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β in assembly of the B cell antigen receptor (BCR)
Immunology Letters 112 (2007) 47–57
Role of the extracellular and transmembrane domain of Ig-␣/ in assembly of the B cell antigen receptor (BCR) Janis Dylke, Jared Lopes 1 , May Dang-Lawson, Steve Machtaler, Linda Matsuuchi ∗ Cell Biology Group, Department of Zoology, Cell and Developmental Biology Research Group, Life Sciences Institute, Infection, Inflammation and Immunity (I3) Research Group, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada Received 21 September 2006; received in revised form 26 June 2007; accepted 27 June 2007 Available online 23 July 2007
Abstract The B cell antigen receptor (BCR) is expressed on the surface of B-lymphocytes where it binds antigen and transmits signals that regulate B cell activation, growth and differentiation. The BCR is composed of membrane IgM (mIgM) and two signaling proteins, Ig-␣ and Ig-. If either of the signaling proteins is not expressed, the incomplete mIgM-containing BCR will not traffic to the cell surface. Our hypothesis is that specific protein:protein interactions between both the extracellular and transmembrane (TM) regions of Ig-␣ and Ig- are necessary for receptor assembly, cell surface expression and effective signaling to support the proper development of B cells. While previous work has shown the importance of the TM region in BCR assembly, this study indicates that a heterodimer of the extracellular domains of Ig-␣ and Ig- are also required for proper association with mIgM. Cell lines expressing mutated Ig-␣ proteins that did not heterodimerize with Ig- in the extracellular and TM domains were unable to properly assemble the BCR. Conversely, an Ig-␣ mutant with an Ig- cytoplasmic tail (C (␣/␣/)) was able to assemble with the rest of the BCR, in particular with Ig-, and traffic to the cell surface. Thus, both the extracellular and TM regions of the Ig-␣/Ig- must be properly associated in order for the BCR to assemble. © 2007 Elsevier B.V. All rights reserved. Keywords: B cell antigen receptor; Assembly
1. Introduction The B cell antigen receptor (BCR) is a key regulator of B lymphocyte activation, growth and differentiation. It is expressed on the surface of B lymphocytes and is composed of a ligand binding receptor (membrane immunoglobulin; mIg) and a signaling component (Ig-␣/) [1,2]. B cell development in the bone marrow requires that the BCR signals to the cell when a functional receptor has trafficked to the cell surface and if the BCR binds to self antigen. This prevents non-functional and self-reactive B cells from escaping into the periphery. Once the B cell enters the periphery, the BCR binds foreign antigens and signals to the cell to proliferate and differentiate into an antibody produc∗
Corresponding author. Tel.: +1 604 822 4881; fax: +1 604 822 2416. E-mail address: [email protected] (L. Matsuuchi). 1 Current address: Department of Immunology, University of Washington, Benaroya Research Institute at Virginia Mason Medical Center, Seattle, WA 98101-2795, USA. 0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.06.005
ing plasma cell or a long-lived memory cell [3,4]. This allows the body to combat novel microorganisms and to keep a B cell repertoire that is reactive to microorganisms that have been previously encountered, allowing a faster immune response upon re-infection. In this sense, the functionality of the BCR is essential for the immune system to successfully eliminate foreign microorganisms. In addition, a non-functional BCR can lead to autoimmune diseases and B cell leukemias and lymphomas [5]. The mIg component of the mIgM BCR is unable to signal due to its short cytoplasmic tail, therefore it must associate with two signaling proteins, Ig-␣ and Ig-, to form the complete functional BCR [6–8]. Ig-␣ and Ig- are disulfide-linked, transmembrane proteins that are non-covalently associated with mIg. The current model suggests that one heterodimer of Ig-␣/ associates with one mIg complex [9]. Ig-␣ and Ig- are found on the surface of B cells during the early stages of B cell development. Antigen binding or cross-linking of the BCR results in signaling mediated by the Ig-␣/ heterodimer [2,10].
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The transmembrane portion of the protein is proposed to be an ␣-helix consisting of 26 amino acids that span the lipid bilayer [11]. Thirteen of those amino acids are highly conserved among different immunoglobulin isotypes and 11 of those 13 are proposed to line up on one side of the helix suggesting that they, or that side of the helix, may be important for interaction with other molecules. The transmembrane region is not composed entirely of hydrophobic amino acids as is seen with many transmembrane proteins. There are 9 polar amino acids in the transmembrane region of mIgM. This suggests that the H chain transmembrane region could be interacting with the transmembrane regions of other proteins since one might predict that polar amino acids should be hidden or shielded in the hydrophobic lipid bilayer [10]. The signaling portion of the BCR is composed of a heterodimer of Ig-␣ and Ig-. Ig-␣ is a transmembrane glycoprotein. The extracellular portion of murine Ig-␣ is composed of 109 amino acids. The amino acid sequence indicates that the extracellular regions of Ig-␣ fold into a barrel domain similar to those seen in the H and L chains. There are two cysteine residues (positions 50 and 101) that form a disulfide bond between the  sheets in the extracellular region. Another cysteine residue (position 113) forms a disulfide bond with Ig-. Ig-␣ also has two glycosylation sites at amino acids 58 and 68 [7,12]. The transmembrane domain is predicted as an ␣-helix consisting of 22 amino acids, and the cytoplasmic tail is predicted to contain 61 amino acids [10]. The cytoplasmic domain has a highly conserved immunoreceptor tyrosine-based activation motif (ITAM) with the amino acid sequence D/Ex7 D/Ex2 Yx2 L/Ix7 Yx2 L/I [13]. Additionally, the cytoplasmic domain has two other tyrosine residues (positions 176 and 204). Phosphorylation of these residues recruits the B cell linker proteins Vav and Grb2. Activated B cells lacking these residues do not effectively present antigen to T cells indicating that tyrosine176, 204 phosphorylation is important for intracellular trafficking of the receptor after antigen cross-linking and in B cell development [14,15]. The residue at position 204 may also have a role in binding the SH2 domain of the adaptor protein BLNK [16,17]. The Ig- protein is similar in structure to Ig-␣. The extracellular region of Ig- is composed of 129 amino acids that form  sheets. There are four cysteine residues that form two intramolecular disulfide bonds (43:124 and 65:120) and an additional cysteine residue (135) that forms the disulfide bond with Ig-␣. The extracellular domain also has three glycosylation sites [8]. The transmembrane region, like Ig-␣, is an ␣-helix composed of 22 amino acids. The cytoplasmic tail consists of 48 amino acids and has an ITAM sequence. The ITAM sequence for both Ig-␣ and Ig- is conserved in different species indicating that protein function requires that the sequence remains unchanged [13]. Tyrosine phosphorylation of the ITAM domains by Src-family tyrosine kinases leads to interaction with the SH2 domain of Syk, a protein tyrosine kinase, resulting in the activation of numerous downstream intracellular signaling events [3,4]. Murine Ig-, unlike Ig-␣, does not contain any additional tyrosine residues. The Ig-␣/ heterodimer makes up the signaling portion of the BCR.
All four components of the membrane IgM (mIgM)containing BCR (H chains, L chains, Ig-␣ and Ig-) must be expressed in the cell in order for assembly and cell surface trafficking to occur [18,19]. However, if only the cytoplasmic tails of Ig-␣ or Ig- are truncated or mutated the mIgM BCR can still traffic to the cell surface, but B cell signaling and development are disrupted [20,21]. The study by Reichlin et al. showed that truncation of the Ig-␣ or Ig- cytoplasmic tail, leaving a heterodimer in the extracellular and transmembrane domains, allowed for BCR formation and cell surface trafficking. In support of this data, a study by Wang et al. [21] showed that an Ig- mutant that heterodimerized with Ig-␣ in the extracellular and transmembrane domains, but homodimerized in the cytoplasmic domain, was able to form a complete BCR and traffic to the cell surface. Both of these studies suggest that heterodimerization in the extracellular and transmembrane domains are both required for BCR assembly and cell surface trafficking. This hypothesis is further examined here using three mutated Ig-␣ constructs (C␣ (//␣), X␣2 (␣//) and C (␣/␣/) to express altered chimeric forms of the protein. These studies will help us to increase our understanding of which portions of the BCR are required for BCR cell surface expression, of particular importance during the different stages of B cell development. This is important since disruptions of the normal levels of surface BCR are associated with various B cell lymphomas and these in turn correlate with mutations in the Ig-␣/ proteins. The idea being that either defects in cell signaling or in cell surface expression which influences the levels of cell signaling are responsible for the cues that the developing B lymphocyte receives to proliferate, become anergic or undergo apoptosis during the process of cancer development [5,22,23]. Thus some of the structural mutations in the Ig-␣/ protein that are outside of the cytoplasmic tail [22] are also important in this regard and our work here will help identify which of these regions to focus on as important regions of protein interaction during BCR assembly. 2. Materials and methods 2.1. Antibodies, immunoprecipitating and immunoblotting reagents The rabbit anti-mouse IgM ( chain specific) antibody was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pennsylvania). The rabbit anti-mouse light chain antibody was from Bethyl Laboratories (Montgomery, Texas). The rabbit antimouse Ig-␣ cytoplasmic tail antibody was described previously [24]. The rabbit anti-mouse Ig-␣ extracellular antibodies, produced with a 30 amino acid peptide to the extracellular domain of Ig-␣ (amino acids 29–58) and the rabbit anti-mouse Ig- extracellular antibody, produced with a 30 amino acid peptide to the extracellular domain of Ig- (amino acids 71–100) were from Dr. Richard Meagher (University of Georgia, Athens, Georgia). The rabbit anti-mouse Ig- cytoplasmic antibody that recognizes the cytoplasmic tail of Ig- was from Dr. Marcus Clark (University of Chicago, Chicago, Illinois). Horseradish peroxidase (HRP)-conjugated protein A was from Amersham Biosciences (Baie d’Urfe, Quebec). The goat anti-rabbit IgG-HRP and the
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fluorescein (FITC)-conjugated goat anti-mouse IgM ( chain specific) antibody were from Jackson Immunoresearch Laboratories, Inc. (West Grove, Pennsylvania). The rhodamine labeled goat anti-mouse IgM ( chain specific) used for cell surface fluorescence was from BD Biosciences (Palo Alto, California). 2.2. Plasmids The pMX-puro retroviral expression vector was from Dr. Alice Mui (Jack Bell Research Centre, Vancouver, BC). The pWZL-Blast1 and pWZL-Blast3 retroviral expression vectors were from Dr. Stephen Robbins (University of Calgary, Calgary, Alberta). The pMSCV-puro retroviral expression vector was purchased from BD Biosciences. The pMIGR1-Xα, pMIGR1-Igβ, pMIGR1-ΔCY and MIGR1p-Cα expression vectors were from Dr. Marcus Clark (University of Chicago, Chicago, Illinois) [21]. 2.3. Plasmids created 2.3.1. pMSCV-puro-Cβ and pWZL-Blast3-Cβ (α/α/β) This plasmid encodes an Ig-␣ protein that is extracellularly and transmembrane Ig-␣ and cytoplasmically Ig- (␣/␣/). Sitedirected mutagenesis was used to insert a BamHI restriction enzyme site into pMX-puro-␣ between the transmembrane and cytoplasmic domains. The primers used for site-directed mutagenesis were JDXA1 (5 G CTG CTA TTC AGG ATC CGG TAA TTG CGG CCG3 ) and JDXA2 (5 CGG CCG CAA TAA CCG GAT CCT GAA TAG CAG C3 ). The Ig-␣ fragment from pMX-puro-␣ was then excised using XhoI and BamHI restriction enzymes and inserted into pMIGRI-X␣ to create pMIGRI-C. The DNA encoding C was then excised from pMIGR1 using XhoI and EcoRI restriction enzymes and ligated into pWZL-Blast3 and pMSCV-puro to create pWZL-Blast3-C and pMSCV-puro-C. 2.3.2. pMSCV-puro-Xα2 and pWZL-Blast3-Xα2 (α/β/β) These plasmids encode a protein that is extracellularly Ig-␣ and transmembrane and cytoplasmically Ig- (␣//). Sitedirected mutagenesis was used to insert a SacII restriction enzyme site into pMIGR1-Ig and pMX-puro-␣ between the extracellular and transmembrane domains. The primers used for pMIGR1-Ig were SacB1 (5 CGG CGG AAC ACA CTG AAC CGC GGC ATT ATC TTG ATC CAG ACC3 ) and SacB2 (5 GGT CTG GAT CAA GAT AAT GCC GCG GTT CAG TGT GTT CCG CCG3 ) and the primers used for pMXpuro-␣ were SacA1 (5 GGG GAA GGT ACC AAG AAC CGC GGC ATC ACA GCA GAA GGG3 ) and SacA2 (5 CCC TTC TGC TGT GAT GCC GCG GTT CTT GGT ACC TTC CCC3 ). The Ig-␣ extracellular domain DNA fragment was then excised from pMX-puro-␣ using the XhoI and SacII restriction enzyme sites and inserted into pMIGR1-Ig, replacing the Ig- extracellular domain and creating pMIGR1-X␣2. The X␣2 DNA fragment was then excised from pMIGR1-X␣2 using XhoI and EcoRI restriction enzymes and ligated into pWZL-Blast3 and pMSCV-puro to create pWZL-Blast3-X␣2 and pMSCV-puro-X␣2.
