Cell Surface Receptor Signaling Research Articles

TIMP-2: an endogenous inhibitor of angiogenesis

Remodeling of the extracellular matrix--regulated by the matrix metalloproteinases (MMPs) and their endogenous inhibitors--is an important component of disease progression in many chronic disease states. Unchecked MMP activity can result in significant tissue damage, facilitate disease progression and is associated with host responses to pathologic injury, such as angiogenesis. The tissue inhibitors of metalloproteinases (TIMPs) have been shown to regulate MMP activity. However, recent findings demonstrate that an MMP-independent effect of TIMP-2 inhibits the mitogenic response of human microvascular endothelial cells to growth factors. This is the first demonstration of a cell-surface signaling receptor for a member of the TIMP family and suggests that TIMP-2 functions to regulate cellular responses to growth factors. These new findings are integrated in a comprehensive model of TIMP-2 function in tissue homeostasis.

The cell cycle associated protein, HTm4, is expressed in differentiating cellsof the hematopoietic and central nervous system in mice

HTm4 is a member of a newly defined family of human and murine proteins, the MS4 (membrane-spanning four) protein group, which has a distinctive four-transmembrane structure. MS4 protein functions include roles as cell surface signaling receptors and intracellular adapter proteins. We have previously demonstrated that HTm4 regulates the function of the KAP phosphatase, a key regulator of cell cycle progression. In humans, the expression of HTm4 is largely restricted to cells of the hematopoietic lineage, possibly reflecting a causal role for this molecule in differentiation/proliferation of hematopoietic lineage cells. In this study, we show that, like the human homologue, murine HTm4 is also predominantly a hematopoietic protein with distinctive expression patterns in developing murine embryos and in adult animals. In addition, we observed that murine HTm4 is highly expressed in the developing and adult murine nervous system, suggesting a previously unrecognized role in central and peripheral nervous system development.

Gene Expression Profiles in Acute Myeloid Leukemias (AML): A Novel Approach Using SAGE and Custom Microarray.

Chromosome translocations are among the most common genetic abnormalities in human leukemia. Each translocation may affect a different pair of genes. The abnormally expressed genes that result from the different translocations provide a rich source for identifying specific markers for clinical diagnosis of each translocation. Microarrays have identified genes differentially expressed in different translocations but the results between laboratories are not always compatible. We used SAGE to quantitate gene expression in bone marrow (BM) samples from 22 patients with four types of AML, namely de novo AML M2 with t(8;21), AML M3 or M3V with t(15;17), AML M4Eo with inv(16), AML M5 with t(9;11) or secondary t(9;11).We generated SAGE libraries from CD15+ leukemic myeloid progenitor cells, collecting over 106 SAGE tags, of which 209,486 were unique tags; 136,010 were known genes and ESTs, and 73,476 were novel transcripts. SAGE tags for further analysis were selected based on a 5-fold difference between patients' samples and normal CD15+ BM; they were also statistically significantly different at the 5 % level. Using these strict criteria, we identified 1,571 unique tags, of which 1,405 were known genes and ESTs, and 166 were novel transcripts that were either specific for each translocation or were common for all four translocations. Changes in expression of these known genes which fall into different gene ontogeny functional categories varied by translocation. For example, those associated with macromolecular biosynthesis, transport and transcription were most altered in the t(8;21); those related to defense response and apoptosis were altered in the t(15;17); cell proliferation genes were most affected by the t(9;11). Cell surface receptor signaling, intracellular signaling and RNA processing were altered in treatment related but not in de novo t(9;11). From this analysis, we identified the functional molecular signature of each translocation. We designed a custom microarray to validate our SAGE data analysis. Our initial pilot microarray experiment with 96 genes that were specific for each translocation or common for all translocations used mononuclear cells from normal and patient BM and translocation cell lines, ME-1, THP-1, Mono Mac-6, Kasumi 1, NB-4; the array data from BM matched the SAGE data for 48-75 % of genes and the majority of cell lines, except ME-1, matched at least 70 % with the SAGE results for the appropriate translocation. We have now designed a full-scale microarray that contains over 400 probes including 250 known genes, 61 ESTs, 45 novel sequences and 48 genes reported by others. We will test at least 100 patients' samples with the four translocations to validate which genes provide a robust, reproducible “fingerprint” for each translocation and for all translocations. We will correlate our microarray data with age, sex, race, response to treatment, survival and other mutations (FLT3, MLL ITD, etc) to identify any transcripts that might reliably define these categories. Our results will provide new insights into genes that collaborate with each translocation to lead to a fully leukemic phenotype.

