Unveiling the B cell receptor structure.

Molecular structures provide a road map for understanding and controlling B cell receptor activation.

Cryo-EM structures of two human B cell receptor isotypes.

The B cell receptor (BCR) complex plays a critical role in B cell development and immune responses. The assembly mechanisms underlying the BCR complex remain unknown. We determined the cryo-electron microscopy (cryo-EM) structures of human IgG-BCR and IgM-BCR, which consist of membrane-bound immunoglobulin molecules (mIg) and Igα/ß subunits at a 1:1 stoichiometry. Assembly of both BCR complexes involves their extracellular domains, membrane-proximal connection peptides, and transmembrane (TM) helices. The TM helices of mIgG and mIgM share a conserved set of hydrophobic and polar interactions with Igα/ß TM helices. By contrast, the IgG-Cγ3 and IgM-Cµ4 domains interact with extracellular Ig-like domains of Igα/ß through head-to-tail and side-by-side modes, respectively. This work reveals the structural basis for BCR assembly and provides insights into BCR triggering.

Cryo-EM structure of the human IgM B cell receptor.

The B cell receptor (BCR) initiates immune responses through antigen recognition. We report a 3.3-angstrom cryo-electron microscopy structure of human immunoglobulin M (IgM)-BCR in the resting state. IgM-BCR comprises two heavy chains, two light chains, and the Igα/Igß heterodimer. The ectodomains of the heavy chains closely stack against those of Igα/Igß, with one heavy chain locked between Igα and Igß in the juxtamembrane region. Extracellular interactions may determine isotype specificity of the BCR. The transmembrane helices of IgM-BCR form a four-helix bundle that appears to be conserved among all BCR isotypes. This structure contains 14 glycosylation sites on the IgM-BCR ectodomains and reveals three potential surface binding sites. Our work reveals the organizational principles of the BCR and may facilitate the design of antibody-based therapeutics.

Tandem-Cleavage Linkers Improve the In Vivo Stability and Tolerability of Antibody-Drug Conjugates.

Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody-drug conjugates (ADCs), the sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, which leads to systemic release of the cytotoxic payload. This action reduces the therapeutic index by causing off-target toxicities that can be dose-limiting. For example, a common side-effect of ADCs made using peptide-cleavable linkers is myelosuppression, including neutropenia. Only a few reports describe methods for optimizing peptide linkers to maintain efficient and potent tumor payload delivery while enhancing circulating stability. Herein, we address these critical limitations through the development of a tandem-cleavage linker strategy, where two sequential enzymatic cleavage events mediate payload release. We prepared dipeptides that are protected from degradation in the circulation by a sterically encumbering glucuronide moiety. Upon ADC internalization and lysosomal degradation, the monosaccharide is removed and the exposed dipeptide is degraded, which liberates the attached payload inside the target cell. We used CD79b-targeted monomethyl auristatin E (MMAE) conjugates as our model system and compared the stability, efficacy, and tolerability of ADCs made with tandem-cleavage linkers to ADCs made using standard technology with the vedotin linker. The results, where rat studies showed dramatically improved tolerability in the hematopoietic compartment, highlight the role that linker stability plays in efficacy and tolerability and also offer a means of improving an ADC's therapeutic index for improved patient outcomes.

B cell receptor accessory molecule CD79 gets involved in response against Streptococcus agalactiae infection and BCR signaling in Nile tilapia (Oreochromis niloticus).

CD79, composed of two distinct chains called CD79a and CD79b, is a transmembrane protein that forms a B cell antigen receptor with membrane immunoglobulin, and generates a signal following antigen recognition by the B cell receptor. In this study, the CD79a (OnCD79a) and CD79b (OnCD79b) were cloned and identified from Nile tilapia (Oreochromis niloticus). The cDNA of ORF for OnCD79a and OnCD79b are 669 and 627 bp, coding 222 and 208 amino acids, respectively. The deduced protein analysis showed that both CD79a andCD79b contain an immunoreceptor tyrosine-based activation motif in their intracellular tails that used to propagate a signal in a B cell. Expression analysis revealed that both CD79a and CD79b expressed at high levels in immune tissues, such as anterior kidney and spleen, and in IgM+ B cells. Upon Streptococcus agalactiae (S. agalactiae) infection, the expressions of OnCD79a and OnCD79b were significantly up-regulated in anterior kidney and spleen. The significant up-regulations of OnCD79a and OnCD79b were also detected in leukocytes after in vitro challenge with S. agalactiae. Further, stimulations of LPS and anti-OnIgM monoclonal antibody induced significant up-regulations of OnCD79a and OnCD79b in leukocytes. Taken together, the results of this study indicated that CD79 molecule, playing roles in BCR signaling, was likely to get involved in host defense against bacterial infection in Nile tilapia.