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2.3.3. pWZL-Blast1-Cα (β/β/α) This plasmid encodes a protein that is extracellularly and transmembrane Ig- and cytoplasmically Ig-␣ (//␣). The DNA encoding C␣ was excised from MIGR1p-C␣ with BglII and XhoI restriction enzymes. It was then ligated into pWZL-Blast1 that had been digested with BglII and SalI (XhoI and SalI have compatible cohesive ends). This created pWZL-Blast1-C␣. 2.4. Molecular biology techniques Restriction enzymes (Invitrogen Life Technologies (Burlington, Ontario) or New England Biolabs (Pickering, Ontario)) were added to the DNA according to manufacturer’s instructions and digested at 37 ◦ C as directed. All molecular biology techniques were performed using standard procedures. DNA fragments were purified using a QIAquick Gel Extraction Kit (Qiagen, Mississauga, Ontario) following manufacturer’s instructions. Small volumes of plasmid DNA were purified using the GenElute Plasmid Miniprep Kit (Sigma–Aldrich Canada, Oakville, Ontario) according to manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using puReTaq Ready-ToGo PCR beads (Amersham Biosciences Canada) according to manufacturer’s instructions. The reactions were run at 95 ◦ C for 45 s, 55 ◦ C for 2 min and 72 ◦ C for 2 min for a total of 35 cycles. Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). All oligonucleotide primers were purchased from the Nucleic Acid Protein Service Unit (UBC) and the reactions were run in a DNA Thermal Cycler (Perkin-Elmer Cetus) according to manufacturer’s instructions. After transformation of the XL1-Blue Supercompetent bacteria, a single colony was used to inoculate LB broth for small scale DNA preparation. The resulting DNA was tested for the presence of the intended mutation by restriction enzyme digestion and DNA sequencing (Nucleic Acid Protein Service Unit). 2.5. Tissue culture cell lines The AtT20 and ASS murine pituitary tumor cell line was from Dr. Regis Kelly (University of California, San Francisco, California) and has been described [25]. The R142 cell line in an AtT20 cell line that expresses mIgM and Ig-␣ [18] and the WT5 cell line is an AtT20 cell line that expresses mIgM and Ig- [21]. The Syk 13 cell line is an AtT20 cell line that expresses mIgM, Ig-␣ and Ig- [26]. The J558 m3 and J558 15-25 murine plasmacytoma cell lines were from Dr. Louis Justement (University of Alabama, Birmingham, Alabama) and from Dr. Marcus Clark (University of Chicago, Chicago, Illinois). The BOSC 23 human fibroblast cell line was from Dr. Warren Pear (University of Pennsylvania, Philadelphia, Pennsylvania). Tissue culture cell lines were grown as described using standard procedures in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Life Technologies) or Roswell Park Memorial Institute (RPMI)-1640 media containing 4.5 g/L glucose, 2 mM l-glutamine, 110 mg/L sodium pyruvate, 5–10%
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heat inactivated fetal calf serum (FCS) (Invitrogen Life Technologies), 50 units/mL penicillin and 50 g/mL streptomycin sulfate (Invitrogen Life Technologies). 2.6. Cell lysis Cells were detergent lysed on ice with 0.5–1.0 mL of cold Triton X-100 lysis buffer (20 mM Tris–HCl pH 8.0, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA (Fisher Scientific Canada, Ottawa, Ontario), 10% glycerol, 10 g/mL leupeptin (Roche Diagnostics, Indianapolis, Indiana), 1 g/mL aproprotin (Roche Diagnostics), 1 mM pepstatin A (Sigma–Aldrich), 1 mM Na3 VO4 , 1 mM phenylmethylsulfonyl fluoride (PMSF) (Roche Diagnostics)) or cold digitonin lysis buffer (1% digitonin (Sigma–Aldrich), 10 mM triethanolamine pH 7.8, 150 mM NaCl, 1 mM EDTA, 10 g/mL leupeptin, 1 g/mL aproprotin,
1 mM pepstatin A, 1 mM PMSF). Lysates were centrifuged at 14,000 rpm for 15 minutes at 4 ◦ C and the protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, Illinois). The samples were frozen at −20 ◦ C for long-term storage. 2.7. Transfections Cells were transfected using retroviral particles or DNA mixtures using standard procedures. Cells were drug selected in media containing 0.4 g/mL puromycin (Calbiochem, La Jolla, California) or 4 g/mL of blasticidin S (Invitrogen Life Technologies), depending on the drug resistance gene that was used in the transfection. Drug resistant populations of cells as well as single stably transfected clones were recovered for further experiments.
Fig. 1. Antibody screening by immunoblotting and expression of new plasmids. Thirty to 50 g of protein from whole cell lysates from untransfected cells or from AtT20 cells stably expressing various components of the BCR, from transiently transfected BOSC fibroblast cells or from transiently transfected and drug selected J558 plasmacytoma cells were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with anti-Ig-␣ or anti-Ig- antibodies (Abs). Panel a, schematic diagram showing wild type and three different chimeric versions of Ig-␣ and Ig-, X␣2 (␣//; denoting the extracellular domain, TM domain and cytoplasmic tail, from left to right), C␣ (//␣) and C (␣/␣/). The white color represents Ig-␣, the grey color represents Ig-. Ig-␣ or Ig- domains appear in parentheses next to the name of the mutated protein in the order from the left, extracellular domain/transmembrane domain/cytoplasmic domain. Panels b–e, tests showing specificity of anti-Ig-␣/ extracellular and anti-cytoplasmic domain antibodies. Panels f–i, tests showing expression of new constructs encoding chimeric Ig-␣/s. Panel b, Abs used for immunoblot recognizing the extracellular domain of Ig-␣. Panel c, Abs used for immunoblot recognizing the cytoplasmic domain of Ig-. Panel d, Abs used for immunoblot recognizing the extracellular domain of Ig-. Panel e, Abs used for immunoblot recognizing the cytoplasmic domain of Ig-␣. Panel f, expression of X␣2 (␣//) and C (␣/␣/) in BOSC fibroblast cells. Panel g, expression of C␣ (//␣) in AtT20 cells expressing the other chains of the BCR. The various sizes are due to differential glycosylation. Panel h, expression of C (␣/␣/) and X␣2 (␣//) in J558 cells. Panel i, expression of C␣ (//␣) in J558 cells. , , ␣, : the components of the BCR that the cell line expresses. IB: immunoblotting antibody below each panel. 䊉: Background band. Grey coloration: Ig-, white coloration: Ig-␣. Molecular weight markers in kilodaltons (kDa) to the left of each panel.
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2.8. Immunoprecipitations All cells to be used for immunoprecipitations were lysed in digitonin lysis buffer. One thousand micrograms of cell lysate was added to 20 L of washed Protein A-Sepharose 4B beads (Sigma–Aldrich) in 1.5 mL tubes (Axygen Scientific). Five microlitres of antibody, previously determined to be in antibody excess for successful co-immunoprecipitation studies, were added to each tube and the mixture was rocked for 1 h at 4 ◦ C. The samples were centrifuged after incubation and the lysate was aspirated. The beads were then washed twice with lysis buffer and the bound proteins were removed from the beads by adding SDS-PAGE reducing sample buffer and boiling for 5 min in a water bath. The samples were then loaded onto an SDS-PAGE mini-gel and analyzed by Western immunoblotting using standard procedures.
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2.9. Surface expression of the BCR by fluorescence activated cell sorting (FACS) The J558 cells were centrifuged at 1500 rpm for 5 min and resuspended in 1 mL of cold sorter buffer (phosphate buffer saline (PBS) with 1% heat inactivated fetal calf serum (FCS)). One million cells were added to each Falcon 2054 FACScan tube and the volume was adjusted to 1 mL with sorter buffer. The cells were then centrifuged at 1500 rpm for 5 min and resuspended in 50 L of FITC-goat anti-IgM solution (30 g/mL of FITC-goat anti-IgM (Jackson ImmunoResearch Laboratories, Inc.) in sorter buffer) and incubated on ice for 30 min. The cells were resuspended in 1 mL of sorter buffer, vortexed and another millilitre of sorter buffer was added. The cells were then centrifuged at 1500 rpm for 5 min in the cold and resuspended in 1 mL of sorter buffer with 4 g/mL of 7-amino actinomycin D to mark the dead
Fig. 2. The association of the C␣ (//␣) protein with wild type (WT) Ig-␣ and mIgM. Stably transfected AtT20 non-lymphoid cells that were already expressing mIgM and wild type Ig-␣ [18,21] were stably transfected with DNA constructs that encoded the C␣ (//␣) protein and drug selected. The association of C␣ with Ig-␣ and mIgM was tested by co-immunoprecipitation assays in order to determine if the receptor would assemble. Panel a, diagrammatic representation of C␣-containing BCRs to show the potential interactions that could occur between C␣ (//␣) and WT Ig-␣ or WT Ig-. Panels b and c, AtT20 cells expressing C␣ were lysed and the BCR components were immunoprecipitated from 1000 g of whole cell lysate using Protein A-Sepharose and anti- heavy chain (IP#1) or anti-Ig-␣ cytoplasmic tail (IP#2) specific antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted as indicated, with either anti-Ig- cytoplasmic tail antibodies (b, upper panel) or anti-Ig- extracellular domain antibodies (c, upper panel) and then stripped and reprobed (S&R) with either anti-␣ cytoplasmic tail antibodies (b and c, lower panels). , , ␣, : the components of the BCR that the cell lines express, IP: immunoprecipitating antibody, IB: immunoblotting antibody, S&R: immunoblotting antibody used to reprobe the filter, 䊉: background band or immunoprecipitating antibody that reacts with the protein A-HRP. *: indicates that the proteins have variable glycosylation patterns. Double headed box arrow indicates the protein interactions. Dotted pattern: membrane IgM, white color: Ig-␣, grey color: Ig-. Molecular weight markers in kilodaltons (kDa) to the left of the panels.
