Structural studies in solution of the recombinant N-terminal pair of short consensus/complement repeat domains of complement receptor type 2 (CR2/CD21) and interactions with its ligand C3dg

Human complement receptor type 2 (CR2, CD21) is a cell surface receptor that binds three distinct ligands (complement C3d, Epstein-Barr virus gp350/220, and the low-affinity IgE receptor CD23) via the N-terminal two of fifteen or sixteen short consensus/complement repeat (SCR) domains. Here, we report biophysical studies of the CR2 SCR 1-2 domain binding to its ligand C3dg. Two recombinant forms of CR2 containing the SCR 1-2 and SCR 1-15 domains were expressed in high yield in Pichia pastoris and baculovirus, respectively. Circular dichroism spectroscopy showed that CR2 SCR 1-2 receptor possessed a beta-sheet secondary structure with a melting temperature of 59 degrees C. Using surface plasmon resonance, kinetic parameters for the binding of either CR2 SCR 1-2 or the full-length SCR 1-15 form of CR2 showed that the affinity of binding to immobilized C3d is comparable for the SCR 1-15 compared to the SCR 1-2 form of CR2. Unexpectedly, both the association and dissociation rates for the SCR 1-15 form were slower than for the SCR 1-2 form. These data show that the SCR 1-2 domains account for the primary C3dg binding site of CR2 and that the additional SCR domains of full-length CR2 influence the ability of CR2 SCR 1-2 to interact with its ligand. Studies of the pH and ionic strength dependence of the interaction between SCR 1-2 and C3d by surface plasmon resonance showed that this is influenced by charged interactions, possibly involving the sole His residue in CR2 SCR 1-2. Sedimentation equilibrium studies of CR2 SCR 1-2 gave molecular weights of 17 000, in good agreement with its sequence-derived molecular weight to show that this was monomeric. Its sedimentation coefficient was determined to be 1.36 S. The complex with C3d gave molecular weights in 50 mM and 200 mM NaCl buffer that agreed closely with its sequence-derived molecular weight of 50 600 and showed that a 1:1 complex had been formed. Molecular graphics views of homology models for the separate CR2 SCR 1 and SCR 2 domains showed that both SCR domains exhibited a distribution of charged groups throughout its surface. The single His residue is located near a long eight-residue linker between the two SCR domains and may influence the linker conformation and the association of C3d and CR2 SCR 1-2 into their complex. Sedimentation modeling showed that the arrangement of the two SCR domains in CR2 SCR 1-2 is highly extended in solution.     

Binding of C3 and C3dg to the CR2 complement receptor induces growth of an Epstein-Barr virus-positive human B cell line

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.     

Expression of CR2 (the C3dg/EBV receptor, CD21) on normal human peripheral blood T lymphocytes.

The expression of CR2 (the C3dg/EBV receptor, CD21) on normal human T lymphocytes was investigated using purified peripheral blood T cells and indirect immunofluorescence with biotinylated anti-CR2 mAb and streptavidin-phycoerythrin. Thirty to 40% of normal peripheral blood T lymphocytes expressed CR2 Ag. The cells expressed three nonoverlapping epitopes of CR2. The specificity of the staining for CR2 epitopes was demonstrated by the ability of unlabeled anti-CR2 mAb but not of anti-CR1 mAb of the same isotype to compete for the binding of biotinylated anti-CR2 mAb to T cells. The intensity of staining of T lymphocytes with anti-CR2 mAb was approximately 10-fold lower than that of peripheral blood B cells. CR2 was immunoprecipitated from purified T lymphocytes as a single protein of apparent Mr 145,000. The presence of CR2 on normal human T lymphocytes suggests that the receptor may modulate the function of T cells in the immune response and the susceptibility of the cells to infection by lymphocytotropic viruses.     

Mutational analysis of the complement receptor type 2 (CR2/CD21)-C3d interaction reveals a putative charged SCR1 binding site for C3d

We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.     

