ABSTRACT
We studied the potential role of seed bank in the dynamics of the understorey in a turkey oak-sessile oak forest (Querceteum petraeae-cerris) in Hungary. We used long-term records of the herb layer (1973-2006) and the seed bank composition of 2006 to assess the role of seed bank in the regeneration of herb layer. The total cover of herb layer decreased from 22% (1973) to 6% (1988), and remained low (<10%) till 2006; coinciding with the increasing cover of secondary canopy dominated by Acer campestre. We found a low density seed bank (ca. 1300 seeds/m2). Altogether 33 species were germinated from the soil samples. A few generalist weed species composed the majority of seed bank. It was possible to assign a seed bank type for 19 species; 14 species out of 19 was long-term persistent. We found that the characteristic perennial forest herbs and grasses had only sparse seed bank. The Jaccard similarity between vegetation and seed bank was low (<30%). Our results suggest that the continuous establishment of forest herbs are not based on local persistent seed bank; it should be based on vegetative spreading and/or seed rain.
Subject(s)
Biodiversity , Quercus , Seeds , Hungary , Plant Weeds , Population DynamicsABSTRACT
The actin cytoskeleton association is required for caspase 8-independent Fas/CD95 receptor internalization, a critical step for an optimal death-inducing signaling complex formation along the endocytic pathway, leading to efficient activation of the caspase cascade and, ultimately, cell death. However, the way in which this initiation phase of Fas receptor signaling is regulated is still unknown. We report herein that, in B cells, upon Fas engagement, the tyrosine phosphatase SHP-1-regulated Vav dephosphorylation, by downmodulating the Fas-ezrin-actin linkage is a fine-tune switch-off mechanism that the cell uses as a way to terminate the receptor internalization, controlling therefore the time and extent of the DISC formation and cell death.
Subject(s)
Apoptosis , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Proto-Oncogene Proteins c-vav/metabolism , fas Receptor/metabolism , Actin Cytoskeleton/metabolism , Animals , B-Lymphocytes/enzymology , B-Lymphocytes/metabolism , Cells, Cultured , Cytoskeletal Proteins/metabolism , Mice , Phosphorylation , Signal TransductionABSTRACT
In the absence of phosphorylated Gab2, immature B cells are unable to maintain a prolonged signal transduction, which is necessary for the transcriptional regulation and activation of the cells. Thus, the lack of Gab2 phosphorylation may drive immature B cells to apoptosis.
Subject(s)
Apoptosis/physiology , B-Lymphocytes/cytology , Cell Survival/physiology , Signal Transduction/physiology , Adaptor Proteins, Signal Transducing , Animals , B-Lymphocytes/immunology , B-Lymphocytes/physiology , Lymphocyte Activation , Mice , Phosphoproteins/physiology , Receptors, Antigen, B-Cell/immunology , Spleen/immunology , Transcription, GeneticABSTRACT
The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type IIb Fcgamma receptor (FcgammaRIIb) is phosphorylated on its tyrosine upon co-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITIM) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcgammaRIIb in the activation of SHP-2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP-2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcgammaRII-SHIP complexes formed in the intact cells may also activate SHP-2. Grb2-associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine-phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 subunit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their activity. Gab1 is a potential substrate of SHP-2, thus its binding to FcgammaRIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcgammaRIIb upon its co-clustering with BCR. Gab1 may recruit SH2 domain-containing molecules to the phosphorylated FcgammaRIIb. SHP-2, activated upon the binding to FcgammaRIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3-K and ultimately in the inhibition of downstream activation pathways in BCR/FcgammaRIIb co-aggregated cells.
Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Antigens, CD/metabolism , Phosphoproteins/metabolism , Receptors, Antigen, B-Cell/metabolism , Receptors, IgG/metabolism , Amino Acid Motifs , Intracellular Signaling Peptides and Proteins , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphopeptides/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/metabolism , Proteins/metabolism , SH2 Domain-Containing Protein Tyrosine Phosphatases , Shc Signaling Adaptor Proteins , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1ABSTRACT
A recombinant soluble form of the human Fc gamma receptor was produced by engineering a cDNA construct containing the extracellular part of the mature protein. After expression in bacteria as inclusion body, the polypeptide was highly purified and was refolded in vitro with a method that was developed for the renaturation of immunoglobulin fragments. With this method oxidation of the disulfide bridges within the domains of the protein is done in the presence of an artificial 'chaperone' which protects the polypeptide molecules from unwanted protein protein interactions thereby inhibiting the incorrect oxidation of the SH-groups. and misfolding of the protein. The refolded recombinant soluble Fc gamma RIIb showed several characteristics of the native receptor: (i) it was recognized by a series of monoclonal antibodies specific for, and in most cases produced against the native cell-surface receptor: (ii) it is bound to its ligand (the Fc-region of different immunoglobulins) under very diverse conditions: and (iii) it is competed strongly and specifically with the native cell surface receptor for both ligand and antibody binding in experiments with distinct read-outs; (iv) monoclonal antibodies produced against the recombinant protein specifically recognized Fc gamma RIIb on different cells. From these data it was concluded that the recombinant soluble Fc-receptor was in a native, functionally active form, and its function was not affected by the lack of glycosylation.
Subject(s)
Antigens, CD/chemistry , Antigens, CD/immunology , Escherichia coli/metabolism , Protein Folding , Receptors, IgG/chemistry , Receptors, IgG/immunology , Animals , Antibodies/immunology , Antibodies, Monoclonal , Antigens, CD/genetics , B-Lymphocytes , Blotting, Western , Calcium Signaling , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Humans , Ligands , Mice , Mice, Inbred BALB C , Receptors, IgG/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SolubilityABSTRACT
Co-clustering B cell receptors (BCR) and type II receptors binding the Fc part of IgG (Fc gamma RIIb) inhibits B cell activation and antibody production. Tyrosine phosphorylation of an intracellular motif of Fc gamma RIIb has been shown to be a prerequisite of the inhibition. After being phosphorylated by BCR-activated tyrosine kinases, the immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of Fc gamma RIIb recruits SH2 domain containing protein tyrosine phosphatase(s) (PTPs) and polyphosphoinositol 5-phosphatase (SHIP) to the vicinity of BCR, which in turn dephosphorylate their specific substrates. This leads to the interruption of signal transduction, consequently to the anergy and/or apoptosis of the cell. The downstream signaling pathways affected by Fc gamma RIIb-BCR co-clustering are not clarified yet, neither the substrates of PTPs are known. We have studied the Fc gamma RIIb mediated B cell inhibition on human Burkitt lymphoma cell line (BL41). From the lysates of BL41 cells SHP-2 and phosphatidylinositol 3-kinase (PI3-K), as well as the protein tyrosine kinase (PTK) Lyn bind both to the BCR-co-clustered Fc gamma RIIb and to its P-ITIM peptide. Lyn hyperphosphorylates the P-ITIM associated molecules, including SHIP in the in vitro protein tyrosine kinase activity assay. The P-ITIM-compelled multi-phosphoprotein complex binds to and activates SHP-2, which in turn dephosphorylates SHIP and Shc and probably other substrates. Subcellular localisation of these signaling molecules is regulated by the phosphotyrosine-SH2 domain interactions, thus dephosphorylation may result in the re-direction of Shc and SHIP within the cell, consequently, in the modulation of their activity. Finally, co-clustering Fc gamma RIIb and BCR or Fc gamma RIIb and CD19 on the intact cells inhibited PI3-K activity as detected in the anti-phosphotyrosine (anti-PY) precipitates. The results indicate that SHP-2 bound to and activated by the BCR co-clustered Fc gamma RIIb, may down-regulate PI3-K activity by dephosphorylating a yet unidentified regulatory molecule, which recruits PI3-K to the cell membrane.