The localization of CD3, CD79a, CD68 and S100 protein immunoreactive cells in hemal nodes of Saanen goat (Capra hircus).

We investigated the structure of hemal nodes in Saanen goats using immunohistochemical staining. We examined the distribution of CD3 positive T lymphocytes, CD79a positive B lymphocytes, CD68 positive macrophages and S100 protein positive follicular dendritic cells. Hemal nodes of six healty adult female goats were used. Hemal nodes were removed from the thoracic and abdominal cavities. The oval to round hemal nodes were observed especially between the abdominal aorta and vena cava, and near the kidneys and adrenal glands. Tissue sections were stained with Crossmon's modified triple stain to demonstrate general histological structure. The avidin-biotin-peroxidase technique using anti-CD3, anti-CD79a, anti-CD68 and anti-S100 primary antibodies was used for immunohistochemistry. Many CD3 positive T lymphocytes were found in the germinal center of the lymph follicles and in the lymphatic cords of hemal nodes; CD3 positive cells also were observed in the sinuses. CD79a and CD68 positive cells were found at the germinal center of the lymph follicles. In the lymph follicles near the subcapsular sinuses, CD79a and CD68 positive cells were found especially in e areas bordering the mantle zone. S100 positive cells were found in the lymph follicles, lymphatic cords and sinuses.

Multiplex sequencing for EZH2, CD79B, and MYD88 mutations using archival cytospin preparations from B-cell non-Hodgkin lymphoma aspirates previously tested for MYC rearrangement and IGH/BCL2 translocation.

BACKGROUND: Gene rearrangements and specific translocations define some B-cell non-Hodgkin lymphoma (NHL) subtypes. Genome-wide mutational studies have revealed recurrent point mutations with prognostic implications. The goals of this study were to evaluate the feasibility of applying a multiplex mutation assay to archival cytospin preparations (CPs) and to investigate the rate of EZH2, CD79B, and MYD88 mutations in B-cell NHL samples previously tested for MYC rearrangement and/or IGH/BCL-2 translocation. METHODS: DNA was extracted from archival CPs of B-cell NHL cases with previous fluorescence in situ hybridization (FISH) assays for MYC rearrangement and/or IGH/BCL-2 translocation. Multiplex sequencing was performed for the detection of EZH2 (Y641), CD79B (Y196), and MYD88 (L265) mutations. Sanger sequencing was applied to samples with positive results and failed assays. RESULTS: Eighty-eight archival CPs were available from 40 patients. Alterations detected by FISH were: MYC rearrangement (10 cases), IGH/BCL-2 translocations (21 cases), dual translocations (6 cases), and other abnormalities for IGH/BCL-2 (23 cases) and for MYC (16 cases). DNA concentration ranged from 1.88 to 62.85 ng/µL (mean, 9.46 ng/µL). Successful results were obtained in 88.0% of the specimens submitted to multiplex sequencing. With Sanger sequencing, 2 additional mutated cases were found, and all cases with mutations were confirmed. Eight specimens showed mutations: 6 for EZH2, 1 for CD79B, and 1 for MYD88. Among them, 5 cases showed concurrent MYC and/or IGH/BCL-2 translocations and 2 revealed abnormal signals of IGH/BCL-2 and MYC. CONCLUSIONS: CPs archived for up to 6 years are a reliable source of high-quality genomic material for multiplex sequencing. Almost all B-cell NHL with point mutations showed concurrent chromosomal abnormalities.

