Models of signal transduction through the B‐cell antigen receptor

From the numerous surface markers of a B lymphocyte, the B-cell antigen receptor (BCR) complex is probably the most powerful marker influencing the developmental processes of the cell. The BCR consists of the membrane-bound immunoglobulin (mIg) but, depending on the state of differentiation, may be associated with a couple of other transmembrane proteins, most notably Igα (CD79a) and Igβ (CD79b).1 The N-terminal end of the mIg harbours the antigen-binding site, characterized by an incredibly high potential for diversification and built up by the variable regions of heavy and light chains. Followed by three or four constant domains, depending on the selected immunoglobulin-isotype, the mIg finally expresses two further domains: a transmembrane domain and a cytoplasmic tail, both of which vary in their isotype-specific amino acid composition.2 So far, the sheath proteins Igα and Igβ are known as the signal transduction component of the BCR complex, connecting the antigen receptor to the tyrosine phosphorylation pathways in the cell. All isotypes of mIg can form a complex with Igα and Igβ,3 indicating an involvement of all isotypes in the signal transduction pathway. Venkitaraman et al.3 showed that for mIgG and mIgD the Igα/Igβ sheath is not required for surface expression. However, Igα/Igβ is the minimum requirement for signal transduction4 and, in the case of IgM, is responsible for the release from intracellular retention sites.5 Igα is expressed by the mb-1 gene and is a 32 000 MW glycoprotein. Igβ (B29 gene) can be expressed in two different isoforms of 37 000 and 39 000 MW, respectively. Interestingly, Igα can be differentially glycosylated. Pogue and Goodnow6 suggested that the extracellular spacer domain of mIgD is necessary and sufficient to confer the mIgD-specific glycosylation pattern of the mb-1 gene.6 However, it remains to be elucidated if and how alternative glycosylation of mb-1 may affect signalling competence or internalization. Based on original studies, the transmembrane domain of mIgs was identified as a stretch of 25 uncharged amino acids between charged residues of the putative extracellular and intracellular regions.7 This implies a very short cytoplasmic tail of only three amino acids lysine (K), valine (V), lysine (K) for the μ and δ heavy chains. However, according to the neural network protein prediction method (PHDtopology)8 the transmembrane domain is assumed to be six amino acids shorter, resulting in cytoplasmic tails six amino acids in length for IgM and IgD. A third algorithm,9 even suggests a μ-cytoplasmic tail of 11 amino acid residues. Undoubtedly, this would have an effect on the strength of the immunoglobulin tails to interact with signal-transducing components, as supposed by Cambier and colleagues.2 Most of the functional data concerning the transmembrane domain are deduced from point mutation studies. It could be shown that two polar motifs, the –YSTTVT- and the –TTAST- patches within the μ transmembrane domain are of crucial importance for correct functioning of the BCR. Amino acid residues of these regions contain structural information sufficient for association of μ chains with the Igα/Igβ sheath.10 This assembly may be additionally affected by the glycosylation pattern of the extracellular domain of the BCR.11 Pleiman et al.12 reported, that distinct point mutations within the –YSTTVT- motif totally inhibited antigen-induced signal transduction, though the interaction with Igα was not impaired. However, the use of polyclonal anti-μ serum restored signal transduction probably as a result of more efficient cross-linking of the BCR. Pleiman et al.12 further hypothesized that the presence of Igα/Igβ, although absolutely required, is not sufficient for signal induction, suggesting the existence of a kinase prebound to the μ heavy chain. This assumption supports the finding of Williams et al.13 who identified a serine/threonine kinase associated with the IgM-BCR independently of the Igα/Igβ sheath. Mitchell et al.14 mutated the tyrosine residue within the –YSTTVT- to phenylalanine and showed that the signalling competence was not influenced, but efficient processing and presentation of antigen was affected. The mutation did not affect the association with the Igα/Igβ sheath, pointing to a role of the mIg in intracellular trafficking to class II-rich processing vesicles. Whether these two polar motifs also influence the selective binding of the BCR-associated proteins (BAPs) remains to be investigated. Five BAPs have been identified so far, three of which exclusively bind mIgM (BAP32, BAP37 and BAP41)15 and two interact with mIgD (BAP29 and BAP31).16 The –TTAST- and –YSTTVT- patches have also been suggested to be critical for endoplasmic reticulum retention of the BCR. In addition to the transmembrane