Blymphocytes persist in a resting state until they are stimulated by polyvalent Ags recognized by their B cell Ag receptors (BCRs). Cross-linking of the BCR initiates a cascade of signals resulting in B lymphocyte activation, differentiation, and Ab secretion. The presence of Igs on the surface of B cells and their roles in mediating “blast transformation” were first suspected in 1965, when Sell and Gell reported the stimulation of rabbit lymphocytes by an allotype-specific antiserum (1). In the early 1980s, analysis of membrane-bound IgM (mIgM)2 revealed that it included the specialized membrane isoform of μ-chains (μm), which resulted from alternative mRNA splicing at discrete stages of B cell development (2, 3, 4). The μm-chains shared extracellular domains in common with μ-chains of secreted IgM; however, μm-chains possessed hydrophobic transmembrane (TM) and short cytoplasmic domains (three residues). The cytoplasmic tails of μm-chains lacked the intrinsic enzymatic activities found in many other cell surface receptors. This suggested that additional proteins were required for the transmission of signals in response to BCR cross-linking. As described in the featured Pillars of Immunology article by Michael Reth and colleagues (5), it took nearly a decade to identify the proteins that interact directly with μm-chains: Ig-α and its partner, Ig-β.

In 1988, the Reth laboratory attempted to assemble functional BCRs in B cell tumor lines (6). In mIg+ B lymphoma cells, they detected the display of exogenous μm-chains on the cell surface using an anti-idiotype antibody. In contrast, in mIg plasmacytoma cells, exogenous μm-chains were sequestered with endogenous light chains in the rough endoplasmic reticulum and were not displayed on the cell surface. In light of analogies with studies of T cell receptor complexes, these results indicated requirements for accessory proteins to bring Ig molecules to the surface of B cells.

In 1989, Fritz Melchers’s laboratory used subtracted cDNA libraries to identify transcripts restricted to B lymphocytes. Using a library generated from a pre-B cell line, Nobuo Sakaguchi isolated the m-mb-1 cDNA, which encoded a putative membrane protein, MB-1 (7). The open reading frame of the cDNA included putative leader and extracellular domains comprising an Ig fold as well as TM and cytoplasmic (61 aa) domains. The presence of consensus N-glycosylation sites suggested a 26- to 35-kDa membrane glycoprotein. Dot blot hybridization detected the restricted expression of mb-1 gene transcripts in cells representing pre-B and immature and mature B cells, but not in plasma cells, T cells, and nonlymphoid cells. Structural similarities between the putative MB-1 protein and the γ-chain of the T cell receptor CD3 complex suggested that MB-1 was part of a homologous complex on B cells.

In an accompanying paper, the Reth laboratory identified the protein missing from plasmacytoma cells as B34, a 34-kDa disulfide-linked glycoprotein associated with membrane-bound IgM (8). 558LμM cells were created by expressing (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific μm-chains stably in J558L plasmacytoma cells. The majority of 558LμM cells failed to display μm-chains efficiently on the plasma membrane; however, rare cells were mIgM+ (6). A clone of these rare cells, 558LμM3, displayed the transfected μm-chains homogeneously on the cell surface. Biosynthetically labeled (35S) 558LμM3 cells were lysed using digitonin (a mild detergent), and mIgM complexes were purified using NP-hapten affinity chromatography and fractionated by SDS-PAGE. The analysis revealed the association of μm-chains with B34. B34 was detected similarly in mIg complexes in a pre-B cell line. Importantly, the gain of mIgM on surfaces of 558LμM3 cells correlated with expression of mb-1 gene transcripts, which were not detected in parent mIgM 558LμM cells.