Therapeutic targeting of the BCR-associated protein CD79b in a TCR-based approach is hampered by aberrant expression of CD79b.

Immunotherapy of B-cell malignancies using CD19-targeted chimeric antigen receptor-transduced T cells or CD20-targeted therapeutic monoclonal antibodies has shown clinical efficacy. However, refractory disease and the emergence of antigen-loss tumor escape variants after treatment demonstrate the need to target additional antigens. Here we aimed to target the B-cell receptor-associated protein CD79b by a T-cell receptor (TCR)-based approach. Because thymic selection depletes high-avidity T cells recognizing CD79b-derived peptides presented in self-HLA molecules, we aimed to isolate T cells recognizing these peptides presented in allogeneic HLA. Peptide-HLA tetramers composed of CD79b peptides bound to either HLA-A2 or HLA-B7 were used to isolate T-cell clones from HLA-A*0201 and B*0702-negative individuals. For 3 distinct T-cell clones, CD79b specificity was confirmed through CD79b gene transduction and CD79b-specific shRNA knockdown. The CD79b-specific T-cell clones were highly reactive against CD79b-expressing primary B-cell malignancies, whereas no recognition of nonhematopoietic cells was observed. Although lacking CD79b-cell surface expression, intermediate reactivity toward monocytes, hematopoietic progenitor cells, and T-cells was observed. Quantitative reverse transcriptase polymerase chain reaction revealed low CD79b gene expression in these cell types. Therefore, aberrant gene expression must be taken into consideration when selecting common, apparently lineage-specific self-antigens as targets for TCR-based immunotherapies.

Tyrosine phosphorylation within the intrinsically disordered cytosolic domains of the B-cell receptor: an NMR-based structural analysis.

Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b in vitro before and after phosphorylation. The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.

Time-resolved multidimensional NMR with non-uniform sampling.

Time-resolved experiments demand high resolution both in spectral dimensions and in time of the studied kinetic process. The latter requirement traditionally prohibits applications of the multidimensional experiments, which, although capable of providing invaluable information about structure and dynamics and almost unlimited spectral resolution, require too lengthy data collection. Our work shows that the problem has a solution in using modern methods of NMR data collection and signal processing. A continuous fast pulsing three-dimensional experiment is acquired using non-uniform sampling during full time of the studied reaction. High sensitivity and time-resolution of a few minutes is achieved by simultaneous processing of the full data set with the multi-dimensional decomposition. The method is verified and illustrated in realistic simulations and by measuring deuterium exchange rates of amide protons in ubiquitin. We applied the method for characterizing kinetics of in vitro phosphorylation of two tyrosine residues in an intrinsically disordered cytosolic domain of the B cell receptor protein CD79b. Signals of many residues including tyrosines in both phosphorylated and unmodified forms of CD79b are found in a heavily crowded region of 2D ¹H-¹5N correlation spectrum and the significantly enhanced spectral resolution provided by the 3D time-resolved approach was essential for the quantitative site-specific analysis.

B cell receptor accessory molecule CD79α: characterisation and expression analysis in a cartilaginous fish, the spiny dogfish (Squalus acanthias).

CD79α (also known as Igα) is a component of the B cell antigen receptor complex and plays an important role in B cell signalling. The CD79α protein is present on the surface of B cells throughout their life cycle, and is absent on all other healthy cells, making it a highly reliable marker for B cells in mammals. In this study the spiny dogfish (Squalus acanthias) CD79α (SaCD79α) is described and its expression studied under constitutive and stimulated conditions. The spiny dogfish CD79α cDNA contains an open reading frame of 618 bp, encoding a protein of 205 amino acids. Comparison of the SaCD79α gene with that of other species shows that the gross structure (number of exons, exon/intron boundaries, etc.) is highly conserved across phylogeny. Additionally, analysis of the 5' flanking region shows SaCD79α lacks a TATA box and possesses binding sites for multiple transcription factors implicated in its B cell-specific gene transcription in other species. Spiny dogfish CD79α is most highly expressed in immune tissues, such as spleen, epigonal and Leydig organ, and its transcript level significantly correlates with those of spiny dogfish immunoglobulin heavy chains. Additionally, CD79α transcription is up-regulated, to a small but significant degree, in peripheral blood cells following stimulation with pokeweed mitogen. These results strongly indicate that, as in mammals, spiny dogfish CD79α is expressed by shark B cells where it associates with surface-bound immunoglobulin to form a fully functional BCR, and thus may serve as a pan-B cell marker in future shark immunological studies.

Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

Syk is a dual-specificity kinase that self-regulates the signal output from the B-cell antigen receptor.

Upon B-cell activation, the signaling subunits Ig-α and Ig-ß of the B-cell antigen receptor become phosphorylated not only on tyrosines but also on serine residues. Using a specific antibody, we show that serine 197 (S197) in the cytoplasmic tail of Ig-α is phosphorylated upon B-cell antigen receptor activation, and that this modification inhibits the signal output of the B-cell antigen receptor. Surprisingly, we found that the well-known protein tyrosine kinase Syk (spleen tyrosine kinase) phosphorylates S197 on Ig-α, thus not only activating but also inhibiting signaling from the B-cell antigen receptor. This finding identifies Syk as a dual-specificity kinase and establishes a previously unexplored paradigm for the self-regulation of biological signaling processes.

Structural and functional studies of Igalphabeta and its assembly with the B cell antigen receptor.

The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Igbeta form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Igalpha displays a similar fold as Igbeta. An Igalphabeta heterodimer model was generated based on the unique disulfide-bonded Igbeta dimer. Solution binding studies showed that the extracellular domains of Igalphabeta preferentially recognize the constant region of BCR with mu chain specificity, suggesting a role for Igalphabeta to enhance BCRmu chain signaling. Cluster mutations on Igalpha, Igbeta, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the Cmu4 domain of mIgM. These studies provide the first structural model for understanding BCR function.

Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

In vivo effects of targeting CD79b with antibodies and antibody-drug conjugates.

Antibodies directed against B cells are in use for the treatment of non-Hodgkin's lymphoma and autoimmune disorders. The B-cell-restricted surface antigen CD79b, a signaling component of the B-cell receptor, has been shown as a promising antibody target in mouse efficacy models of systemic lupus erythematosus. Anti-CD79b antibody-drug conjugates (ADC), cytotoxic drugs linked through specialized chemical linkers to antibodies, are effective in mouse xenograft models of non-Hodgkin's lymphoma. We were interested in evaluating the systemic effects of anti-CD79b antibodies and ADCs in normal animals as a step toward the development of these molecules as therapeutics. As we were unable to identify any cell surface binding anti-human CD79b antibodies that were cross-reactive to other species, we developed an antibody to cynomolgus monkey (Macaca fascicularis) CD79b (anti-cyCD79b). The anti-cynomolgus antibody, anti-cyCD79b (10D10), and the maytansine (tubulin inhibitor)-conjugated ADC, anti-cyCD79b (10D10)-MCC-DM1, were administered to cynomolgus monkeys at approximately 30 mg/kg (6,000 microg DM1/m(2)) for two doses 3 weeks apart. Anti-cyCD79b and anti-cyCD79b-MCC-DM1 resulted in peripheral blood B-cell depletion of approximately 65% and approximately 94%, respectively. In addition, anti-cyCD79b-MCC-DM1 resulted in near-complete absence of splenic germinal centers, an observation supporting an effect on dividing B cells. Both molecules were well tolerated, with minimal findings for the antibody and findings for the ADC limited to the lymphoid and hematopoietic systems, liver, and peripheral nerves. These preclinical data suggest that targeting CD79b with antibodies or ADCs may provide safe and effective therapies for B-cell malignancies and autoimmune diseases.

B cell receptor accessory molecules in the channel catfish, Ictalurus punctatus.

