Jürgen Wienands

Abnormal Development and Function of B Lymphocytes in Mice Deficient for the Signaling Adaptor Protein SLP-65

During signal transduction through the B cell antigen receptor (BCR), several signaling elements are brought together by the adaptor protein SLP-65. We have investigated the role of SLP-65 in B cell maturation and function in mice deficient for SLP-65. While the mice are viable, B cell development is affected at several stages. SLP-65-deficient mice show increased proportions of pre-B cells in the bone marrow and immature B cells in peripheral lymphoid organs. B1 B cells are lacking. The mice show lower IgM and IgG3 serum titers and poor IgM but normal IgG immune responses. Mutant B cells show reduced Ca2+ mobilization and reduced proliferative responses to B cell mitogens. We conclude that while playing an important role, SLP-65 is not always required for signaling from the BCR.

Interaction of SLP adaptors with the SH2 domain of Tec family kinases

Activation of lymphocytes through their antigen receptors leads to mobilization of intracellular Ca2+ ions. This process requires expression of SLP adaptors and involves phosphorylation of phospholipase C‐γ isoforms by the Tec‐related protein tyrosine kinase Btk in B cells and Itk in T cells. The SH2 domain of Btk and Itk is essential for phospholipase C‐γ phosphorylation and mutations in this domain lead to the X‐linked agammaglobulinemia immuno deficiency in humans. Here we show that, in contrast to SH2 domains from other signaling proteins, the Btk and Itk SH2 domains exhibit a restricted binding specificity. They bind selectively to tyrosine‐phosphorylated SLP‐65 and SLP‐76 in activated B and T cells, respectively. Our findings suggest that Btk / Itk and phospholipase C‐γ both bind via their SH2 domain to phosphorylated SLP adaptors, and that this association is required for the activation of phospholipase C‐γ.

Non-T Cell Activation Linker (NTAL): A Transmembrane Adaptor Protein Involved in Immunoreceptor Signaling

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non–T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcγ- and Fcɛ-receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non–T cells.

The B-cell antigen receptor complex

Abstract Here, Michael Reth, John Cambier and colleagues review the genetics, biochemistry and cell biology o f the recently-identified accessory components o f the B-cell antigen receptor.

Seroprevalence of autoantibodies against brain antigens in health and disease.

We previously reported an unexpectedly high seroprevalence (∼10%) of N‐methyl‐D‐aspartate‐receptor subunit‐NR1 (NMDAR1) autoantibodies (AB) in healthy and neuropsychiatrically ill subjects (N = 2,817). This finding challenges an unambiguous causal relationship of serum AB with brain disease. To test whether similar results would be obtained for other brain antigen‐directed AB previously connected with pathological conditions, we systematically screened serum samples of 4,236 individuals.

Association of SLP-65 / BLNK with the B cell antigen receptor through a non-ITAM tyrosine of Ig-α

The cytoplasmic adaptor protein SLP‐65 (BLNK or BASH) is a cricital downstream effector of the B cell antigen receptor (BCR). Tyrosine‐phosphorylated SLP‐65 assembles intracellular signaling complexes such as the Ca2 + initiation complex encompassing phospholipase C‐γ2 and Bruton′s tyrosine kinase. It is, however, unclear how the SLP‐65 signaling module can be recruited to the plasma membrane. Here we show that following B cell stimulation, SLP‐65 associates directly with the BCR signaling subunit, the Ig‐α / Ig‐β heterodimer. The interaction is mediated by theSrc homology 2 domain of SLP‐65 and the phosphorylated Ig‐α tyrosine 204, which is located outside of the immunoreceptor tyrosine‐based activation motif. Our data identify an unexpected BCR phosphorylation pattern and indicate that Ig‐α has the capability to serve as transmembrane adaptor in BCR signaling.

Recruitment of the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor–intrinsic costimulation to class-switched B cells

The improved antibody responses of class-switched memory B cells depend on enhanced signaling from their B cell antigen receptors (BCRs). However, BCRs on both naive and antigen-experienced B cells use the canonical immunoglobulin-associated α and β-protein signaling subunits. Here we identified a BCR isotype–specific signal-amplification mechanism. Whereas immunoglobulin M (IgM)-containing BCRs initiated intracellular signals exclusively through immunoglobulin-associated α- and β-proteins, IgG- and IgE-containing BCRs also used a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. When phosphorylated, this tyrosine recruited the adaptor Grb2, resulting in sustained protein kinase activation and prolonged generation of second messengers, which together culminated in enhanced B cell proliferation. Hence, membrane-bound IgG and IgE exert antigen recognition as well as costimulatory functions, thereby rendering memory B cells less dependent on T cell help.

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM.

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD‐alpha) and 39 kd (Ig‐beta), respectively. The two novel proteins are not found in the IgD‐expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane‐bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD‐associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.

SH3P7 Is a Cytoskeleton Adapter Protein and Is Coupled to Signal Transduction from Lymphocyte Antigen Receptors

ABSTRACT Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

Induction of the antigen receptor expression on B lymphocytes results in rapid competence for signaling of SLP‐65 and Syk

The binding of antigen to the B cell antigen receptor (BCR) results in the activation of protein tyrosine kinases (PTKs) such as Lyn and Syk, and the phosphorylation of several substrate proteins including HS1 and SLP‐65. How these signaling elements are connected to the BCR is not well understood. Using an expression vector for a tamoxifen‐regulated Cre recombinase, we have developed a method that allows the inducible expression of the BCR. Disruption of the VH leader reading frame of the immunoglobulin heavy chain by two loxP sites is overcome by Cre‐mediated DNA recombination and results in the cell surface expression of the BCR starting 4 h after exposure of transfected B cells to tamoxifen. This method can, in principle, be employed for the inducible expression of any secreted or type I transmembrane protein. By monitoring the activation of signaling elements in pervanadate‐stimulated B cells expressing different levels of the BCR, we show here that phosphorylation of SLP‐65 and Syk, but not of Lyn, is strictly dependent on the expression of the BCR on the cell surface. These data suggest that the BCR reorganizes its signaling molecules as soon as it appears on the cell surface.