Immunofluorescence studies of the immunoglobulin receptors of B-lymphocytes]

The surface membrane Ig receptors of B lymphocytes in patients with atopic pollen asthma were studied by the direct immunofluorescence method. Use was made of labelled sera against human IgE, and IgG. It was shown that on the average 2.1 +/- 0.33% lymphocytes had IgE receptors, 8.4 +/- 0.63% IgG receptors and 18.7 +/- 1.16% human globulin receptors. A specific nature of B lymphocytes is suggested.     

Role of cholesterol in the capping of surface immunoglobulin receptors on murine lymphocytes

Previously, we have shown that the capping of surface immunoglobulins on murine lymphocytes can be affected by modulating the lipid environment of the surface membrane with free fatty acids. In the present study, murine lymphocytes were depleted of cholesterol by incubation with phospholipid vesicles. As the cellular cholesterol:phospholipid ratio decreased, the capping of the surface immunoglobulin was seen to decrease. This inhibition of capping could not be reversed by calcium and is not accompanied by changes in either the cytoskeletal element alpha-actinin or cellular ATP levels. Incubation of the cholesterol-depleted cells with cholesterol-containing phospholipid vesicles raised both the cholesterol:phospholipid ratio and capping levels to values close to those of untreated control cells. Remarkably, stearic acid, a saturated fatty acid, could also restore the capping levels in the cholesterol-depleted cells. On the basis of the present data and measurements of the fluorescence polarization of the probe diphenyl hexatriene, we propose a model in which the protein(s) involved in capping is located in a gel-like lipid domain, and that removal of cholesterol makes this domain less gel-like and inhibits capping. Restoration of the gel-like nature of this domain by the addition of either cholesterol or stearic acid enables the protein(s) to function normally.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes.

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24 degrees C. Within 20 min most of the cell lines destroyed 20% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37 degrees C. Bmax and KD for the different lymphocytes was 5.3--19 . 10(11) M and 1.8--18,5 . 10(11) M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Serum evaluation of the balance between soluble interleukin-2 and interleukin-4 receptors

To elucidate the usefulness of the simultaneous analysis of the multiple kinds of soluble cytokine receptors, we determined both the soluble interleukin 2 receptor (sIL-2R, Th1-type cytokine receptor) and the soluble interleukin 4 receptor (sIL-4R, Th2-type cytokine receptor) levels in the sera of healthy subjects as reference values and preliminarily applied to evaluate the patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameter of the severity. Both sIL-2R and sIL-4R levels in the sera of healthy children were significantly higher than those of healthy adults (p<0.01). The serum sIL-2R level of the patients with severe HUS (n=4) was higher than that of the patients with mild/moderate HUS (n=6) at the initial stage (p<0.01) or healthy children (n=51, p<0.01). Whereas, the serum sIL-4R level of both the severe and mild/moderate groups was lower than that of the healthy control children, although there was no significant difference among the three groups. Namely, the soluble receptor balance (sIL-2R/sIL-4R) in the patients with severe HUS may shift. We considered that the evaluation of the balance between soluble cytokine receptors might be informative for the evaluation of the immune states, as well as the conventional cytokine balance (Th1/Th2).     

Cross-talk between RON receptor tyrosine kinase and other transmembrane receptors

RON is a transmembrane receptor tyrosine kinase that mediates biological activities of Macrophage Stimulating Protein (MSP). MSP is a multifunctional factor regulating cell adhesion, motility, growth and survival. MSP binding to RON causes receptor tyrosine phosphorylation leading to up-regulation of RON catalytic activity and subsequent activation of downstream signaling molecules. Recent studies show that RON is spatially and functionally associated with other transmembrane molecules including adhesion receptors integrins and cadherins, and cytokine and growth factor receptors IL-3 betac, EPOR and MET. For example, MSP-induced cell shape change is mediated via RON-activated IL-3 betac receptor. Activation of integrins causes MSP-independent RON phosphorylation, and the integrin/RON collaboration regulates cell survival. Thus, RON can be activated without MSP by ligand stimulation of RON-associated receptors, and MSP-activated RON can cause ligand-independent activation of RON-associated receptors. As a result of the receptor cross-activation RON-specific pathways become a part of a signal transduction network of other receptors, and conversely signaling pathways activated by other receptors can be used by RON. This receptor collaboration extends the spectrum of cellular responses generated by MSP and by putative ligands of RON-associated receptors. However signaling pathways involved in the receptor cross-talk and underlying activation mechanisms remain to be investigated. The purpose of this review is to summarize data and to discuss a role of cross-talk between RON and other transmembrane receptors.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