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cells. The cells were then examined on the Becton Dickinson FACScan (BD Biosciences) according to manufacturers directions and the data was analyzed using CellQuest Software (BD Biosciences). 3. Results 3.1. Screening antibodies for immunoblotting and immunoprecipitation The immunoprecipitation experiments to determine protein:protein interactions required antibodies that were specific to the extracellular or cytoplasmic domains of Ig-␣ and Ig-. Additional domain-specific anti-Ig-␣ and anti-Ig- antibodies were obtained from Dr. Richard Meagher and Elizabeth McKinney (University of Georgia, Athens, GA). These rabbit antibodies
that recognized either the Ig-␣ extracellular domain (Fig. 1b; antibody #689) or the Ig- extracellular domain (Fig. 1d; antibody #624) were effective when used for immunoblotting and they also immunoprecipitated the proteins from cell lysates (data not shown). The ability of previously described anti-Ig- cytoplasmic tail antibodies and anti-Ig-␣ cytoplasmic tail antibodies [21,22] are shown in Fig. 1c and e, respectively and Fig. 1f–i. 3.2. Association of wild type Ig-α and Ig-β with the Cα protein (β/β/α) In a previous study [21] we showed that a BCR containing mIgM, wild type Ig-␣ and the C␣ (//␣) protein would be expressed on the cell surface, signal and support B cell development. When these components of the BCR were expressed together, mIgM associated with C␣ (Fig. 2a, con-
Fig. 3. The association of the C␣ (//␣) protein with wild type Ig- and mIgM. J558 plasmacytoma cells that were already expressing mIgM and wild type Ig- were transiently transfected with DNA constructs that encoded the C␣ (//␣) protein and drug selected. The association of C␣ with Ig- and mIgM were tested by co-immunoprecipitation assays in order to determine if a homodimer of the extracellular and transmembrane region of Ig- would interfere with receptor assembly. Panel a, center and right drawing, diagrammatic representations of two C␣-containing BCRs to show the potential interactions that could occur between C␣ (//␣)and WT Ig-␣ or WT Ig-. Panels b and c, J558 cells expressing C␣ were lysed and the BCR components were immunoprecipitated from 1000 g of protein from whole cell lysate using Protein A-Sepharose and anti- heavy chain (IP#1) or anti-Ig-␣ cytoplasmic tail (IP#2) specific antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with either anti-Ig-␣ cytoplasmic tail antibodies (b, upper panel) or anti-Ig- cytoplasmic tail antibodies (c, upper panel) and then stripped and reprobed (S&R) with either anti- chain antibodies (b, lower panel) or anti-Ig-␣ cytoplasmic tail antibodies (c, lower panels). , , ␣, : the components of the BCR that the cell line expresses, IP: immunoprecipitating antibody, IB: immunoblotting antibody, S&R: immunoblotting antibody used to reprobe the filter, 䊉: background band, *: immunoprecipitating antibody that reacts with the protein A-HRP. Dotted pattern: membrane IgM, white color: Ig-␣, grey color: Ig-. Molecular weight markers in kilodaltons (kDa) to the left of the panels.
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figuration shown in the center drawing; Fig. 2b, upper panel) and with Ig-␣ (Fig. 2b, lower panel). Thus a heterodimer of Ig-␣ and Ig- extracellular and transmembrane domains, but not the cytoplasmic tail, is sufficient to support Ig-␣/ association and BCR surface expression ([21] and data not shown). Similar associations or lack thereof were shown using an antiIg- extracellular domain antibody (Fig. 2c) as well as the previously mentioned anti-Ig- cytoplasmic domain antibody (Fig. 2b). However, the C␣ (//␣) protein failed to associate with mIgM (Fig. 3a, configuration shown in the rightmost drawing; Fig. 3b, upper panel) and did not co-immunoprecipitate with wild type Ig- (Fig. 3c). Thus, homodimers of the Ig extracellular and transmembrane domains impeded Ig-␣/ association and assembly with mIgM. The most likely interpretation of this data is that the sequences in the transmembrane region of Ig-␣/ are required for stable association at the cell surface.
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3.3. Association of wild type Ig-β with the Xα2 protein (α/β/β) The X␣2 protein was co-expressed with Ig- in order to see if a heterodimer of the extracellular domains (one Ig- and one Ig-␣), while retaining an Ig- homodimer of the transmembrane region, would allow for receptor assembly (Fig. 4a, configuration on the right). Membrane IgM and Ig- both failed to associate with X␣2 (Fig. 4b, upper panel, Fig. 4c, respectively). We went on to express soluble forms of both Ig-␣ and Ig- transiently and stably in J558 cells with the other chains of the BCR (data not shown). And while intracellular protein could be detected, it was expressed at low levels and we were unable to show the association of just the extracellular domains with the rest of the receptor proteins. This is consistent with the idea that the transmembrane regions of the Ig-␣/ are key for efficient receptor assembly.
Fig. 4. The association of the X␣2 (␣//) protein with wild type Ig- and mIgM. J558 plasmacytoma cells that were already expressing mIgM and wild type Ig- were transiently transfected with DNA constructs that encoded the X␣2 (␣//) protein and drug selected. The association of X␣2 with Ig- and mIgM were tested by co-immunoprecipitation assays in order to determine if a homodimer of the transmembrane region of Ig- would interfere with receptor assembly. Panel a, a diagrammatic representation of the X␣2-containing BCR showing potential interactions that could occur between the different components. Panels b and c, J558 cells expressing X␣2 were lysed and the BCR components were immunoprecipitated from 1000 g of whole cell lysate using Protein A-Sepharose and anti- heavy chain (IP#1) or anti-Ig- extracellular domain (IP#2) specific antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with anti-Ig-␣ extracellular domain antibodies (b and c, upper panels) and then stripped and reprobed (S&R) with either anti- chain antibodies (b, lower panel) or anti-Ig- cytoplasmic tail antibodies (c, lower panel). , , ␣, : the components of the BCR that the cell line expresses, IP: immunoprecipitating antibody, IB: immunoblotting antibody, S&R: immunoblotting antibody used to reprobe the filter, 䊉: background bands. The various sizes of Ig- are due to a range of glycosylated forms, *: possible degradation product of the Ig-␣ protein. Dotted pattern: membrane IgM, white color: Ig-␣, grey color: Ig-. Molecular weight markers in kilodaltons (kDa) to the left of the panels.
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Fig. 5. The association of the C (␣/␣/) protein with wild type Ig- and mIgM. J558 plasmacytoma cells that were already expressing mIgM and wild type Ig- were transiently transfected with DNA constructs that encoded the C (␣/␣/) protein and drug selected. Similarly, stably transfected AtT20 cells expressing mIgM and wild type Ig- (WT5 cells) were stably re-transfected with C (␣/␣/). The association of C with Ig- and with mIgM was tested by co-immunoprecipitation assays in order to determine if a heterodimer (as opposed to homodimers) of the transmembrane region and extracellular domain of Ig- would allow for BCR assembly. Panel a, a diagrammatic representation of the C-containing BCR showing potential interactions that could occur between the different components. Panels b and c, J558 cells expressing C were lysed and the BCR components were immunoprecipitated from 1000 g of whole cell lysate using Protein A-Sepharose and anti- heavy chain (IP#1) or anti-Ig- extracellular domain (IP#2) specific antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with anti-Ig-␣ extracellular domain antibodies (b and c, upper panels) and then stripped and reprobed (S&R) with either anti- chain antibodies (b, lower panel) or anti-Ig- cytoplasmic tail antibodies (c, lower panel). Panels d and e, non-lymphoid AtT20 cells expressing the components of the BCR listed on the top of each gel were lysed and the BCR chains were immunoprecipitated as described above, with either anti- chain antibodies or anti-Ig- extracellular domain antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with anti-Ig-␣ extracellular domain antibodies (d and e, upper panels) and then stripped and reprobed with either anti- chain antibodies (d, lower panel) or anti-Ig- cytoplasmic tail antibodies (e, lower panel). , , ␣, : the components of the BCR that the cell line expresses. Ig-@ indicates the less glycosylated form of Ig-. C# represents C that was not removed completely after the strip and reprobe. IP: immunoprecipitating antibody, IB: immunoblotting antibody, S&R: immunoblotting antibody used to reprobe the filter, 䊉: background bands, *: the various sizes of Ig- are due to a range of glycosylated forms. Dotted pattern: membrane IgM, white color: Ig-␣, grey color: Ig-. Molecular weight markers in kilodaltons (kDa) to the left of the panels.
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3.4. Association of wild type Ig-β with the Cβ protein (α/α/β) and expression on the cell surface A simple swap of the transmembrane domain of X␣2 (␣//) to that of Ig-␣, generates the C protein (␣/␣/) (Fig. 5a, configurations in center and right drawing). C co-immunoprecipitates
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with mIgM (Fig. 5b, upper panel) and it co-immunoprecipitates with wild type Ig- (Fig. 5c, upper panel) when co-expressed in populations of transfected J558 lymphoid cells. Analogous associations between the chains were seen when these constructs were expressed in our stably transfected non-lymphoid cell system using AtT20 pituitary cells, where the amount
Fig. 6. Surface expression of BCRs containing C␣, X␣2 or C. J558 cells already expressing membrane IgM and wild type Ig- were transiently transfected with constructs encoding WT Ig-␣ (␣/␣/␣) or mutated chimeric constructs encoding C␣ (//␣), X␣2 (␣//) or C (␣/␣/). Transfected cells were selected in drug media and the cell surface BCRs were detected by staining intact cells with FITC-goat anti-mouse IgM heavy chain antibodies. Cells were also incubated with 7-amino actinomycin D (7-AAD) to label the dead cells. Cell populations were then analyzed by flow cytometry and the percentage cells containing surface heavy chain quantified as an indication of surface BCR levels. , , Ig-␣, Ig-: indicate the components of the BCR that the cell line expresses. The Greek ␣ or  letters in parentheses represent the extracellular domain, transmembrane domain and cytoplasmic tail, in sequence from left to right, found in the Ig-␣/ chain of interest. Quantification of the percentage of total cells in each quadrant is shown in the corners of each quadrant. For example, the BCR containing wild type Ig-␣ (␣/␣/␣) has 57% of the counted cells showing surface BCR (number shown in the lower right quadrant). The BCR containing C (␣/␣/) has 29% of the counted cells showing surface BCR. In the image showing the expressed protein, white coloration represents Ig-␣, grey coloration represents Ig-.
