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J C Cambier

Publications and source records attributed to J C Cambier.

At least 91 records · Page 5Linked to original sources
PubMed1994

Signal transduction by the B cell antigen receptor and its coreceptors.

B lymphocyte antigen receptors, membrane immunoglobulins (mIg), function in focusing and internalization of antigen for subsequent presentation to T cells and in transmembrane transduction of signals leading to cell activation, anergy, or deletion. Until quite recently, the ability of this receptor to transduce signals in spite of a virtual lack of cytoplasmic structure, left a significant gap in our understanding of how it is coupled to cytoplasmic signal propagators. Studies conducted during the past five years have defined a mIg-associated protein complex homologous to the CD3 complex associated with the T cell antigen receptor. Components of this disulfide linked heterodimeric complex, Ig-alpha and Ig-beta, contain an approximately 26 residue sequence motif termed ARH1, also known as TAM, which binds to cytoplasmic effectors, including src-family tyrosine kinases, and contains all structural information needed for signal transduction. Receptor associated src-family kinases which are activated following receptor cross-linking, also associate with downstream effectors, including phospholipase C gamma (PLC gamma), p21ras. GTPase activating protein (GAP), phosphatidylinositol 3-kinase (PI3-k) and microtubule associate protein kinase (MAPk2). In some cases, these associations are induced by receptor cross-linking and lead directly to effector activation. The current literature indicates that these interactions may occur in sequence and culminate in the activation of three major pathways of signal propagation including those mediated by PLC gamma, p21ras and PI3-k. This chapter reviews various molecular aspects of the B cell antigen receptor complex, including extended structure of the complex, and receptor-effector interactions and their biologic consequences. Finally, an integrated model of antigen receptor signaling is presented.

Amino Acid Sequence↗
PubMed1993

Signaling-defective mutants of the B lymphocyte antigen receptor fail to associate with Ig-alpha and Ig-beta/gamma.

The B cell receptor for antigen, the membrane immunoglobulin (mIg) molecule, is normally expressed in association with at least two other proteins, mb-1 (Ig-alpha) and B29 (Ig-beta/gamma). We have previously described a set of murine B cell clones transfected with wild-type and mutated forms of membrane-bound IgM (mIgM) that vary in their ability to transduce signals to the cell interior. Analysis of the B cell receptor complex in signaling and nonsignaling mutants of mIgM reveals complete concordance between intact signaling ability and ability of the mIgM to associate with Ig-alpha and Ig-beta/gamma. These results point to the importance of the mIgM transmembrane region in mediating binding to accessory molecules, and provide support for a role of Ig-alpha and Ig-beta/gamma that extends beyond surface expression to signal transduction in B lymphocytes.

Amino Acid Sequence↗
PubMed1993

B cell antigen receptor cross-linking triggers rapid protein kinase C independent activation of p21ras1.

Membrane Ig (mIg) functions in binding and internalization of Ag for subsequent processing and presentation to T cells, as well as in transmembrane transduction of signals that lead to cell activation, proliferation, and differentiation. Tyrosine kinase activation and subsequent phosphatidylinositol hydrolysis and Ca2+ mobilization are clearly important intermediary events in receptor-mediated B cell activation. However, many details of the cellular signal transduction pathways utilized by this receptor are not resolved. Recent studies that demonstrated co-capping of mIg and the proto-oncoprotein p21ras suggested that this low m.w. GTP-binding protein may function in mIg-mediated signal transduction. p21ras has been implicated in some but not all protein tyrosine kinase/phospholipase C involving signaling pathways. To explore the potential role of p21ras in B cell Ag receptor-mediated signaling, we assessed the effect of Ag receptor ligation on the proportion of p21ras in the active GTP-bound state. We present evidence that p21ras is activated by mIgM and mIgG cross-linking by anti-receptor antibodies as well as by Ag. Depending upon the stimulus employed, this response is detectable within 1 min and occurs with similar kinetics as inductive protein tyrosine phosphorylation and Ca2+ mobilization. Ag dose dependence of this response is similar to that of inductive protein tyrosine phosphorylation. In these cells p21ras is also activated by PMA suggesting that p21ras activation after receptor cross-linking may be mediated by an effector molecule that functions downstream from protein kinase C (PKC). However, the kinetics of p21ras activation after mIg cross-linking are inconsistent with the possibility that PKC functions as the sole mediator of p21ras activation in this system. Finally, under conditions in which the PKC inhibitor calphostin C blocks PMA-induced p21ras activation, it does not inhibit Ag-induced p21ras activation. These data suggest that PKC effector mechanisms play a negligible role in p21ras activation during mIg-mediated signaling.

