Natural proteolytic processing of hemofiltrate CC chemokine 1 generates a potent CC chemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties.

Hemofiltrate CC chemokine (HCC)-1 is a recently described human chemokine that is constitutively expressed in numerous tissues and is present at high concentrations in normal plasma. Using a cell line expressing CC chemokine receptor (CCR)5 as a bioassay, we isolated from human hemofiltrate an HCC-1 variant lacking the first eight amino acids. HCC-1[9-74] was a potent agonist of CCR1, CCR3, and CCR5 and promoted calcium flux and chemotaxis of T lymphoblasts, monocytes, and eosinophils. It also blocked entry of HIV-1 strains using CCR5 as coreceptor. Limited tryptic digestion of HCC-1 generated the active variant. Conditioned media from several tumor cell lines activated HCC-1 with a high efficiency, and this activity could be inhibited by serine protease inhibitors. Our results indicate that HCC-1 represents a nonfunctional precursor that can be rapidly converted to the active chemokine by proteolytic processing. This process represents an additional mechanism by which tumor cells might generate chemoattractant molecules and recruit inflammatory cells. It might also affect HIV-1 replication in infected individuals and play an important role in AIDS pathogenesis.

Search for a third susceptibility gene for maturity-onset diabetes of the young. Studies with eleven candidate genes.

Maturity-onset diabetes of the young (MODY) is a model for genetic studies of non-insulin-dependent diabetes mellitus. We have identified 15 MODY families in which diabetes is not the result of mutations in the glucokinase gene. This cohort of families will be useful for identifying other diabetes-susceptibility genes. Nine other candidate genes potentially implicated in insulin secretion or insulin action have been tested for linkage with MODY in these families, including glucokinase regulatory protein, hexokinase II, insulin receptor substrate 1, fatty acid-binding protein 2, glucagon-like peptide-1 receptor, apolipoprotein C-II, glycogen synthase, adenosine deaminase (a marker for the MODY gene on chromosome 20), and phosphoenolpyruvate carboxykinase. None of these loci showed evidence for linkage with MODY, implying that mutations in these genes do not make a major genetic contribution to the development of MODY. In addition to these linkage analyses, one or two affected subjects from each family were screened for the presence of the A to G mutation at nucleotide 3,243 of the mitochondrial tRNA(Leu(UUR)) gene. This mutation was not found in any of these subjects. Finally, we report the localization of the gene encoding the regulatory protein of glucokinase to chromosome 2, band p22.3 and the identification of a restriction fragment length polymorphism at this locus.

Heterologous expression of an active rat regulatory protein of glucokinase.

The cDNA presumed to encode the rat liver regulatory protein of glucokinase has been expressed in Escherichia coli and a partially soluble protein has been obtained. This recombinant protein was partially purified and found to have the same apparent molecular mass as the regulatory protein purified from rat liver. Like the latter, it inhibited rat liver glucokinase competitively with respect to glucose and its effect was sensitive to fructose 6-phosphate and fructose 1-phosphate.

Cloning and sequencing of rat liver cDNAs encoding the regulatory protein of glucokinase.

cDNAs encoding the rat liver regulatory protein of glucokinase were cloned and sequenced. The deduced protein contains 568 amino acids for a molecular mass of 62,867 Da. Northern blot analysis showed the presence of a major RNA species of 2.35 kb in rat liver. No signal was observed with muscle, brain, heart, testis, intestine or spleen RNA. Recombinant regulatory protein expressed in Escherichia coli was insoluble and inactive, and was presumably contained in inclusion bodies. Western blot analysis showed that the recombinant protein was recognized by antibodies raised against regulatory protein purified from rat liver.

Agonist and antagonist activities on human NPFF(2) receptors of the NPY ligands GR231118 and BIBP3226.

Neuropeptide FF (NPFF) is a part of a neurotransmitter system acting as a modulator of endogenous opioid functions. At this time, no non-peptide or peptide NPFF-antagonists have been discovered. Here, we demonstrate that Neuropeptide Y (NPY) ligands, in fact possess significant ability to interact with the human NPFF(2) receptors. NPY Y(1) antagonist BIBP3226 and mixed Y(1) antagonist/Y(4) agonist GR231118 are able to displace with low affinity, 50 -- 100 nM, the specific binding on NPFF receptors expressed in CHO cells as well as in rat dorsal spinal cord, an affinity however superior to those determined against Y(2), Y(4) or Y(5) receptors. Furthermore, BIBP3226 which is unable to inhibit the forskolin-stimulated cyclic AMP production mediated by NPFF(2) receptors, antagonizes the effect of NPFF, revealing the first antagonist of NPFF receptors. These properties of NPY ligands on Neuropeptide FF receptors must be considered when evaluating pharmacological activities of these drugs.