B cell receptor (BCR) cross-talk: CD40 engagement creates an alternate pathway for BCR signaling that activates I kappa B kinase/I kappa B alpha/NF-kappa B without the need for PI3K and phospholipase C gamma

BCR signaling is propagated by a series of intermediaries and eventuates in NF-kappaB activation, among other outcomes. Interruption of several mediators that constitute the signalosome, such as PI3K and phospholipase Cgamma2, completely blocks BCR signaling for NF-kappaB. We show here that this accepted, conventional paradigm is, in fact, limited to naive B cells. CD40L treatment reprograms normal B cells such that a novel, alternate pathway for BCR signaling is created. Through this alternate pathway BCR triggering induces nuclear NF-kappaB without the need for PI3K or for phospholipase Cgamma2. Induction of NF-kappaB via the alternate pathway is accompanied by IkappaB kinase beta (IKKbeta) phosphorylation, IkappaBalpha phosphorylation, and IkappaBalpha degradation, and inhibition of IKKbeta blocked IkappaBalpha degradation. Several key events in the conventional pathway, including early protein tyrosine phosphorylation, were unimpeded by generation of the alternate pathway which appears to operate in parallel, rather than in competition, with classical BCR signaling. These results demonstrate cross-talk between CD40 and BCR, such that the requirements for BCR signaling are altered by prior B cell exposure to CD40L. The alternate BCR signaling pathway bypasses multiple signalosome elements and terminates in IKKbeta activation.     

Isolating the Epstein-Barr virus gp350/220 binding site on complement receptor type 2 (CR2/CD21)

Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by x-ray crystallography, and the CR2 binding site on this protein has been characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed toward the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged residues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, and Arg-83). These data appear to complement the CR2 binding site on gp350, which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2-gp350 complex, we also provide evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as monoclonal antibodies 171 and 1048 whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding.     

Epitope mapping using the X-ray crystallographic structure of complement receptor type 2 (CR2)/CD21: identification of a highly inhibitory monoclonal antibody that directly recognizes the CR2-C3d interface.

Complement receptor type 2 (CR2)/CD21 is a B lymphocyte cell membrane C3d/iC3b receptor that plays a central role in the immune response. Human CR2 is also the receptor for the EBV viral membrane glycoprotein gp350/220. Both C3d and gp350/220 bind CR2 within the first two of 15-16 repetitive domains that have been designated short consensus/complement repeats. Many mAbs react with human CR2; however, only one currently available mAb is known to block both C3d/iC3b and gp350/220 binding. We have used a recombinant form of human CR2 containing the short consensus/complement repeat 1-2 ligand-binding fragment to immunize Cr2(-/-) mice. Following fusion, we identified and further characterized four new anti-CR2 mAbs that recognize this fragment. Three of these inhibited binding of CR2 to C3d and gp350/220 in different forms. We have determined the relative inhibitory ability of the four mAbs to block ligand binding, and we have used overlapping peptide-based approaches to identify linear epitopes recognized by the inhibitory mAbs. Placement of these epitopes on the recently solved crystal structure of the CR2-C3d complex reveals that each inhibitory mAb recognizes a site either within or adjacent to the CR2-C3d contact site. One new mAb, designated 171, blocks CR2 receptor-ligand interactions with the greatest efficiency and recognizes a portion of the C3d contact site on CR2. Thus, we have created an anti-human CR2 mAb that blocks the C3d ligand by direct contact with its interaction site, and we have provided confirmatory evidence that the C3d binding site seen in its crystal structure exists in solution.     