Subject(s)
Antigens, CD/physiology , B-Lymphocytes/enzymology , Phosphatidylinositol 3-Kinases/physiology , Phosphoric Monoester Hydrolases/physiology , Protein Tyrosine Phosphatases/physiology , Receptors, IgG/physiology , Antigens, CD/metabolism , B-Lymphocytes/physiology , Binding, Competitive/immunology , Humans , Inositol Polyphosphate 5-Phosphatases , Intracellular Signaling Peptides and Proteins , Macromolecular Substances , Phosphoinositide-3 Kinase Inhibitors , Phosphopeptides/metabolism , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, B-Cell/physiology , Receptors, IgG/metabolism , SH2 Domain-Containing Protein Tyrosine Phosphatases , Signal Transduction/immunologyABSTRACT
Co-clustering of the type II receptors binding the Fc part of IgG (FcgammaRIIb) and B cell receptors results in the translocation of cytosolic, negative regulatory molecules to the phosphorylated immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of the FcgammaRIIb. SH2 domain-containing protein tyrosine phosphatases (SHP-1 and SHP-2), and the polyphosphoinositol 5'-phosphatase (SHIP) have been reported earlier to bind to murine FcgammaRIIb P-ITIM. However, neither the functional substrates of these enzymes, nor the mechanism of the inhibition are fully resolved. We show here that the human FcgammaRIIb binds SHP-2 when co-clustered with the B cell receptors, whereas its synthetic P-ITIM peptide bindes SHP-2 and SHIP in lysates of the Burkitt's lymphoma cell line BL41. The P-ITIM peptide binding enhances SHP-2 activity, resulting in dephosphorylation and release of P-ITIM-bound SHIP and Shc. Moreover, P-ITIM-bound SHP-2 dephosphorylates synthetic peptides corresponding to the sites of tyrosine phosphorylation on SHIP and Shc, indicating that these proteins are its potential substrates. Thus SHP-2-induced dephosphorylation may modulate the intracellular localization and/or activity of SHIP and Shc, thereby inhibiting further activation pathways which they mediate.
Subject(s)
B-Lymphocytes/enzymology , B-Lymphocytes/immunology , Protein Tyrosine Phosphatases/metabolism , Receptors, IgG/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Humans , Intracellular Signaling Peptides and Proteins , Mice , Molecular Sequence Data , Peptides/genetics , Peptides/immunology , Peptides/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , SH2 Domain-Containing Protein Tyrosine Phosphatases , Tyrosine/metabolism , src-Family Kinases/metabolismABSTRACT
Fc gammaRIIb (CD32) controls antibody production by down-regulating cell activation, when co-clustered with B cell antigen receptors (BCR) in vivo, via immune complexes consisting of secreted IgG and antigen. Fc gammaRIIb-BCR co-ligation in vitro was shown to inhibit the Ca2+ influx from the extracellular space, the mechanism of which is not fully understood. Human B cells express Fc gammaRIIb1 and Fc gammaRIIb2, differing only in a 19 amino acid long insert in the cytoplasmic tail of the former. To elucidate whether Fc gammaRIIb1 and Fc gammaRIIb2 isoforms show any difference in the down-regulation of B cells, we have studied the effect of co-clustering of BCR and Fc gammaRIIb1 or Fc gammaRIIb2 on the Ca2+ signaling in a Burkitt's lymphoma cell line, ST486, transfected with the two isoforms respectively. We have shown here, for the first time, that co-aggregation of BCR and Fc gammaRIIb may also inhibit Ca2+ release from the endoplasmic reticulum pool of human B cells. Both isoforms mediated this inhibition and the inhibitory effect depended on the ratio of BCR to Fc gammaRIIb cross-linking. In contrast to Fc gammaRIIb, the CD21/CD19 complex was shown to up-regulate B cell response by lowering the activation threshold. We have shown here that co-clustering of Fc gammaRIIb with CD19 inhibited the CD19-induced Ca2+ influx. Furthermore, the three party co-aggregation of Fc gammaRIIb with BCR and CD19 resulted in a decreased Ca2+ response, as compared to the BCR- plus CD19-induced one, indicating that Fc gammaRIIb may inhibit CD19-induced enhancement of B cell activation. On the basis of these data we suggest that IgG-containing and C3d-fixing immune complexes may down-regulate the B cell response by interfering with both BCR- and CD19-mediated Ca2+ mobilization.