B cell receptor (BCR) accessory molecules CD79a and CD79b homologs were identified in the channel catfish, Ictalurus punctatus. Both are found as single copy genes that encode proteins containing a signal peptide, an extracellular immunoglobulin domain, a transmembrane region and a cytoplasmic tail containing an immune-receptor tyrosine-dased activation motif (ITAM). IpCD79a and IpCD79b transcripts correlate well with IgM message expression. They are highly expressed in peripheral blood leukocytes (PBL) enriched in membrane (m) IgM+ cells and catfish clonal B cell lines, but not in catfish clonal T cells, indicating that IpCD79a and IpCD79b expression is B cell restricted. Studies using catfish clonal B cells (3B11) transfected with constructs encoding epitope-tagged IpCD79a and IpCD79b revealed that IpCD79a was expressed as a 45 kDa protein and IpCD79b was expressed as a 32 kDa protein. Furthermore, co-immunoprecipitations of epitope-tagged CD79 proteins demonstrate that these molecules are non-covalently associated with mIgM. These data correlate with some of the previous immunoprecipitation data demonstrating that catfish mIgM associates with proteins of 45 and 32 kDa.

MHC class II structural requirements for the association with Igalpha/beta, and signaling of calcium mobilization and cell death.

Emerging evidence indicates that in addition to their well-characterized role in antigen presentation, MHC II molecules transmit signals that induce death of APCs. Appropriately timed APC death is important for prevention of autoimmunity. Though the exact mechanism of MHC II-mediated cell death signaling is unknown, the response appears independent of caspase activation and does not involve Fas-FasL interaction. Here we investigated MHC II structural requirements for mediation of cell death signaling in a murine B cell lymphoma. We found that neither the transmembrane spanning regions nor the cytoplasmic tails of MHC II, which are required for MHC II-mediated cAMP production and PKC activation, are required for the death response. However, mutations in the connecting peptide region of MHC II alpha chain (alphaCP), but not the beta chain (betaCP), resulted in significant impairment of the death response. The alphaCP mutant was also unable to mediate calcium mobilization responses, and did not associate with Igalpha/beta. Knock-down of Igbeta by shRNA eliminated the MHC II-mediated calcium response but not cell death. We propose that MHC II mediates cell death signaling via association with an undefined cell surface protein(s), whose interaction is partially dependent on alphaCP region.

Signaling status of IgG B cell receptor (IgG BCR) is indicative for an activated state of circulating B cells in multiple myeloma.

Circulating post-switch B cells have been proposed as proliferative and disseminating progenitors in multiple myeloma. It is unclear whether the class-switched antigen receptor expressed at the surface of these cells plays a role in their expansion. In this work, the signaling status of IgG B cell receptor (BCR) isolated from the lysates of peripheral blood lymphocytes of 32 patients with IgG multiple myeloma, at the time of diagnosis, was investigated by examining whether phosphorylation of BCR Igalpha and Igbeta signal transducer factors (co-receptors) or other signaling molecules was abnormal in these cells when compared with healthy controls. In IgG BCR of normal controls, weak phosphorylation of 56 and 61 kDa Src kinase-related proteins and unphosphorylated co-receptors were found. In myeloma, p56 and p61 kDa proteins, co-receptors, and other IgG BCR-associated proteins from the signal cascade were phosphorylated. Myeloma patients can be classified into subgroups by IgG BCR phosphorylation profiles which characteristically coordinated with the level of IgG paraprotein in serum and the stage of disease. There was a correlative trend between the extent of phosphorylation reduction and advanced stage of disease. Reduced phosphorylation was more pronounced with advanced stages of multiple myeloma.

Role of the extracellular and transmembrane domain of Ig-alpha/beta in assembly of the B cell antigen receptor (BCR).

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-alpha and Ig-beta. 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-alpha and Ig-beta 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-alpha and Ig-beta are also required for proper association with mIgM. Cell lines expressing mutated Ig-alpha proteins that did not heterodimerize with Ig-beta in the extracellular and TM domains were unable to properly assemble the BCR. Conversely, an Ig-alpha mutant with an Ig-beta cytoplasmic tail (Cbeta (alpha/alpha/beta)) was able to assemble with the rest of the BCR, in particular with Ig-beta, and traffic to the cell surface. Thus, both the extracellular and TM regions of the Ig-alpha/Ig-beta must be properly associated in order for the BCR to assemble.