Animals↗
PubMed1993

The gamma subunit of the B cell antigen-receptor complex is a C-terminally truncated product of the B29 gene.

The predominant Ag-receptor complex of B cells consists of mIgM or mIgD noncovalently associated with glycosylated heterodimers of Ig-alpha and Ig-beta or Ig-alpha and Ig-gamma. Upon B cell stimulation the associated proteins are phosphorylated, giving rise to pp32/33 (alpha), pp37 (beta), and pp34 (previously designated gamma). Ig-alpha and Ig-beta contain extended cytoplasmic structure (61 and 48 amino acids, respectively) and associate with cytoplasmic effectors indicating that they are directly involved in signal transduction. Here we report analysis of the structural relationship of mIgM- and mIgD-associated Ig-beta and Ig-gamma chains from mice. N-terminal sequence, immunoblotting, and physicochemical analyses show that both Ig-beta and Ig-gamma are products of the B cell-specific B29 gene and demonstrate that the 37-kDa Ig-beta protein is the full length predicted product of the B29 gene. The Ig-associated protein that migrates in the 34-kDa range is actually two distinct species. The minor species is a phosphorylatable and underglycosylated form of full length Ig-beta, and the major species is a C-terminally truncated form of B29, which we now designate Ig-gamma. This conclusion is based on the observations that Ig-gamma is composed of a core protein which is 3 to 4 kDa smaller than deglycosylated Ig-beta, it is not phosphorylated, unlike Ig-beta, and it does not react with an antiserum raised against a peptide of the seven C-terminal amino acids of B29. Based on these findings we estimate that Ig-gamma is truncated by about 30 to 36 amino acid residues and hypothesize that the most 3' B29 exon, which encodes the 32 C-terminal residues, may not be expressed in Ig-gamma. All of the documented B29 products are found in association with both mIgM and mIgD. Interestingly, Ig-gamma is found in intermediate and low density splenic B cells, but is not detectable in resting B cells. This raises the possibility that it may confer some distinct signaling function on the Ag receptors of these cells.

Amino Acid Sequence↗
PubMed1993

Structural compartmentalization of MHC class II signaling function.

Major histocompatibility complex (MHC) class II molecules are critical restricting elements in the generation of thymus-dependent immune responses. Recent studies indicate that in addition to providing a composite epitope for recognition by T-cell antigen receptors, MHC class II molecules function in signal transduction through interaction with other cellular proteins. Mutational analyses indicate that structural information necessary for these functions is compartmentalized in different aspects of the molecular complex. Here, William Wade and colleagues review the structural basis of this MHC class II function as defined in the I-A alpha and -beta chains.

Amino Acid Sequence↗
PubMed1993

Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.

Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.

Animals↗
PubMed1992

Human pre-B and B cell membrane mu-chains are noncovalently associated with a disulfide-linked complex containing a product of the B29 gene.

B cell activation after Ag binding to membrane Ig (mIg) is mediated by a complex series of events that involves proximal activation of a tyrosine kinase and phospholipase C. Until recently it was unclear how mIgM and mIgD, with their limited cytoplasmic domains (three amino acids on each H chain), were able to couple to these secondary signal transducers. Studies of murine B cells conducted in several laboratories, including our own, suggest that products of the mb-1 (IgM-alpha or IgD-alpha) and B29 (Ig-beta, Ig-gamma) genes occur as disulfide-linked alpha/beta and alpha/gamma heterodimers that are noncovalently associated with mIgM and mIgD. Although studies utilizing Daudi and Raji cell lines indicate that human mIgM is also associated with a dimer containing the mb-1 gene product, the other molecules associated with the human receptor have not been identified. In this report we characterize the phosphoproteins that are noncovalently associated with mIgM on human tonsillar B cells and human pre-B cell lines. mIgM is noncovalently associated with a disulfide-linked heterodimer composed of variably glycosylated forms of two core proteins with apparent molecular mass of 26.5 and 27 kDa. Western blotting analysis reveals that the lower m.w. component of each of the mIgM-associated heterodimers and its 27-kDa deglycosylated core protein are reactive with antibodies against the murine B29 gene product. Thus, a product of the B29 gene is a component of the AgR complex in human and murine B cells, occurring as a disulfide linked dimer with product(s) of the mb-1 gene. Interestingly, mb-1 and B29 gene products expressed on human cells are much more heterogenously N-glycosylated than their murine B cell counterparts.