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of protein expressed was larger and co-immunoprecipitation recovered more of the interacting chains. Note that C coimmunoprecipitated with mIgM (Fig. 5d, upper panel) and with Ig-␣ (Fig. 5e, upper panel). These data taken together indicate that lymphoid specific chaperone proteins were not necessary for receptor assembly and trafficking. More importantly, when C, X␣2 and C␣ are all individually reconstituted into J558 cells expressing mIgM and wild type Ig, and compared, only C was capable of rescuing the complete BCR from the secretory pathway and allowing for cell surface expression as detected by flow cytometry (FACS) using a fluorescently tagged antibody to the H chain of mIgM (Fig. 6). In the wild type BCR panel the two clusters of data points represent either cells that were fluorescently labeled and were expressing the BCR on their cell surface (lower right quadrant) or cells that were not (lower left quadrant). The upper quadrants represent dead cells. Of the three constructs tested by this method, the only one that had data points in the lower right quadrant was C. This indicates that BCRs containing C are expressed on the cell surface while the BCRs containing C␣ or X␣2 are not expressed on the cell surface. Thus, we have shown using the reciprocal construct C (compared with C␣ in Fig. 3, which failed to associate and assemble into BCRs), that a heterodimer of the Ig-␣/ extracellular and transmembrane domains were sufficient to promote Ig-␣/ association, BCR assembly and cell surface expression. 4. Discussion BCR assembly and trafficking to the cell surface is dependent upon the proper association of all four components of the BCR. This study has shown that heterodimerization between Ig-␣ and Ig- in the extracellular and transmembrane domains in necessary for BCR assembly. Assembly of the BCR within the cell is dependent upon chaperone proteins that retain BCR components in the ER until assembly is complete [18,19]. The sequence of events leading to BCR assembly by chaperone proteins may require Ig-␣/ heterodimerization in the extracellular domain. The chaperone proteins bound to Ig-␣ or Ig- may remain bound to the signaling part of the BCR until disulfide bond formation occurs between Ig-␣ and Ig-. Disulfide bonds could potentially occur between homodimers, but this may be prevented due to conformational differences or due to interactions between the regions of the proteins where carbohydrate addition occurs. Ig␣ and Ig- have a different number of extracellular glycosylation sites, two and four, respectively, and these may assist or impede disulfide bond formation between heterodimers. This study has shown that a key interaction between mIgM and Ig-␣ appears to occur in the transmembrane domain. As mentioned, mIgM contains nine polar amino acids in its transmembrane domain. This is unusual for a membrane spanning protein and suggests that these amino acids are likely interacting with another transmembrane region [10,27]. Taking into consideration that both Ig-␣ and Ig- also have polar amino acids in their transmembrane region it is possible that Ig-␣ and/or Ig shield the polar amino acids of mIgM from the hydrophobic lipid bilayer by way of hydrogen bonding between the residues.
Additional interactions between Ig-␣/ and mIgM may occur by in the extracellular regions and its state of glycosylation may be important. A study done by Li et al. [28] indicated that the secreted form of IgM that is lacking the transmembrane domain is still able to associate with Ig-␣ and Ig- and, when IgM is deglycosylated Ig-␣/ binding is reduced. The experiments showing that murine Ig-␣ protein expressed by itself with mIgM can easily associate with the rest of the receptor [29] suggest a preference to the assembly process. Reciprocal experiments were we expressed the Ig- protein by itself with mIgM did not result in Ig- association with the rest of the BCR (data not shown), and with this it is tempting to propose that Ig-␣ preferentially binds first with mIgM before Ig-. However, in B lymphoma variants that lack mIgM expression, the Ig-␣/ efficiently assemble with each other [30]. Thus it is unclear in the murine system, what the exact order of BCR assembly might be and this is in contrast to studies of the human BCR where the belief that the human Ig-␣/ heterodimer must form before associating with human mIgM [31]. The formation of the Ig-␣/ heterodimer seems to require heterodimerization in the extracellular and transmembrane domains. The extracellular interactions are through disulfide bonds and potentially through other non-covalent interactions or contact between other extracellular regions. The heterodimerization in the transmembrane domain is conceivably by way of hydrogen bonding between two particular amino acid residues. The fifth residue in the Ig-␣ transmembrane region is glutamic acid, a negatively charged polar amino acid. This amino acid could be interacting with the sixth amino acid in the Ig transmembrane domain, a glutamine residue. Glutamic acid and glutamine are both strongly polar residues and could form strong ionic and hydrogen bonds. Interactions could be strengthened in the transmembrane environment which does not contain water molecules that compete with these residues for hydrogen bonding [32]. We have found that the extracellular and transmembrane domains of Ig-␣/ need to be heterodimeric in order for BCR assembly and cell surface trafficking to occur. Dimerization in the cytoplasmic domain may also be required, but previous studies showed that homodimers of the cytoplasmic tail, or a missing cytoplasmic tail have no effect on assembly and secretion of the BCR [20,21]. Our studies will allow us to identify mutated forms of the BCR to use in future studies, in particular those with changes in the cytoplasmic tails, in order to better define their roles in cell signaling and the downstream effects on cytoskeletal rearrangements that affect cell adhesion, cell spreading, receptor internalization/endocytosis and the formation of actin rich immune synapse formation [33–36].
Acknowledgements The work has been supported by grants to L.M. from the Canadian Institute of Health Research (CIHR) and the National Science and Engineering Council of Canada (NSERC). The work is submitted as partial fulfillment of a M.Sc. degree to J. Dylke from the University of British Columbia.
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References [1] Hombach J, Lottspeich F, Reth M. Identification of the genes encoding the IgM-␣ and Ig- components of the IgM antigen receptor complex by amino-terminal sequencing. Eur J Immunol 1990;20:2795–9. [2] Reth M, Wienands J, Tsubata T, Hombach J. Identification of components of the B cell antigen receptor complex. Adv Exp Med Biol 1991;292: 207–14. [3] Dal Porto JM, Gauld SB, Merrell KT, Mills D, Pugh-Bernard AE, Cambier JC. B cell antigen receptor signaling 101. Mol Immunol 2004;41:599–613. [4] Gold MR. To make antibodies or not: signaling by the B-cell antigen receptor. Trends Pharm Sci 2002;23:316–24. [5] Thompson AA, Talley JA, Do NH, Kagan HL, Kunkel L, Berenson J, et al. Aberrations of the B Cell receptors B29 (CD79b) gene in chronic lymphocytic leukemia. Blood 1997;90:1387–94. [6] Reth M, Hombach J, Wienands J, Campbell KS, Chien N, Justement LB, et al. The B-cell antigen receptor complex. Immunol Today 1991;12:196–201. [7] Hombach J, Leclercq L, Radbruch A, Rajewsky K, Reth M. A novel 34-kd protein co-isolated with the IgM molecule in surface IgM-expressing cells. EMBO J 1988;7:3451–6. [8] Hermanson GG, Eisenberg D, Kincade PW, Wall R. B29: a member of the immunoglobulin gene superfamily exclusively expressed on B-lineage cells. Proc Natl Acad Sci USA 1988;85:6890–4. [9] Schamel WWA, Reth M. Monomeric and oligomeric complexes of the B cell antigen receptor. Immunity 2000;13:5–14. [10] Reth M. Antigen receptors on B lymphocytes. Ann Rev Immunol 1992;10:97–121. [11] Rogers J, Early P, Carter C, Calame K, Bond M, Hood L, et al. Two mRNAs with different 3 ends encode membrane-bound and secreted forms of immunoglobulin chain. Cell 1980;20:303–12. [12] Sakaguchi N, Kashiwamura SI, Kimoto M, Thalmann P, Melchers F. B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties. EMBO J 1988;7:3457–64. [13] Reth M. Antigen receptor tail clue. Nature 1989;338:383–4. [14] Patterson HC, Kraus M, Kim YM, Ploegh H, Rajewsky K. The B cell receptor promotes B cell activation and proliferation through a non-ITAM tyrosine in the Ig-␣ cytoplasmic domain. Immunity 2006;25:55–65. [15] Siemasko K, Skaggs BJ, Kabak S, Williamson E, Brown BK, Song W, et al. Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Ig-. J Immunol 2002;168:2127–38. [16] Kabak S, Skaggs BJ, Gold MR, Affolter M, West K, Foster M, et al. The direct recruitment of BLNK to Ig-␣ couples the B cell antigen receptor to distal signaling pathway. Molec Cell Biol 2002;22:2524–35. [17] Engels N, Wollscheid B, Wienands J. Association of SLP-65/BLNK with the B cell antigen receptor through a non-ITAM tyrosine of Ig-␣. Eur J Immunol 2001;31:2126–34. [18] Matsuuchi L, Gold MR, Travis A, Grosschedl R, DeFranco AL, Kelly RB. The membrane IgM-associated proteins MB-1 and Ig- are sufficient to promote surface expression of a partially functional B-cell antigen receptor in a nonlymphoid cell line. Proc Natl Acad Sci USA 1992;89: 3404–8. [19] Venkitaraman AR, Williams GT, Dariavach P, Neuberger MS. The B-cell antigen receptor of the five immunoglobulin classes. Nature 1991;352:777–82.
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[20] Reichlin A, Gazumyan A, Nagaoka H, Kirsch KH, Kraus M, Rajewsky K, et al. B cell receptor with two Ig-␣ cytoplasmic domains supports development of mature but not anergic B cells. J Exp Med 2004;199:855–65. [21] Wang LD, Lopes J, Cooper AB, Dang-Lawson M, Matsuuchi L, Clark MR. Selection of B lymphocytes in the periphery is determined by the functional capacity of the B cell antigen receptor. Proc Natl Acad Sci USA 2004;101:1027–32. [22] Thompson AA, Do NH, Saxon A, Wall R. Widespread B29 (CD79b) gene defects and loss of expression in chronic lymphocytic lymphoma. Leukemia Lymphoma 1999;32:561–9. [23] Gordon MS, Kato RM, Lansigan F, Thompson AA, Wall R, Rawlings DJ. Aberrant B cell receptor signaling from B29 (Ig-, CD79b) gene mutations of chronic lymphocytic leukemia B cells. Proc Natl Acad Sci USA 2000;97:5504–9. [24] Gold MR, Matsuuchi L, Kelly RB, DeFranco AL. Tyrosine phosphorylation of components of the B cell antigen receptor following receptor crosslinking. Proc Natl Acad Sci USA 1991;88:3436–40. [25] Matsuuchi L, Kelly RB. Constitutive and basal release from the endocrine cell line AtT20. J Cell Biol 1991;112:843–52. [26] Richards JD, Gold MR, Hourihane SL, DeFranco AL, Matsuuchi L. Reconstitution of B cell antigen receptor-induced signaling events in a nonlymphoid cell line by expressing the Syk protein-tyrosine kinase. J Biol Chem 1996;271:6458–66. [27] Banfic H, Tang X, Batty IH, Downes CP, Chen C, Rittenhouse SE. A novel integrin-activated pathway forms PKB/Akt-stimulatory phosphatidylinositol 3,4-bisphosphate via phosphatidylinositol 3-phosphate in platelets. J Biol Chem 1998;273:13–6. [28] Li Q, Santini R, Rosenspire AR. Glycosylated extracellular domains of membrane immunoglobulin contribute to its association with mb1/B29 gene products and the B cell receptor complex. Immunol Invest 1998;27:57–72. [29] Foy SP, Matsuuchi L. Association of B lymphocyte antigen receptor polypeptides with multiple chaperone proteins. Immunol Lett 2001;78:149–60. [30] Condon C, Hourihane SL, Dang-Lawson M, Escribano J, Matsuuchi L. Aberrant trafficking of the B cell antigen receptor Ig-␣/ subunit in a B lymphoma cell line. J Immunol 2000;165:1427–37. [31] Brouns GS, de Vries E, Borst J. Assembly and intracellular transport of the human B cell antigen receptor complex. Int Immunol 1995;7:359– 75. [32] Partridge AW, Melnyk RA, Deber CM. Polar residues in membrane domains of proteins: molecular basis for helix:helix association in a mutant CFTR TM segment. Biochemistry 2002;41:3647–53. [33] Batista FD, Iber D, Neuberger MS. B cells acquire antigen from target cells after synapse formation. Nature 2001;411:489–94. [34] Fleire SJ, Goldman JP, Carrasco YR, Weber M, Bray D, Batista FD. B cell ligand discrimination through a spreading and contraction response. Science 2006;312:738–41. [35] Carrasco YR, Fleire SJ, Cameron T, Dustin ML, Batista FD. LFA-1/ICAM1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 2004;20:589–99. [36] Carrasco YR, Batista FD. B-cell activation by membrane-bound antigens is facilitated by the interaction of VLA-4 with VCAM-1. EMBO J 2006;25:889–99.
