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The Journal of Immunology, 2002, 168: 5612-5620.
Copyright © 2002 by The American Association of Immunologists

AP2 Adaptor Complex-Dependent Internalization of CD5: Differential Regulation in T and B Cells1

Xianghuai Lu2,*, Robert C. Axtell*, James F. Collawn{dagger}, Andrew Gibson*, Louis B. Justement{ddagger} and Chander Raman3,*

* Division of Clinical Immunology and Rheumatology, Departments of Medicine, {dagger} Cell Biology, and {ddagger} Microbiology, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD5 is a key regulator of Ag receptor-mediated activation, selection, and differentiation in both T and B cells. Accumulating evidence indicates that lymphocyte activation and selection are sensitive to variations in levels of CD5 on the cell surface. We now show that CD5 expression on the surface of B and T cells is regulated posttranslationally by direct interaction with the µ2 subunit of the AP2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD5 is rapidly internalized from the cell surface in lymphoid cell lines, mature splenic T and B cells, and peritoneal CD5+ B cells following monovalent or bivalent ligation of the receptor. We mapped the µ2 subunit binding site on CD5 to Y429 and determined that the integrity of this site was necessary for CD5 internalization. Cross-linking of the Ag receptor with intact Abs inhibited CD5 internalization in B cells, but had the opposite effect in T cells. However, if F(ab')2 Abs were used to stimulate the Ag receptor in B cells, the effect on CD5 internalization was now similar to that observed in T cells, indicating that signals through the Ag receptor and FcR regulate CD5 endocytosis in B cells. This was confirmed using an Fc{gamma}RIIB1-deficient B cell line. The ability to differentially alter posttranslational CD5 expression in T and B cells is likely to be key in regulation of Ag receptor signaling and generation of tolerance in T and B lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD5 is a 67-kDa glycoprotein belonging to the scavenger receptor family of proteins that is constitutively expressed on all T cells and a subset of B cells known as B1a B cells (1, 2, 3, 4). Structurally, CD5 is closely related to CD6, each having three extracellular scavenger receptor cysteine-rich (4) domains (5). Although the identity of a CD5 counter receptor/ligand remains controversial, potential ligands include CD72 (6), gp40–80 (7, 8), and Ig-framework region sequences (9). A good candidate for a CD5 ligand is a 150-kDa glycoprotein that is expressed on monocytes, lymphocytes, and from proteolytic cleavage in serum (10).

CD5 is associated both physically and functionally with the Ag receptor complexes in both T and B cells. Based on data from CD5-deficient mice, it has been suggested that CD5 exerts a net negative effect on TCR signaling in T cells (11, 12). Immature T cells in CD5-/- mice are hyperresponsive to TCR stimulation and exhibit altered positive and negative selection (11). However, other studies suggest that CD5 stimulation enhances negative selection in CD4+ or CD8+ single positive semimature thymocytes (13, 14). In mature T cells, CD5 is primarily considered to be a costimulatory molecule. Costimulation experiments indicate that CD5 signaling is synergistic with TCR/CD3 or CD28 stimulation in T cells from mice or humans (15, 16, 17).

In contrast to T cells, very little is known regarding the function of CD5 in B cells. As with T cells, the data indicate that the effect of CD5 on B cell Ag receptor (BCR)4-mediated signaling can be inhibitory or costimulatory. The study showing that B1a (CD5+) B cells can be induced to proliferate in response to anti-µ stimulation when CD5 is sequestered away from the BCR or deleted as in B1 B cells from CD5-deficient mice indicates an inhibitory role for CD5 (18). This conclusion is also supported by the observation that coligation of CD5 and BCR leads to inhibition of calcium mobilization, and this inhibitory effect requires the pseudo-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of CD5 (19). However, B cell expansion and splenic hyperplasia associated with elevated CD5 expression as in aged CD5 or CD19 transgenic mice is consistent with a stimulatory role for CD5 in the B lineage (20, 21). Overall, these data suggest that the net effect of CD5 signaling is likely to be contextual and dependent on the differentiation and/or activation stage of the cell.

In both the B and T cell compartments, the expression of CD5 in vivo is directly associated with strength of Ag receptor-induced intracellular signals (22, 23, 24). CD5 expression and the frequency of CD5+ B cells is elevated in mice carrying naturally occurring or induced mutations that enhance the strength of BCR-induced intracellular signals (21, 25, 26, 27). In contrast, mutations that lead to diminished ability to signal via the BCR result in either a dramatic decrease in the number of CD5+ B cells or a complete loss of this population (28, 29). Similarly, the level of CD5 expressed on T cells is altered in mice that have mutations that affect the strength of TCR/CD3-mediated signals (30). These observations suggest that the regulation of CD5 expression is likely to be a key event in the maintenance of homeostasis in these lymphoid compartments. Although CD5 expression is regulated at the transcriptional level (31), it appears that posttranslational processes also regulate the expression of CD5 (20). The posttranslational regulation of CD5 expression is likely to be involved in the regulation of early signaling initiated by Ag receptor ligation in T and B cells.

Using intact mAb, F(ab')2, or Fab of anti-CD5 mAb to treat T and B cells, we show that CD5 is rapidly removed from the cell surface at a rate significantly greater than constitutive turnover of the receptor. To identify the proteins that may be involved in this process, we used the yeast two-hybrid system and screened a human PBL library with the cytoplasmic tail of CD5 as "bait". Using this approach, we determined that the µ2 (AP50) subunit of the AP2 adaptor complex associates with CD5. This finding was confirmed based on coimmunoprecipitation experiments using cell lines. Subsequently, the AP2 binding site was mapped to a tyrosine-based internalization motif, which contains the first tyrosine of the pseudo-immunoreceptor tyrosine-based activation motif, and we determined that this motif was required for CD5 internalization. We show that CD5 internalization was enhanced by Ag receptor signaling in primary T cells, but inhibited in B cells. The inhibition of CD5 internalization in B cells was dependent on coligation of FcR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6-CD5 transgenic mice (20) were a gift from J. F. Kearney (Department of Microbiology, University of Alabama, Birmingham, AL) and BALB/C and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were used at 9–16 wk of age. The CD5 gene in CD5 transgenic mice is under the control of the IgH chain promoter and enhancer.

Cell lines and reagents

The human T leukemia cell line, Jurkat, murine T cell line, EL4, and B cell lines, A20 and IIA1.6, were maintained in RPMI 1640 medium supplemented with 10% FCS (Life Technologies, Gaithersburg, MD). COS-7 cells were grown in DMEM supplemented with 10% FBS. Monoclonal anti-{alpha}-adaptin (AP-2{alpha}, clone 100/2) and mouse IgG1 (MOPC21) were obtained from Sigma-Aldrich (St. Louis, MO). PE anti-human CD5 (UCHT2), anti-human CD3 (UCHT1), PE anti-mouse CD90.2 (Thy 1.2, G7), FITC anti-mouse CD19 (1D3), biotin anti-mouse IgG1 (Igh-4a), and streptavidin-allophycocyanin were obtained from BD PharMingen (San Diego, CA). Anti-CD3 (145-2C11) and anti-CD5 (53-7.313) mAb were purified from hybridoma cells obtained from American Type Culture Collection (Manassas, VA) and purified by National Cell Culture Center (Minneapolis, MN). Biotinylated anti-CD5 and anti-CD22 was generated using EZ-Link Biotinylation kit from Pierce (Rockford, IL). Anti-µ (B76) and anti-CD22 (Cy34.1) were purified from culture supernatants. Fab anti-CD5 was prepared as per protocol using the ImmunoPure Fab Preparation kit from Pierce and labeled with Alexa 488 (Molecular Probes, Eugene, OR). The purity of the Fab preparation was confirmed by SDS-PAGE analysis under nonreducing conditions and reducing conditions followed by silver staining. Routine SDS-PAGE analysis also established that both conjugated and unconjugated Fab preparations did not aggregate during storage. F(ab')2 anti-CD5 was prepared using immobilized pepsin from Pierce and then conjugated to Alexa 488. The purity of F(ab')2 was confirmed by reducing and nonreducing SDS-PAGE analysis. LY294002 was obtained from Calbiochem (La Jolla, CA), protein A-agarose and protein G-agarose were from Life Technologies, and SuperSignal chemiluminescence substrate was obtained from Pierce. Peroxidase-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA).

