution of LABFL-GFP and PAGFL-GFP in isolated DRMs. We could not directly visualize the DRM distribution of LABFL-GFP relative to endogenous PAG or vice versa because the mixed orientation of DRM vesicles (46) precludes reliable immunostaining after isolation. Therefore, we examined the distribution of the fluorescent adaptors relative to CD20 or CD59 labeled on intact cells before lysis. A very high degree of co-localization was observed between PAGFL-GFP and CD20 (Fig. 4A (upper panel) and D). The relative intensity of CD20 (Cy3) and PAG (GFP) in each DRM particle was similar, indicating a stoichiometric relationship. In sharp contrast, only a low level of co-localization was detected between LABFL-GFP and CD20 and tended to occur between bright points of CD20 staining and dim points of LABFL-GFP, and vice versa (Fig. 4A (lower panel) and D). The degree of co-localization of PAGFL-GFP with CD59 was lower than with CD20, but similar to that of CD20-GFP with CD59, whereas co-localization of LABFL-GFP with CD59 was significantly lower (Fig. 4B and D).


Figure 4
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  		Fig. 4 LAB-GFP and PAG-GFP localize to distinct membrane microdomains. BJAB cells stably transfected with either LABFL-GFP or PAGFL-GFP were labeled for (A) CD20 or (B) CD59 using specific murine mAbs and goat anti-mouse IgG(Cy3). The cells were then lysed with cold 1% Triton lysis buffer and subjected to sucrose density centrifugation. The buoyant membrane fractions were collected, concentrated and imaged by immunofluorescence microscopy. For the CD59 counter-stained samples, BJAB cells transfected with CD20-GFP were also examined. The images shown are representative of nine images for each sample and a graphical representation of the mean relative co-localization for each set of images ± standard error is presented (shown in D). (C) BJAB cells stably transfected with either LABFL-GFP or PAGFL-GFP and counter-stained for CD20 were lysed without detergent and subjected to Optiprep density centrifugation for imaging as above. Co-localization was estimated at 78 ± 7% and 26 ± 3% for PAGFL-GFP/CD20 and LABFL-GFP/CD20, respectively (mean of six images each ± standard error), as shown in (E).

 
The parallel behavior of two dissimilar raft-associated proteins, CD20 and CD59, relative to LAB and PAG in these DRM experiments suggests that LAB and PAG are localized in predominantly distinct microdomains in the cells from which the DRMs were derived. However, a common concern is that detergents may cause coalescence of rafts or even induce the formation of raft-like domains. To address this, we also imaged buoyant or ‘light’ membranes (LMs) isolated using a non-detergent method (40). LM imaging of LABFL-GFP and PAGFL-GFP relative to CD20 also revealed differential distribution of LAB and PAG (Fig. 4C and E). Co-localization between LABFL-GFP or PAGFL-GFP and CD20 was estimated at 26 ± 3% and 78 ± 7%, respectively, using this technique. This difference could not be attributed to different levels of expression of the constructs because expression was monitored by flow cytometry and was consistently ~50% lower for PAGFL-GFP (MFI = 81 compared with MFI = 150 for LABFL-GFP in a typical experiment). Thus, the higher degree of co-localization of PAGFL-GFP with CD20, compared with LABFL-GFP with CD20, in membrane fragments isolated by both detergent and non-detergent methods, supports the conclusion that LABFL-GFP and PAGFL-GFP are physically separated in the plasma membrane.

The cytoplasmic domain of LAB confers internalization following BCR stimulation
If LAB and PAG localize to distinct raft microdomains on the surface of B lymphocytes, as the data in Fig. 4 suggest, it is possible that raft localization could have a mechanistic role in the internalization of LAB but not PAG following BCR stimulation. In that case, the transmembrane and juxtamembrane regions of the proteins would be expected to be critical determinants of their differential internalization. Alternatively, selective internalization of LAB may be driven by its cytoplasmic domain. To test these possibilities, we created a series of chimeric fusion constructs. Truncated LABTR-GFP included the extracellular, transmembrane and juxtamembrane residues of LAB but lacked most of the cytoplasmic domains (Fig. 5A); this construct localized to DRMs (fractions 3 and 4 in Fig. 5B) and co-capped with stimulated BCR, but failed to internalize (Fig. 5C). Replacing the cytoplasmic domain of LAB with that of PAG (LAB-PAG-GFP) did not allow internalization of the construct (Fig. 5D). The inability of both LABTR-GFP and LAB-PAG-GFP to internalize following BCR stimulation demonstrated an absolute requirement for the cytoplasmic domain of LAB. To determine whether the cytoplasmic domain of LAB was sufficient to allow internalization of a full-length adaptor protein that lacked the transmembrane and juxtamembrane sequences of LAB, we replaced the cytoplasmic domain of PAG with that of LAB (PAG-LAB-GFP) and found that this construct was internalized following BCR stimulation (Fig. 5E).