Characterization of the human glomerular C3 receptor as the C3b/C4b complement type one (CR1) receptor

The functional and immunochemical characteristics of the human glomerular C3 receptor were investigated by adherence of sheep erythrocytes (Es) coated with defined C3 fragments and by using polyclonal and/or monoclonal antibodies directed against epitopes expressed on complement receptors CR1, CR2, and CR3. C3b-bearing Es (EsC3b) strongly adhered to glomeruli in frozen kidney sections in a reaction that was selectively inhibited by F(ab')2 anti-CR1 antibodies. There was no adherence of EsC3dg, EsC3d, and EsC3bi in the presence or absence of Ca++ and Mg++ under physiologic buffer conditions. The weak glomerular binding of EsC3bi, which was observed in half-isotonic buffer was selectively suppressed by anti-CR1 antibodies. By indirect immunofluorescence, anti-CR1 antibodies stained all podocytes in glomeruli, whereas no staining of kidney sections was seen with OKM1 and anti-Mol antibodies directed against the alpha-chain of CR3 and with anti-CR2 antibodies anti-B2 and BL13. Solubilization of membrane glycoproteins from freshly isolated glomeruli from three human kidneys, in the presence of 0.1% Nonidet P-40, yielded a material that bound to lentil lectin Sepharose and could accelerate the decay of preformed cell-bound amplification C3 convertase sites in a reaction that was inhibited by anti-CR1 antibodies. The material containing CR1 activity was labeled with 125I, immunoprecipitated with anti-CR1, and analyzed by SDS-PAGE and autoradiography. Anti-CR1 immunoprecipitated a form of CR1 of Mr 205,000 in solubilized glomeruli from three donors, and an additional form of Mr 160,000 in glomeruli from two of the donors. Immunoprecipitation of CR1 from surface-labeled erythrocytes from these individuals demonstrated them to be homozygous for the 205,000 Mr form of the receptor. Whether the 160,000 band represents in vitro or in vivo proteolytic cleavage of CR1, or cell specific-modulation of gene expression of glomerular CR1, remains unclear. Thus, CR1 is the only type of C3 receptor expressed in the human kidney. Glomerular CR1 shares the functional antigenic and biochemical properties of the C3b/C4b CR1 receptor of peripheral blood cells.     

Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350

The binding of the Epstein-Barr virus glycoprotein gp350 by complement receptor type 2 (CR2) is critical for viral attachment to B lymphocytes. We set out to test hypotheses regarding the molecular nature of this interaction by developing an enzyme-linked immunosorbent assay (ELISA) for the efficient analysis of the gp350-CR2 interaction by utilizing wild-type and mutant forms of recombinant gp350 and also of the CR2 N-terminal domains SCR1 and SCR2 (designated CR2 SCR1-2). To delineate the CR2-binding site on gp350, we generated 17 gp350 single-site substitutions targeting an area of gp350 that has been broadly implicated in the binding of both CR2 and the major inhibitory anti-gp350 monoclonal antibody (MAb) 72A1. These site-directed mutations identified a novel negatively charged CR2-binding surface described by residues Glu-21, Asp-22, Glu-155, Asp-208, Glu-210, and Asp-296. We also identified gp350 amino acid residues involved in non-charge-dependent interactions with CR2, including Tyr-151, Ile-160, and Trp-162. These data were supported by experiments in which phycoerythrin-conjugated wild-type and mutant forms of gp350 were incubated with CR2-expressing K562 cells and binding was assessed by flow cytometry. The ELISA was further utilized to identify several positively charged residues (Arg-13, Arg-28, Arg-36, Lys-41, Lys-57, Lys-67, Arg-83, and Arg-89) within SCR1-2 of CR2 that are involved in the binding interaction with gp350. These experiments allowed a comparison of those CR2 residues that are important for binding gp350 to those that define the epitope for an effective inhibitory anti-CR2 MAb, 171 (Asn-11, Arg-13, Ser-32, Thr-34, Arg-36, and Tyr-64). The mutagenesis data were used to calculate a model of the CR2-gp350 complex using the soft-docking program HADDOCK.     

Uncommon endocytic and trafficking pathway of the natural killer cell CD94/NKG2A inhibitory receptor

The CD94/NKG2A inhibitory receptor, expressed by natural killer and T cells, is constantly exposed to its HLA-E ligand expressed by surrounding cells. Ligand exposure often induces receptor downregulation. For CD94/NKG2A, this could potentiate activation receptor(s) induced responses to normal bystander cells. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent macropinocytic- like process; however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1⁺, Rab5⁺ early endosomes. It does appear in Rab4⁺ early/sorting endosome, but, in the time period examined, fails to reach Rab11⁺ recycling or Rab7⁺ late endosomes or lysosome-associated membrane protein-1⁺ lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression.     