Functional characterization of a human receptor for neuropeptide FF and related peptides.

1. Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. 2. Binding experiments were performed with a new radioiodinated probe, [(125)I]-EYF, derived from the EFW-NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [(125)I]-EYF with a K(d) of 0.06 nM. Various NPFF analogues and related peptides inhibited [(125)I]-EYF specific binding with the following rank order (K(i)): human NPAF (0.22 nM), SQA-NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW-NPSF (0.32 nM), QFW-NPSF (0.35 nM), 3D (1.12 nM), Met-enk-RF-NH(2) (3.25 nM), FMRF-NH(2) (10.5 nM) and NPSF (12.1 nM). 3. The stimulatory activity of the same set of peptides was measured by a functional assay based on the co-expression of NPFFR, G(alpha 16) and apoaequorin. The rank order of potency was consistent with the results of the binding assay. 4. Membranes from NPFFR expressing CHO cells bound GTP gamma[(35)S] in the presence of SQA-NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of G(i) family members. 5. SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre-treatment. 6. RT -- PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

Distribution of a glucocorticoid-induced orphan receptor (JP05) mRNA in the central nervous system of the mouse.

JP05 (originally referred to as glucocorticoid-induced receptor gene or cDNA clone 4.2) designates a gene originally isolated from murine thymoma WEHI-7TG cells after being treated with glucocorticoids and forskolin. This gene is also induced by dexamethasone (a potent glucocorticoid receptor agonist) in isolated normal murine thymocytes. The predicted amino acid sequence was found to share significant similarity to the family of G-protein-coupled receptors, in particular to the tachykinin receptors NK-1, NK-2 and NK-3, with which it has an overall identity of 32%, 31% and 33%, respectively. The results of the present in situ hybridization analysis reveal that JP05 mRNA containing cells are extensively distributed throughout the rostrocaudal extension of the brain and spinal cord. However, the vast majority of the areas with high to moderate levels of JP05 mRNA were localized in the forebrain, primarily within limbic system structures, the dorsal and ventral striatum and in some hypothalamic nuclei. These results are discussed in relation to the central nervous system distribution of glucocorticoid receptor-containing cells and to the tachykinin system.

Distribution of an orphan G-protein coupled receptor (JP05) mRNA in the human brain.

JP05, also called GPR72 or GIR, is an orphan G-protein-coupled receptor, GPCR, showing significant structural similarity to the tachykinin receptors. The anatomical distribution of JP05 mRNA was first described in the central nervous system of the mouse, and recently the human JP05 orphan receptor gene has been cloned. In the present study the distribution of JP05 mRNA was examined in the human forebrain using in situ hybridization analysis. The results revealed a wide but discrete distribution of the transcript with strongly JP05 mRNA expressing cells, presumably neurons, present in the cerebral cortex (layer II), hippocampus (pyramidal CA3 neurons and granule cells), amygdala (basal and periamygdaloid cortical nuclei), in the endopiriform nucleus, diagonal band of Broca, thalamus (nucleus reuniens, parafascicular nucleus) and hypothalamus (posterior, dorsal, and around the medial mammillary). Weaker signals were detected in the deeper cortical layers and throughout the striatum. A few positive cells were evident in the raphe but not in the substantia nigra or pontine nuclei. The results indicate significant similarities between human and mouse brain with regard to JP05 mRNA expression. The distribution patterns of JP05 mRNA in the human brain suggest involvement in control of emotions and of neuroendocrine, cognitive and motor functions.

The regulatory protein of liver glucokinase.

Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by fructose 1-phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of fructose 6-phosphate, the regulatory protein binds to, and inhibits, liver glucokinase. Fructose 1-phosphate antagonizes this inhibition by causing dissociation of the glucokinase-regulatory protein complex. Both phosphate esters act by binding to the regulatory protein, and by presumably causing changes in its conformation. The regulatory protein behaves as a fully competitive inhibitor. It inhibits liver glucokinase from various species, and rat islet glucokinase, but has no effect on hexokinases from mammalian tissues or from yeast, or on glucokinase from microorganisms. Kinetic studies indicate that the regulatory protein binds to glucokinase at a site distinct from the catalytic site. Several phosphate esters, mainly polyol-phosphates, were found to mimick the effect of fructose 6-phosphate. The most potent is sorbitol 6-phosphate, suggesting that fructose 6-phosphate is recognized by the regulatory protein in its open-chain configuration. Other phosphate esters and Pi have a fructose 1-phosphate-like effect. The stimulatory effect of fructose on glucose phosphorylation is observed not only in isolated hepatocytes but also in the livers of anesthetized rats. This suggests that fructose could be a nutritional signal causing an increase in the hepatic glucose uptake.