B1Bradykinin Receptor Homo-Oligomers in Receptor Cell Surface Expression and Signaling: Effects of Receptor Fragments

The human B(1) bradykinin receptor is an inducible and constitutively active G protein-coupled receptor that is involved in the inflammatory and pain responses to injury. Here, we investigated the role of B(1) receptor homo-oligomerization in cell surface receptor expression. B(1) receptors tagged with either the FLAG or hemagglutinin epitope were monitored immunologically and by radio-ligand binding, biotinylation, and phosphoinositide hydrolysis in human embryonic kidney 293 cells. Selective immunoprecipitation, immunoblotting, and immunoelectron microscopy with epitope-specific antibodies together provided evidence for constitutively formed cell surface receptor homo-oligomers. Truncation of the receptor from the N- and C-terminal ends indicated that the epitope for oligomerization seems to be located between Leu(26) on top of transmembrane helix 1 and Val(71) at the bottom of helix 2. A receptor construct terminating at Asp(134) at the bottom of helix 3, B1stop135, was expressed in the cell. It is interesting that this construct behaved as a dominant-negative mutant by competitively preventing formation of intact B(1) receptor homo-oligomers, and redistributing B(1) receptors from the cell surface to a common intracellular compartment. In contrast, expression of a construct containing the residues downstream of Asp(134), B1del(2-134), was inactive in this regard. Together, these results are consistent with a mechanism where constitutive B(1) receptor homooligomerization is required for expression of receptors on the cell surface and subsequent constitutive receptor signaling. This may be a novel mechanism by which the cell regulates the presentation of this constitutively highly active receptor at various stages of injury.

Transcriptional regulation of FGF-2 gene expression in cardiac myocytes

Fibroblast growth factor-2 (FGF-2) exerts its cardioprotective effect through cell surface receptor signaling and may play a role in the normal maintenance of a healthy myocardium. One mechanism of FGF-2 release from contracting cardiomyocytes is through transient sarcolemmal disruption, with accumulation in the extracellular matrix. Continuous FGF-2 release would require a link to synthesis and, thus, we examined regulation of FGF-2 promoter activity in cardiomyocytes as a potential target for optimizing cardioprotection. To investigate autoregulation, neonatal rat cardiomyocytes, (NRCM), were transfected with approximately 1 or 0.1 kb of rat FGF-2 promoter sequences linked to luciferase, -1058FGF-2p.luc and -110FGF-2p.luc, and treated with or without FGF-2. FGF-2 promoter activity was significantly increased approximately 2.5-fold with both genes. The proximal promoter region of rat FGF-2 contains putative binding sites for the early growth response-1 (Egr-1) and stimulating protein 1 (Sp1) transcription factors. Overexpression of Egr-1 and Sp1 increased -1058FGF-2p.luc expression by 4.4- and 8.7-fold, respectively. Mutation of Egr-1 and overlapping Sp1 sites did not blunt the response of -110FGF-2p.luc to FGF-2 treatment but did significantly reduce basal promoter activity. Transgenic mice expressing -1058FGF-2p.luc were treated with isoproterenol (IsP) to increase heart rate and endogenous FGF-2 release. FGF-2 promoter activity was stimulated significantly at 6 h, and increases in both FGF-2 and its receptor mRNA levels were also detected. In contrast, no effect of IsP was seen on -1058FGF-2p.luc or -110FGF-2p.luc in transfected NRCMs. FGF-2 released from cardiomyocytes may act to regulate its own synthesis at the transcriptional level. The mechanism does not appear to require an intact Egr-1 site in the proximal promoter region. This may, however, reflect redundancy in the control of FGF-2 promoter activity as our data support a stimulatory role for Egr-1 and Sp1.