Role of the extracellular and transmembrane domain of Ig-␣/ in assembly of the B cell antigen receptor (BCR) Janis Dylke, Jared Lopes 1 , May Dang-Lawson, Steve Machtaler, Linda Matsuuchi ∗ Cell Biology Group, Department of Zoology, Cell and Developmental Biology Research Group, Life Sciences Institute, Infection, Inflammation and Immunity (I3) Research Group, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada Received 21 September 2006; received in revised form 26 June 2007; accepted 27 June 2007 Available online 23 July 2007
Abstract The B cell antigen receptor (BCR) is expressed on the surface of B-lymphocytes where it binds antigen and transmits signals that regulate B cell activation, growth and differentiation. The BCR is composed of membrane IgM (mIgM) and two signaling proteins, Ig-␣ and Ig-. If either of the signaling proteins is not expressed, the incomplete mIgM-containing BCR will not traffic to the cell surface. Our hypothesis is that specific protein:protein interactions between both the extracellular and transmembrane (TM) regions of Ig-␣ and Ig- are necessary for receptor assembly, cell surface expression and effective signaling to support the proper development of B cells. While previous work has shown the importance of the TM region in BCR assembly, this study indicates that a heterodimer of the extracellular domains of Ig-␣ and Ig- are also required for proper association with mIgM. Cell lines expressing mutated Ig-␣ proteins that did not heterodimerize with Ig- in the extracellular and TM domains were unable to properly assemble the BCR. Conversely, an Ig-␣ mutant with an Ig- cytoplasmic tail (C (␣/␣/)) was able to assemble with the rest of the BCR, in particular with Ig-, and traffic to the cell surface. Thus, both the extracellular and TM regions of the Ig-␣/Ig- must be properly associated in order for the BCR to assemble. © 2007 Elsevier B.V. All rights reserved. Keywords: B cell antigen receptor; Assembly
1. Introduction The B cell antigen receptor (BCR) is a key regulator of B lymphocyte activation, growth and differentiation. It is expressed on the surface of B lymphocytes and is composed of a ligand binding receptor (membrane immunoglobulin; mIg) and a signaling component (Ig-␣/) [1,2]. B cell development in the bone marrow requires that the BCR signals to the cell when a functional receptor has trafficked to the cell surface and if the BCR binds to self antigen. This prevents non-functional and self-reactive B cells from escaping into the periphery. Once the B cell enters the periphery, the BCR binds foreign antigens and signals to the cell to proliferate and differentiate into an antibody produc∗
Corresponding author. Tel.: +1 604 822 4881; fax: +1 604 822 2416. E-mail address: [email protected] (L. Matsuuchi). 1 Current address: Department of Immunology, University of Washington, Benaroya Research Institute at Virginia Mason Medical Center, Seattle, WA 98101-2795, USA. 0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.06.005
ing plasma cell or a long-lived memory cell [3,4]. This allows the body to combat novel microorganisms and to keep a B cell repertoire that is reactive to microorganisms that have been previously encountered, allowing a faster immune response upon re-infection. In this sense, the functionality of the BCR is essential for the immune system to successfully eliminate foreign microorganisms. In addition, a non-functional BCR can lead to autoimmune diseases and B cell leukemias and lymphomas [5]. The mIg component of the mIgM BCR is unable to signal due to its short cytoplasmic tail, therefore it must associate with two signaling proteins, Ig-␣ and Ig-, to form the complete functional BCR [6–8]. Ig-␣ and Ig- are disulfide-linked, transmembrane proteins that are non-covalently associated with mIg. The current model suggests that one heterodimer of Ig-␣/ associates with one mIg complex [9]. Ig-␣ and Ig- are found on the surface of B cells during the early stages of B cell development. Antigen binding or cross-linking of the BCR results in signaling mediated by the Ig-␣/ heterodimer [2,10].
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The transmembrane portion of the protein is proposed to be an ␣-helix consisting of 26 amino acids that span the lipid bilayer [11]. Thirteen of those amino acids are highly conserved among different immunoglobulin isotypes and 11 of those 13 are proposed to line up on one side of the helix suggesting that they, or that side of the helix, may be important for interaction with other molecules. The transmembrane region is not composed entirely of hydrophobic amino acids as is seen with many transmembrane proteins. There are 9 polar amino acids in the transmembrane region of mIgM. This suggests that the H chain transmembrane region could be interacting with the transmembrane regions of other proteins since one might predict that polar amino acids should be hidden or shielded in the hydrophobic lipid bilayer [10]. The signaling portion of the BCR is composed of a heterodimer of Ig-␣ and Ig-. Ig-␣ is a transmembrane glycoprotein. The extracellular portion of murine Ig-␣ is composed of 109 amino acids. The amino acid sequence indicates that the extracellular regions of Ig-␣ fold into a barrel domain similar to those seen in the H and L chains. There are two cysteine residues (positions 50 and 101) that form a disulfide bond between the  sheets in the extracellular region. Another cysteine residue (position 113) forms a disulfide bond with Ig-. Ig-␣ also has two glycosylation sites at amino acids 58 and 68 [7,12]. The transmembrane domain is predicted as an ␣-helix consisting of 22 amino acids, and the cytoplasmic tail is predicted to contain 61 amino acids [10]. The cytoplasmic domain has a highly conserved immunoreceptor tyrosine-based activation motif (ITAM) with the amino acid sequence D/Ex7 D/Ex2 Yx2 L/Ix7 Yx2 L/I [13]. Additionally, the cytoplasmic domain has two other tyrosine residues (positions 176 and 204). Phosphorylation of these residues recruits the B cell linker proteins Vav and Grb2. Activated B cells lacking these residues do not effectively present antigen to T cells indicating that tyrosine176, 204 phosphorylation is important for intracellular trafficking of the receptor after antigen cross-linking and in B cell development [14,15]. The residue at position 204 may also have a role in binding the SH2 domain of the adaptor protein BLNK [16,17]. The Ig- protein is similar in structure to Ig-␣. The extracellular region of Ig- is composed of 129 amino acids that form  sheets. There are four cysteine residues that form two intramolecular disulfide bonds (43:124 and 65:120) and an additional cysteine residue (135) that forms the disulfide bond with Ig-␣. The extracellular domain also has three glycosylation sites [8]. The transmembrane region, like Ig-␣, is an ␣-helix composed of 22 amino acids. The cytoplasmic tail consists of 48 amino acids and has an ITAM sequence. The ITAM sequence for both Ig-␣ and Ig- is conserved in different species indicating that protein function requires that the sequence remains unchanged [13]. Tyrosine phosphorylation of the ITAM domains by Src-family tyrosine kinases leads to interaction with the SH2 domain of Syk, a protein tyrosine kinase, resulting in the activation of numerous downstream intracellular signaling events [3,4]. Murine Ig-, unlike Ig-␣, does not contain any additional tyrosine residues. The Ig-␣/ heterodimer makes up the signaling portion of the BCR.
All four components of the membrane IgM (mIgM)containing BCR (H chains, L chains, Ig-␣ and Ig-) must be expressed in the cell in order for assembly and cell surface trafficking to occur [18,19]. However, if only the cytoplasmic tails of Ig-␣ or Ig- are truncated or mutated the mIgM BCR can still traffic to the cell surface, but B cell signaling and development are disrupted [20,21]. The study by Reichlin et al. showed that truncation of the Ig-␣ or Ig- cytoplasmic tail, leaving a heterodimer in the extracellular and transmembrane domains, allowed for BCR formation and cell surface trafficking. In support of this data, a study by Wang et al. [21] showed that an Ig- mutant that heterodimerized with Ig-␣ in the extracellular and transmembrane domains, but homodimerized in the cytoplasmic domain, was able to form a complete BCR and traffic to the cell surface. Both of these studies suggest that heterodimerization in the extracellular and transmembrane domains are both required for BCR assembly and cell surface trafficking. This hypothesis is further examined here using three mutated Ig-␣ constructs (C␣ (//␣), X␣2 (␣//) and C (␣/␣/) to express altered chimeric forms of the protein. These studies will help us to increase our understanding of which portions of the BCR are required for BCR cell surface expression, of particular importance during the different stages of B cell development. This is important since disruptions of the normal levels of surface BCR are associated with various B cell lymphomas and these in turn correlate with mutations in the Ig-␣/ proteins. The idea being that either defects in cell signaling or in cell surface expression which influences the levels of cell signaling are responsible for the cues that the developing B lymphocyte receives to proliferate, become anergic or undergo apoptosis during the process of cancer development [5,22,23]. Thus some of the structural mutations in the Ig-␣/ protein that are outside of the cytoplasmic tail [22] are also important in this regard and our work here will help identify which of these regions to focus on as important regions of protein interaction during BCR assembly. 2. Materials and methods 2.1. Antibodies, immunoprecipitating and immunoblotting reagents The rabbit anti-mouse IgM ( chain specific) antibody was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pennsylvania). The rabbit anti-mouse light chain antibody was from Bethyl Laboratories (Montgomery, Texas). The rabbit antimouse Ig-␣ cytoplasmic tail antibody was described previously [24]. The rabbit anti-mouse Ig-␣ extracellular antibodies, produced with a 30 amino acid peptide to the extracellular domain of Ig-␣ (amino acids 29–58) and the rabbit anti-mouse Ig- extracellular antibody, produced with a 30 amino acid peptide to the extracellular domain of Ig- (amino acids 71–100) were from Dr. Richard Meagher (University of Georgia, Athens, Georgia). The rabbit anti-mouse Ig- cytoplasmic antibody that recognizes the cytoplasmic tail of Ig- was from Dr. Marcus Clark (University of Chicago, Chicago, Illinois). Horseradish peroxidase (HRP)-conjugated protein A was from Amersham Biosciences (Baie d’Urfe, Quebec). The goat anti-rabbit IgG-HRP and the
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fluorescein (FITC)-conjugated goat anti-mouse IgM ( chain specific) antibody were from Jackson Immunoresearch Laboratories, Inc. (West Grove, Pennsylvania). The rhodamine labeled goat anti-mouse IgM ( chain specific) used for cell surface fluorescence was from BD Biosciences (Palo Alto, California). 2.2. Plasmids The pMX-puro retroviral expression vector was from Dr. Alice Mui (Jack Bell Research Centre, Vancouver, BC). The pWZL-Blast1 and pWZL-Blast3 retroviral expression vectors were from Dr. Stephen Robbins (University of Calgary, Calgary, Alberta). The pMSCV-puro retroviral expression vector was purchased from BD Biosciences. The pMIGR1-Xα, pMIGR1-Igβ, pMIGR1-ΔCY and MIGR1p-Cα expression vectors were from Dr. Marcus Clark (University of Chicago, Chicago, Illinois) [21]. 2.3. Plasmids created 2.3.1. pMSCV-puro-Cβ and pWZL-Blast3-Cβ (α/α/β) This plasmid encodes an Ig-␣ protein that is extracellularly and transmembrane Ig-␣ and cytoplasmically Ig- (␣/␣/). Sitedirected mutagenesis was used to insert a BamHI restriction enzyme site into pMX-puro-␣ between the transmembrane and cytoplasmic domains. The primers used for site-directed mutagenesis were JDXA1 (5 G CTG CTA TTC AGG ATC CGG TAA TTG CGG CCG3 ) and JDXA2 (5 CGG CCG CAA TAA CCG GAT CCT GAA TAG CAG C3 ). The Ig-␣ fragment from pMX-puro-␣ was then excised using XhoI and BamHI restriction enzymes and inserted into pMIGRI-X␣ to create pMIGRI-C. The DNA encoding C was then excised from pMIGR1 using XhoI and EcoRI restriction enzymes and ligated into pWZL-Blast3 and pMSCV-puro to create pWZL-Blast3-C and pMSCV-puro-C. 2.3.2. pMSCV-puro-Xα2 and pWZL-Blast3-Xα2 (α/β/β) These plasmids encode a protein that is extracellularly Ig-␣ and transmembrane and cytoplasmically Ig- (␣//). Sitedirected mutagenesis was used to insert a SacII restriction enzyme site into pMIGR1-Ig and pMX-puro-␣ between the extracellular and transmembrane domains. The primers used for pMIGR1-Ig were SacB1 (5 CGG CGG AAC ACA CTG AAC CGC GGC ATT ATC TTG ATC CAG ACC3 ) and SacB2 (5 GGT CTG GAT CAA GAT AAT GCC GCG GTT CAG TGT GTT CCG CCG3 ) and the primers used for pMXpuro-␣ were SacA1 (5 GGG GAA GGT ACC AAG AAC CGC GGC ATC ACA GCA GAA GGG3 ) and SacA2 (5 CCC TTC TGC TGT GAT GCC GCG GTT CTT GGT ACC TTC CCC3 ). The Ig-␣ extracellular domain DNA fragment was then excised from pMX-puro-␣ using the XhoI and SacII restriction enzyme sites and inserted into pMIGR1-Ig, replacing the Ig- extracellular domain and creating pMIGR1-X␣2. The X␣2 DNA fragment was then excised from pMIGR1-X␣2 using XhoI and EcoRI restriction enzymes and ligated into pWZL-Blast3 and pMSCV-puro to create pWZL-Blast3-X␣2 and pMSCV-puro-X␣2.
