Cytoplasmic Domains Of Ig-alpha Research Articles

The avian B-cell receptor complex: distinct roles of Igalpha and Igbeta in B-cell development.

The bursa of Fabricius has evolved in birds as a gut-associated site of B-cell lymphopoiesis that is segregated from the development of other hematopoietic lineages. Despite differences in the developmental progression of chicken as compared to murine B-cell lymphopoiesis, cell-surface immunoglobulin (sIg) expression has been conserved in birds as an essential checkpoint in B-cell development. B-cell precursors that express an sIg complex that includes the evolutionarily conserved Igalpha/beta heterodimer colonize lymphoid follicles in the bursa, whereas B-cell precursors that fail to express sIg due to non-productive V(D)J recombination are eliminated. Productive retroviral gene transfer has allowed us to introduce chimeric receptor constructs into developing B-cell precursors in vivo. Chimeric proteins comprising the extracellular and transmembrane regions of murine CD8alpha fused to the cytoplasmic domain of chicken Igalpha efficiently supported B-cell development in precursors that lacked endogenous sIg expression. By contrast, expression of an equivalent chimeric receptor containing the cytoplasmic domain of Igbeta actively inhibited B-cell development. Consequently, the cytoplasmic domains of Igalpha and Igbeta play functionally distinct roles in chicken B-cell development.

Cooperativity and Segregation of Function within the Ig-α/β Heterodimer of the B Cell Antigen Receptor Complex

The B cell antigen receptor complex contains heterodimers of Ig-alpha and Ig-beta. The cytoplasmic tails of each of these chains contain two conserved tyrosines, phosphorylation of which initiates the signal transduction cascades activated by the receptor complex. Although the cytoplasmic domains of Ig-alpha and Ig-beta have been expressed individually and demonstrated to be competent signal transduction units, we postulated that within the context of a heterodimer, Ig-alpha and Ig-beta could have new, complementary or even synergistic functions. Therefore we developed a system to compare the signal transducing capacities of dimers of Ig-alpha/Ig-alpha, Ig-beta/Ig-beta, or Ig-alpha/Ig-beta. This was done by fusing the extracellular and transmembrane domains of either human platelet-derived growth factor receptor (PDGFR) alpha or beta to the cytoplasmic tail of either Ig-alpha or Ig-beta. Three cell lines expressing PDGFRbeta/Ig-alpha, PDGFRbeta/Ig-beta, or PDGFRalpha/Ig-beta together with PDGFRbeta/Ig-alpha were established in the murine B cell line A20 IIA1.6. While aggregation of each dimer by itself could induce the tyrosine phosphorylation of cellular substrates, only aggregation of the heterodimer induced the phosphorylation of substrates similar in range and intensity to that induced by the endogenous B cell antigen receptor complex. Interestingly, Ig-beta remarkably enhanced the rapidity (Tmax decreased from 5 to 1 min) and intensity (greater than 10-fold enhancement) of Ig-alpha phosphorylation. Conversely, the phosphorylation of Ig-beta was reduced to undetectable levels when co-aggregated with Ig-alpha. The enhancement of Ig-alpha phosphorylation by Ig-beta correlated with a lowering of the stimulation threshold for tyrosine kinase activation.

The cytoplasmic domains of immunoglobulin (Ig) alpha and Ig beta can independently induce the precursor B cell transition and allelic exclusion.

In mature B cells, signals transduced through membrane immunoglobulin (Ig) produce cellular activation, yet the same receptor can also mediate deletion and silencing of autoreactive B cells. In addition, Ig expression during the antigen-independent phase of B cell development regulates the precursor B (pre-B) cell transition and allelic exclusion. To account for the diverse regulatory functions induced by membrane Ig, it has been proposed that individual receptor components have independent physiologic activities. Here we establish a role for Ig alpha in the pre-B cell transition and allelic exclusion. We find that the cytoplasmic domain of Ig alpha contains sufficient information to trigger both of these antigen-independent events. Direct comparisons of the cytoplasmic domains of Ig alpha and Ig beta show that the two are indistinguishable in the induction of the pre-B cell transition and allelic exclusion. Our experiments suggest that, despite the reported differences in certain biochemical assays, Ig alpha and Ig beta have redundant functions in the developing B cell.

Reconstitution of the B Cell Antigen Receptor Signaling Components in COS Cells

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.