Ligand-induced coupling versus receptor pre-association: cellular automaton simulations of FGF-2 binding

The binding of basic fibroblast growth factor (FGF-2) to its cell surface receptor (CSR) and subsequent signal transduction is known to be enhanced by heparan sulfate proteoglycans (HSPGs). HSPGs bind FGF-2 with low affinity and likely impact CSR-mediated signaling via stabilization of FGF-2–CSR complexes via association with both the ligand and the receptor. What is unknown is whether HSPG associates with CSR in the absence of FGF-2. In this paper, we determine conditions by which pre-association would impact CSR–FGF-2–HSPG triad formation assuming diffusion-limited surface reactions. Using mean-field rate equations, we show that (i) when [HSPG] is much higher than [CSR], the presence of pre-formed complexes does not affect the steady state of FGF-2 binding, and (ii) when the concentrations are comparable, the presence of pre-formed complexes substantially increases the steady-state concentration of FGF-2 bound to CSR. These findings are supported by explicit cellular automaton simulations, which justify the mean-field treatment. We discuss the advantages of such a two-receptor system compared to a single-receptor model, when the parameters are comparable. Further, we speculate that the observed high concentration of HSPG in intact cells ([HSPG]∼100[CSR]) provides a way to ensure that the binding levels of FGF-2 to its signaling receptor remains high, irrespective of the presence of pre-formed CSR–HSPG complexes on the cell surface, while allowing the cell to finely tune the response to FGF-2 via down-regulation of the signaling receptor.

Modulation of prostate cancer growth in bone microenvironments.

Bone remains one of the major sites, and most lethal host organs, for prostate cancer metastasis. Prostate cell spread and establishment in bone depends on multiple reciprocal modifications of bone stromal and epithelial cancer cell behaviors. This review focuses on recent advances in the characterization of cell-cell and cell-matrix interplay, effects on cell growth, adhesion and invasion, and several therapeutic possibilities for co-targeting prostate cancer cells and bone stroma. We address the topic from three main perspectives: (1) the normal and aging bone stromal environment, (2) the "reactive" bone stromal environment, and (3) the cancerous prostate epithelial cells themselves. First, normal, and especially aging, bones provide uniquely rich and "fertile soil" for roaming cancer cells. The interactions between prostate cancer cells and insoluble extracellular matrices, soluble growth factors, and/or sex steroid hormones trigger bone remodeling, through increased osteoclastogenesis and furthur matrix metalloproteinase activity. Second, after cancer cell arrival and establishment in the bone, host stromal cells respond, becoming "reactive" in a process again involving extracellular matrix remodeling, together with growth factor and steroid receptor signaling this process ultimately enhances cancer cell migration, stromal transdifferentiation, and invasion of the cancer tissues by stromal, inflammatory, and immune-responsive cells. Third, prostate cancer cells also respond to supportive bone microenvironments, where soluble and matrix-associated molecules affect cancer cell growth and gene expression, especially altering cancer cell surface receptor and integrin-mediated cell signaling. We discuss both integrin cell-matrix and gap junctional cell-cell communication between cancer cells and their microenvironments during prostate cancer progression.

Convergent Mechanisms for Recognition of Divergent Cytokines by the Shared Signaling Receptor gp130

Gp130 is a shared cell-surface signaling receptor for at least ten different hematopoietic cytokines, but the basis of its degenerate recognition properties is unknown. We have determined the crystal structure of human leukemia inhibitory factor (LIF) bound to the cytokine binding region (CHR) of gp130 at 2.5 Å resolution. Strikingly, we find that the shared binding site on gp130 has an entirely rigid core, while the LIF binding interface diverges sharply in structure and chemistry from that of other gp130 ligands. Dissection of the LIF-gp130 interface, along with comparative studies of other gp130 cytokines, reveal that gp130 has evolved a “thermodynamic plasticity” that is relatively insensitive to ligand structure, to enable crossreactivity. These observations reveal a novel and alternative mechanism for degenerate recognition from that of structural plasticity.