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2.3.3. pWZL-Blast1-Cα (β/β/α) This plasmid encodes a protein that is extracellularly and transmembrane Ig- and cytoplasmically Ig-␣ (//␣). The DNA encoding C␣ was excised from MIGR1p-C␣ with BglII and XhoI restriction enzymes. It was then ligated into pWZL-Blast1 that had been digested with BglII and SalI (XhoI and SalI have compatible cohesive ends). This created pWZL-Blast1-C␣. 2.4. Molecular biology techniques Restriction enzymes (Invitrogen Life Technologies (Burlington, Ontario) or New England Biolabs (Pickering, Ontario)) were added to the DNA according to manufacturer’s instructions and digested at 37 ◦ C as directed. All molecular biology techniques were performed using standard procedures. DNA fragments were purified using a QIAquick Gel Extraction Kit (Qiagen, Mississauga, Ontario) following manufacturer’s instructions. Small volumes of plasmid DNA were purified using the GenElute Plasmid Miniprep Kit (Sigma–Aldrich Canada, Oakville, Ontario) according to manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using puReTaq Ready-ToGo PCR beads (Amersham Biosciences Canada) according to manufacturer’s instructions. The reactions were run at 95 ◦ C for 45 s, 55 ◦ C for 2 min and 72 ◦ C for 2 min for a total of 35 cycles. Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). All oligonucleotide primers were purchased from the Nucleic Acid Protein Service Unit (UBC) and the reactions were run in a DNA Thermal Cycler (Perkin-Elmer Cetus) according to manufacturer’s instructions. After transformation of the XL1-Blue Supercompetent bacteria, a single colony was used to inoculate LB broth for small scale DNA preparation. The resulting DNA was tested for the presence of the intended mutation by restriction enzyme digestion and DNA sequencing (Nucleic Acid Protein Service Unit). 2.5. Tissue culture cell lines The AtT20 and ASS murine pituitary tumor cell line was from Dr. Regis Kelly (University of California, San Francisco, California) and has been described [25]. The R142 cell line in an AtT20 cell line that expresses mIgM and Ig-␣ [18] and the WT5 cell line is an AtT20 cell line that expresses mIgM and Ig- [21]. The Syk 13 cell line is an AtT20 cell line that expresses mIgM, Ig-␣ and Ig- [26]. The J558 m3 and J558 15-25 murine plasmacytoma cell lines were from Dr. Louis Justement (University of Alabama, Birmingham, Alabama) and from Dr. Marcus Clark (University of Chicago, Chicago, Illinois). The BOSC 23 human fibroblast cell line was from Dr. Warren Pear (University of Pennsylvania, Philadelphia, Pennsylvania). Tissue culture cell lines were grown as described using standard procedures in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Life Technologies) or Roswell Park Memorial Institute (RPMI)-1640 media containing 4.5 g/L glucose, 2 mM l-glutamine, 110 mg/L sodium pyruvate, 5–10%
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heat inactivated fetal calf serum (FCS) (Invitrogen Life Technologies), 50 units/mL penicillin and 50 g/mL streptomycin sulfate (Invitrogen Life Technologies). 2.6. Cell lysis Cells were detergent lysed on ice with 0.5–1.0 mL of cold Triton X-100 lysis buffer (20 mM Tris–HCl pH 8.0, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA (Fisher Scientific Canada, Ottawa, Ontario), 10% glycerol, 10 g/mL leupeptin (Roche Diagnostics, Indianapolis, Indiana), 1 g/mL aproprotin (Roche Diagnostics), 1 mM pepstatin A (Sigma–Aldrich), 1 mM Na3 VO4 , 1 mM phenylmethylsulfonyl fluoride (PMSF) (Roche Diagnostics)) or cold digitonin lysis buffer (1% digitonin (Sigma–Aldrich), 10 mM triethanolamine pH 7.8, 150 mM NaCl, 1 mM EDTA, 10 g/mL leupeptin, 1 g/mL aproprotin,
1 mM pepstatin A, 1 mM PMSF). Lysates were centrifuged at 14,000 rpm for 15 minutes at 4 ◦ C and the protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, Illinois). The samples were frozen at −20 ◦ C for long-term storage. 2.7. Transfections Cells were transfected using retroviral particles or DNA mixtures using standard procedures. Cells were drug selected in media containing 0.4 g/mL puromycin (Calbiochem, La Jolla, California) or 4 g/mL of blasticidin S (Invitrogen Life Technologies), depending on the drug resistance gene that was used in the transfection. Drug resistant populations of cells as well as single stably transfected clones were recovered for further experiments.
Fig. 1. Antibody screening by immunoblotting and expression of new plasmids. Thirty to 50 g of protein from whole cell lysates from untransfected cells or from AtT20 cells stably expressing various components of the BCR, from transiently transfected BOSC fibroblast cells or from transiently transfected and drug selected J558 plasmacytoma cells were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with anti-Ig-␣ or anti-Ig- antibodies (Abs). Panel a, schematic diagram showing wild type and three different chimeric versions of Ig-␣ and Ig-, X␣2 (␣//; denoting the extracellular domain, TM domain and cytoplasmic tail, from left to right), C␣ (//␣) and C (␣/␣/). The white color represents Ig-␣, the grey color represents Ig-. Ig-␣ or Ig- domains appear in parentheses next to the name of the mutated protein in the order from the left, extracellular domain/transmembrane domain/cytoplasmic domain. Panels b–e, tests showing specificity of anti-Ig-␣/ extracellular and anti-cytoplasmic domain antibodies. Panels f–i, tests showing expression of new constructs encoding chimeric Ig-␣/s. Panel b, Abs used for immunoblot recognizing the extracellular domain of Ig-␣. Panel c, Abs used for immunoblot recognizing the cytoplasmic domain of Ig-. Panel d, Abs used for immunoblot recognizing the extracellular domain of Ig-. Panel e, Abs used for immunoblot recognizing the cytoplasmic domain of Ig-␣. Panel f, expression of X␣2 (␣//) and C (␣/␣/) in BOSC fibroblast cells. Panel g, expression of C␣ (//␣) in AtT20 cells expressing the other chains of the BCR. The various sizes are due to differential glycosylation. Panel h, expression of C (␣/␣/) and X␣2 (␣//) in J558 cells. Panel i, expression of C␣ (//␣) in J558 cells. , , ␣, : the components of the BCR that the cell line expresses. IB: immunoblotting antibody below each panel. 䊉: Background band. Grey coloration: Ig-, white coloration: Ig-␣. Molecular weight markers in kilodaltons (kDa) to the left of each panel.
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2.8. Immunoprecipitations All cells to be used for immunoprecipitations were lysed in digitonin lysis buffer. One thousand micrograms of cell lysate was added to 20 L of washed Protein A-Sepharose 4B beads (Sigma–Aldrich) in 1.5 mL tubes (Axygen Scientific). Five microlitres of antibody, previously determined to be in antibody excess for successful co-immunoprecipitation studies, were added to each tube and the mixture was rocked for 1 h at 4 ◦ C. The samples were centrifuged after incubation and the lysate was aspirated. The beads were then washed twice with lysis buffer and the bound proteins were removed from the beads by adding SDS-PAGE reducing sample buffer and boiling for 5 min in a water bath. The samples were then loaded onto an SDS-PAGE mini-gel and analyzed by Western immunoblotting using standard procedures.
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2.9. Surface expression of the BCR by fluorescence activated cell sorting (FACS) The J558 cells were centrifuged at 1500 rpm for 5 min and resuspended in 1 mL of cold sorter buffer (phosphate buffer saline (PBS) with 1% heat inactivated fetal calf serum (FCS)). One million cells were added to each Falcon 2054 FACScan tube and the volume was adjusted to 1 mL with sorter buffer. The cells were then centrifuged at 1500 rpm for 5 min and resuspended in 50 L of FITC-goat anti-IgM solution (30 g/mL of FITC-goat anti-IgM (Jackson ImmunoResearch Laboratories, Inc.) in sorter buffer) and incubated on ice for 30 min. The cells were resuspended in 1 mL of sorter buffer, vortexed and another millilitre of sorter buffer was added. The cells were then centrifuged at 1500 rpm for 5 min in the cold and resuspended in 1 mL of sorter buffer with 4 g/mL of 7-amino actinomycin D to mark the dead
Fig. 2. The association of the C␣ (//␣) protein with wild type (WT) Ig-␣ and mIgM. Stably transfected AtT20 non-lymphoid cells that were already expressing mIgM and wild type Ig-␣ [18,21] were stably transfected with DNA constructs that encoded the C␣ (//␣) protein and drug selected. The association of C␣ with Ig-␣ and mIgM was tested by co-immunoprecipitation assays in order to determine if the receptor would assemble. Panel a, diagrammatic representation of C␣-containing BCRs to show the potential interactions that could occur between C␣ (//␣) and WT Ig-␣ or WT Ig-. Panels b and c, AtT20 cells expressing C␣ were lysed and the BCR components were immunoprecipitated from 1000 g of whole cell lysate using Protein A-Sepharose and anti- heavy chain (IP#1) or anti-Ig-␣ cytoplasmic tail (IP#2) specific antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted as indicated, with either anti-Ig- cytoplasmic tail antibodies (b, upper panel) or anti-Ig- extracellular domain antibodies (c, upper panel) and then stripped and reprobed (S&R) with either anti-␣ cytoplasmic tail antibodies (b and c, lower panels). , , ␣, : the components of the BCR that the cell lines express, IP: immunoprecipitating antibody, IB: immunoblotting antibody, S&R: immunoblotting antibody used to reprobe the filter, 䊉: background band or immunoprecipitating antibody that reacts with the protein A-HRP. *: indicates that the proteins have variable glycosylation patterns. Double headed box arrow indicates the protein interactions. Dotted pattern: membrane IgM, white color: Ig-␣, grey color: Ig-. Molecular weight markers in kilodaltons (kDa) to the left of the panels.