This issue’s Pillars of Immunology paper by Joachim Hombach and colleagues (5) expanded upon observations in the papers described above by confirming that the presence of B34 (renamed IgM-α) in mIgM complexes was dependent on mb-1 gene expression. Furthermore, they demonstrated the association of IgM-α with its partner Ig-β in disulfide-linked heterodimers. To validate that IgM-α was encoded by mb-1, 558LμM cells were transfected stably to express mb-1 transcripts. All mb-1 transfectants displayed mIgM as efficiently as the 558LμM3 clone. 35S-labeled mIgM complexes were affinity purified from digitonin lysates of mb-1-transfected 558LμM cells as before; however, in these experiments the purified proteins were fractionated using two-dimensional nonreducing/reducing SDS-PAGE. This method discriminates monomeric proteins, which track in a diagonal pattern, from disulfide-linked proteins, which migrate off of the diagonal. IgM-α was purified similarly from mb-1-transfected 558LμM cells and mIgM+ 558LμM3 cells, but not from parent 558LμM cells. These results demonstrated that IgM-α is linked with mb-1 gene expression. In addition, detection of an additional 39-kDa species, termed Ig-β, on two-dimensional SDS-PAGE gels required IgM-α expression. Comigration of Ig-β with IgM-α in the nonreducing dimension suggested their association in disulfide-linked heterodimers. Copurification of mIgM from 558LμM3 cells following cell surface labeling with 125I further confirmed that IgM-α and Ig-β colocalized in mIgM complexes on B cells. The specificity of these interactions was verified by the inability of IgM-α or Ig-β to coimmunoprecipitate with μm-chains in which the μ TM domain was substituted with the TM domain of the MHC class I (H-2Kk) protein, which allowed for constitutive display of the hybrid μ-chains on the cell surface.

Michael Reth also drew attention to similarities between the cytoplasmic tails of various TM proteins suspected of participating in signal transduction in lymphocytes (9), including CD3-γ, -δ, and -ζ of the TCR complex, the envelope protein of the bovine leukemia virus (gp30), and FcRεRI-γ and -β. Each of their cytoplasmic domains features the consensus motif D/EX7D/EX2YX2LX7YX2L/I. These sequences, later termed immunoreceptor tyrosine-based activation motifs or ITAMs (10), were conspicuously present in the putative proteins encoded by mb-1 (and B29; see below) mRNAs in B cells. It was suggested that cross-linking by antireceptor antibodies would align these motifs to facilitate the assembly of the higher order complexes necessary for TM signaling. The first direct evidence of the signaling functions of IgM-α (now termed Ig-α) came from the studies of Campbell and Cambier, who showed that proteins of the accessory complex were phosphorylated inducibly on tyrosine residues (11).

The presence of Ig-α, Ig-β, and a third species, Ig-γ, in pre-BCRs, mIgM, and other mIg complexes in mouse and human B cells was soon supported by several studies (11, 12, 13, 14, 15). N-terminal amino acid sequencing (16, 17, 18) and coimmunoprecipitation (19) confirmed that they were encoded by transcripts of mb-1 (Ig-α) and the B29 gene (Ig-β and Ig-γ), which was defined previously as a gene with B cell-restricted expression (20). Differential glycosylation of Ig-α was found to account for differences between proteins associated with mIgM or mIgD (14, 17).

In addition to B lineage cells, subsequent studies demonstrated that Ig heavy and light chains, together with Ig-α and Ig-β, were sufficient for the assembly of mIg complexes on nonlymphoid cells (14, 21). However, these complexes were not sufficient to transduce activating signals, including the mobilization of intracellular Ca2+, in these cells or in plasmacytomas (21, 22). This is largely due to requirements for Src family tyrosine kinases (e.g. Lyn), which phosphorylate tyrosine residues in ITAMs of the Ig-α cytoplasmic tail in response to BCR ligation (18, 23, 24, 25, 26, 27). Subsequently, it was determined that ITAM-phosphorylated Ig-α and Ig-β serve as scaffolding for coordinating the multiple signaling cascades necessary for BCR signaling (reviewed in Ref. 28). Finally, the essential requirements for ITAMs within Ig-α were demonstrated elegantly by defective B cell maturation and Ab responses in mice that expressed Ig-α lacking its cytoplasmic domain (29). In summary, the initial work by Reth and colleagues foreshadowed the rapid expansion of knowledge concerning mechanisms of signal transduction through the BCR and its roles in B cell biology.