Yeast two-hybrid screen

The yeast two-hybrid screen was performed using the GAL4 binding domain (BD) CD5 cytoplasmic domain (BD-CD5) fusion construct as described previously (32).

Constructs

To generate BD-CD5 deletion mutants, we used the QuikChange mutagenesis kit from Stratgene (La Jolla, CA) as described previously (32). The eight primers used to generate all the mutations are as follows: 1) 5'-CTCCGACAGTGACGCTGATCTGCATGG-3', 2) 5'-CCATGCAGATCAGCGTCACTGTCGGAG-3', 3) 5'-AGCTCTTCAGTCTCGGACGGTTGCCGT-3', 4) 5'-TCCTCTTCTGACTATGATCTGCATGGG-3', 5) 5'-CTAGTGAAGTAATTCCGCCAG-3', 6) 5'-CTGGCGGAATTACTTCACTAG-3', 7) 5'-CAGGAACTCCTGACTGTAAGCTTATCCAG-3', 8) 5'-CTGGATAAGCTTACAGTCAGGAGTTCCTG-3'. The BD-CD5-463Xconstruct was generated using primers 1 and 2, the BD-CD5{Delta}423-461 construct was generated using primers 3 and 4, the BD-CD5-384X construct was generated using primers 5 and 6, and the BD-CD5-437X construct was generated using primers 7 and 8. Primers 5 and 6 were also used to generate pCDNA3 CD5-384X and primers 7 and 8 were used to generate pCDNA3 CD5-437X. To generate pCDNA3 CD5 Y429F, Y441F, and Y463F (3Y-F), six primers were used as follow: 1) 5'-GACAGTGACTTTGATCTGCA-3', 2) 5'-TGCAGATCAAAGTCACTGTC-3', 3) 5'-CCCTTCCAGAGCTGGAAAAGCTGCAGG-3', 4) 5'-CCTGTCAGCTTTTCCAGCTCTGGAAGGG-3', 5) 5'-GGGAGGTTGGCTGAATTCGTTATCCACGTG-3', 6) 5'-CACGTGGATAACGAATTCAGCCAACCTCCC-3'. This construct was made sequentially by generating Y429F, then Y441F, and finally, Y463F. To generate GST CD5 fusion protein, the CD5 cytoplasmic domain DNA was amplified using sense primer 5'-GTGGATCCCCGAAATTCCGCCAG-3' and the antisense primer 5'-CTGGCGGAATTTCGGGGATCCAC-3', and cloned in frame into the pGEX-4T-3 vector (Amersham Pharmacia Biotech, Piscataway, NJ). The absence of PCR-introduced artifacts, and the presence of desired nucleotide changes were established by bidirectional nucleotide sequencing using dye terminator chemistry (Applied Biosystems, Foster City, CA). Fusion proteins were prepared using B-PER Bacterial Protein Extraction Reagent (Pierce) as directed by the manufacturer. The fusion proteins were analyzed by SDS-PAGE, and the preparations containing a single band of the appropriate molecular mass were used for subsequent experiments. Protein concentration was quantitated using the Bio-Rad protein assay (Bio-Rad, Hercules, CA).

Transfections

COS-7 cells (1 x 106) were seeded into 100-mm dishes, and 24 h later the cells were transfected with 4 µg of plasmid DNA/dish using the LipofectAMINE Plus kit (Life Technologies) according to the manufacturer’s instructions. For generation of stable transfectants, transfected COS-7 and Jurkat cells were selected using 700 µg/ml and 1 mg/ml Geneticin (Life Technologies), respectively. The transfectants were sorted for equivalent expression after staining with anti-CD5 or anti-CD8 Abs. In some experiments, COS-7 cells were analyzed 48 h following transfection in the absence of any selection (transient transfection).

Internalization assay

To measure CD5 internalization, we used a modified flow cytometric internalization assay (33, 34). Briefly, cells (1 x 106/sample) were incubated with PE-conjugated intact anti-human or anti-mouse CD5 mAb (1 µg/ml) for 15 min at 4°C. Following two washes to remove unbound Ab, one set of samples was rapidly warmed to 37°C, while the other was left at 4°C. At different time points (0–30 min), cells were chilled rapidly and all samples were stained with biotin-conjugated anti-human or anti-mouse IgG followed by streptavidin-allophycocyanin staining to detect anti-CD5 mAb remaining on the surface of the cells. All samples were analyzed in a flow cytometer (FACSCalibur; BD Biosciences, Mountain View, CA) to detect PE (FL2) and allophycocyanin (FL4) fluorescence (mean fluorescence intensity; MFI). The percent endocytosis was calculated using MFI values of allophycocyanin (FL4) channel as follows: 100 - [(MFI at 37°C/MFI at 4°C) x 100]. In experiments that assessed internalization using Fab of anti-mouse CD5 mAb or when stimulation was involved, the initial wash to remove excess Ab was skipped. The cells were exposed to excess Ab throughout the assay. This was necessary because prebound Fab anti-CD5 dissociated from cell surface at 37°C. For stimulation, cells were treated with anti-CD3 (145-2C11), anti-µ (B76), or F(ab')2 fragment of goat anti-mouse µ as indicated, followed by the addition of intact anti-CD5 or Fab for an additional 15 min. In some experiments, CD5 internalization was assessed in the presence of brefeldin A (BFA) at 10 µg/ml or cyclohexamide (50 µg/ml) as described by Liu et al. (35).

The use of Alexa 488 (FL1) or PE-labeled (FL2) anti-CD5 mAb provided an internal control for loss of Ab from cell surface, since these dyes are detectable within cells even under acidic conditions, as might be expected in lysosomal vesicles. The MFI of total CD5 did not change throughout the treatment period, establishing that decrease in anti-CD5, as detected by anti-human or anti-mouse IgG, was a function of loss of CD5 from cell surface by internalization rather than dissociation from cell surface pool of CD5.