Signal transduction by the B-cell antigen receptor.

The antigen receptor of B lymphocytes (BCR) plays important roles in recognition of foreign antigens and self-components to allow the immune system to make appropriate antibody responses. The BCR is a complex between membrane immunoglobulin and the Ig-alpha and Ig-beta heterodimer. Site-directed mutagenesis experiments have shown that the mu heavy chain transmembrane domain plays a key role in the association of mIgM with Ig-alpha/Ig-beta. In the absence of complex formation, mIgM is retained in the endoplasmic reticulum, and this function is also specified by the mu chain transmembrane domain. The ability of various mutant mIgM molecules to associate with Ig-alpha/Ig-beta correlates well with their ability to induce signal transduction reactions such as protein tyrosine phosphorylation and phosphoinositide breakdown. Thus, the signaling ability of the BCR appears to reside in the Ig-alpha/Ig-beta heterodimer. The cytoplasmic domains of Ig-alpha and Ig-beta each contain an ITAM sequence, which is defined by its limited homology with subunits of the T-cell antigen receptor and of Fc receptors. Moreover, chimeric proteins containing these ITAMs and surrounding sequences from the cytoplasmic domains of Ig-alpha or Ig-beta exhibit signaling function characteristics of the intact BCR. The Ig-alpha and Ig-beta chimeras are each capable of inducing all of the BCR signaling events tested and thus represent redundant functions. Cross-linking these chimeras leads to their phosphorylation and to binding of the intracellular tyrosine kinases Lyn and Syk. The BCR expressed in the nonlymphoid AtT20 cells, which express the Src-family tyrosine kinase Fyn but not Syk, was not able to trigger vigorous signaling reactions. Introduction of the active form of Syk into these cells restored some signaling events. These results are consistent with a model in which the ITAMs act to initiate the BCR signaling reactions by binding and activating tyrosine kinases.

The B cell antigen receptor complex: mechanisms and implications of tyrosine kinase activation.

The B cell receptor is a multimeric receptor complex whose constituent chains appear to mediate distinct and possibly interrelated functions. In this review we have focused on how one chain, immunoglobulin (Ig)-alpha, may function to activate tyrosine kinases and the consequences of that activation. The cytoplasmic domain of Ig-alpha contains a consensus sequence, the antigen recognition homology 1 (ARH 1) motif, which is found in Ig-beta and other antigen recognition receptor associated chains. We argue that this conserved structure reflects an underlying conserved mechanism of secondary effector activation. Our data also indicates that the specificity of each motif (i.e., the elements which restrict secondary effector binding to particular motifs) is encoded within divergent sequences found in each ARH 1 motif. In the particular case of kinase activation by Ig-alpha, the subsequent phosphorylation of multiple tyrosines on Ig-alpha, Ig-beta, CD19, CD22 and possibly other functionally related chains form recruitment sites for a myriad of secondary signal transducers. In this model, proximal tyrosine kinases and phosphatases do not function so much to mediate the linear transfer of information as to establish and modulate an interrelated network of signal transducers capable of driving complicated cellular responses.

Signal transduction by immunoglobulin is mediated through Ig alpha and Ig beta.

Immunoglobulin (Ig) antigen receptors are composed of a noncovalently-associated complex of Ig and two other proteins, Ig alpha and Ig beta. The cytoplasmic domain of both of these Ig associated proteins contains a consensus sequence that is shared with the signaling proteins of the T cell and Fc receptor. To test the idea that Ig alpha-Ig beta heterodimers are the signaling components of the Ig receptor, we have studied Ig mutations that interfere with signal transduction. We find that specific mutations in the transmembrane domain of Ig that inactivate Ca2+ and phosphorylation responses also uncouple IgM from Ig alpha-Ig beta. These results define amino acid residues that are essential for the assembly of the Ig receptor. Further, receptor activity can be fully reconstituted in Ca2+ flux and phosphorylation assays by fusing the cytoplasmic domain of Ig alpha with the mutant Igs. In contrast, fusion of the cytoplasmic domain of Ig beta to the inactive Ig reconstitutes only Ca2+ responses. Thus, Ig alpha and Ig beta are both necessary and sufficient to mediate signal transduction by the Ig receptor in B cells. In addition, our results suggest that Ig alpha and Ig beta can activate different signaling pathways.