Deorphanization of G-protein-coupled receptors.

G-protein-coupled receptors constitute one of the major families of drug targets. Orphan receptors, for which the ligands and function are still unknown, are an attractive set of future targets for presently unmet medical needs. Screening strategies have been developed over the years in order to identify the natural ligands of these receptors. Natural or chimeric G-proteins that can redirect the natural coupling of receptors toward intracellular calcium release are frequently used. Potential problems include poor expression or trafficking to the cell surface, constitutive activity of the receptors, or the presence of endogenous receptors in the cell types used for functional expression, leading to nonspecific responses. Many orphan receptors characterized over the last 10 years have been associated with previously known bioactive molecules. However, new and unpredicted biological mediators have also been purified from complex biological sources. A few old and recent examples, including nociceptin, chemerin, and the F2L peptide are illustrated. Future challenges for the functional characterization of the remaining orphan receptors include the potential requirement of specific proteins necessary for quality control, trafficking or coupling of specific receptors, the possible formation of obligate heterodimers, and the possibility that some constitutively active receptors may lack ligands or respond only to inverse agonists. Adapted expression and screening strategies will be needed to deal with these issues.

Effect of sulphur mustard on the expression of urokinase in cultured 3T3 fibroblasts.

The expression of plasminogen activator (PA), a serine proteinase involved in the degradation of extracellular matrix proteins, has been investigated in 3T3 fibroblasts after in vitro exposure to sulphur mustard (SM). Expression of the cell-associated enzyme has been assessed with a synthetic substrate assay and at the mRNA level. Twenty-four hours after 100 microM SM, cell viability (monitored by MTT assay) was not significantly affected, but protein synthesis (tritiated leucine incorporation) was reduced to < 20% of the control value. PA activity was significantly increased compared to control cells with a 20-fold increase after 24 h. This up-regulation was independent of the cell density, occurred maximally between days 1 and 4 and persisted for at least 6 days after exposure. Lower concentrations of SM (< or = 10 microM) did not significantly affect PA activity. Northern blotting experiments revealed an increased expression of urokinase (u-PA) transcripts in cells treated with 100 microM SM, with a peak at 10 h after exposure. Conditioned culture medium from cell cultures treated with 100 microM SM did not affect the expression of PA activity in naive or SM-treated cultures. Thiodiglycol (100 microM), the main metabolite of SM, did not influence the expression of PA in the same system. Different compounds were tested for modulation of the PA upregulation after SM exposure. Nicotinamide (5 mM), vitamin D3 (10(-10)M), extracellular calcium (2 mM) or EGTA (5 mM) had no effect. Ryanodine (10 microM) amplified the PA up-regulation by a factor of 2 and vanadate (500 microM) reduced it by approximately 50%. Dexamethasone (1 microM) added directly after SM treatment almost completely prevented the induction of PA at both the protein and mRNA levels. Overall these results demonstrate an up-regulation of urokinase in 3T3 fibroblasts after treatment with SM, which is possibly mediated by intracellular calcium mobilization. Further studies are needed to identify the significance of this proteolytic response in the pathogenesis of blistering and/or DNA repair mechanisms.

Binding of sorbitol 6-phosphate and of fructose 1-phosphate to the regulatory protein of liver glucokinase.

Using a binding assay in which the ligand-protein complex is separated from free ligand by precipitation with poly(ethylene glycol) 6000, we found that the regulatory protein of rat liver glucokinase bound close to 1 mol of radiolabelled sorbitol 6-phosphate, a negative effector, or of fructose 1-phosphate, a positive effector, per mol of regulatory protein. Scatchard plots were linear, the dissociation constant being 0.3 microM for both phosphate esters. Sorbitol 6-phosphate and fructose 1-phosphate competed with each other for the binding. Competition was also observed with psicose 1-phosphate, ribitol 5-phosphate, arabitol 5-phosphate and 3-phosphoglycerate, all of which are known to affect the inhibition exerted by the regulatory protein. At a concentration of 10%, poly(ethylene glycol) 6000 decreased the concentration of regulatory protein causing 50% inhibition to a larger extent in the absence (12-fold) than in the presence (3-fold) of a saturating concentration of fructose 6-phosphate, another negative effector. Furthermore, it increased by about 3-fold the apparent affinity for inhibitory phosphate esters, indicating that it induced conformational changes of the regulatory protein.

Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation.