Immunoprecipitation and immunoblotting

Cells (2 x 107) were lysed in 1 ml of lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% v/v Nonidet P-40, 1 mM Na3VO4, 10 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. The cell debris was removed by centrifugation. For GST coprecipitations, GST and GST fusion proteins were incubated with lysates, and then precipitated by the addition of glutathione agarose (Amersham Pharmacia Biotech) equilibrated in lysis buffer containing 1 mg/ml of BSA. For coimmunoprecipitation, lysates were incubated with anti-human CD8 (OKT8) followed by precipitation with protein G-agarose. All the precipitates were separated by SDS-PAGE gel electrophoresis. Proteins were transferred onto nitrocellulose membranes and probed with monoclonal anti-{alpha}-adaptin (AP-2{alpha}) followed by peroxidase-conjugated goat anti-mouse IgG and SuperSignal chemiluminescence substrate. The filters were then stripped (2% SDS, 62.5 mM Tris-HCl (pH 6.8), 100 mM 2-ME) for 30 min at 60°C and reprobed with rabbit antiserum to CD5 cytoplasmic tail (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD5 internalization induced by receptor ligation

Previous studies have shown that Abs to CD5 induce internalization of CD5 in T and B cell lines and human PBL; however, the mechanism responsible for CD5 internalization has not been defined (36, 37, 38). We measured CD5 internalization in Jurkat cells and in COS-7 cells transfected with human CD5 following incubation with a fluorescent-tagged anti-CD5 Ab (Fig. 1GoA). The kinetics of CD5 internalization in both cell lines showed that endocytosis peaked within 15 min after transfer of cells from 4 to 37°C. To determine whether CD5 is internalized in normal cells, endocytosis was assessed in mature splenic T cells, peritoneal B1a B cells, and splenic B cells from CD5 transgenic mice following incubation with fluorescent-tagged intact anti-mouse CD5 mAb. In each case, CD5 was rapidly internalized, reaching a plateau within 20 min similar to that observed in cell lines (Fig. 1GoB and data not shown). Internalization of CD5 following incubation with F(ab')2 anti-CD5 was comparable to that obtained with intact anti-CD5 (Fig. 1GoB). To determine whether dimerization or cross-linking of CD5 was necessary to induce internalization of CD5, we generated Fab of anti-CD5. We observed that in both T cells and B cells, monovalent ligation with Fab anti-CD5 was sufficient to induce rapid internalization of CD5 (Fig. 1GoB). The observed internalization of CD5 was not due to dissociation of anti-CD5 mAb from extracellular CD5 or due to small amounts of aggregated Fab anti-CD5 (see Materials and Methods).



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  		FIGURE 1. Ab-dependent internalization of CD5. A, Kinetics of CD5 internalization using intact anti-CD5 mAb in Jurkat cells ({blacktriangleup}) and human CD5-expressing COS-7 cells ({blacksquare}). Data represent mean ± SD from three independent experiments. B, CD5 internalization in mouse splenic T and B cells and peritoneal B cells. Percentage of endocytosis of CD5 was determined in mature T and B cells from mouse spleen incubated with intact, F(ab')2 or Fab anti-mouse CD5 for 15 min at 37°C. Endocytosis of CD5 in peritoneal B cells (Perit. B) was measured after 15 min incubation with intact anti-CD5 only. CD5 expressing mature splenic B cells were obtained from CD5 transgenic mice. Data represent mean ± SD of triplicate values from a representative experiment (n = 5).

 
The internalization of CD5 following Ab binding may represent constitutive turnover of CD5 from cell surface rather than ligand-induced endocytosis because our assay does not distinguish between these processes. To directly assess the constitutive turnover rate of CD5, we used BFA that blocks expression of newly synthesized proteins by inhibiting transport of proteins from endoplasmic reticulum (ER) to Golgi and also reexpression (recycling) of endocytosed proteins by causing fusion of early endosomes with the trans-Golgi network (39, 40). The protein synthesis inhibitor, cyclohexamide, was used to distinguish between effects of BFA on ER to Golgi transport and endosomes trans-Golgi network fusion. The transferrin receptor (CD71) is known to be rapidly recycled and its expression was reduced by ~50% within 2 h following BFA treatment (Fig. 2Go and Ref. 41). Some of the reduction in surface expression is a result of blockade in transport of newly synthesized protein from ER to Golgi as demonstrated by cyclohexamide treatment. In contrast to transferrin receptor, the turnover rate of CD5 was very slow from both T and B cells (Fig. 2Go). Treatment of lymphocytes with BFA or cyclohexamide separately or together did not significantly reduce surface expression of CD5 throughout the 4-h time period of the assay, indicating that the cells surface expression of CD5 is relatively stable. The expression of CD45 and Thy1 were slightly reduced by either BFA or cyclohexamide, showing that the turnover rate of these proteins was higher than that of CD5, but still lower than that of CD71, and is consistent with a previous report (35). The rapid loss of CD5 expression from the cell surface after incubation with monovalent and bivalent Ab in the absence of high constitutive turnover demonstrates that CD5 is actively endocytosed following receptor ligation and this process does not require receptor dimerization.



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  		FIGURE 2. Slow constitutive turnover of cell surface CD5. Changes in surface expression of CD5, CD45, CD90 (Thy-1), and CD71 (transferrin receptor) were assessed following treatment with BFA (10 µg/ml) or cyclohexamide (50 µg/ml). Percentage of surface expression was calculated by determining the ratio of MFI of treated cells compared with MFI of untreated cells at each time point and extrapolated as a percentage. Data represent mean ± SD of triplicate determination from a representative experiment (n = 3).

 
Effect of BFA on Ab-mediated endocytosis of CD5

CD5, once internalized, may traffic to lysosomal vesicles for degradation or be recycled back to the surface. If CD5 is recycled following endocytosis, we would predict that loss of surface CD5 following receptor ligation would be much greater in the presence of BFA that also inhibits reexpression of endocytosed proteins. We observed that decrease in surface CD5 levels in T cells induced by intact anti-CD5, F(ab')2 anti-CD5 or Fab anti-CD5 was similar in the presence or absence of BFA (Fig. 3Go). In B cells, there was a general trend of greater reduction of CD5 in the presence of BFA and the difference was significant if Fab anti-CD5 was used to induce internalization of CD5. Overall, the results suggest that the majority of CD5 is trafficked to lysosomal degradation pathway rather than recycled back to the cell surface.



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  		FIGURE 3. Effect of BFA on Ab-dependent internalization of CD5. Internalization of CD5 in T and B cells was determined 15 min following incubation with intact F(ab')2 or Fab anti-CD5 at 37°C in the presence or absence of BFA (10 µg/ml). Data represent mean ± SD of triplicate values from a representative experiment (n = 3).

 
The cytoplasmic tail of CD5 associates with the AP2 adaptor complex

The kinetics of CD5 internalization and the ability of monovalent ligands to induce CD5 internalization suggest that CD5 modulation does not result from homeostatic turnover of the plasma membrane but rather is an active response facilitated by an associated molecule. To identify the protein(s) that may be involved in this process, we used the yeast two-hybrid system and screened a human GAL4 activation domain (AD) PBL library with the GAL4 DNA-BD fused to the cytoplasmic tail of CD5 as bait. One cDNA isolated from the screen had a sequence identical with the µ2 subunit (also known as AP50) of the AP2 adaptor complex (42). This cDNA represented 20% of 500 potential CD5 interacting clones (32). None of the AD-µ2 clones sequenced were full-length. The longest cDNA clone represented aa 2–436, and the shortest represented aa 121–436, indicating that the first 121 aa were not necessary for interaction with CD5 (Fig. 4GoA). To determine the specificity of the interaction, we selected two transmembrane proteins, Ig{alpha} and PirB, that contain potential tyrosine-based internalization motifs in their cytoplasmic tail (42). BD-cytoplasmic tail fusion constructs of these proteins were unable to interact with AD-µ2 (clone AD-489; Table IGo). Similar results were obtained with another AD-µ2, clone AD-500 (data not shown).