P21-activated Kinase 1 (PAK1) Interacts with the Grb2 Adapter Protein to Couple to Growth Factor Signaling

A variety of intracellular signaling pathways are linked to cell surface receptor signaling through their recruitment by Src homology 2 (SH2)/SH3-containing adapter molecules. p21-activated kinase 1 (PAK1) is an effector of Rac/Cdc42 GTPases that has been implicated in the regulation of cytoskeletal dynamics, proliferation, and cell survival signaling. In this study, we describe the specific interaction of PAK1 with the Grb2 adapter protein both in vitro and in vivo. We identify the site of this interaction as the second proline-rich SH3 binding domain of PAK1. Stimulation of the epidermal growth factor receptor (EGFR) in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. A cell-permeant TAT-tagged peptide encompassing the second proline-rich SH3 binding domain of PAK1 simultaneously blocked Grb2 and activated EGFR association with PAK1, in vitro and in vivo, indicating that Grb2 mediates the interaction of PAK1 with the activated EGFR. Blockade of this interaction decreased the epidermal growth factor-induced extension of membrane lamellae. Thus Grb2 may serve as an important mechanism for linking downstream PAK signaling to various upstream pathways.

O-fucose modifications of epidermal growth factor-like repeats and thrombospondin type 1 repeats: unusual modifications in unusual places.

Recent discoveries revealing that carbohydrate modifications play critical roles in a wide variety of biological processes have brought wide recognition to the field of glycobiology. Growing attention has focused on the function of unusual O-linked carbohydrate modifications such as O-fucose. O-fucose modifications have been described in several different protein contexts, including epidermal growth factor-like repeats and thrombospondin type 1 repeats. The O-fucose modifications on thrombospondin type 1 repeats have only recently been described, but the site of modification occurs in a region proposed to play a role in cell adhesion. O-fucose modifications on epidermal growth factor-like repeats have been described as important players in several signal transduction systems. For instance, Notch, a cell-surface signaling receptor required for many developmental events, bears multiple O-fucose saccharides on the epidermal growth factor-like repeat of its extracellular domain. The O-fucose moieties serve as a substrate for the beta1,3 N-acetylglucosaminyltransferase activity of Fringe, a known modifier of Notch function. The alteration of O-fucose structures by Fringe influences the ability of Notch ligands to activate the receptor and provides a means to regulate Notch signaling. Thus, O-fucose and Fringe provide a clear example of how carbohydrate modifications can have direct functional consequences on the proteins they modify.

Novel PET and SPECT radioligands for visualization of diseased regulatory pathways in breast carcinoma

Abstract Many new breast-cancer treatment strategies are not based on traditional anti-proliferative effects and thus, standard clinical methods for assessing the efficacy of these agents may not be useful. Consequently, new methodologies are necessary which will enable on the one hand, researchers to explore the full potential of novel anti-cancer strategies and on the other hand, clinicians to monitor more accurately treatment response based on more directive and specific endpoints. Functional imaging by means of positron emission tomography (PET) or single photon emission computerized tomography (SPECT) facilitates the evaluation of tumour physiology, metabolism, and proliferation, providing parameters more closely related to the site of action of these anti-cancer drugs. Alterations in these parameters will do often precede volume changes observed with effective cancer therapy and thus may provide more sensitive and earlier markers of tumour cell death or growth inhibition than gross anatomical changes as currently measured by X-rays, CT or MRI. This paper describes the potential role of recently developed radioligands for both SPECT and PET imaging as molecular imaging markers for the in vivo assessment of the following diseased regulatory pathways in breast cancer: cell surface receptor signalling pathways; oestrogen action; cell death pathways; multidrug resistance and invasion-, metastasis and angiogenesis.

Serum-dependence of LPS-induced neurotoxicity in rat cortical neurons.

Previous studies have shown that the bacterial endotoxin, lipopolysaccharide (LPS), is neurotoxic both in vitro and in vivo. The rate of binding of LPS to a target cell is greatly enhanced by serum in general and by LPS binding protein (LBP) in particular. The purpose of the study described in this paper was to determine if microglia activation and LPS-induced neurotoxicity is serum or LBP dependent. A murine microglial cell line, BV2, was used to assess the serum dependence of nitric oxide production and tumor necrosis factor a release in microglia. Embryonic rat cortical neuron/glia mixed cultures were used to determine the serum dependence of LPS-induced neurotoxicity. Our results from both cell culture systems show that LPS-induced inflammatory responses are serum dependent at lower doses of LPS and progressively become serum independent above 10 ng/ml. Purified human recombinant LBP reconstitutes the lost LPS-induced inflammatory responses in primary and immortalized cell cultures treated with heat-denatured serum and appears to account for the serum dependence. These data suggest that the cell surface signaling receptor for LPS at the low and high concentrations are likely to differ, consistent with the existence of a variety of LPS receptors.