The author has no financial conflict of interest.

2

Abbreviations used in this paper: mIgM, membrane-bound IgM; μm, μ-chain membrane isoform; NP, (4-hydroxy-3-nitrophenyl)acetyl-hapten; TM, transmembrane.

1
Sell, S., P. G. H. Gell.
1965
. Studies on rabbit lymphocytes in vitro. I. Stimulation of blast transformation with an antiallotype serum.
J. Exp. Med.
122
:
423
-440.
2
Alt, F. W., A. L. Bothwell, M. Knapp, E. Siden, E. Mather, M. Koshland, D. Baltimore.
1980
. Synthesis of secreted and membrane-bound immunoglobulin μ heavy chains is directed by mRNAs that differ at their 3′ ends.
Cell
20
:
293
-301.
3
Early, P., J. Rogers, M. Davis, K. Calame, M. Bond, R. Wall, L. Hood.
1980
. Two mRNAs can be produced from a single immunoglobulin μ gene by alternative RNA processing pathways.
Cell
20
:
313
-319.
4
Rogers, J., P. Early, C. Carter, K. Calame, M. Bond, L. Hood, R. Wall.
1980
. Two mRNAs with different 3′ ends encode membrane-bound and secreted forms of immunoglobulin μ chain.
Cell
20
:
303
-312.
5
Hombach, J., T. Tsubata, L. Leclerq, H. Stappert, M. Reth.
1990
. Molecular components of the B-cell antigen receptor complex of the IgM class.
Nature
343
:
760
-762.
6
Hombach, J., F. Sablitzky, K. Rajewsky, M. Reth.
1988
. Transfected plasmacytoma cells do not transport the membrane form of IgM to the cell surface.
J. Exp. Med.
167
:
652
-657.
7
Sakaguchi, N., S.-I. Kashiwamura, M. Kimoto, P. Thalmann, F. Melchers.
1988
. B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties.
EMBO J.
7
:
3457
-3464.
8
Hombach, J., L. Leclercq, A. Radbruch, K. Rajewsky, M. Reth.
1988
. A novel 34-kd protein co-isolated with the IgM molecule in surface IgM-expressing cells.
EMBO J.
7
:
3451
-3456.
9
Reth, M..
1989
. Antigen receptor tail clue.
Nature
338
:
383
-384.
10
Cambier, J. C..
1995
. New nomenclature for the Reth motif (or ARH1/TAM/ARAM/YXXL).
Immunol. Today
16
:
110
11
Campbell, K. S., J. C. Cambier.
1990
. B lymphocyte antigen receptors (mIg) are non-covalently associated with a disulfide linked, inducibly phosphorylated glycoprotein complex.
EMBO J.
9
:
441
-448.
12
Chen, J. Z., A. M. Stall, L. A. Herzenberg.
1990
. Differences in glycoprotein complexes associated with IgM and IgD on normal murine B cells potentially enable transduction of different signals.
EMBO J.
9
:
2117
-2124.
13
Parkhouse, R. M. E..
1990
. Three B-cell surface molecules associating with membrane immunoglobulin.
Immunology
69
:
298
-302.
14
Venkitaraman, J., G. Williams, M. Dariavach, M. Neuberger.
1991
. The B-cell antigen receptor of the five immunoglobulin classes.
Nature
352
:
777
-781.
15
Clark, M. R., R. J. Friedrich, K. S. Campbell, J. C. Cambier.
1992
. Human pre-B and B cell membrane μ-chains are noncovalently associated with a disulfide-linked complex containing a product of the B29 gene.
J. Immunol.
149
:
2857
-2863.
16
Hombach, J., F. Lottspeich, M. Reth.
1990