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cells. The cells were then examined on the Becton Dickinson FACScan (BD Biosciences) according to manufacturers directions and the data was analyzed using CellQuest Software (BD Biosciences). 3. Results 3.1. Screening antibodies for immunoblotting and immunoprecipitation The immunoprecipitation experiments to determine protein:protein interactions required antibodies that were specific to the extracellular or cytoplasmic domains of Ig-␣ and Ig-. Additional domain-specific anti-Ig-␣ and anti-Ig- antibodies were obtained from Dr. Richard Meagher and Elizabeth McKinney (University of Georgia, Athens, GA). These rabbit antibodies
that recognized either the Ig-␣ extracellular domain (Fig. 1b; antibody #689) or the Ig- extracellular domain (Fig. 1d; antibody #624) were effective when used for immunoblotting and they also immunoprecipitated the proteins from cell lysates (data not shown). The ability of previously described anti-Ig- cytoplasmic tail antibodies and anti-Ig-␣ cytoplasmic tail antibodies [21,22] are shown in Fig. 1c and e, respectively and Fig. 1f–i. 3.2. Association of wild type Ig-α and Ig-β with the Cα protein (β/β/α) In a previous study [21] we showed that a BCR containing mIgM, wild type Ig-␣ and the C␣ (//␣) protein would be expressed on the cell surface, signal and support B cell development. When these components of the BCR were expressed together, mIgM associated with C␣ (Fig. 2a, con-
Fig. 3. The association of the C␣ (//␣) protein with wild type Ig- and mIgM. J558 plasmacytoma cells that were already expressing mIgM and wild type Ig- were transiently transfected with DNA constructs that encoded the C␣ (//␣) protein and drug selected. The association of C␣ with Ig- and mIgM were tested by co-immunoprecipitation assays in order to determine if a homodimer of the extracellular and transmembrane region of Ig- would interfere with receptor assembly. Panel a, center and right drawing, diagrammatic representations of two C␣-containing BCRs to show the potential interactions that could occur between C␣ (//␣)and WT Ig-␣ or WT Ig-. Panels b and c, J558 cells expressing C␣ were lysed and the BCR components were immunoprecipitated from 1000 g of protein from whole cell lysate using Protein A-Sepharose and anti- heavy chain (IP#1) or anti-Ig-␣ cytoplasmic tail (IP#2) specific antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with either anti-Ig-␣ cytoplasmic tail antibodies (b, upper panel) or anti-Ig- cytoplasmic tail antibodies (c, upper panel) and then stripped and reprobed (S&R) with either anti- chain antibodies (b, lower panel) or anti-Ig-␣ cytoplasmic tail antibodies (c, lower panels). , , ␣, : the components of the BCR that the cell line expresses, IP: immunoprecipitating antibody, IB: immunoblotting antibody, S&R: immunoblotting antibody used to reprobe the filter, 䊉: background band, *: immunoprecipitating antibody that reacts with the protein A-HRP. Dotted pattern: membrane IgM, white color: Ig-␣, grey color: Ig-. Molecular weight markers in kilodaltons (kDa) to the left of the panels.
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figuration shown in the center drawing; Fig. 2b, upper panel) and with Ig-␣ (Fig. 2b, lower panel). Thus a heterodimer of Ig-␣ and Ig- extracellular and transmembrane domains, but not the cytoplasmic tail, is sufficient to support Ig-␣/ association and BCR surface expression ([21] and data not shown). Similar associations or lack thereof were shown using an antiIg- extracellular domain antibody (Fig. 2c) as well as the previously mentioned anti-Ig- cytoplasmic domain antibody (Fig. 2b). However, the C␣ (//␣) protein failed to associate with mIgM (Fig. 3a, configuration shown in the rightmost drawing; Fig. 3b, upper panel) and did not co-immunoprecipitate with wild type Ig- (Fig. 3c). Thus, homodimers of the Ig extracellular and transmembrane domains impeded Ig-␣/ association and assembly with mIgM. The most likely interpretation of this data is that the sequences in the transmembrane region of Ig-␣/ are required for stable association at the cell surface.
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3.3. Association of wild type Ig-β with the Xα2 protein (α/β/β) The X␣2 protein was co-expressed with Ig- in order to see if a heterodimer of the extracellular domains (one Ig- and one Ig-␣), while retaining an Ig- homodimer of the transmembrane region, would allow for receptor assembly (Fig. 4a, configuration on the right). Membrane IgM and Ig- both failed to associate with X␣2 (Fig. 4b, upper panel, Fig. 4c, respectively). We went on to express soluble forms of both Ig-␣ and Ig- transiently and stably in J558 cells with the other chains of the BCR (data not shown). And while intracellular protein could be detected, it was expressed at low levels and we were unable to show the association of just the extracellular domains with the rest of the receptor proteins. This is consistent with the idea that the transmembrane regions of the Ig-␣/ are key for efficient receptor assembly.
Fig. 4. The association of the X␣2 (␣//) protein with wild type Ig- and mIgM. J558 plasmacytoma cells that were already expressing mIgM and wild type Ig- were transiently transfected with DNA constructs that encoded the X␣2 (␣//) protein and drug selected. The association of X␣2 with Ig- and mIgM were tested by co-immunoprecipitation assays in order to determine if a homodimer of the transmembrane region of Ig- would interfere with receptor assembly. Panel a, a diagrammatic representation of the X␣2-containing BCR showing potential interactions that could occur between the different components. Panels b and c, J558 cells expressing X␣2 were lysed and the BCR components were immunoprecipitated from 1000 g of whole cell lysate using Protein A-Sepharose and anti- heavy chain (IP#1) or anti-Ig- extracellular domain (IP#2) specific antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with anti-Ig-␣ extracellular domain antibodies (b and c, upper panels) and then stripped and reprobed (S&R) with either anti- chain antibodies (b, lower panel) or anti-Ig- cytoplasmic tail antibodies (c, lower panel). , , ␣, : the components of the BCR that the cell line expresses, IP: immunoprecipitating antibody, IB: immunoblotting antibody, S&R: immunoblotting antibody used to reprobe the filter, 䊉: background bands. The various sizes of Ig- are due to a range of glycosylated forms, *: possible degradation product of the Ig-␣ protein. Dotted pattern: membrane IgM, white color: Ig-␣, grey color: Ig-. Molecular weight markers in kilodaltons (kDa) to the left of the panels.
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Fig. 5. The association of the C (␣/␣/) protein with wild type Ig- and mIgM. J558 plasmacytoma cells that were already expressing mIgM and wild type Ig- were transiently transfected with DNA constructs that encoded the C (␣/␣/) protein and drug selected. Similarly, stably transfected AtT20 cells expressing mIgM and wild type Ig- (WT5 cells) were stably re-transfected with C (␣/␣/). The association of C with Ig- and with mIgM was tested by co-immunoprecipitation assays in order to determine if a heterodimer (as opposed to homodimers) of the transmembrane region and extracellular domain of Ig- would allow for BCR assembly. Panel a, a diagrammatic representation of the C-containing BCR showing potential interactions that could occur between the different components. Panels b and c, J558 cells expressing C were lysed and the BCR components were immunoprecipitated from 1000 g of whole cell lysate using Protein A-Sepharose and anti- heavy chain (IP#1) or anti-Ig- extracellular domain (IP#2) specific antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with anti-Ig-␣ extracellular domain antibodies (b and c, upper panels) and then stripped and reprobed (S&R) with either anti- chain antibodies (b, lower panel) or anti-Ig- cytoplasmic tail antibodies (c, lower panel). Panels d and e, non-lymphoid AtT20 cells expressing the components of the BCR listed on the top of each gel were lysed and the BCR chains were immunoprecipitated as described above, with either anti- chain antibodies or anti-Ig- extracellular domain antibodies. Immunoprecipitates were analyzed by SDS-PAGE. Proteins from the gels were transferred to nitrocellulose and immunoblotted with anti-Ig-␣ extracellular domain antibodies (d and e, upper panels) and then stripped and reprobed with either anti- chain antibodies (d, lower panel) or anti-Ig- cytoplasmic tail antibodies (e, lower panel). , , ␣, : the components of the BCR that the cell line expresses. Ig-@ indicates the less glycosylated form of Ig-. C# represents C that was not removed completely after the strip and reprobe. IP: immunoprecipitating antibody, IB: immunoblotting antibody, S&R: immunoblotting antibody used to reprobe the filter, 䊉: background bands, *: the various sizes of Ig- are due to a range of glycosylated forms. Dotted pattern: membrane IgM, white color: Ig-␣, grey color: Ig-. Molecular weight markers in kilodaltons (kDa) to the left of the panels.
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3.4. Association of wild type Ig-β with the Cβ protein (α/α/β) and expression on the cell surface A simple swap of the transmembrane domain of X␣2 (␣//) to that of Ig-␣, generates the C protein (␣/␣/) (Fig. 5a, configurations in center and right drawing). C co-immunoprecipitates
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with mIgM (Fig. 5b, upper panel) and it co-immunoprecipitates with wild type Ig- (Fig. 5c, upper panel) when co-expressed in populations of transfected J558 lymphoid cells. Analogous associations between the chains were seen when these constructs were expressed in our stably transfected non-lymphoid cell system using AtT20 pituitary cells, where the amount
Fig. 6. Surface expression of BCRs containing C␣, X␣2 or C. J558 cells already expressing membrane IgM and wild type Ig- were transiently transfected with constructs encoding WT Ig-␣ (␣/␣/␣) or mutated chimeric constructs encoding C␣ (//␣), X␣2 (␣//) or C (␣/␣/). Transfected cells were selected in drug media and the cell surface BCRs were detected by staining intact cells with FITC-goat anti-mouse IgM heavy chain antibodies. Cells were also incubated with 7-amino actinomycin D (7-AAD) to label the dead cells. Cell populations were then analyzed by flow cytometry and the percentage cells containing surface heavy chain quantified as an indication of surface BCR levels. , , Ig-␣, Ig-: indicate the components of the BCR that the cell line expresses. The Greek ␣ or  letters in parentheses represent the extracellular domain, transmembrane domain and cytoplasmic tail, in sequence from left to right, found in the Ig-␣/ chain of interest. Quantification of the percentage of total cells in each quadrant is shown in the corners of each quadrant. For example, the BCR containing wild type Ig-␣ (␣/␣/␣) has 57% of the counted cells showing surface BCR (number shown in the lower right quadrant). The BCR containing C (␣/␣/) has 29% of the counted cells showing surface BCR. In the image showing the expressed protein, white coloration represents Ig-␣, grey coloration represents Ig-.