Aluminum↗
PubMed1992

The B cell antigen receptor complex: association of Ig-alpha and Ig-beta with distinct cytoplasmic effectors.

The B cell antigen receptor complex is a hetero-oligomeric structure composed of antigen binding, membrane immunoglobulin, and transducer-transporter substructures. The transducer-transporter substructure is composed of disulfide-linked dimers of immunoglobulin (Ig)-alpha and Ig-beta/gamma subunits that are products of the mb-1(alpha) and B29 (beta/gamma) genes. Although the receptor complex associates with Src family kinases that are activated after receptor ligation, the site of interaction of these and other cytoplasmic effector molecules with receptor subunits is unknown. The cytoplasmic tails of Ig-alpha and Ig-beta chains were found to associate with distinct sets of effector molecules. The Ig-alpha chain cytoplasmic domain bound to the Src family kinases Lyn and Fyn, phosphatidylinositol-3 kinase (PI-3 kinase), and an unidentified 38-kilodalton phosphoprotein; the cytoplasmic tail of Ig-beta bound PI-3 kinase and unidentified 40- and 42-kilodalton phosphoproteins. Binding activity was found to occur within a 26-amino acid sequence of Ig-alpha and Ig-beta that contains a motif [(Asp or Glu)-(any amino acid)7-(Asp or Glu)-Tyr-(any amino acid)3-Leu-(any amino acid)7-Tyr-(any amino acid)2-(Leu or Ile)] previously implicated in signal transduction via other receptors including the Fc epsilon receptor I and the T cell antigen receptor. These findings indicate that the subunits act independently to activate distinct second messenger pathways.

Amino Acid Sequence↗
PubMed1992

CD4 binding to major histocompatibility complex class II antigens induces LFA-1-dependent and -independent homotypic adhesion of B lymphocytes.

T helper cells recognize processed antigen (Ag) in the context of major histocompatibility complex (MHC) class II antigens present on the surface of B cells and other Ag-presenting cells. This interaction is mediated through the T cell receptor complex with associate recognition of class II molecules by the CD4 molecule. In this study, the binding of a soluble recombinant CD4/Ig heavy chain fusion protein (CD4-gamma 3) or monoclonal antibody (mAb) to class II antigens on human B cells was shown to induce rapid and specific homotypic adhesion of B cells and most B lymphoblastoid cell lines. mAb reactive with CD4 inhibited CD4-gamma 3-induced adhesion and a mutant B lymphoblastoid cell line deficient in class II antigens failed to respond. Induction of homotypic adhesion was dependent on energy metabolism and a functional cytoskeleton, and class II+ pre-B cells did not exhibit adhesion in response to these stimuli, suggesting that cross-linking of class II molecules generated a transmembrane signal and did not simply aggregate cells. In addition, MHC class II-induced adhesion was Fc receptor independent, as 15 mAb of different Ig isotypes reactive with HLA-D or HLA-DQ gene products induced adhesion. Anti-class II mAb and CD4-gamma 3 were able to induce adhesion at concentrations as low as 10 ng/ml and 100 ng/ml, respectively. Suboptimal stimulation of B cell lines through HLA-D antigens induced homotypic adhesion that was dependent on the activation of LFA-1 (CD11a/CD18), and which could be blocked by specific mAb. However, at greater signal strengths, adhesion was not blocked by mAb against the known adhesion receptors, suggesting the induction of a novel adhesion pathway. Consistent with this, homotypic adhesion induced by engagement of MHC class II antigens was observed with LFA-1-deficient B cell lines, and was independent of CD49d or CD18 expression. Thus, the direct engagement of B cell class II antigens by CD4 is likely to generate transmembrane signals which trigger both LFA-1-dependent and LFA-1-independent adhesion pathways.

B-Lymphocytes↗
PubMed1992

Signal transduction by T- and B-cell antigen receptors: converging structures and concepts.

Recent evidence demonstrates that the antigen receptor complexes of T and B lymphocytes are very similar in general architecture and primary and secondary structure of component polypeptides, and that they use common mechanisms for transmembrane signal transduction. Most importantly, multiple subunits of each receptor (Ig-alpha, Ig-beta and Ig-gamma, CD3 gamma, CD3 delta and CD3 epsilon, and T-cell receptor zeta and eta) possess a motif of approximately 26 amino-acids, denoted ARH1, which appears to carry sufficient structural information for receptor-mediated lymphocyte activation.