domain, the exoplasmic constant region was shown also to play a role in endoplasmic reticulum retention: deletion of the first constant exon CH1 of the μ heavy chain allowed surface expression in the absence of mb-1.17 It is likely that immunoglobulin chains are retained in the endoplasmic reticulum by chaperons such as calnexin,18 BiP and GRP94, preventing surface expression until their binding domains are masked by the Igα/Igβ dimer.19 This mechanism may provide correct assembly of the BCR and degradation of incorrectly folded or incomplete complexes.20 To date, most studies were performed using the μ heavy chain. However, neither the –YSTTVT- nor the –TTAST- patch is conserved among the different isotypes, suggesting further motifs to be responsible for Igα association. Furthermore, there is a remarkable clonal variation among the cell lines transfected with immunoglobulin heavy chain constructs concerning expression and surface transport, complicating a precise interpretation of the cellular and molecular events leading to correct assembly of functional BCRs.3,21 All immunoglobulin cytoplasmic tails differ in size and amino acid composition. However, with the exception of IgA, the first three amino acids (KVK; Kyte algorithm) are conserved among the different isotypes (Fig. 1). Probably, the putative three amino acid residues KVK of the mIgM and mIgD would be far too short to interact with any signal transducers, whereas a tail of six amino acids (as predicted by PHDtopology) or 11 amino acids (Kyte and Doolittle7) could be sufficient for active protein–protein interactions. Apparently, the charge of the tail rather than the precise amino acid composition of the KVK tail (at least in human μ chains) is responsible for proper signalling and internalization. An exchange of KVK to RIR in transfected cell lines impairs neither calcium mobilization nor antigen internalization. On the other hand, a negatively charged mIgM tail prevents the surface expression of the receptor, probably by interfering with the negatively charged phospholipids within the membrane.22 However, deletion of the KVK tail of the μ chain results in the generation of a phosphatidyl-inositol-linked membrane protein, lacking any competence for signalling and antigen presentation in transfected cell lines.23 The dramatic effects of removing or exchanging the cytoplasmic tail could indicate that the tail is strongly involved in the interaction with immunoglobulin-associated proteins. Alternatively, the deletion of the cytoplasmic domain could induce an inactivating allosteric conformational change in the structure of the mIgM molecule because of the covalent attachment to the membrane lipids. Amino acid alignment of the C-terminal domains of mouse mIg-isotypes. Light shading, domains as predicted by the Kyte and Doolittle algorithm; medium shading, domains as predicted by PHDtopology; dark shading, domains as predicted by the Klein algorithm. In the case of mIgA, the transmembrane and cytoplasmic domains are encoded by just one exon. Concerning the extracellular domain, light and dark shade are coherent. In contrast to mIgM, the tails of the other isotypes (γ, ε and α) are strongly suggested to interact with additional signalling units. Truncation of the murine ε and γ1 tails from 28 amino acids to the conserved KVK sequence in knock-out mice result in severely reduced quantity and affinity of the respective secreted antibody in both primary and secondary responses, though class switch was not affected.24,25 In a recent work Luger et al.26 additionally described that somatic diversity of the immunoglobulin repertoire is influenced by the cytoplasmic tails of mIgs. Furthermore, transfectants of γ1 tail-truncated constructs25 exhibit a dramatic reduction in surface expression. Weiser et al.27 showed that mIgG2a, without the Igα/Igβ sheath, is efficiently internalized after antigen binding, demonstrating the capacity of the tail to interact with the internalization machinery independently from sheath proteins. According to Patel and Neuberger,28 other isotypes require the Igα/Igβ sheath for antigen internalization, though the process of internalization may be driven independently on the tyrosine residues within the sheath. For correct intracellular targeting of the BCR however, Igα/Igβ may not be sufficient.14 Martin and Goodnow29 could show that an exchange of the transmembrane and cytoplasmic domain of mIgM for mIgG1 strongly enhances plasma cell formation by increasing clonal expansion and decreasing cell loss during the germinal centre reaction. They suggested that the longer tail is responsible for the enhanced secondary immune response. Summarizing, one can speculate whether the reduced serum response in tail-truncated mice is simply the result of reduced surface expression, or if the tail serves as the actual docking site for additional