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  		FIGURE 4. CD5 interacts with AP2 adaptor complex. A, Schematic diagram of representative human µ2 cDNA and CD5-interacting µ2 cDNA clones isolated from the yeast two-hybrid screen. Solid lines represent identity to human µ2 (H-µ2) nucleotide sequence. Dotted lines represent nucleotide sequence of GAL4 AD. Numbers above solid lines indicate amino acid positions relative to human µ2. B, Coimmunoprecipitation of AP2{alpha} with CD5. Lysates from transfected Jurkat cells expressing chimeric CD8-CD5 protein were immunoprecipitated with anti-CD8 mAb (OKT8). The presence of AP2 was detected in Western blots using anti-AP2{alpha} mAb (100/2). The blot was stripped and reprobed with polyclonal anti-CD5. Only chimeric protein was detected, since the lysates were immunoprecipitated with anti-CD8. The bands representing AP2{alpha}, CD8-CD5 chimeric protein, and H chain are indicated. C, AP2 interacts with Y429 in CD5 cytoplasmic tail. GST-CD5 cytoplasmic tail fusion proteins representing wild-type CD5 (GST-WT), Y429F mutant CD5 (GST-3Y-F), and Y429A mutant CD5 (GST-Y429A) were used to precipitate AP2 from Jurkat cell lysates, and the blot was probed with anti-AP2{alpha} mAb as described (B). GST protein was used as control. A parallel Coomassie stained 12% SDS-PAGE (lower panel) of the three fusion proteins and GST control show that equivalent protein was used for each of the precipitations.

 

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  		Table I. Specificity of interaction between AD-AP50 and BD-CD5

 
The AP2 adaptor complex is a heterotetramer consisting of two large subunits ({alpha}2 and {beta}2), an intermediate subunit (µ2), and a small subunit ({delta}2) (42). None of the subunits exist within cells as free molecules. To determine whether the association of the AP2 adaptor complex with CD5 could be demonstrated in mammalian cells, we performed coimmunoprecipitation experiments. For these studies, we used Jurkat cells that were transfected with chimeric CD8-CD5 protein in which the extracellular region was human CD8 and the transmembrane and cytoplasmic tail are from CD5. Nonidet P-40 lysates from CD8-CD5 expressing and untransfected Jurkat cells were immunoprecipitated with anti-CD8 mAb (OKT8) and Western blots were probed with anti-AP2{alpha} mAb (clone 100/2). A band representing AP2{alpha} was found only in the lane representing lysates from CD8-CD5-expressing Jurkat cells, but not untransfected Jurkat cells (Fig. 4GoB). Because Jurkat cells do not express endogenous CD8, the use of CD8-CD5-expressing Jurkat cells provided an internal control for nonspecific coprecipitation of AP2 by anti-CD8. The ability of AP2 to interact with CD5 was also demonstrated by coprecipitation of AP2 by GST-WT CD5, but not GST alone from Jurkat cell lysates (Fig. 4GoC). Taken together these data demonstrate that the AP2 adaptor complex specifically interacts with the cytoplasmic tail of CD5, and that the association is mediated by the µ2 subunit.

The AP2 binding site maps to Y429 in the CD5 cytoplasmic tail

The AP2 adaptor complex binds to internalization motifs in the cytoplasmic tail of transmembrane proteins and mediates recruitment to clathrin-coated pits (42). The internalization sequences are comprised of either tyrosine-based motifs, NPxY and Yxx[/{phi}], or di-leucine (LL) motif. CD5 cytoplasmic tail has four tyrosines, three of which have the consensus motif Yxx[/{phi}], Y378, Y429, and Y441. Nevertheless, Y378 is an unlikely candidate since it represents either the last amino acid of the transmembrane domain, or the first amino acid of the cytoplasmic tail. To determine which of these tyrosines mediate the interaction with µ2, we generated a panel of truncation and deletion mutations in the CD5 cytoplasmic tail that lack one or more of these motifs and tested them in the yeast two-hybrid assay (Fig. 5GoA). We found that while the deletion of Y463 did not affect the ability of µ2 to interact with CD5, in contrast, BD-CD5 constructs that lacked both Y429 and Y441 with unaltered Y378 exhibited no interaction with µ2. This result indicated that Y429, Y441, or both were involved in the interaction with CD5. To identify which of these tyrosines comprise the binding site of µ2, we generated a truncated BD-CD5 construct, BD-CD5-437X, which lacked Y441 and tested its ability along with controls to interact with µ2 in the yeast two-hybrid assay. Based on a liquid lacZ assay, we found that BD-CD5-437X interacted as efficiently as BD-CD5-463X mutant and with slightly lower levels of lacZ than "wild-type" BD-CD5 (Fig. 5GoB). This observation suggested that the tyrosine most likely to be involved in the interaction with µ2 was Y429. To test this directly, we generated two additional mutants, BD-CD5{Delta}429-433 (deletes Y429-P433) and BD-CD5-Y429A. Both of these constructs when cotransformed with AD-µ2 (clone AD489) into yeast cells exhibited very low lacZ activity comparable to that obtained BD-CD5-384X, which lacks most of the cytoplasmic tail (Fig. 5GoB). The yeast two-hybrid data correlated with the ability to coprecipitate AP2{alpha} from Jurkat cell lysates with wild-type, but not Y429A GST fusion proteins of CD5 cytoplasmic tail (Fig. 4GoC). Mutation of all three tyrosines to phenylalanine (GST-3Y-F) did not affect the ability to coprecipitate AP2{alpha}. Overall, these data confirm that Y429 is required for the interaction with the AP2 adaptor complex.



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  		FIGURE 5. Mapping of AP2 µ2 binding site on CD5. A, Wild-type BD-CD5 and deletion/truncation constructs were screened for interaction with AD-µ2 clone, AD489, in the yeast two-hybrid assay for growth on histidine-deficient plates. B, Wild-type BD-CD5 and deletion/truncation/substitution constructs were screened for interaction with AD-µ2 clone, AD489, in a yeast two-hybrid assay for lacZ activity. Data represent mean ± SD of triplicate values from a representative experiment (n = 3)

 
Y429 is associated with ligand-dependent and -independent CD5 internalization

Mapping studies using the yeast two-hybrid assay established that Y429 was necessary for the interaction with µ2. To determine whether this interaction is important for CD5 internalization, we generated a panel of human CD5 mutant constructs in which the Y429 codon was deleted or substituted with that encoding phenylalanine or alanine. We transiently transfected wild-type and mutant CD5 encoding constructs into COS-7 cells and measured their ability to be internalized following ligation with anti-CD5 mAb (Fig. 6Go). Within 15 min, 40% of wild-type CD5 had been internalized. In contrast, the internalization was inhibited by ~70% in CD5 mutants that lack Y429 (CD5{Delta}429-433 and CD5-384X). Mutation of all three tyrosines in the cytoplasmic tail of CD5 to phenylalanine (CD5–3Y-F) did not alter CD5 internalization, but mutation of Y429 to alanine dramatically inhibited CD5 endocytosis (Fig. 6Go). The ~10–12% internalization observed with CD5 constructs containing Y429A mutation or lacking Y429 occurred rapidly (5 min) and remained at the same level up to 30 min (data not shown).



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  		FIGURE 6. Tyr429 is necessary for CD5 internalization. CD5 internalization was measured in COS-7 cells transiently transfected with constructs that encode wild-type or deletion/substitution mutants of CD5. Internalization was assessed 15 min after incubation with intact anti-CD5 at 37°C. Data represent mean ± SD of triplicate transfections from a representative experiment (n = 3).

 
The cell surface expression of CD5 on COS-7 cells transiently transfected with constructs in which Y429 was mutated to alanine or deleted was consistently higher than those transfected with wild-type CD5 (data not shown). This result was confirmed by transfection with different clones containing the same mutation and/or different preparations of DNA. These data suggested to us that the AP2 adaptor complex and Y429 may also be involved in ligand-independent down modulation of CD5, thereby regulating steady state expression of cell surface CD5. To directly test this possibility, COS-7 cells were transiently transfected with wild-type and CD5Y429A constructs and 48 h later, the constitutive turnover rate of CD5 was determined using BFA and cyclohexamide. The surface expression of wild-type CD5 was slightly reduced (9%) within 30 min, and by 1 h the level of expression was 77.5% of BFA untreated cells (Fig. 7Go). In contrast, BFA treatment resulted in only slight decrease in surface expression of Y429A mutant CD5 over the 2-h period of the assay. The loss of surface expression of wild-type CD5 was largely a result of blockade in recycling of CD5 since cyclohexamide treatment only marginally reduced surface CD5 expression. The kinetics of CD5 reduction from cell surface following cyclohexamide treatment was similar for wild-type and Y429A CD5 protein, indicating that the half lives of both these proteins were similar and independent of AP2 binding (Fig. 7Go). Based on these data, we conclude that the AP2 binding site, Y429, is necessary for ligand-dependent and -independent CD5 internalization.