Amino Acid Sequence↗
PubMed1992

Membrane immunoglobulin and its accomplices: new lessons from an old receptor.

B lymphocytes express multisubunit receptors for antigen that play multiple roles in generation of the immune response. These receptors act as transmembrane signal transducers but also act to capture, concentrate, and internalize antigen for subsequent proteolytic processing and presentation to T cells. During the past decade great progress has been made in our understanding of the extended structure of the receptor and the molecular basis by which it transduces signals. It is now clear that the B cell antigen receptor is a complex structure composed of antigen binding and transducer/transporter substructures. The antigen binding substructure is composed of disulfide-linked immunoglobulin H and L chains, and is noncovalently associated with transducer/transporter substructures composed of disulfide-linked heterodimers of alpha, beta, and gamma chain products of the mb-1 (alpha) and B29 (beta and gamma) genes. The cytoplasmic tails of these chains associate with src-family tyrosine kinases including fyn, lyn, blk, and lck and other SH2-containing molecules such as phosphatidylinositol 3-kinase. Available evidence indicates that these interactions are mediated via an amino acid sequence motif of approximately 22 amino acids that is found in both alpha and beta chains. Receptor ligation triggers the activation of multiple receptor-associated src-family kinases leading to phosphorylation and activation of PLC gamma 1 and PLC gamma 2. Subsequent phosphoinositide hydrolysis and calcium mobilization, presumably acting in concert with other tyrosine kinase-activated mechanisms, leads to transcriptional activation of a number of immediate early genes and, ultimately, to B cell proliferation.

Amino Acid Sequence↗
PubMed1992

B-cell proliferation initiated by Ia cross-linking and sustained by interleukins leads to class switching but not somatic mutation in vitro.

Somatic mutations that are acquired by antibody V genes of antigen-stimulated B cells ultimately provide the clonal diversity from which memory B cells are selected during immune responses to T-cell-dependent antigens. Somatic mutations apparently are not acquired when B cells are stimulated by mitogens nor when they participate in immune responses to T-cell-independent antigens. Since the basis of T-cell-dependent humoral immunity is T-cell recognition of processed antigen in the context of class II major histocompatibility glycoproteins (Ia) on the B-cell surface, we sought to determine whether the ligation of Ia on B cells induces somatic mutation. B cells were stimulated in vitro by a procedure in which their proliferation was dependent upon ligation of surface Ia with antibody. Sequences of hybridoma V genes derived from these B cells revealed no somatic mutations despite prolonged stimulation in vitro and the induction of immunoglobulin secretion and switching to isotypes characteristic of T cell-dependent humoral immunity. We infer that Ia-mediated signalling and isotype switching are not causally related to somatic mutation. The avenue of differentiation that leads to somatic mutation in memory B cells is apparently separable from that leading to proliferation, immunoglobulin secretion and switching.

Animals↗
PubMed1991

Alpha-chains of IgM and IgD antigen receptor complexes are differentially N-glycosylated MB-1-related molecules.

The major B cell Ag receptors, membrane (m) IgM and mIgD, are noncovalently associated with disulfide-linked heterodimers of alpha, beta, and gamma glycoproteins. The beta and gamma chains have apparent molecular masses of 37 and 34 kDa, respectively, and are associated with both mIgM and mIgD. Receptor alpha chains, however, exhibit Ig isotype specificity. IgM-alpha and IgD-alpha have apparent molecular masses of 32 and 33 kDa, respectively. Recently, the alpha chain of the IgM Ag receptor complex was identified as the product of the mb-1 gene, and the beta and gamma chains were characterized as products of the B29 gene. The failure of mb-1 cDNA to hybridize with mRNA from J558 delta m2.6 plasmacytomas expressing surface mIgD in association with IgD-alpha has led to the conclusion that IgM-alpha and IgD-alpha are not closely related. In this report we have used protein biochemical methods to characterize differences in the mIgM- and mIgD-associated alpha chains. In addition to a slightly greater apparent m.w., IgD-alpha was slightly more acidic than IgM-alpha. The alpha chains had nearly identical proteolytic peptide maps, and were also noted to have multiple loci of identity with MB-1 based on amino terminal sequencing and immunoblotting. In an attempt to determine whether the alpha chains differed as a result of differential posttranslational modification, they were compared after deglycosylation with N-glycanase. The results indicate that the apparent m.w. as well as isoelectric point differences are primarily due to differential N-linked glycosylation. These studies indicate that IgM-alpha and IgD-alpha are products of the mb-1 gene or closely related genes.