signalling components or adaptor proteins, thus affecting BCR-mediated signalling or antigen processing and presentation.30 One could argue that only the transmembrane regions are important for surface expression, as previously discussed, but regarding the slight variations in the transmembrane domain of the ε and γ subtypes, the longer tails could be supposed to compensate for a diminished potency of the transmembrane domain for Igα association. One of the first events after receptor engagement is the subsequent phosphorylation of the ITAMs (immunoreceptor tyrosine-based activation motif with the sequence –YxxLx7YxxI/L-, which are present as single copy in the cytoplasmic domains of Igα and Igβ (Fig. 2). This phosphorylation is mediated by members of the Src-family kinases Lyn, Blk, Fyn and/or Lck. The phosphorylated ITAMs provide a binding site for the SH2-containing kinase Syk, which in turn is subsequently activated by Src-family kinases, resulting in an enhanced binding capacity towards the phosphorylated ITAMs (src-family kinases also bear SH2 domains). This activation cascade leads to the recruitment of additional effector and scaffold molecules, most notably BLNK (SLP65 or BASH), HS1 and SHC which are further substrates for the protein tyrosine kinases (PTKs) leading to the formation of a putative stable signalling complex, the The signalling pathways activated receptor engagement are characterized and most notably protein kinases, by calcium and the All these pathways are is known how the phosphorylation of the ITAMs within the Igα/Igβ sheath is In two (Fig. for the of BCR signalling are the allosteric activation of cytoplasmic kinases antigen binding, and the activation of a cytoplasmic tyrosine kinase of two or more on the A further of with the presence of the kinases leading to The kinases could be or the antigen-induced of could result in their signalling for the allosteric as as the two further can be the and the signalling domain of the BCR The mIg two immunoglobulin heavy two immunoglobulin light the transmembrane region and the cytoplasmic domain, is associated with the two proteins Igα and sheath proteins, their intracellular tail an On antigen the of the ITAMs are phosphorylated by members of the Src-family for the phosphorylation of the ITAMs within the Igα/Igβ sheath. In two for the of BCR signalling are the allosteric activation of cytoplasmic kinases antigen binding and the activation of cytoplasmic kinases of two or more on the membrane However, the kinases leading to the phosphorylation could be to the cytoplasmic tails of Igα/Igβ or the as a result of the are to membrane domain or first suggested by Cambier et antigen binding could induce a conformational change in the structure of the cytoplasmic of the a prebound protein tyrosine kinase that the cytoplasmic signalling Alternatively, that the conformational change of the mIg antigen binding may to the of which the receptor kinases by selective of A third is that the conformational change the of the BCR distinct signalling domains within the In this that the of two or more the activation of cytoplasmic the surface of a B According to this a signal is by cross-linking of antigen or as a result of of by on the cell The of signal activation that a receptor interaction with the of the of a receptor is In other the antigen the of membrane of the depending on The the affinity of the BCR for the the more the of one or more on the cell surface and the the signal This of signalling was for the but it that BCR signal may in a because molecular of can be from B-cell even in the absence of The is that is with the BCR so that surface of to the ITAMs of the BCR the conformational change receptor engagement could to on the According to this to Igα/Igβ without receptor however, it remains whether the binding of the to Igα is mediated by the domain as a result of phosphorylation of the ITAMs or if the of interaction is from Alternatively, the could be with the mIg without the presence of the Igα/Igβ sheath, as predicted by two However, there is also for a complex prebound to the BCR as by and This complex may the BAPs and is assumed to bind to ITAMs of the Igα/Igβ sheath, preventing the phosphorylation of the BCR engagement could result in the release of the complex from the BCR and subsequent the for phosphorylation by the kinases resulting in the of signalling it was that is present in membrane or whereas the BCR to be from On cross-linking by the BCR This that the of the BCR within would the phosphorylation of ITAMs by important finding is that is not active but In two tyrosine residues and within the domain to the of of is whereas phosphorylation of the association with the SH2 domain, kinase of is negatively by tyrosine kinase kinase or and influenced by the is a transmembrane protein tyrosine that is expressed in all However, cell a distinct in molecular from 000 to The isoforms all the intracellular domain, but differ in their extracellular domain concerning length and pattern of The for the B-cell is because of molecular of It is suggested that can on a of different the of receptor in a or Most to the that enhances BCR signalling by of tyrosine of the et that B exhibit of both the and tyrosine In of show tyrosine phosphorylation of cellular proteins the state by calcium release BCR to have both and in BCR signalling and have been by selective of from or in of receptor The of signalling is most likely by it could be shown that the of was phosphorylation by leading to the interaction of the kinase with a pattern of of and or from the BCR. 