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  		FIGURE 7. Tyr429 is associated with regulation of constitutive turnover of CD5. CD5 expression on cell surface was measured at various time points in COS-7 cells transiently transfected with wild-type ({square}) or Y429A ({circ}) CD5 constructs in the presence or absence of BFA or cyclohexamide as described in Fig. 2Go. Data represent mean ± SD of triplicate transfections from a representative experiment (n = 3).

 
Effect of TCR or BCR cross-linking on CD5 internalization

Previous studies showed that the µ2 subunit of the AP2 adaptor complex is unable to bind to tyrosine-based internalization motifs in which the critical tyrosine is phosphorylated (43, 44, 45). Those biochemical and cell biological studies were confirmed when the crystal structure of the µ2 subunit bound to peptide was solved showing that the substrate-binding pocket of the µ2 subunit lacks the space for binding of tyrosine-based internalization motifs in which the tyrosine is phosphorylated (46). Ag receptor cross-linking in both B and T cells induces rapid tyrosine phosphorylation of CD5 (22, 24). Therefore, we tested the possibility that Ag receptor stimulation would ablate CD5 internalization by blocking the ability of the AP2 adaptor complex to interact with it. For these experiments, we used normal T cells from mouse spleens, a mouse B cell line, A20, that expresses relatively high levels of CD5, and a mouse T cell line, EL4. B cells were obtained from spleens of CD5 transgenic mice in which CD5 is constitutively and selectively expressed on all B cells at levels equivalent to that expressed on endogenous B1a B cells (20). Cross-linking of the BCR on splenic B cells with intact anti-µ Ab completely inhibited CD5 internalization measured using Fab anti-CD5 mAb (Fig. 8Go). In contrast, CD3 stimulation of splenic T cells with anti-CD3 mAb (145-2C11) consistently augmented CD5 internalization. The apparent dichotomy in the effect of Ag receptor stimulation on CD5 internalization between T and B cells was also reproduced in the A20 B cell line and the EL4 T cell line (Fig. 8Go). In fact, cross-linking of the Ag receptor on A20 B cells with anti-IgG resulted in 18.3% increase in net expression of surface CD5 within 15 min, most likely from an endogenous presynthesized pool (Fig. 8Go). Similar results were also obtained if intact anti-CD5 mAb was used to assess internalization, indicating that the effect of Ag receptor stimulation on CD5 endocytosis was independent of CD5 cross-linking.



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  		FIGURE 8. BCR and TCR-dependent regulation of CD5 internalization in primary and transformed B and T cells. CD5 internalization mediated by Fab anti-CD5 was measured at 15 min in mature B cells or T cells with or without stimulation with: 1) anti-µ or F(ab')2 anti-µ (for splenic B cells); 2) anti-IgG or F(ab')2 anti-IgG (for A20 B cells); and 3) anti-CD3 (for splenic T cells and EL4 cells). Data represent mean ± SD of triplicate transfections from a representative experiment (n = 5).

 
Use of intact Abs to stimulate the BCR induces coaggregation of the low affinity FcR, Fc{gamma}RIIB1, and delivery of an inhibitory signal (47). To determine whether signals through the FcR might be responsible for the inhibition of CD5 internalization following BCR stimulation with intact Abs to IgM or IgG, we used the F(ab')2 fraction of Abs to stimulate CD5+ B cells and the A20 cell line. We found that stimulation of B cells with F(ab')2 anti-µ or A20 cells with F(ab')2 anti-IgG resulted in enhanced internalization paralleling that observed in T cells following CD3 stimulation (Fig. 8Go). To directly test whether FcRs on B cells were responsible for the inhibition of CD5 internalization following BCR stimulation, we used the FcR-deficient A20 B cell line, IIA1.6. The kinetics of CD5 internalization were assessed following incubation with intact anti-CD5 mAb in the presence or absence of BCR stimulation with intact Abs to IgG. CD5 was rapidly internalized in A20 and IIA1.6 cells reaching a plateau at 15 min for both cell lines (Fig. 9Go). However, in the presence of BCR stimulation, CD5 internalization was completely inhibited in A20 cells, but not in IIA1.6 cells in which a slight increase was noted. Even though IIA1.6 cells exhibited similar kinetics of CD5 internalization when compared with A20 cells, the amount of internalization was greater in IIA1.6. The difference in net internalization is most likely due to intrinsic differences between these two cell lines. We observed no difference whether we used F(ab')2 anti-CD5 instead of intact anti-CD5 to induce CD5 internalization, indicating that the Fc portion of anti-CD5 did not stimulate the FcR (data not shown). Overall, these results show that signals through either the BCR or TCR can modulate CD5 internalization. In B cells, depending on whether FcRs are coaggregated or not, the net effect can be inhibition or enhancement of CD5 endocytosis.



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  		FIGURE 9. Regulation of CD5 internalization by Fc{gamma}RIIB1. CD5 internalization induced by intact anti-CD5 in A20 (squares) or IIA1.6 (circles) in the presence (filled) or absence (open) of stimulation with intact goat anti-mouse IgG. Data represent mean ± SD of triplicate values from a representative experiment (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have defined the mechanism of CD5 internalization and demonstrated that it can be differentially regulated in T and B cells. Endocytosis of CD5 occurred rapidly in response to monovalent or bivalent Ab ligation in cell lines expressing either endogenous or transfected CD5, in normal mature T cells, and in mature primary B cells that express CD5 endogenously (peritoneal B1a B cells) or induced as in CD5-transgenic mice. We determined, using BFA and cyclohexamide, that CD5, unlike the TCR:CD3 complex (35) exhibits a low constitutive rate of turnover; and therefore, the rapid internalization of CD5 in response to receptor dimerization or cross-linking primarily represents ligand-induced endocytosis. Using BFA, we were also able to show that in both normal T and B cells, CD5 was not recycled back to surface following endocytosis with reagents that would dimerize the receptor. However, if endocytosis was initiated with monomeric Ab, ~10% of CD5 was recycled back to the surface in B cells, but none in T cells.

In an effort to define the mechanism responsible for CD5 endocytosis, we determined that the AP2 adaptor complex directly associates with the cytoplasmic tail of CD5 via its µ2 subunit. Because the AP2 adaptor complex and clathrin triskelions form the major structural components of plasma membrane-coated pits and vesicles, our results indicate that CD5 internalization is mediated via the clathrin-coated pit-dependent pathway. The AP2 adaptor complex is a heterotetramer consisting of four subunits, two large, {alpha}2 and {beta}2 (110–115 kDa), a medium, µ2 (50 kDa) and a small, {delta}2 (17 kDa). Of the four recognized endocytic signals (48), the tyrosine-based motifs, NPxY or Yxx[/{phi}] (42), and the dileucine motif, LL, interact with AP2, and only the Yxx[/{phi}] motif binds to the µ2 subunit (46, 48). The cytoplasmic tail of CD5 has two tyrosines (Y429 and Y441) that lie within an Yxx{phi} motif and we mapped the interaction to Y429 in the motif 429YSQP432. The interaction of µ2 with CD5 was essentially abrogated when the YSQP motif was deleted or the critical tyrosine was mutated to alanine. The lack of any effect on the ability of µ2 to bind CD5 in which Y429 was mutated to F can be expected since the Fxx{phi} motif can fit into the hydrophobic pocket of µ2 and has been shown to efficiently bind AP2 (41, 49). The requirement of Y429 for CD5 endocytosis functionally links AP2 to internalization of CD5 via clathrin-coated pits (Fig. 6Go). The reason for differences between the current study, which demonstrates a requirement for Y429 for CD5 endocytosis and a previous report showing that the region which includes this amino acid was not necessary for internalization is not completely clear (50). In this study, we assessed the mechanism of rapid down modulation (within first 30 min) of CD5 whereas in the other report internalization was assessed at 3 and 6 h following Ab binding, and it is possible that immediate vs late internalization of CD5 may be regulated differently. Additionally, in the previous study some of the internalization may reflect changes in surface expression that are related to the half-life of CD5, which is not dependent on Y429 (Fig. 7Go).