Subject(s)
Antigens, CD19/physiology , B-Lymphocytes/immunology , Calcium/metabolism , Receptors, Antigen, B-Cell/metabolism , Receptors, IgG/physiology , Humans , Receptor Aggregation , Signal Transduction , TransfectionABSTRACT
Co-clustering of Fc gamma RIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of Fc gamma RIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human Fc gamma RIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human Fc gamma RIIb, synthetic peptides were designed to cover almost the entire intracellular Fc gamma RIIb domain, including Fc gamma RIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53-56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating Fc gamma RIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both Fc gamma RIIb1 and Fc gamma RIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to Fc gamma RIIb1 and Fc gamma RIIb2 specific sequences. Synthetic peptide representing Fc gamma RIIb1 and Fc gamma RIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to Fc gamma RIIb1 or Fc gamma RIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human Fc gamma RIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SHIP and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the Fc gamma RIIb co-clustered BCR are responsible for the Fc gamma RIIb mediated negative regulation of human B-cell activation.
Subject(s)
Antigens, CD/metabolism , B-Lymphocytes/metabolism , Inositol/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Tyrosine Phosphatases/metabolism , Receptors, IgG/metabolism , Signal Transduction , Amino Acid Sequence , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoproteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , src-Family Kinases/metabolismABSTRACT
Fc gamma receptors type IIb1 (Fc gammaRIIb1) inhibit B-cell activation when co-ligated with B-cell antigen receptors (BCR) by immune complexes. In murine B-cells the inhibition is mediated by the interaction of the phosphorylated immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of Fc gammaRIIb1 with the SH2 domain containing protein tyrosine phosphatase. SHP1. To clarify the mechanism of Fc gammaRIIb mediated inhibition of human B-cells we have studied the association of signaling molecules with human Fc gammaRIIb1 after co-ligating with BCR. Fc gammaRIIb1 were affinity purified from the Burkitt lymphoma cell line, BL41. Several tyrosine phosphorylated proteins were co-isolated with Fc gammaRIIb1 at 145, 110, and 50 60 kDa, which were not present in Fc gammaRIIb1 free immune complexes. Among these molecules we have identified the p52 Shc adaptor protein. Furthermore, we have shown that the insolubilised synthetic peptide corresponding P-ITIM bound Shc, Lyn and the p75 and p 10 unidentified tyrosine phosphorylated proteins. Here we describe that the cell membrane associated Shc is partially dephosphorylated in BCR-Fc gammaRIIb1 co-ligated samples, suggesting that its function in regulating p21ras monomeric G protein is impaired. Indeed, we have detected a lower p21ras activity in BCR-Fc gammaRIIb1 co-crosslinked samples. These data indicate that co-ligation of BCR and Fc gammaRIIb1 interrupts signal transduction between protein tyrosine kinase activation and p21ras mediated activation pathway. Since in contrast to the mouse B-cells both Fc gammaRIIb1 and Fc gammaRIIb2 are expressed in human B-cells, we have investigated the inhibitory function of the two receptors in Fc gammaRIIb negative Burkitt lymphoma cell line ST486 transfected with Fc gammaRIIb1 and Fc gammaRIIb2, respectively. Both Fc gammaRIIb1 and Fc gammaRIIb2 inhibited the rise of intracellular Ca2+ induced by the crosslinking of BCR. The rate of the inhibition depended on the ratio of the co-crosslinked receptors (BCR-Fc gammaRIIb1) to the crosslinked BCR (BCR-BCR). Co-crosslinking of the two receptors inhibited not only the capacitive Ca2+ entry but rather the total Ca2+ response in both Fc gammaRIIb1 and Fc gammaRIIb2 transfected human B-cells. CD19 represents the signal transduction unit of complement receptor, CR2 (CD21), and is responsible for the complement activating IgM-immune complex induced enhancement of B-cell activation. Co-crosslinking of CD19 and BCR was shown to enhance B-cell activation due to the recruitment of further signaling molecules to the activator complex by the phosphorylated tyrosine residues of CD19. Here we show a novel finding that co-ligation of CD19 with Fc gammaRIIb1 inhibits the CD19-induced upregulation of Ca2+ response. The results indicate that IgG plus complement containing immune complexes may inhibit B-cell activation in vivo, due to the Fc gammaRIIb1-mediated interruption of signal transduction via both BCR and CD19.