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In mice with mutations in the cytoplasmic tails of and to the cytoplasmic tail of IgM, a dramatic of the serum of the respective was be the result of signalling proteins with distinct as a couple of proteins have been identified that interact with mIgM (BAP32, BAP37 and mIgD (BAP29 and and human in identified a potential the kinase as interaction for the cytoplasmic tails of and as the glycosylation pattern of Igα may be distinct signalling to the the of the respective isotypes on the cell membrane could to alternative signal that the of μ and δ expression on B is strongly on whether the cell is or of the the data the that signal transduction pathways in with the signal transduction pathways the Igα/Igβ sheath proteins. 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germinal centre B and in the formation of in that signalling proteins with the and it that could on the site of antigen by the to the formation of B with affecting BCR signalling in a the also strongly the B-cell a of conserved transmembrane proteins, first in the the release of The were to distinct of and from which the immune to a response in are expressed on B the of antigen by the BCR and engagement of the a signal that leads to enhanced B-cell activation and plasma cell In contrast to and of which only affect BCR-mediated signalling there is a of with a potential to interact with the BCR within the B-cell membrane to the signal strength and probably signal antigen This the and In of the with the BCR results in subsequent tyrosine phosphorylation of their cytoplasmic tails by BCR-associated This leads to the of a signal that is or depending on whether the tail of the an or an (immunoreceptor tyrosine-based ITAMs provide a binding site for tyrosine kinases, whereas such as domain protein tyrosine and domain to the BCR complex, the activation that is mediated by the murine have been and which are from the gene by alternative On B only is a stable receptor complex within the membrane with and of to the mIg by antigen with the for BCR-mediated signalling and B-cell A for is a membrane protein on the surface of B and An important on BCR-mediated signalling is also performed by the receptor for acid which are present on of to the mIg responses, as in a to BCR could be the binding to in supports the of on BCR This mechanism to the of However, was shown to the signalling IgM and but not that because the longer tail of prevents phosphorylation of However, this is in to studies, cross-linking of on to The immunoglobulin A and B are probably the They are to the though this remains to be a effect on BCR a for secreted are among immune They are on and for and have been and most of the immune response by antigen for subsequent presentation to However, only two are on B the and In contrast to the other an within cytoplasmic tail, thus to the of Most negatively BCR signalling not to of but to from is to B and but can also be on a of by an results in the release of which is to bind and the role of during an immune response been a of in mice was shown to be or However, further studies have to the of the response by BCR signalling and/or increasing antigen for processing and The of a B is the of However, antibody is in complex by the that B be to to an of The between the and the is by the B-cell antigen receptor and to that the B cell pathway. The antigen receptor is a protein complex of the membrane form of the selected immunoglobulin and least two further proteins Igα and So far, the membrane immunoglobulin been as the of the receptor complex, the cytoplasmic tails of the proteins were identified as the signal connecting the antigen receptor to the tyrosine phosphorylation in the cell. However, expression of additional and signalling is important for the and of leading to a plasma cell or cell It is not whether the cytoplasmic tails of the immunoglobulin or the cytoplasmic tails of the proteins or both tails are necessary for these It that BCR-mediated signalling different distinct However, there are very data the of to these a for in the in these pathways and work and were by the the and the