Expression, molecular association, and functions of C3 complement receptors CR1 (CD35) and CR2 (CD21) on the human T cell line HPB-ALL.

We have investigated the expression, molecular association, ligand binding properties, and ability to transduce intracellular signals of CR1 and CR2 C3 receptors on cells of the human HPB-ALL T cell line. CR1 and CR2 on HPB-ALL cells bound polymeric C3b and C3dg and several anti-CR1 and anti-CR2 mAb recognizing different epitopes of the receptors on normal peripheral blood cells. Immunoprecipitated CR1 and CR2 exhibited similar m.w. to those of the receptors on normal peripheral blood T and B lymphocytes. CR1 and CR2 were partially associated in the form of CR1/CR2 complexes in the cell membrane as assessed by the ability of the receptors to cocap and cointernalize and to form a detergent-sensitive complex upon immunoprecipitation analysis. Triggering of CR2 with mAb OKB7 that recognizes an epitope associated with the ligand binding site of the receptor induced an increase in intracellular free calcium concentration in HPB-ALL cells. The signal provided by mAb OKB7 did not synergize with that triggered by anti-CD3 mAb UCHT1. Triggering of CR1 did not result in changes in intracellular free calcium concentration. Our observations have significance for the biology of normal human T cells because the majority of peripheral blood T cells that express CR1 also expressed CR2 and because a change in (Ca2+)i was induced by mAb OKB7 in purified normal T cells. These functions may be relevant for the regulatory role of C3 fragments on the immune response to T-dependent Ag and for the penetration into T cells of lymphocytotropic viruses.     

B cell receptor (BCR) cross-talk: CD40 engagement enhances BCR-induced ERK activation

Bystander B cells may be initially stimulated through CD40, which enhances susceptibility to Fas-mediated apoptosis, before encountering Ag, which produces Fas resistance. A key issue in this process is to what extent CD40 cross-talk might affect subsequent BCR signaling. It has previously been shown that CD40 engagement bypasses or mitigates the need for Bruton's tyrosine kinase in subsequent BCR signaling for NF-kappaB activation. However, the full extent of the effects of CD40 on BCR signaling has not been delineated. In the present study we evaluated the possibility that CD40-mediated cross-talk also affects another principal outcome of BCR signaling: MAPK activation. We found that prior stimulation of primary murine B cells with CD40L markedly enhanced the level of ERK and JNK (but not p38 MAPK) phosphorylation produced by subsequently added anti-Ig Ab, and much, but not all, of this enhancement was independent of PI3K and phospholipase C. CD40L treatment similarly enhanced BCR-induced MAPK kinase (MEK) phosphorylation, and MEK was required for enhancement of ERK. Although BCR-induced c-Raf phosphorylation was also enhanced by prior CD40L treatment, c-Raf was not required for MEK/ERK phosphorylation. These results identify a novel system of receptor cross-talk between CD40 and BCR and indicate that the effects of CD40 engagement on subsequent BCR stimulation spread beyond NF-kappaB to involve the MAPK pathway.     

Characterization of human complement receptor type 2 (CR2/CD21) as a receptor for IFN-alpha: a potential role in systemic lupus erythematosus

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands, C3d/iC3b, EBV-gp350, and CD23, a previous study has identified CR2 as a potential receptor for IFN-alpha. IFN-alpha, a multifunctional cytokine important in the innate immune system, has recently been proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study, we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-alpha in the same affinity range as the other three well-characterized ligands studied in parallel. In addition, we show that IFN-alpha interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b, EBV-gp350, and CD23. Finally, we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-alpha-responsive genes. Thus, IFN-alpha represents a fourth class of extracellular ligands for CR2 and interacts with the same domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-alpha receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction.     

Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21)

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between B cell and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established. The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electronegative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2. Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d. By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d. Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity.     