The GoLoco motif: heralding a new tango between G protein signaling and cell division.

The Galpha and Gbetagamma components of heterotrimeric G proteins, typically associated with cell-surface receptor signaling, also partake in the macromolecular interactions that underlie cell polarity and cell division. Proteins with Galpha-binding GoLoco motifs, such as Drosophila melanogaster Pins (for Partner of Inscuteable) and its mammalian counterpart LGN, participate in multi-protein complexes that maintain cellular asymmetry and orderly segregation of chromosomal content and daughter cell bodies. The GoLoco motif was recently identified as a selective Galpha-binding partner: the GoLoco-Galpha interaction can displace Gbetagamma and inhibit guanine nucleotide release from the bound Galpha subunit. Recent x-ray crystallographic studies suggest ways in which GoLoco-motif peptides may modulate heterotrimeric G protein signaling. Such peptides could be exploited to help dissect the signals that underpin cell polarity and cell division processes.

Progress in understanding autoimmune exocrinopathy using the non-obese diabetic mouse: an update.

Sjögren's Syndrome (SS) is a chronic autoimmune disease characterized by histological and functional alterations of salivary and lacrimal glands that result in a severe dryness of the mouth and the eyes. The etiology of SS has remained undefined despite investigators' significant efforts to identify the mechanisms of initiation. Based on histopathology, several animal models are available--such as MRL/lpr, NZW/NZB, NFS/sld, graft vs. host, transgenic mouse expressing viral surface antigen, and the non-obese diabetic (NOD) mouse--for investigation of the etiology of SS. Biochemical and immunological similarities between human SS and autoimmune exocrinopathy (AEC) in the NOD mouse, including the loss of secretory function, establish the NOD mouse as an appropriate model to unravel the underlying pathophysiology of SS. Recently, several NOD congenic partner strains have been developed to investigate the roles of genetic intervals, cytokines, and autoantibodies in the disease pathogenesis. Studies on NOD-scid suggest that the pathogenesis of SS occurs in two phases: an asymptomatic phase, in which epithelial cells of exocrine tissues undergo dedifferentiation accompanied by elevated apoptosis; and a second phase in which autoaggression is mounted against target organ autoantigens, resulting in the activation of T- and B-cells, and the generation of autoantibodies. The presence of autoantibodies on the cell-surface signaling receptor, the muscarinic(3) receptor, in both SS patients and the NOD mice correlates with the hallmark clinical symptom of secretory dysfunction. Additionally, the NOD mouse model provides an important example of how both Th1 and Th2 cytokines, as well as non-immune genetic loci, are involved in the maintenance of and progression to the overt disease state. Ultimately, analysis of these data provides insight into potentially novel therapeutic interventions.

Crystal Structure of Rhodopsin: Implication for Vision and Beyond. Mechanisms of Acti.

INTRODUCTION. The G-protein-coupled receptor (GPCRs) superfamily comprises one of the most diverse groups of signaling molecules involved in numerous physiological processes. Binding of specific ligands to the extracellular or transmembrane domains causes conformational changes that act as a switch to relay the signal to heterotrimeric guanine nucleotide-binding proteins (G-proteins), which in turn evoke subsequent intracellular responses. More than 400 nonsensory GPCRs are also of great interest in medicine, because a vast number of therapeutic agents (>50%) are directed toward these receptors. The largest subfamily, constituting ~90% of all G-protein-coupled receptors (GPCRs), includes rhodopsins, cone pigments, and adrenergic and other receptors. Rhodopsin is involved in photon absorption in retinal rod cells and activation of transducin, a photoreceptor specific G-protein. Understanding of the conformational transformation of inactive GPCR into an activated form capable of interacting with G-protein and signal transduction is a key problem in elucidating molecular steps of cell-surface receptor signaling. The resolution of the X-ray structure of the first GPCR, rhodopsin, provides new insights on the molecular mechanism of activation[1-5].