CD5 endocytosis, in addition to being mediated via clathrin-coated pits, is also dependent on interaction with the cytoskeleton. Reorganization of the cytoskeleton and polymerization of actin was shown to be required for CD5 endocytosis (37). In fact, the L chain complex of the dynein complex, Tctex-1, associates with the membrane proximal region of CD5 (51). The H chains of the dynein complex interact with the cytoskeleton, whereas the L chains are involved in linking transmembrane proteins to membrane transport vesicles.

The Ab-dependent endocytosis, especially Fab anti-CD5-mediated endocytosis, is likely to represent ligand-dependent endocytosis under physiological conditions. Though the clear identity of the CD5 ligand has not been established, a good candidate is the recently identified 150-kDa protein that occurs as both membrane-associated form on monocytes and lymphocytes, and as a proteolytically cleaved soluble protein (10). In addition to ligand-dependent endocytosis, the AP2 adaptor complex also seems to be involved in regulating the constitutive turnover of CD5 by a mechanism that is ligand-independent as observed for CTLA4 (Refs. 43, 44, 45 ; Fig. 7Go). Physiologically, this is likely to be very important for lymphocyte homeostasis because elevated CD5 expression is associated with inhibiting induction of B cell tolerance in immature B cells and costimulation in mature peripheral B cells (Ref. 52 ; X. Liang, H. Zhao, and C. Raman, manuscript in preparation). In T cells, CD5 overexpression may lead to depressed negative selection in the thymus allowing for the selection of autoreactive T cell clones since loss of CD5 expression results in dramatically enhanced negative selection (11). Unlike in primary cells, some of CD5 in cell lines is recycled back to the surfaced after endocytosis, and this may reflect differences between transformed cells and normal lymphocytes.

The finding that Ag receptor cross-linking on both B and T cells enhanced CD5 endocytosis was unexpected. This is because Y429 is a major site of Ag-receptor-induced phosphorylation of CD5 (53) and tyrosine phosphorylation of the internalization motif is expected to preclude interaction with µ2 (43, 44, 45, 54). The lack of inhibition may in part be due to rapid dephosphorylation of Y429 and the fact that not all of the available CD5 is phosphorylated following Ag receptor stimulation. We have not completely resolved the mechanism underlying enhanced endocytosis of CD5 following Ag receptor stimulation in the absence of FcR coligation. A possible mechanism is via the Ag receptor-induced activation of phosphatidylinsitol 3-kinase (PI3-kinase) that will catalyze the conversion of phosphatidylinositol 4,5-biphosphate to phosphatidylinositol 3,4,5-triphosphate (PI3,4,5P3). The plekstrin homology (PH) domain in the {alpha}2 subunit of the AP2 adaptor complex binds with high affinity to PI3,4,5P3 and localizes AP2 complex to the membrane (55, 56). This interaction also mediates an increase in the affinity of µ2 binding to tyrosine-motifs to facilitate enhanced receptor endocytosis. Preliminary experiments using the pharmacological agent LY294002, a PI3-kinase inhibitor, indicated that PI3 kinase is involved in the Ag receptor-induced enhancement of CD5 internalization (our unpublished observation).

The inhibition of CD5 internalization following cross-linking of BCR with intact Abs may be a function of Src homology 2 domain-containing 5' inositol phosphatase (SHIP) activity (Fig. 8Go). Stimulation of BCR with intact anti-receptor Abs coengages the inhibitory Fc{gamma}R on B cells, Fc{gamma}RIIBI, resulting in tyrosine phosphorylation of its cytoplasmic tail and recruitment of SHIP (47). The involvement of Fc{gamma}RIIBI in inhibition of CD5 endocytosis following BCR stimulation was confirmed by the lack of inhibition of CD5 internalization in Fc{gamma}RIIBI-deficient IIA1.6 cells and when F(ab')2 Abs were used to stimulate BCR on normal B cells or A20 cells. Because the {alpha}2 subunit of the AP2 adaptor complex is localized to the membrane by its interaction with PI3,4,5P3, the catalysis of PI3,4,5P3 to phosphatidylinositol 3,4-diphosphate (PI3,4P2) by SHIP will inhibit the translocation of AP2 to membrane. Consistent with this hypothesis, studies have demonstrated that mutation of the phosphoinositide binding site on AP2{alpha}2 blocks AP2-mediated endocytosis, demonstrating an essential role for PI3,4,5P3 in endocytosis (57).

The ability to differentially regulate CD5 internalization in T and B cells could allow for different net effects of CD5 signaling that may be cell type, developmental stage, and/or activation stage specific. In fact, our recent in vivo studies indicate that CD5 has a net negative regulatory role in immature and mature T cells, whereas in B cells, the net effect of CD5 is inhibitory in immature B cells but can be either costimulatory or inhibitory in mature B cells. The lack of clear knowledge as to how CD5 regulates Ag receptor signaling in normal cells makes it difficult to predict the role of CD5 internalization in this process. One possibility is that the interaction of AP2 with CD5 is required for localizing CD5 into sphingolipid-rich microdomains or rafts in lymphocytes, as has been recently reported (58). Therefore, the inhibition of CD5 internalization by SHIP would inhibit its recruitment into lipid rafts and result in a net positive effect on Ag receptor signaling. Conversely, the enhancement of AP2 association with membrane lipids would facilitate greater recruitment of CD5 into rafts, resulting in greater attenuation of TCR/BCR signaling. It is also possible that CD5 functions as a "sink" either to colocalize or segregate activation/inhibitory molecules with respect to TCR/BCR complex. In such a situation, the rapid removal of CD5 from the cell surface or its retention could have opposite effects on Ag receptor signaling. In summary, the ability to posttranslationally regulate the cell surface CD5 expression allows for this receptor to function with opposite effects on TCR/BCR signaling without the need to invoke a distinct set of signaling pathways for different functions. This property may also explain why the published literature often conflicts as to the physiological role of CD5 in regulation of Ag receptor signaling.

The mechanism by which AP2-mediated CD5 internalization is regulated is likely to be important because CD5 plays a fundamental and important role in B and T lymphocyte selection, activation, and generation of tolerance. In addition to SHIP described above, another key regulator of AP2-mediated endocytosis may be CK2, an important regulator of receptor endocytosis (59). We and others have shown that CK2 constitutively associates with and is activated by CD5 (32, 60). The development of transgenic mice that express CD5 on B and/or T lymphocytes lacking the ability to interact with the AP2 adaptor complex will be key in defining the physiological role of posttranslational regulation of CD5 expression.


   Acknowledgments
 
We thank Dr. Robert Kimberly for critical review of the manuscript and Anling Kuo for technical help.