Subject(s)
Antigens, CD/immunology , B-Lymphocytes/immunology , Receptors, Antigen, B-Cell/immunology , Receptors, IgG/immunology , Signal Transduction , Antigens, CD19/immunology , Calcium/immunology , Humans , Lymphocyte Activation , Proto-Oncogene Proteins p21(ras)/immunology , Tumor Cells, Cultured , src Homology Domains/immunologyABSTRACT
Co-ligation of antigen receptors and type II Fcgamma receptors (FcgammaRIIb) on B cells interrupts signal transduction and ultimately inhibits antibody production. We have identified p52 Shc in the FcgammaRIIb1-specific immunoprecipitates isolated from the membrane fraction of BL41 Burkitt lymphoma cells following B cell receptor-FcgammaRIIb1 co-ligation. The insolubilized synthetic peptide representing the phosphorylated form of the tyrosine-based inhibitory motif of FcgammaRIIb also binds Shc from the lysates of activated but not from resting BL41 cells. This suggests that the binding does not depend on the interaction of FcgammaRIIb1-phosphotyrosine with the SH2 domain of Shc. Tyr phosphorylation of FcgammaRIIb1-associated Shc is low, indicating an impaired function. Shc is implicated in regulating p21(ras) activation; thus, we have compared p21(ras) activities in BL41 cells treated in different ways. p21(ras) activity is reduced when B cell receptor and FcgammaRIIb1 are co-ligated. p21(ras) couples protein-tyrosine kinase-dependent events to the Ser/Thr kinase-mediated signaling pathway leading to the activation of mitogen-activated protein kinases (MAPK). Our results show that B cell receptor-FcgammaRIIb1 co-cross-linking partially inhibits mitogen-activated protein kinase activity. We conclude that FcgammaRIIb1-dependent inhibition of human B cell activation may be based on interrupting signal transduction between protein-tyrosine kinases and the p21(ras)/mitogen-activated protein kinase-dependent activation pathway.
Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Antigens, CD/physiology , B-Lymphocytes/immunology , Lymphocyte Activation , Mitogen-Activated Protein Kinases , Proto-Oncogene Proteins p21(ras)/physiology , Receptors, Antigen, B-Cell/physiology , Receptors, IgG/physiology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Humans , Mitogen-Activated Protein Kinase 3 , Proteins/metabolism , Receptors, IgG/immunology , Shc Signaling Adaptor Proteins , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1 , src Homology DomainsABSTRACT
To test the suggested structural relationship between the electrogenic H+ transporting system and the NADPH oxidase of phagocytes, the existence of the enzyme and the transport process was investigated in human tonsillar T lymphocytes. It is shown that tonsillar T cells possess an arachidonic acid activatable, Cd(2+)- and Zn(2+)-sensitive electrogenic H+ efflux pathway with similar properties as reported earlier in various phagocytic cells. The presence of cytochrome b558, the membrane component of the oxidase, could not be detected in tonsillar T lymphocytes either by immunoblot or by flow cytometric analysis. It is suggested that the electrogenic H+ transporting pathway is structurally independent of the NADPH oxidase complex.