Figure 5
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  		Fig. 5 The cytoplasmic domain of LAB is essential for internalization. The configuration of LABTR-GFP, PAG-LAB-GFP and LAB-PAG-GFP constructs is illustrated in (A). The LABTR-GFP chimeric protein comprises amino acid residues 1–35 of LAB fused to the N-terminus of GFP (TR = truncated). The remaining cytoplasmic domain of LAB (residues 36–243) was fused to residues 1–47 of PAG at the amino-terminus of GFP. The inverse chimeric protein had the cytoplasmic domain of PAG fused to the truncated LAB sequence. (B) The raft localization of the constructs was checked by sucrose density gradient centrifugation of 1% Triton lysates as described in Fig. 1. BJAB cells transiently transfected with (C) LABTR-GFP, (D) LAB-PAG-GFP or (E) PAG-LAB-GFP were labeled with F(ab')2 anti-IgM(Cy3) either following fixation or at 37°C for 10 min prior to fixation. Note that all constructs co-capped with the BCR on the cell surface following stimulation but that only PAG-LAB-GFP internalized. Results are representative of at least 90% of >100 cells in at least three independent experiments for each construct.

 
Although the transmembrane and juxtamembrane regions of LAB were dispensable for LAB internalization (i.e. replaceable by the equivalent regions of PAG, which is not internalized), it remained possible that raft localization is necessary. An attempt was made to disrupt LAB localization to rafts by mutating the cysteine residues that are the sites of palmitoylation on the molecule. The mutations caused a complete defect in transport of LAB to the cell surface, making it impossible to use these cells to track the internalization of LAB from the plasma membrane (data not shown). In an alternate approach, the cytoplasmic domain of LAB was fused to the extracellular, transmembrane and juxtamembrane residues of LAX (Fig. 6A). LAX does not contain a juxtamembrane CXXC motif for palmitoylation and thus localizes to non-raft membranes (47). As shown in Fig. 6B, truncated LAXTR-GFP and LAX-LAB-GFP were both detected in the detergent-soluble fractions 7 and 8. Truncated LAXTR-GFP co-capped with the BCR but did not internalize (Fig. 6C); however, the LAX-LAB-GFP chimera co-capped and co-internalized with the BCR following BCR stimulation (Fig. 6), suggesting that raft localization may not be essential.


Figure 6
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  		Fig. 6 Raft localization may not be required for internalization of LAB. The configuration of LAXTR-GFP and LAX-LAB-GFP constructs is illustrated in (A). The LAXTR-GFP chimeric protein comprises amino acid residues 1–68 of LAX ligated to the N-terminus of GFP. The cytoplasmic domain of LAB (residues 36–243) was fused to the truncated LAX sequence. (B) The raft localization of the constructs was checked by sucrose density gradient centrifugation of 1% Triton lysates as described in Fig. 1. (C) BJAB cells transiently transfected with LAXTR-GFP or LAX-LAB-GFP were labeled with F(ab')2 anti-IgM(Cy3) either following fixation or at 37°C for 10 min prior to fixation. Note that LAXTR-GFP co-capped with the BCR on the cell surface following stimulation but did not internalize, whereas LAX-LAB-GFP internalized. Results are representative of at least 90% of >100 cells in at least three independent experiments for each construct.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
This is the first report of receptor-activated endocytosis of a transmembrane adaptor protein. An intracellular pool of LAT has been described, but in contrast to LAB, the endocytic pool of LAT is present in resting cells and is recruited to the plasma membrane following TCR engagement (48). Internalization of LAB is driven by its cytoplasmic domain, which includes a number of motifs implicated in endocytosis mediated by clathrin or ubiquitin (49), and may not absolutely require localization to rafts. Truncated LAB, comprised of only the transmembrane/juxtamembrane regions, co-capped with BCRs following stimulation but remained on the cell surface and could not direct internalization of the cytoplasmic domain of PAG (in the LAB-PAG chimera). On the other hand, the cytoplasmic domain of LAB was able to direct internalization of the transmembrane/juxtamembrane regions of both PAG (PAG-LAB-GFP) and LAX (LAX-LAB-GFP). LAX-LAB-GFP remained in the Triton-soluble fractions of sucrose gradients even after BCR stimulation (data not shown), suggesting that raft localization is not required for its internalization. However, we cannot exclude the possibility that LAX-LAB-GFP might be localized to raft membranes defined by other criteria.