Amino Acid Sequence↗
PubMed1991

Regulation of B cell antigen receptor signal transduction and phosphorylation by CD45.

CD45 is a member of a family of membrane proteins that possess phosphotyrosine phosphatase activity, and is the source of much of the tyrosine phosphatase activity in lymphocytes. In view of its enzymatic activity and high copy number, it seems likely that CD45 functions in transmembrane signal transduction by lymphocyte receptors that are coupled to activation of tyrosine kinases. The B cell antigen receptor was found to transduce a Ca(2+)-mobilizing signal only if cells expressed CD45. Also, both membrane immunoglobulin M (mIgM) and CD45 were lost from the surface of cells treated with antibody to CD45, suggesting a physical interaction between these proteins. Finally, CD45 dephosphorylated a complex of mIg-associated proteins that appears to function in signal transduction by the antigen receptor. These data indicate that CD45 occurs as a component of a complex of proteins associated with the antigen receptor, and that CD45 may regulate signal transduction by modulating the phosphorylation state of the antigen receptor subunits.

Animals↗
PubMed1991

IgM antigen receptor complex contains phosphoprotein products of B29 and mb-1 genes.

Membrane immunoglobulin M (mIgM) and mIgD are major B-lymphocyte antigen receptors, which function by internalizing antigens for processing and presentation to T cells and by transducing essential signals for proliferation and differentiation. Although ligation of mIgM or mIgD results in rapid activation of a phospholipase C and a tyrosine kinase(s), these receptors have cytoplasmic tails of only three amino acid residues (Lys-Val-Lys), which seem ill suited for direct physical coupling with cytoplasmic signal transduction structures. In this report, we identify the alpha, beta, and gamma components of the mIgM-associated phosphoprotein complex, which may play a role in signal transduction. Proteolytic peptide mapping demonstrated that the IgM-alpha chain differs from Ig-beta and Ig-gamma. The chains were purified, and amino-terminal sequencing revealed identity with two previously cloned B-cell-specific genes. One component, IgM-alpha, is a product of the mb-1 gene, and the two additional components, Ig-beta and Ig-gamma, are products of the B29 gene. Immunoblotting analysis using rabbit antibodies prepared against predicted peptide sequences of each gene product confirmed the identification of these mIgM-associated proteins. The deduced sequence indicates that these receptor subunits lack inherent protein kinase domains but include common tyrosine-containing sequence motifs, which are likely sites of induced tyrosine phosphorylation.

Amino Acid Sequence↗
PubMed1991

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.

Animals↗
PubMed1991

Ligation of membrane Ig leads to calcium-mediated phosphorylation of the proto-oncogene product, Ets-1.

Recent studies have demonstrated that the nuclear protein, Ets-1, which is preferentially expressed in lymphocytes, binds to the long terminal repeat of Moloney murine sarcoma virus and HTLV-1 and regulates gene expression. The association of Ets-1 with DNA has been shown to be lost when the protein is phosphorylated. Thus, Ets-1 may regulate gene expression in lymphocytes and this activity may be determined by its phosphorylation state. To address the possibility that Ets-1 activity may be altered by membrane (m) Ig-mediated signal transduction, we analyzed the effect of mIgM and mIgD ligation on the phosphorylation state of Ets-1. Monoclonal anti-IgM or anti-IgD antibody stimulation of normal mouse B cells led to increased phosphorylation of Ets-1 within 2 min. This response was absolutely dependent on calcium mobilization and could be induced by elevation of intracellular free calcium using the calcium ionophore, ionomycin. Calcium release from intracellular stores was sufficient to mediate the phosphorylation of Ets-1. Treatment of resting B cells with IL-4, TGF beta-1, IFN-gamma, anti-class I, or anti-class II antibodies did not induce Ets-1 phosphorylation. In summary, calcium mobilization from intracellular stores after mIgM or mIgD ligation provides a necessary and sufficient signal for activation of Ets-1 phosphorylation. This phosphorylation event may act in the alteration of gene expression during B cell activation.

Animals↗