. Identification of the genes encoding the IgM-α and Ig-β components of the IgM antigen receptor complex by amino-terminal sequencing.
Eur. J. Immunol.
20
:
2795
-2799.
17
Campbell, K. S., E. J. Hager, J. C. Cambier.
1991
. Alpha-chains of IgM and IgD antigen receptor complexes are differentially N-glycosylated MB-1-related molecules.
J. Immunol.
147
:
1575
-1580.
18
Campbell, K. S., E. J. Hager, R. J. Friedrich, J. C. Cambier.
1991
. IgM IgM antigen receptor complex contains phosphoprotein products of B29 and mb-1 genes.
Proc. Natl. Acad. Sci. USA
88
:
3982
-3986.
19
Ishihara, K., W. J. Wood, Jr, M. Damore, G. G. Hermanson, R. Wall, P. W. Kincade.
1992
. B29 gene products complex with immunoglobulins on B lymphocytes.
Proc. Natl. Acad. Sci. USA
89
:
633
-637.
20
Hermanson, G. G., D. Eisenberg, P. W. Kincade, R. Wall.
1988
. B29: a member of the Ig superfamily exclusive expressed on B-lineage cells.
Proc. Natl. Acad. Sci. USA
85
:
6890
-6894.
21
Matsuuchi, L., M. R. Gold, A. Travis, R. Grosschedl, A. L. DeFranco, K. R.B. .
1992
. The membrane IgM-associated proteins MB-1 and Ig-β are sufficient to promote surface expression of a partially functional B-cell antigen receptor in a nonlymphoid cell line.
Proc. Natl Acad. Sci. USA
89
:
3404
-3408.
22
Justement, L. B., J. Weinands, J. Hombach, M. Reth, J. C. Cambier.
1990
. Membrane IgM and IgD molecules fail to transduce Ca2+ mobilizing signals when expressed on differentiated B lineage cells.
J. Immunol.
144
:
3272
-3280.
23
Gold, M. R., L. Matsuuchi, R. B. Kelly, A. L. DeFranco.
1991
. Tyrosine phosphorylation of components of the B-cell antigen receptors following receptor crosslinking.
Proc. Natl Acad. Sci. USA
88
:
3436
-3440.
24
Lin, J., L. B. Justement.
1992
. The MB-1/B29 heterodimer couples the B cell antigen receptor to multiple src family protein tyrosine kinases.
J. Immunol.
149
:
1548
-1555.
25
Clark, M. R., K. S. Campbell, A. Kazlauskas, S. A. Johnson, M. Hertz, T. A. Potter, C. Pleiman, J. C. Cambier.
1992
. The B cell antigen receptor complex: association of Ig-α and Ig-β with distinct cytoplasmic factors.
Science
258
:
123
-126.
26
Matsuo, T., J. Nomura, K. Kuwahara, H. Igarashi, S. Inui, M. Hamaguchi, M. Kimoto, N. Sakaguchi.
1993
. Cross-linking of B cell receptor-related MB-1 molecule induces protein tyrosine phosphorylation in early B lineage cells.
J. Immunol.
150
:
3766
-3775.
27
Kim, K. M., G. Alber, P. Weiser, M. Reth.
1993
. Differential signaling through the Ig-α and Ig-β components of the B cell antigen receptor.
Eur. J. Immunol.
23
:
911
-916.
28
Dal Porto, J. M., S. B. Gauld, K. T. Merrell, D. Mills, A. E. Pugh-Bernard, J. Cambier.
2004
. B cell antigen receptor signaling 101.
Mol. Immunol.
41
:
599
-613.
29
Torres, R. M., H. Flaswinkel, M. Reth, K. Rajewsky.
1996
. Aberrant B cell development and immune response in mice with a compromised BCR complex.
Science
272
:
1804
-1808.