   Footnotes
 
1 This work was supported by grants from Arthritis Foundation AI, National Institutes of Health Grants AG16221 and AI44836 (to C.R.), the Cystic Fibrosis Foundation (COLLAWGO), and the American Heart Association (Established Investigator Grant; to J.F.C.). Back

2 Current address: Department of Surgery, Emory University School of Medicine, 5211 Woodruff Memorial Building, Atlanta, GA 30322. Back

3 Address correspondence and reprint requests to Dr. Chander Raman, Department of Medicine, University of Alabama, LHRB 463, 1530 3rd Avenue South, Birmingham, AL 35294-0007. E-mail address: craman{at}uab.edu Back

4 Abbreviations used in this paper: BCR, B cell Ag receptor; BD, binding domain; AD, activation domain; MFI, mean fluorescence intensity; PI3,4,5P3, phosphatidylinositol 3,4,5-triphosphate; SHIP, Src homology 2 domain-containing 5' inositol phosphatase; BFA, brefeldin A; ER, endoplasmic reticulum; PI3 kinase, phosphatidylinsitol 3-kinase. Back

Received for publication February 7, 2001. Accepted for publication April 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Resnick, D., A. Pearson, M. Krieger. 1994. The SRCR family: a family reminiscent of the Ig superfamily. Trends Biochem. Sci. 19:5.[Medline]
  2. Reinherz, E. L., P. C. Kung, G. Goldstein, S. F. Schlossman. 1979. A monoclonal antibody with selective reactivity with functionally mature thymocytes and all peripheral human T lymphocytes. J. Immunol. 123:1312.[Abstract/Free Full Text]
  3. Hardy, R. R.. 1989. B cell ontogeny and B cell subsets. Curr. Opin. Immunol. 2:189.[Medline]
  4. Kantor, A.. 1991. A new nomenclature for B cells. Immunol. Today 12:388.[Medline]
  5. Aruffo, A., M. B. Melnick, P. S. Linsley, B. Seed. 1991. The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristic of a new family of cell surface and secreted proteins. J. Exp. Med. 174:949.[Abstract/Free Full Text]
  6. Van de Velde, H., I. von Hoegen, W. Luo, J. R. Parnes, K. Thielemans. 1991. The B cell surface protein CD72/Lyb-2 is the ligand for CD5. Nature 351:662.[Medline]
  7. Biancone, L., M. A. Bowen, A. Lim, A. Aruffo, G. Andres, I. Stamenkovic. 1996. Identification of a novel inducible cell-surface ligand for CD5 on activated lymphocytes. J. Exp. Med. 184:811.[Abstract/Free Full Text]
  8. Bikah, G., F. M. Lynd, A. A. Aruffo, J. A. Ledbetter, S. Bondada. 1998. A role for CD5 in cognate interactions between T cells and B cells, and identification of a novel ligand for CD5. Int. Immunol. 10:1185.[Abstract/Free Full Text]
  9. Pospisil, R., M. G. Fitts, R. G. Mage. 1996. CD5 is a potential selecting ligand for B cell surface immunoglobulin framework region sequences. J. Exp. Med. 184:1279.[Abstract/Free Full Text]
  10. Calvo, J., L. Places, O. Padilla, J. M. Vial, J. Vives, M. A. Bowen, F. Lozano. 1999. Interaction of recombinant and natural soluble CD5 forms with an alternative cell surface ligand. Eur. J. Immunol. 29:2119.[Medline]
  11. Tarakhovsky, A., S. B. Kanner, J. Hombach, J. A. Ledbetter, W. Müller, N. Killeen, K. Rajewsky. 1995. A role for CD5 in TCR-mediated signal transduction and thymocyte selection. Science 269:535.[Abstract/Free Full Text]
  12. Chan, S., C. Waltzinger, A. Tarakhovsky, C. Benoist, D. Mathis. 1999. An influence of CD5 on the selection of CD4-lineage T cells. Eur. J. Immunol. 29:2916.[Medline]
  13. Kishimoto, H., J. Sprent. 1999. Several different cell surface molecules control negative selection of medullary thymocytes. J. Exp. Med. 190:65.[Abstract/Free Full Text]
  14. Page, D. M.. 1999. Cutting edge: thymic selection and autoreactivity are regulated by multiple coreceptors involved in T cell activation. J. Immunol. 163:3577.[Abstract/Free Full Text]
  15. Vandenberghe, P., J. L. Ceuppens. 1991. Immobilized anti-CD5 together with prolonged activation of protein kinase C induce interleukin 2-dependent T cell growth: evidence for signal transduction through CD5. Eur. J. Immunol. 21:251.[Medline]
  16. Vandenberghe, P., J. Verwilghen, F. Van Vaeck, J. L. Ceuppens. 1993. Ligation of the CD5 or CD28 molecules on resting human T cells induces expression of the early activation antigen CD69 by a calcium- and tyrosine kinase-dependent mechanism. Immunology 78:210.[Medline]
  17. Kroesen, B. J., A. Bakker, R. A. van Lier, H. T. The, L. de Leij. 1995. Bispecific antibody-mediated target cell-specific costimulation of resting T cells via CD5 and CD28. Cancer Res. 55:4409.[Abstract/Free Full Text]
  18. Bikah, G., J. Carey, J. R. Ciallella, A. Tarakhovsky, S. Bondada. 1996. CD5-mediated negative regulation of antigen receptor-induced growth signals in B-1 B cells. Science 274:1906.[Abstract/Free Full Text]
  19. Gary-Gouy, H., P. Bruhns, C. Schmitt, A. Dalloul, M. Daeron, G. Bismuth. 2000. The pseudo-immunoreceptor tyrosine-based activation motif of CD5 mediates its inhibitory action on B-cell receptor signaling. J. Biol. Chem. 275:548.[Abstract/Free Full Text]
  20. Chen, X., Y. Matsuura, J. F. Kearney. 1995. CD5 transgenic mice. Curr. Top. Microbiol. Immunol. 194:209.[Medline]
  21. Sato, S., N. Ono, D. A. Steeber, E. S. Pisetsky, T. F. Tedder. 1996. CD19 regulates B lymphocyte signaling thresholds critical for development of B-1 lineage cells in autoimmunity. J. Immunol. 157:4371.[Abstract]
  22. Beyers, A. D., L. L. Spruyt, A. F. Williams. 1992. Molecular associations between T-lymphocyte antigen receptor complex and the surface antigen CD2, CD4, or CD8 and CD5. Proc. Natl. Acad. Sci. USA 89:2945.[Abstract/Free Full Text]
  23. Osman, N., A. I. Lazarovits, M. J. Crumpton. 1993. Physical association of CD5 and the T cell receptor/CD3 antigen complex on the surface of human T lymphocytes. Eur. J. Immunol. 23:1173.[Medline]
  24. Lankester, A. C., G. M. W. van Schijndel, J. L. Cordell, C. J. M. van Noesel, R. A. W. van Lier. 1994. CD5 is associated with the human B cell antigen receptor complex. Eur. J. Immunol. 24:812.[Medline]
  25. Tsui, H. W., K. A. Siminovitch, L. de Souza, F. W. Tsui. 1993. Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene. Nat. Genet. 4:124.[Medline]
  26. Chan, V. W., F. Meng, P. Soriano, A. L. DeFranco, C. A. Lowell. 1997. Characterization of the B lymphocyte populations in lyn-deficient mice and role of lyn in signal initiation and down-regulation. Immunity 7:69.[Medline]