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein.

Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.

Characterization of the Human B Cell RAG-associated Gene,hBRAG, as a B Cell Receptor Signal-enhancing Glycoprotein Dimer That Associates with Phosphorylated Proteins in Resting B Cells

Affinity-purified polyclonal antibodies against the hBRAG (human B cell RAG-associated gene) protein were generated to characterize hBRAG at the biochemical level. Immunoblotting and immunoprecipitation experiments with these antibody reagents demonstrate that this protein can be expressed in B cells as a membrane-integrated glycoprotein disulfide-linked dimer. However, both glycosylated and unglycosylated isoforms of hBRAG are detectable with these reagents. Additionally, their use in cell surface biotinylation and flow cytometry reveals subcellular hBRAG pools both at cell surface and intracellular locations. Co-immunoprecipitation experiments with hBRAG antisera detected the association of hBRAG with phosphorylated proteins in resting B cells, including the protein tyrosine kinase Hck, which is subsequently dephosphorylated upon B cell receptor (BCR) ligation. Consistent with its cell surface expression and possible link to BCR signaling, experiments in which alpha-hBRAG antibodies were used to generate early activation signals suggest a modest but specific element of tyrosine phosphorylation occurring through a putative hBRAG receptor. Additional experiments also suggest that hBRAG may be involved in positively enhancing BCR ligation-mediated early activation events. Collectively, these results are consistent with a function for hBRAG as a B cell surface signaling receptor molecule. Coupled with the earlier observation that hBRAG expression correlates with early and late B cell-specific RAG expression, we submit that hBRAG may mediate regulatory signals key to B cell development and/or regulation of B cell-specific RAG expression.

Metalloproteinases Are Involved in Lipopolysaccharide– and Tumor Necrosis Factor-α–Mediated Regulation of CXCR1 and CXCR2 Chemokine Receptor Expression

AbstractThe neutrophil-specific G-protein–coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-α–induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF-α was most dramatically inhibited by metalloproteinase inhibitors; 1,10-phenanthroline and EDTA significantly attenuated LPS- and TNF-α–induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-α–stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF-α inhibited IL-8 receptor–mediated calcium mobilization and IL-8–directed neutrophil chemotaxis, both 1,10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8–mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses.

Metalloproteinases Are Involved in Lipopolysaccharide– and Tumor Necrosis Factor-–Mediated Regulation of CXCR1 and CXCR2 Chemokine Receptor Expression

The neutrophil-specific G-protein–coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor- (TNF-) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-–induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF- was most dramatically inhibited by metalloproteinase inhibitors; 1,10-phenanthroline and EDTA significantly attenuated LPS- and TNF-–induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-–stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF- inhibited IL-8 receptor–mediated calcium mobilization and IL-8–directed neutrophil chemotaxis, both 1,10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8–mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses.

The Core Protein of the Chondroitin Sulfate Proteoglycan Phosphacan Is a High-affinity Ligand of Fibroblast Growth Factor-2 and Potentiates Its Mitogenic Activity

Using a radioligand binding assay we have demonstrated that phosphacan, a chondroitin sulfate proteoglycan of nervous tissue that also represents the extracellular domain of a receptor-type protein tyrosine phosphatase, shows saturable, reversible, high-affinity binding (Kd approximately 6 nM) to fibroblast growth factor-2 (FGF-2). Binding was reduced by only approximately 35% following chondroitinase treatment of the proteoglycan, indicating that the interaction is mediated primarily through the core protein rather than the glycosaminoglycan chains. Immunocytochemical studies also showed an overlapping localization of FGF-2 and phosphacan in the developing central nervous system. At concentrations of 10 microg protein/ml, both native phosphacan and the core protein obtained by chondroitinase treatment potentiated the mitogenic effect of FGF-2 (5 ng/ml) on NIH/3T3 cells by 75-90%, which is nearly the same potentiation as that produced by heparin at an equivalent concentration. Although studies on the role of proteoglycans in mediating the binding and mitogenic effects of FGF-2 have previously focused on cell surface heparan sulfate, our results indicate that the core protein of a chondroitin sulfate proteoglycan may also regulate the access of FGF-2 to cell surface signaling receptors in nervous tissue.