The CR2 receptor (CD21) shows increased expression in the more differentiated cells of an antigen-specific B cell line.

The complement receptor 2 (CR2; CD21), a 145,000 MW glycoprotein, has been useful as a marker of B lymphocyte maturation. It is expressed on the 1:13 monoclonal, EBV-transformed, B cell line which produces TNP-specific IgM-kappa and displays an in vitro capacity for differentiation. The line expresses the CD20+CD21+ phenotype. We studied whether CR2 receptor surface expression varied in relation to the cell cycle or state of differentiation in the 1:13 line. High CD21 and IgM expression occurred in the G1 phase of the cell cycle. In contrast to CD21, there were no distinctly brighter subpopulations of CD20 positive cells in the G1, S, or G2M compartments of the cell cycle. When sorted according to size, smaller cells were predominantly in G1, whereas a greater proportion of the larger cells were in the G2M phase of the cell cycle. The smaller 1:13 cells expressed more CD21 surface antigen and IgM than the larger cells. Cells which formed stable rosettes with TNP-SRBC expressed more surface IgM and CD21 antigen than nonrosetting cells. We have previously shown that the TNP-SRBC rosetting cells were more differentiated, resided in G1, and secreted more immunoglobulin than the nonrosetting cells. Thus increased CR2 expression occurred in the more differentiated cells of this human monoclonal B cell line.     

Structural requirements for C3d,g/Epstein-Barr virus receptor (CR2/CD21) ligand binding, internalization, and viral infection.

The structure of CR2, the human C3d,g/EBV receptor (CR2/CD21) consists of 15 or 16 60-70 amino acid repeats called short consensus repeats (SCRs) followed by a transmembrane and a 34-amino acid intracytoplasmic domain. Functions of CR2 include binding the human complement component C3d,g when it is covalently attached to targets or cross-linked in the fluid phase. In addition, CR2 binds the Epstein-Barr virus (EBV) and mediates internalization of EBV and subsequent infection of cells. In order to explore functional roles of the repetitive extracytoplasmic SCR structure and the intracytoplasmic domain of CR2, we have created truncated CR2 (rCR2) mutants bearing serial deletions of extracytoplasmic SCRs and also the intracytoplasmic tail. We then stably transfected these rCR2 mutants into two cell lines, murine fibroblast L cells and human erythroleukemic K562 cells. Phenotypic analysis of these expressed mutants revealed that 1) The C3d,g- and EBV-binding sites are found in the two amino-terminal SCRs of CR2, 2) expression of SCRs 3 and 4 is further required for high affinity binding to soluble cross-linked C3d,g, 3) the intracytoplasmic domain of CR2 is not required for binding C3d,g or EBV but is necessary for internalization of cross-linked C3d,g as well as for EBV infection of cells, 4) monoclonal anti-CR2 antibodies with similar activities react with single widely separated epitopes, and 5) no functional roles can yet be clearly assigned to SCRs 5-15, as rCR2 mutants not containing these SCRs show no major differences from wild-type rCR2 in binding or internalizing cross-linked C3d,g or mediating EBV binding and infection.     

NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244)

2B4 is an NK cell activation receptor that can provide a co-stimulatory signal to other activation receptors and whose mode of signal transduction is still unknown. We show that cross-linking of 2B4 on NK cells results in its rapid tyrosine phosphorylation, implying that this initial step in 2B4 signaling does not require coligation of other receptors. Ligation of 2B4 in the context of an NK cell-target cell interaction leads to 2B4 tyrosine phosphorylation, target cell lysis, and IFN-gamma release. Coligation of 2B4 with the inhibitory receptors killer cell Ig-like receptor (KIR)2DL1 or CD94/NKG2 completely blocks NK cell activation. The rapid tyrosine phosphorylation of 2B4 observed upon contact of NK cells with sensitive target cells is abrogated when KIR2DL1 or CD94/NKG2 are engaged by their cognate MHC class I ligand on resistant target cells. These results demonstrate that NK inhibitory receptors can interfere with a step as proximal as phosphorylation of an activation receptor.