CD20, like PAG, is not internalized after BCR stimulation (27) and data shown here suggest that it resides in the same rafts as PAG; the latter result is consistent with our previous detection of PAG in CD20 immunoprecipitates (50). We speculate that segregation of LAB into different rafts facilitates its selective internalization (leaving PAG and CD20 on the cell surface), even though rafts may not be mechanistically essential for LAB's internalization. Additionally, it remains possible that localization of LAB and PAG to distinct rafts compartmentalizes their unique signaling functions. In order to test these ideas, it would be necessary to redirect LAB and PAG to different rafts, and this will require an understanding of the sequence information that controls their distinct raft localization. We also examined the localization of BCRs in DRMs after stimulation and found very low co-localization with both PAG-GFP and CD20-GFP (data not shown). This result was expected based on our data showing that PAG and CD20 co-localize in microdomains and that PAG does not internalize with the BCR. We have also previously shown that after receptor stimulation BCRs and CD20 are in distinct DRMs (27). Unexpectedly, however, BCRs also did not co-localize with LAB-GFP in DRMs. From these results, it appears that BCRs and LAB internalize in distinct microdomains that remain predominantly associated in the same intracellular aggregates. The nature and composition of the microdomains harboring BCRs requires further study.

The effect of LAB deficiency on internalization of engaged BCRs indicates a role for LAB in mediating this complex process. Previous analyses revealed no negative effects of LAB deficiency in signaling or cellular functions of murine B cells (12). If anything, there was a slight enhancing effect on calcium influx, B cell proliferation and humoral immune responses to low doses of antigen. Our results are thus the first indication of a specific role for LAB in primary B cells and could explain the slight enhancing effects of LAB deficiency, as internalization of engaged receptors down-regulates signaling from the BCR (24, 26, 51). However, the precise signaling role of LAB in BCR internalization is not yet clear. BCR internalization occurs primarily through clathrin-coated pits in concert with re-organization of the actin cytoskeleton (23, 24, 26, 34). A role for rafts in BCR internalization is controversial, but where it has been shown to occur, there is a requirement for either clathrin or actin (26). LAB could thus be a component of a BCR-activated signaling pathway leading to either clathrin or actin re-organization. A potential function of LAB in one of these pathways is currently under investigation.

The requirement for LAB in BCR internalization does not readily explain why it is itself internalized after receptor stimulation. One possibility is that it is internalized for rapid degradation. LAB was shown to be polyubiquitylated after BCR signaling (7); however, we observed no loss of LAB protein for at least an hour after signal initiation. An alternative explanation is that internalized LAB may be involved in intracellular signaling events, perhaps those regulating BCR trafficking to MHC-rich late endosomes (22). Although intracellular signaling by other receptors has been clearly documented (52), there is limited evidence for signals from the BCR continuing after internalization. Several reports show that preventing BCR internalization enhances or prolongs tyrosine kinase activation, calcium mobilization and ERK phosphorylation (24, 26, 51), consistent with the generally accepted idea that BCR internalization attenuates signaling. However, it remains possible that specific signaling events are initiated after internalization of the BCR. Indeed, Niiro et al. (31) found that BCR-activated phosphorylation of JNK was reduced when BCR internalization was blocked. Interestingly, Bam32 regulates both BCR internalization and JNK activation, and, like LAB, co-localizes with internalized BCRs (31). It will be important to determine whether LAB and Bam32 function together in an intracellular signaling activity. Finally, a non-exclusive possibility is that LAB is internalized as part of the means by which it regulates internalization of engaged BCRs. An understanding of how it contributes to receptor internalization should help to resolve this issue.


   Acknowledgements
 
This study was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) and by personnel awards from the Alberta Heritage Foundation for Medical Research to J.P.D. (Heritage Senior Scholar), C.M.M. (Heritage Graduate Studentship) and L.G. (Heritage Summer Studentship) and the Natural Sciences and Engineering Research Council of Canada (C.M.M.). The Live Cell Imaging Facility at the University of Calgary is supported by CIHR. The authors gratefully acknowledge the expert assistance of Pina Colarusso, Manager of the Live Cell Imaging Facility, the operating room staff at the Alberta Children's Hospital for their help in providing tonsil tissue, Elizabeth Long and Alex Klimowicz for helpful comments on the manuscript and André Veillette, Masato Okada, Burkhart Schraven and Luc Berthiaume for their generous gifts of antibodies.


   Abbreviations
 
BCR, B cell receptor
Cbp, Csk-binding protein
CIHR, Canadian Institutes of Health Research
DRM, detergent-resistant membrane
FBS, fetal bovine serum
GFP, green fluorescent protein
LAB, linker for activation of B cell
LM, light membrane
MCS, multiple cloning site
MFI, mean fluorescence intensity
NTAL, non-T cell activation linker
PAG, phosphoprotein associated with glycophospholipid-enriched membrane microdomain
PKC, protein kinase C
PLC, phospholipase C
TRAP, transmembrane adaptor protein

   Notes
 
Transmitting editor: C. Paige

Received 13 July 2006, accepted 5 October 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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M. J. Polyak, H. Li, N. Shariat, and J. P. Deans
CD20 Homo-oligomers Physically Associate with the B Cell Antigen Receptor: DISSOCIATION UPON RECEPTOR ENGAGEMENT AND RECRUITMENT OF PHOSPHOPROTEINS AND CALMODULIN-BINDING PROTEINS
J. Biol. Chem., July 4, 2008; 283(27): 18545 - 18552.
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