Analysis of binding properties between 20 kDa human growth hormone (hGH) and hGH receptor (hGHR): the binding affinity for hGHR extracellular domain and mode of receptor dimerization.
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It has recently been shown that 20 kDa human growth hormone (hGH) forms the 1:2 hGH:hGH receptor (hGHR) complex and expresses full agonistic activity, although it hardly forms the 1:1 GH:GHR complex as compared with 22 kDa hGH. To clarify this mechanism, we analyzed the mode of receptor dimerization of 20 kDa hGH using the intact form and mutants. Complex formation analysis between hGHR extracellular domain (hGHBP) and either site1 mutant (K157A) or site2 mutant (G105R) by gel-filtration showed that the site1 mutant apparently formed no 1:1 complex and that the site2 mutant formed only the 1:1 complex. Cell proliferation analysis revealed that the activity curve (vs ligand concentration) of 20 kDa hGH showed a bell-shaped pattern. This indicates that the receptor dimerization of 20 kDa hGH proceeds in a sequential manner. Based on this sequential binding we have produced a mathematical model for receptor dimerization as a function of [hGH], [hGHBP], K(d) values for the first hGHBP binding (K(d1)) and the second hGHBP binding (K(d2)). The result of 20 kDa hGH binding to (S201C) hGHBP immobilized on biosensor tip showed that the K(d1) value was 1. 6x10(-8) M. Adopting this value as a constant in the function described above, we have obtained calculative hGHR dimerization curves vs hGH concentration. Since the K(d2) value could not be experimentally determined, the curves were simulatively obtained with varied K(d2) values. The simulated curve pattern coincided with the experimental result of the cell proliferation in Ba/F3-hGHR when the value 2.5x10(-10) M was adopted as K(d2). In conclusion, although the affinity of 20 kDa hGH for the first hGHR binding is reduced to one-tenth, that for the second binding is increased ten-fold in comparison with those of 22 kDa hGH, indicating that 20 kDa hGH can be an effective hGH isoform in the presence of hGHBP. (+info)
Convergence of Fc gamma receptor IIA and Fc gamma receptor IIIB signaling pathways in human neutrophils.
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Human neutrophils (PMNs) express two receptors for the Fc domain of IgG: the transmembrane FcgammaRIIA, whose cytosolic sequence contains an immunoreceptor tyrosine-based activation motif, and the GPI-anchored FcgammaRIIIB. Cross-linking of FcgammaRIIIB induces cell activation, but the mechanism is still uncertain. We have used mAbs to cross-link selectively each of the two receptors and to assess their signaling phenotypes and functional relation. Cross-linking of FcgammaRIIIB induces intracellular Ca2+ release and receptor capping. The Ca2+ response is blocked by wortmannin and by N,N-dimethylsphingosine, inhibitors of phosphatidylinositol 3-kinase and sphingosine kinase, respectively. Identical dose-response curves are obtained for the Ca2+ release stimulated by cross-linking FcgammaRIIA, implicating these two enzymes in a common signaling pathway. Wortmannin also inhibits capping of both receptors, but not receptor endocytosis. Fluorescence microscopy in double-labeled PMNs demonstrates that FcgammaRIIA colocalizes with cross-linked FcgammaRIIIB. The signaling phenotypes of the two receptors diverge only under frustrated phagocytosis conditions, where FcgammaRIIIB bound to substrate-immobilized Ab does not elicit cell spreading. We propose that FcgammaRIIIB signaling is conducted by molecules of FcgammaRIIA that are recruited to protein/lipid domains induced by clustered FcgammaRIIIB and, thus, are brought into juxtaposition for immunoreceptor tyrosine-based activation motif phosphorylation and activation of PMNs. (+info)
The transmembrane mutation G380R in fibroblast growth factor receptor 3 uncouples ligand-mediated receptor activation from down-regulation.
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A point mutation, Gly380Arg, in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leads to achondroplasia, the most common form of genetic dwarfism in humans. This substitution was suggested to enhance mutant receptor dimerization, leading to constitutive, ligand-independent activation. We found that dimerization and activation of the G380R mutant receptor are predominantly ligand dependent. However, using both transient and stable transfections, we found significant overexpression only of the mutant receptor protein. Metabolic pulse-chase experiments, cell surface labeling, and kinetics of uptake of radiolabeled ligand demonstrated a selective delay in the down-regulation of the mutant receptor. Moreover, this receptor was now resistant to ligand-mediated internalization, even at saturating ligand concentrations. Finally, transgenic mice expressing the human G380R mutant receptor under the mouse receptor transcriptional control demonstrated a markedly expanded area of FGFR3 immunoreactivity within their epiphyseal growth plates, compatible with an in vivo defect in receptor down-regulation. We propose that the achondroplasia mutation G380R uncouples ligand-mediated receptor activation from down-regulation at a site where the levels and kinetics of FGFR3 signals are crucial for chondrocyte maturation and bone formation. (+info)
Effects of Src homology domain 2 (SH2)-containing inositol phosphatase (SHIP), SH2-containing phosphotyrosine phosphatase (SHP)-1, and SHP-2 SH2 decoy proteins on Fc gamma RIIB1-effector interactions and inhibitory functions.