Subject(s)
Arachidonic Acid/pharmacology , Cytochrome b Group/metabolism , Proton-Translocating ATPases/metabolism , T-Lymphocytes/metabolism , Cadmium/pharmacology , Cytochrome b Group/analysis , Cytochrome b Group/deficiency , Dicyclohexylcarbodiimide/pharmacology , Flow Cytometry , Granulocytes/immunology , Granulocytes/metabolism , Humans , Kinetics , Masoprocol/pharmacology , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidases , Palatine Tonsil , Proton-Translocating ATPases/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Zinc/pharmacologyABSTRACT
The low-affinity type-IIb IgG Fc-binding receptors (Fc gamma RIIb) are expressed on B cells. When cross-linked with mIgM Fc gamma RIIb are known to down-regulate B-cell activation by interrupting signal transduction upstream from G-protein-activated events. We have studied Fc gamma RII isoforms expressed on resting and activated B cells and the interaction of Fc gamma RIIb1 with molecules transducing the antigen receptor-mediated signals. Expression of Fc gamma RII isoforms was studied by reverse transcription and polymerase chain reaction. Resting B cells express both Fc gamma RIIb2 and Fc gamma RIIb1 isoforms. Activation with anti-IgM or IL-4 induces the splicing of Fc gamma RIIb1 mRNA, while the alternative splicing of Fc gamma RIIb2 mRNA is down-regulated, resulting in the surface expression of Fc gamma RIIb1. Functional differences were found between the two isoforms in inhibiting B-cell activation, suggesting that Fc gamma RIIb2 might influence the threshold of signals necessary for activation of resting B cells, while Fc gamma RIIb1 may regulate in later phases of antibody response. To explore the mechanism by which Fc gamma RII may uncouple antigen receptor-mediated signal transduction, we have investigated the association of signaling molecules with Fc gamma RII. Beside the protein tyrosine kinase (PTK) fyn, protein kinase C (PKC) was found to be co-isolated with Fc gamma RIIb1, suggesting a tight connection between these kinases and Fc gamma RII. We suggest that PKC might be responsible for the activation-induced phosphorylation of Fc gamma RII on serine residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Antigens, CD , B-Lymphocytes/immunology , Receptors, IgG/metabolism , Signal Transduction , Base Sequence , Humans , Lymphocyte Activation , Models, Immunological , Molecular Sequence Data , Precipitin Tests , Protein Binding , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fyn , Receptors, IgG/geneticsABSTRACT
The human type two IgG binding receptors (Fc gamma RII) are encoded by three genes (Fc gamma RIIA, -B and C) resulting in at least six protein isoforms generated by alternative mRNA splicing. Surface expression of Fc gamma RII has been shown to be modulated during B cell activation, although data characterizing the isoform(s) expressed are not available. The extracellular as well as the transmembrane domains of various Fc gamma RII are highly homologous. Only the intracellular domains vary between the different Fc gamma RII isoforms, suggesting differences in signal transduction. Using reverse transcriptase and polymerase chain reaction of mRNA obtained from resting tonsil B cells, we show that the majority of Fc gamma RII mRNA species to be of b2 type, although b1 type and a low level of Fc gamma RIIa type are also present. Culturing the cells for 18 h in the presence of 2.5 U/ml interleukin-4 or 10 micrograms/ml affinity-purified anti-IgM F(ab')2 fragments induced a switch in alternative splicing, resulting in a significant increase of Fc gamma RIIb1 mRNA expression, while the synthesis of Fc gamma RIIb2 mRNA was down-regulated. Stimulation of B cells with 100 ng/ml phorbol 12-myristate 13-acetate induced similar alteration, although only after 48-h treatment. The accumulation of Fc gamma RIIb1 and the reduction of both Fc gamma RIIb2 and Fc gamma RIIa mRNA in activated cells is accompanied by the enhanced expression of Fc gamma RII on the cell surface, representing most probably the Fc gamma RIIb1 isoform. Heat-aggregated IgG inhibited the anti-IgM-induced proliferation of resting but not that of activated B cells, suggesting that aggregation of Fc gamma RIIb2 constitutively expressed on resting B cells might be responsible for the prevention of inadequate activation of resting B cells.