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of protein expressed was larger and co-immunoprecipitation recovered more of the interacting chains. Note that C coimmunoprecipitated with mIgM (Fig. 5d, upper panel) and with Ig-␣ (Fig. 5e, upper panel). These data taken together indicate that lymphoid specific chaperone proteins were not necessary for receptor assembly and trafficking. More importantly, when C, X␣2 and C␣ are all individually reconstituted into J558 cells expressing mIgM and wild type Ig, and compared, only C was capable of rescuing the complete BCR from the secretory pathway and allowing for cell surface expression as detected by flow cytometry (FACS) using a fluorescently tagged antibody to the H chain of mIgM (Fig. 6). In the wild type BCR panel the two clusters of data points represent either cells that were fluorescently labeled and were expressing the BCR on their cell surface (lower right quadrant) or cells that were not (lower left quadrant). The upper quadrants represent dead cells. Of the three constructs tested by this method, the only one that had data points in the lower right quadrant was C. This indicates that BCRs containing C are expressed on the cell surface while the BCRs containing C␣ or X␣2 are not expressed on the cell surface. Thus, we have shown using the reciprocal construct C (compared with C␣ in Fig. 3, which failed to associate and assemble into BCRs), that a heterodimer of the Ig-␣/ extracellular and transmembrane domains were sufficient to promote Ig-␣/ association, BCR assembly and cell surface expression. 4. Discussion BCR assembly and trafficking to the cell surface is dependent upon the proper association of all four components of the BCR. This study has shown that heterodimerization between Ig-␣ and Ig- in the extracellular and transmembrane domains in necessary for BCR assembly. Assembly of the BCR within the cell is dependent upon chaperone proteins that retain BCR components in the ER until assembly is complete [18,19]. The sequence of events leading to BCR assembly by chaperone proteins may require Ig-␣/ heterodimerization in the extracellular domain. The chaperone proteins bound to Ig-␣ or Ig- may remain bound to the signaling part of the BCR until disulfide bond formation occurs between Ig-␣ and Ig-. Disulfide bonds could potentially occur between homodimers, but this may be prevented due to conformational differences or due to interactions between the regions of the proteins where carbohydrate addition occurs. Ig␣ and Ig- have a different number of extracellular glycosylation sites, two and four, respectively, and these may assist or impede disulfide bond formation between heterodimers. This study has shown that a key interaction between mIgM and Ig-␣ appears to occur in the transmembrane domain. As mentioned, mIgM contains nine polar amino acids in its transmembrane domain. This is unusual for a membrane spanning protein and suggests that these amino acids are likely interacting with another transmembrane region [10,27]. Taking into consideration that both Ig-␣ and Ig- also have polar amino acids in their transmembrane region it is possible that Ig-␣ and/or Ig shield the polar amino acids of mIgM from the hydrophobic lipid bilayer by way of hydrogen bonding between the residues.
Additional interactions between Ig-␣/ and mIgM may occur by in the extracellular regions and its state of glycosylation may be important. A study done by Li et al. [28] indicated that the secreted form of IgM that is lacking the transmembrane domain is still able to associate with Ig-␣ and Ig- and, when IgM is deglycosylated Ig-␣/ binding is reduced. The experiments showing that murine Ig-␣ protein expressed by itself with mIgM can easily associate with the rest of the receptor [29] suggest a preference to the assembly process. Reciprocal experiments were we expressed the Ig- protein by itself with mIgM did not result in Ig- association with the rest of the BCR (data not shown), and with this it is tempting to propose that Ig-␣ preferentially binds first with mIgM before Ig-. However, in B lymphoma variants that lack mIgM expression, the Ig-␣/ efficiently assemble with each other [30]. Thus it is unclear in the murine system, what the exact order of BCR assembly might be and this is in contrast to studies of the human BCR where the belief that the human Ig-␣/ heterodimer must form before associating with human mIgM [31]. The formation of the Ig-␣/ heterodimer seems to require heterodimerization in the extracellular and transmembrane domains. The extracellular interactions are through disulfide bonds and potentially through other non-covalent interactions or contact between other extracellular regions. The heterodimerization in the transmembrane domain is conceivably by way of hydrogen bonding between two particular amino acid residues. The fifth residue in the Ig-␣ transmembrane region is glutamic acid, a negatively charged polar amino acid. This amino acid could be interacting with the sixth amino acid in the Ig transmembrane domain, a glutamine residue. Glutamic acid and glutamine are both strongly polar residues and could form strong ionic and hydrogen bonds. Interactions could be strengthened in the transmembrane environment which does not contain water molecules that compete with these residues for hydrogen bonding [32]. We have found that the extracellular and transmembrane domains of Ig-␣/ need to be heterodimeric in order for BCR assembly and cell surface trafficking to occur. Dimerization in the cytoplasmic domain may also be required, but previous studies showed that homodimers of the cytoplasmic tail, or a missing cytoplasmic tail have no effect on assembly and secretion of the BCR [20,21]. Our studies will allow us to identify mutated forms of the BCR to use in future studies, in particular those with changes in the cytoplasmic tails, in order to better define their roles in cell signaling and the downstream effects on cytoskeletal rearrangements that affect cell adhesion, cell spreading, receptor internalization/endocytosis and the formation of actin rich immune synapse formation [33–36].
Acknowledgements The work has been supported by grants to L.M. from the Canadian Institute of Health Research (CIHR) and the National Science and Engineering Council of Canada (NSERC). The work is submitted as partial fulfillment of a M.Sc. degree to J. Dylke from the University of British Columbia.
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References [1] Hombach J, Lottspeich F, Reth M. Identification of the genes encoding the IgM-␣ and Ig- components of the IgM antigen receptor complex by amino-terminal sequencing. Eur J Immunol 1990;20:2795–9. [2] Reth M, Wienands J, Tsubata T, Hombach J. Identification of components of the B cell antigen receptor complex. Adv Exp Med Biol 1991;292: 207–14. [3] Dal Porto JM, Gauld SB, Merrell KT, Mills D, Pugh-Bernard AE, Cambier JC. B cell antigen receptor signaling 101. Mol Immunol 2004;41:599–613. [4] Gold MR. To make antibodies or not: signaling by the B-cell antigen receptor. Trends Pharm Sci 2002;23:316–24. [5] Thompson AA, Talley JA, Do NH, Kagan HL, Kunkel L, Berenson J, et al. Aberrations of the B Cell receptors B29 (CD79b) gene in chronic lymphocytic leukemia. Blood 1997;90:1387–94. [6] Reth M, Hombach J, Wienands J, Campbell KS, Chien N, Justement LB, et al. The B-cell antigen receptor complex. Immunol Today 1991;12:196–201. [7] Hombach J, Leclercq L, Radbruch A, Rajewsky K, Reth M. A novel 34-kd protein co-isolated with the IgM molecule in surface IgM-expressing cells. EMBO J 1988;7:3451–6. [8] Hermanson GG, Eisenberg D, Kincade PW, Wall R. B29: a member of the immunoglobulin gene superfamily exclusively expressed on B-lineage cells. Proc Natl Acad Sci USA 1988;85:6890–4. [9] Schamel WWA, Reth M. Monomeric and oligomeric complexes of the B cell antigen receptor. Immunity 2000;13:5–14. [10] Reth M. Antigen receptors on B lymphocytes. Ann Rev Immunol 1992;10:97–121. [11] Rogers J, Early P, Carter C, Calame K, Bond M, Hood L, et al. Two mRNAs with different 3 ends encode membrane-bound and secreted forms of immunoglobulin chain. Cell 1980;20:303–12. [12] Sakaguchi N, Kashiwamura SI, Kimoto M, Thalmann P, Melchers F. B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties. EMBO J 1988;7:3457–64. [13] Reth M. Antigen receptor tail clue. Nature 1989;338:383–4. [14] Patterson HC, Kraus M, Kim YM, Ploegh H, Rajewsky K. The B cell receptor promotes B cell activation and proliferation through a non-ITAM tyrosine in the Ig-␣ cytoplasmic domain. Immunity 2006;25:55–65. [15] Siemasko K, Skaggs BJ, Kabak S, Williamson E, Brown BK, Song W, et al. Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Ig-. J Immunol 2002;168:2127–38. [16] Kabak S, Skaggs BJ, Gold MR, Affolter M, West K, Foster M, et al. The direct recruitment of BLNK to Ig-␣ couples the B cell antigen receptor to distal signaling pathway. Molec Cell Biol 2002;22:2524–35. [17] Engels N, Wollscheid B, Wienands J. Association of SLP-65/BLNK with the B cell antigen receptor through a non-ITAM tyrosine of Ig-␣. Eur J Immunol 2001;31:2126–34. [18] Matsuuchi L, Gold MR, Travis A, Grosschedl R, DeFranco AL, Kelly RB. The membrane IgM-associated proteins MB-1 and Ig- are sufficient to promote surface expression of a partially functional B-cell antigen receptor in a nonlymphoid cell line. Proc Natl Acad Sci USA 1992;89: 3404–8. [19] Venkitaraman AR, Williams GT, Dariavach P, Neuberger MS. The B-cell antigen receptor of the five immunoglobulin classes. Nature 1991;352:777–82.
57
[20] Reichlin A, Gazumyan A, Nagaoka H, Kirsch KH, Kraus M, Rajewsky K, et al. B cell receptor with two Ig-␣ cytoplasmic domains supports development of mature but not anergic B cells. J Exp Med 2004;199:855–65. [21] Wang LD, Lopes J, Cooper AB, Dang-Lawson M, Matsuuchi L, Clark MR. Selection of B lymphocytes in the periphery is determined by the functional capacity of the B cell antigen receptor. Proc Natl Acad Sci USA 2004;101:1027–32. [22] Thompson AA, Do NH, Saxon A, Wall R. Widespread B29 (CD79b) gene defects and loss of expression in chronic lymphocytic lymphoma. Leukemia Lymphoma 1999;32:561–9. [23] Gordon MS, Kato RM, Lansigan F, Thompson AA, Wall R, Rawlings DJ. Aberrant B cell receptor signaling from B29 (Ig-, CD79b) gene mutations of chronic lymphocytic leukemia B cells. Proc Natl Acad Sci USA 2000;97:5504–9. [24] Gold MR, Matsuuchi L, Kelly RB, DeFranco AL. Tyrosine phosphorylation of components of the B cell antigen receptor following receptor crosslinking. Proc Natl Acad Sci USA 1991;88:3436–40. [25] Matsuuchi L, Kelly RB. Constitutive and basal release from the endocrine cell line AtT20. J Cell Biol 1991;112:843–52. [26] Richards JD, Gold MR, Hourihane SL, DeFranco AL, Matsuuchi L. Reconstitution of B cell antigen receptor-induced signaling events in a nonlymphoid cell line by expressing the Syk protein-tyrosine kinase. J Biol Chem 1996;271:6458–66. [27] Banfic H, Tang X, Batty IH, Downes CP, Chen C, Rittenhouse SE. A novel integrin-activated pathway forms PKB/Akt-stimulatory phosphatidylinositol 3,4-bisphosphate via phosphatidylinositol 3-phosphate in platelets. J Biol Chem 1998;273:13–6. [28] Li Q, Santini R, Rosenspire AR. Glycosylated extracellular domains of membrane immunoglobulin contribute to its association with mb1/B29 gene products and the B cell receptor complex. Immunol Invest 1998;27:57–72. [29] Foy SP, Matsuuchi L. Association of B lymphocyte antigen receptor polypeptides with multiple chaperone proteins. Immunol Lett 2001;78:149–60. [30] Condon C, Hourihane SL, Dang-Lawson M, Escribano J, Matsuuchi L. Aberrant trafficking of the B cell antigen receptor Ig-␣/ subunit in a B lymphoma cell line. J Immunol 2000;165:1427–37. [31] Brouns GS, de Vries E, Borst J. Assembly and intracellular transport of the human B cell antigen receptor complex. Int Immunol 1995;7:359– 75. [32] Partridge AW, Melnyk RA, Deber CM. Polar residues in membrane domains of proteins: molecular basis for helix:helix association in a mutant CFTR TM segment. Biochemistry 2002;41:3647–53. [33] Batista FD, Iber D, Neuberger MS. B cells acquire antigen from target cells after synapse formation. Nature 2001;411:489–94. [34] Fleire SJ, Goldman JP, Carrasco YR, Weber M, Bray D, Batista FD. B cell ligand discrimination through a spreading and contraction response. Science 2006;312:738–41. [35] Carrasco YR, Fleire SJ, Cameron T, Dustin ML, Batista FD. LFA-1/ICAM1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 2004;20:589–99. [36] Carrasco YR, Batista FD. B-cell activation by membrane-bound antigens is facilitated by the interaction of VLA-4 with VCAM-1. EMBO J 2006;25:889–99.