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Coaggregation of Fc gamma RIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated signaling via recruitment of Src homology domain 2 (SH2)-containing phosphatases. In vitro peptide binding experiments using phosphotyrosine-containing sequences derived from the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to mediate Fc gamma RIIB1 effects suggest that the receptor uses SH2-containing inositol phosphatase (SHIP) and SH2-containing phosphotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant Fc gamma RIIB1 effector protein is SHIP. However, biologically significant interactions may be lost in such studies if reactants' dissociation rates (Kd) are high. Thus, it is unclear to what extent these assays reflect the relative recruitment of SHIP, SHP-1, and SHP-2 to the receptor in vivo. As an alternative approach to this question, we have studied the effects of ectopically expressed SHIP, SHP-1, or SHP-2 SH2-containing decoy proteins on Fc gamma RIIB1 signaling. Results demonstrate the SHIP is the predominant intracellular ligand for the phosphorylated Fc gamma RIIB1 ITIM, although the SHP-2 decoy exhibits some ability to bind Fc gamma RIIB1 and block Fc receptor function. The SHIP SH2, while not affecting Fc gamma RIIB1 tyrosyl phosphorylation, blocks receptor-mediated recruitment of SHIP, SHIP phosphorylation, recruitment of p52 Shc, phosphatidylinositol 3,4,5-trisphosphate hydrolysis, inhibition of mitogen-activated protein kinase activation, and, albeit more modestly, Fc gamma RIIB1 inhibition of Ca2+ mobilization. Taken together, results implicate ITIM interactions with SHIP as a major mechanism of Fc gamma RIIB1-mediated inhibitory signaling. (+info)
Ebola virus secretory glycoprotein (sGP) diminishes Fc gamma RIIIB-to-CR3 proximity on neutrophils.
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Previous studies have shown that Ebola virus' secretory glycoprotein (sGP) binds to Fc gamma RIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and Fc gamma RIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and Fc gamma RIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and Fc gamma RIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-Fc gamma RIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and Fc gamma RIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between Fc gamma RIIIB and CR3. (+info)
Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation.
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Recombinant annexin V (rAnV) has been used to identify apoptotic cells based on its ability to bind phosphatidylserine (PS), a lipid normally restricted to the cytoplasmic face of the plasma membrane, but externalized early during apoptosis. However, this association of rAnV binding and apoptosis is not an obligatory one. We demonstrate that rAnV binds to a large fraction of murine B cells bearing selectable Ag receptors despite the fact that these cells are not apoptotic. Phosphatidylserine, which is uniformly distributed on resting B cells, is mobilized to co-cap with IgM on anti-IgM-treated B cells and to colocalize with GM1, a marker of lipid rafts. Cross-linking PS before anti-IgM treatment sequesters this lipid and alters signaling through IgM. Thus, PS exposed on the majority of B cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor-mediated signaling events. (+info)
The high-affinity IgE receptor (FcepsilonRI) blocks apoptosis in normal human monocytes.
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Monocytes have a limited life span, and their homeostasis is regulated by apoptosis in vivo. When cultured in the absence of appropriate exogenous stimuli, they undergo apoptosis, but under the influence of survival signals, these cells differentiate into macrophages or dendritic cells. Here we show that ligation of the high-affinity IgE receptor (FcepsilonRI) on human monocytes from nonatopic individuals markedly reduces apoptosis induced by serum deprivation or by CD95/Fas ligation. Aggregation of FcepsilonRI reduces its own expression but fails to modulate CD95/Fas expression. In contrast, FcepsilonRI ligation enhances the expression of the antiapoptotic molecules Bcl-2 and Bcl-xL, but not Mcl-1, in monocytes. Incubation of unstimulated cells with culture supernatants of FcepsilonRI-activated monocytes prolongs their life span, whereas CD95/Fas expression remains unaffected. The incidence of apoptosis is restored considerably when the supernatant is depleted of TNF-alpha, whereas elimination of IL-1beta, GM-CSF, or IL-12 has no effect. These results indicate that FcepsilonRI mediates signals preventing monocyte apoptosis directly by increasing the levels of Bcl-2 and Bcl-xL, and indirectly by means of TNF-alpha in an autocrine and paracrine fashion. This process may contribute to the establishment of chronic allergic disorders such as atopic dermatitis. (+info)
Synthesis of end-labeled multivalent ligands for exploring cell-surface-receptor-ligand interactions.
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BACKGROUND: Ring-opening metathesis polymerization (ROMP) is a powerful synthetic method for generating unique materials. The functional group tolerance of ruthenium ROMP initiators allows the synthesis of a wide range of biologically active polymers. We generated multivalent ligands that inhibit cell surface L-selectin, a protein that mediates lymphocyte homing and leukocyte recruitment in inflammation. We hypothesized that these ligands function through specific, multivalent binding to L-selection. To examine this and to develop a general method for synthesizing multivalent materials with end-labels, we investigated functionalized enol ethers as capping agents in ruthenium-initiated ROMP. RESULTS: We synthesized a bifunctional molecule that introduces a unique end group by terminating ruthenium-initiated ROMP reactions. This agent contains an enol ether at one end and a masked carboxylic acid at the other. We conjugated a fluorescein derivative to an end-capped neoglycopolymer that had previously been shown to inhibit L-selection function. We used fluorescence microscopy to visualize neoglycopolymer binding to cells displaying L-selectin. Our results suggest that the neoglycopolymers bind specifically to cell surface L-selectin through multivalent interactions. CONCLUSIONS: Ruthenium-initiated ROMP can be used to generate biologically active, multivalent ligands terminated with a latent functional group. The functionalized polymers can be labeled with a variety of molecular tags, including fluorescent molecules, biotin, lipids or antibodies. The ability to conjugate reporter groups to ROMP polymers using this strategy has broad applications in the material and biological sciences. (+info)