  27. Pappu, R., A. M. Cheng, B. Li, Q. Gong, C. Chiu, N. Griffin, M. White, B. P. Sleckman, A. C. Chan. 1999. Requirement for B cell linker protein (BLNK) in B cell development. Science 286:1949.[Abstract/Free Full Text]
  28. Rickert, R. C., K. Rajewsky, J. Roes. 1995. Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice. Nature 376:352.[Medline]
  29. Ahearn, J. M., M. B. Fischer, D. Croix, S. Goerg, M. Ma, J. Xia, X. Zhou, R. G. Howard, T. L. Rothstein, M. C. Carrol. 1996. Disruption of the Cr2 locus results in reduction of B-1a cells and in impaired B cell response to T-independent antigen. Immunity 4:251.[Medline]
  30. Azzam, H. S., A. Grinberg, K. Lui, H. Shen, E. W. Shores, P. E. Love. 1998. CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity. J. Exp. Med. 188:2301.[Abstract/Free Full Text]
  31. Berland, R., H. H. Wortis. 1998. An NFAT-dependent enhancer is necessary for anti-IgM-mediated induction of murine CD5 expression in primary splenic B cells. J. Immunol. 161:277.[Abstract/Free Full Text]
  32. Raman, C., A. Kuo, D. Litchfield, R. P. Kimberly. 1998. Regulation of casein kinase 2 by direct interaction with cell surface receptor CD5. J. Biol. Chem. 273:19183.[Abstract/Free Full Text]
  33. Barak, L. S., M. Tiberi, N. J. Freedman, M. M. Kwatra, R. J. Lefkowitz, M. G. Caron. 1994. A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated {beta}2-adrenergic receptor sequestration. J. Biol. Chem. 269:2790.[Abstract/Free Full Text]
  34. Cefai, D., H. Schneider, O. Matangkasombut, H. Kang, J. Brody, C. E. Rudd. 1998. CD28 receptor endocytosis is targeted by mutations that disrupt phosphatidylinositol 3-kinase binding and costimulation. J. Immunol. 160:2223.[Abstract/Free Full Text]
  35. Liu, H., M. Rhodes, D. L. Wiest, D. A. Vignali. 2000. On the dynamics of TCR: CD3 complex cell surface expression and downmodulation. Immunity 13:665.[Medline]
  36. Shawler, D. L., D. E. Johnson, T. J. McCallister, R. M. Bartholomew, R. O. Dillman. 1988. Mechanisms of human CD5 modulation and capping induced murine monoclonal antibody T101. Clin. Immunol. Immunopathol. 47:219.[Medline]
  37. Alberola-Ila, J., L. Places, V. Fabregat, J. Vives, F. Lozano. 1993. Different mechanisms regulate the monoclonal antibody-induced modulation of CD2, CD3, and CD5 in human lymphocytes. Cell Immunol. 147:247.[Medline]
  38. Jamin, C., P. Lydyard, P. Youinou. 1995. CD5+ B cells: Differential capping and modulation of IgM and CD5. In 9th International Congress of Immunology 2651:447.
  39. Wood, S. A., J. E. Park, W. J. Brown. 1991. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell 67:591.[Medline]
  40. Lippincott-Schwartz, J., L. Yuan, C. Tipper, M. Amherdt, L. Orci, R. D. Klausner. 1991. Brefeldin A’s effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell 67:601.[Medline]
  41. Trowbridge, I. S., J. F. Collawn, C. R. Hopkins. 1993. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu. Rev. Cell Biol. 9:129.
  42. Kirchausen, T.. 1999. Adaptors for clathrin-mediated traffic. Annu. Rev. Cell Biol. 15:705.[Medline]
  43. Zhang, Y., J. P. Allison. 1997. Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein. Proc. Natl. Acad. Sci. USA 94:9273.[Abstract/Free Full Text]
  44. Chuang, E., M. L. Alegre, C. S. Duckett, P. J. Noel, M. G. Vander Heiden, C. B. Thompson. 1997. Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression. J. Immunol. 159:144.[Abstract]
  45. Shiratori, T., S. Miyatake, H. Ohno, C. Nakaseko, K. Isono, J. S. Bonifacino, T. Saito. 1997. Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP-2. Immunity 6:583.[Medline]
  46. Owen, D. J., Y. Vallis, M. E. Noble, J. B. Hunter, T. R. Dafforn, P. R. Evans, H. T. McMahon. 1999. A structural explanation for the binding of multiple ligands by the {alpha}-adaptin appendage domain. Cell 97:805.[Medline]
  47. Coggeshall, K. M.. 1998. Inhibitory signaling by B cell Fc{gamma}RIIb. Curr. Opin. Immunol. 10:306.[Medline]
  48. Marsh, M., H. T. McMahon. 1999. The structural era of endocytosis. Science 285:215.[Abstract/Free Full Text]
  49. Sauter, M. M., A. Pelchen-Matthews, R. Bron, M. Marsh, C. C. LaBranche, P. J. Vance, J. Romano, B. S. Haggarty, T. K. Hart, W. M. Lee, J. A. Hoxie. 1996. An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface. J. Cell Biol. 132:795.[Abstract/Free Full Text]
  50. Vila, J. M., J. Calvo, L. Places, O. Padilla, M. Arman, I. Gimferrer, C. Aussel, J. Vives, F. Lozano. 2001. Role of two conserved cytoplasmic threonine residues (T410 and T412) in CD5 signaling. J. Immunol. 166:396.[Abstract/Free Full Text]
  51. Bauch, A., K. S. Campbell, M. Reth. 1998. Interaction of the CD5 cytoplasmic domain with the Ca2+/calmodulin-dependent kinase II{delta}. Eur. J. Immunol. 28:2167.[Medline]
  52. Hippen, K. L., L. E. Tze, T. W. Behrens. 2000. CD5 maintains tolerance in anergic B cells. J. Exp. Med. 191:883.[Abstract/Free Full Text]
  53. Vila, J. M., I. Gimferrer, O. Padilla, M. Arman, L. Places, M. Simarro, J. Vives, F. Lozano. 2001. Residues Y429 and Y463 of the human CD5 are targeted by protein tyrosine kinases. Eur. J. Immunol. 31:1191.[Medline]
  54. Bonifacino, J. S., E. C. Dell’Angelica. 1999. Molecular bases for the recognition of tyrosine-based sorting signals. J. Cell Biol. 145:923.[Free Full Text]
  55. Rapoport, I., M. Miyazaki, W. Boll, B. Duckworth, L. C. Cantley, S. Shoelson, T. Kirchhausen. 1997. Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes. EMBO J. 16:2240.[Medline]
  56. Jost, M., F. Simpson, J. M. Kavran, M. A. Lemmon, S. L. Schmid. 1998. Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation. Curr. Biol. 8:1399.[Medline]
  57. Gaidarov, I., J. H. Keen. 1999. Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits. J. Cell Biol. 146:755.[Abstract/Free Full Text]
  58. Yashiro-Ohtani, Y., X. Y. Zhou, K. Toyo-Oka, X. G. Tai, C. S. Park, T. Hamaoka, R. Abe, K. Miyake, H. Fujiwara. 2000. Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts. J. Immunol. 164:1251.[Abstract/Free Full Text]
  59. Cotlin, L. F., M. A. Siddiqui, F. Simpson, J. F. Collawn. 1999. Casein kinase II activity is required for transferrin receptor endocytosis. J. Biol. Chem. 274:30550.[Abstract/Free Full Text]
  60. Calvo, J., J. M. Vilda, L. Places, O. S. M., D. Padilla, K. S. Andreu, C. Campbell, C. Aussel, F. Lozano. 1998. Human CD5 signaling and constitutive phosphorylation of C-terminal serine residues by casein kinase II. J. Immunol. 161:6022.[Abstract/Free Full Text]



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