A model of the complex between interleukin-4 and its receptors

A three-dimensional model of interleukin-4 (IL-4) bound to one molecule each of the high- and low-affinity receptors (IL-4R and IL-2Rγ) was built, using the crystal structure of the complex of human growth hormone (HGH) with its receptor (HGHR) as a starting model. The modeling of IL-4 with its receptors was based on the conservation of the sequences and on the predicted structural organization for cytokine receptors, and assuming that the binding mode of the ligands would be similar. Analysis of the interface between IL-4 and both receptor molecules was carried out to reveal which residues are important for complex formation. The modeling procedures showed that there were no major problems in maintaining a reasonable fit of IL-4 with the two receptor molecules, in a manner analogous to the complex of HGH–HGHR. Many of the residues that appear by modeling to be important for binding between IL-4 and the receptors have been previously implicated in that role by different methods. A striking motif of aromatic and positively charged residues on the surface of the C-terminal domains of the receptors is highly conserved in the structure of HGH–HGHR and in the models of IL-4 complexed with its receptors. © 1995 Wiley-Liss, Inc.     

Cross-talk between IRAK-4 and the NADPH oxidase

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.     

Cell surface immunoglobulin XXI. appearance of IgD on murine lymphocytes during differentiation.

The differentiation of Ig-bearing lymphocytes in adult mice was studied by monitoring the appearance of IgD relative to IgM on the surface of splenocytes obtained from lethally irradiated animals reconstituted for various periods of time with adult bone marrow cells, neonatal splenocytes, or Ig- adult splenocytes. It was found that IgM appears before IgD on differentiating lymphocytes. Furthermore, the rate of appearance of IgD during differentiation of adult cells is similar to that observed with neonatal cells.     

Cross-talk between tyrosine kinase and G-protein-linked receptors. Phosphorylation of beta 2-adrenergic receptors in response to insulin.

Protein kinases play a pivotal role in the propagation and modulation of transmembrane signaling pathways. Two major classes of receptors, G-protein-linked and tyrosine kinase receptors not only propagate signals but also are substrates for phosphorylation in response to stimulation by agonist ligands. Insulin (operating via tyrosine kinase receptors) and catecholamines (operating by G-protein-linked receptors) are counterregulatory with respect to lipid and carbohydrate metabolism. How, on a cellular level, these two distinct classes of receptors may cross-regulate each other remains controversial. In the present work we identify a novel cross-talk between members of two distinct classes of receptors, tyrosine kinase (insulin) and G-protein-linked (beta-adrenergic) receptors. Treatment of DDT1 MF-2 hamster vas deferens smooth muscle cells with insulin promoted a marked attenuation (desensitization) of beta-adrenergic receptor-mediated activation of adenylylcyclase. Measured by immune precipitation of beta 2-adrenergic receptors from cells metabolically labeled with [32P]orthophosphate, the basal state of receptor phosphorylation was increased 2-fold by insulin. Phosphoamino acid analysis revealed that for insulin-stimulated cells, the beta 2-adrenergic receptors showed increased phosphorylation on tyrosyl and decreased phosphorylation on threonyl residues. Phosphorylation of the beta-adrenergic receptor was rapid and peaked at 30 min following stimulation of cells by insulin. beta-Adrenergic receptor phosphorylation and attenuation of catecholamine-sensitive adenylylcyclase provide a biochemical basis for the counterregulatory effects of insulin upon catecholamine action.     

Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels

Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X2 induces retargeting and recruitment of mainly intracellular rho1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.     

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains.

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.     

Cross-talk between fMLP and vitronectin receptors triggered by urokinase receptor-derived SRSRY peptide

The urokinase-type plasminogen activator receptor (uPAR) sustains cell migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate chemotactic signaling in response to urokinase. We have characterized the early signaling events triggered by the Ser-Arg-Ser-Arg-Tyr (SRSRY) chemotactic uPAR sequence. Cell exposure to SRSRY peptide promotes directional migration on vitronectin-coated filters, regardless of uPAR expression, in a specific and dose-dependent manner, with maximal effect at a concentration level as low as 10 nm. A similar concentration profile is observed in a quantitative analysis of SRSRY-dependent cytoskeletal rearrangements, mostly consisting of filamentous structures localized in a single cell region. SRSRY analogues with alanine substitutions fail to drive F-actin formation and cell migration, indicating a critical role for each amino acid residue. As with ligand-dependent uPAR signaling, SRSRY stimulates protein kinase C activity and results in ERK1/2 phosphorylation. The involvement of the high affinity N-formyl-Met-Leu-Phe receptor (FPR) in this process is indicated by the finding that 100 nm N-formyl-Met-Leu-Phe inhibits binding of D2D3 to the cell surface, as well as SRSRY-stimulated cell migration and F-actin polarization. Moreover, cell exposure to SRSRY promotes FPR-dependent vitronectin release and increased uPAR.alphavbeta5 vitronectin receptor physical association, indicating that alphavbeta5 activity is regulated by the SRSRY uPAR sequence via FPR. Finally, we provide evidence that alphavbeta5 is required for SRSRY-dependent ERK1/2 phosphorylation, whereas it is not required for protein kinase C activation. The data indicate that the ability of uPAR to stimulate cell migration and cytoskeletal rearrangements is retained by the SRSRY peptide alone and that it is supported by cross-talk between FPR and alphavbeta5.     

Cross-talk between M2 muscarinic and D1 dopamine receptors in the cat adrenal medulla.

In the study reported here we have reached two conclusions. First, the cat adrenal medulla chromaffin cell possesses a dopamine D1 receptor that seems to be coupled to an adenylyl cyclase. Second, this receptor regulates the muscarinic-mediated catecholamine release response through a negative feed-back loop which uses cyclic AMP as a second messenger. These conclusions are supported by the following findings: (i) SKF38393 (a selective D1 receptor agonist), but not quinpirole (a selective D2 agonist), inhibits the methacholine-mediated catecholamine release responses in a concentration-dependent manner (IC50 of around 1-2 microM). (ii) SCH23390 (a selective D1 antagonist), but not sulpiride (a selective D2 antagonist), reversed by 70% the inhibitory effects of SKF38393. (iii) Dibutyril cyclic AMP (500 microM) inhibited by 80% the secretory effects of methacholine.     

Effect of microenvironment on functional activity of murine B-lymphocytes

In contrast to B-splenocytes, murine peritoneal B-cells do not produce or secrete immunoglobulins (Ig). Twenty-four hours after intraperitoneal transfer of splenocytes containing Ig-forming cells (IFC), IFC content in the peritoneal cavity of recipient mice decreases dramatically. This decrease does not depend on the migration of transferred IFC to the spleen (homing); splenectomy has no effect on this process. In order to check whether the lack of IFC in the peritoneal cavity is due to inhibition of synthesis or secretion of Ig, peritoneal cells (contained 4360 IFC/10⁶ cells) of CBA mice were intraperitoneally transferred to CBA/N mice after a 4-day preincubation in vitro. On the next day, ～30% of in vitro transferred IFC were detected in the peritoneal cavity of recipient mice, but on day 4 the IFC content in peritoneum returned to the background level. Repeated in vitro incubation of peritoneal cells of recipient mice restored the IFC number in cultured cells. It means that peritoneal microenvironment inhibits functional activity of murine B-lymphocytes.