In the absence of fructose 6-phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15-fold) lower potency than in the presence of a saturating concentration of fructose 6-phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6-phosphate. In the presence of regulatory protein, sorbitol 6-phosphate, a potent analog of fructose 6-phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between the rates in the absence and in the presence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] at various concentrations of regulatory protein were linear, and demonstrated that the apparent affinity for sorbitol 6-phosphate increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between 1/v in the presence and in the absence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] were also linear and crossed the axis at a value independent of the concentration of regulatory protein. Fructose 1-phosphate released the inhibition exerted by the regulatory protein in a hyperbolic fashion. The concentration of this effector required for a half-maximal effect increased linearly with the concentrations of sorbitol 6-phosphate and of regulatory protein. These results are consistent with a model in which the regulatory protein exists under two conformations, one form which binds inhibitors and glucokinase, and the other which binds activators, although not glucokinase. Sorbitol 6-phosphate, 2-deoxysorbitol 6-phosphate and mannitol 1-phosphate, all analogs of the open-chain configuration of fructose 6-phosphate, inhibited glucokinase in the presence of regulatory protein at lower concentrations than fructose 6-phosphate, whereas fixed analogs of the furanose form of fructose 6-phosphate were inactive or behaved as activators. This indicated that fructose 6-phosphate in its open-chain configuration is recognized by the regulatory protein. A series of compounds exerted an activating effect. These included, in order of decreasing potency: fructose 1-phosphate, psicose 1-phosphate, ribitol 5-phosphate, analogs of fructose 1-phosphate and of ribitol 5-phosphate and, at much higher concentrations, inorganic phosphate.

Short-term control of glucokinase activity: role of a regulatory protein.

Glucokinase is one of the four hexokinases present in mammalian tissues. It is expressed in two cell types that have to respond to changes in the blood glucose concentration, the liver parenchymal cell and the beta-cells of pancreatic islets. The former are responsible for the metabolism and storage of an important part of the ingested glucose, whereas the latter secrete insulin in response to an increase in the blood glucose level. One major characteristic of glucokinase is that it has a relatively low affinity for glucose and displays positive cooperativity for this substrate, despite the fact that it is a monomeric enzyme. Furthermore, unlike other hexokinases, it is not inhibited by micromolar (physiological) concentrations of glucose 6-phosphate but by a regulatory protein that transduces the effect of fructose 6-phosphate and of fructose 1-phosphate. The purpose of this review is to describe these aspects of the regulation of glucokinase.

Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes.

Fructose 1-phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1-phosphate and fructose. The concentration of fructose 1-phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05-1 mM) caused a concentration-dependent and transient increase in the fructose 1-phosphate content. Glucagon (1 microM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1-phosphate in cells incubated with fructose, whereas the addition of 0.1 microM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1-phosphate concentration. Sorbitol had a similar effect to fructose in causing the formation of fructose 1-phosphate. D-Glyceraldehyde was much less potent in this respect than the ketose and its effect disappeared earlier. The effect of D-glyceraldehyde was reinforced by an increase in the glucose concentration and decreased by glucagon. Both fructose and D-glyceraldehyde stimulated the phosphorylation of glucose as estimated by the release of 3H2O from [2-3H]glucose, but the triose was less potent in this respect than fructose and its effect disappeared earlier. Glucagon and ethanol antagonised the effect of low concentrations of fructose or D-glyceraldehyde on the detritiation of glucose. These results support the proposal that fructose 1-phosphate mediates the effects of fructose, D-glyceraldehyde and sorbitol by relieving the inhibition exerted on glucokinase by a regulatory protein.

Urokinase plasminogen activator and plasmin efficiently convert hemofiltrate CC chemokine 1 into its active.

We have previously isolated from human hemofiltrate an N-terminally truncated form of the hemofiltrate CC chemokine 1 (HCC-1), and characterized HCC-1[9-74] as a strong agonist of CCR1, CCR5, and to a lower extent CCR3. In this study, we show that conditioned media from human tumor cell lines PC-3 and 143B contain proteolytic activities that convert HCC-1 into the [9-74] form. This activity was fully inhibited by inhibitors of urokinase-type plasminogen activator (uPA), including PA inhibitor-1, an anti-uPA mAb, and amiloride. Pure preparations of uPA processed HCC-1 with high efficiency, without further degrading HCC-1[9-74]. Plasmin could also generate HCC-1[9-74], but degraded the active product as well. The kinetics of HCC-1 cleavage by uPA and plasmin (Michaelis constant, K(m), of 0.76 +/- 0.4 microM for uPA, and 0.096 +/- 0.05 microM for plasmin; catalytic rate constant, k(cat): 3.36 +/- 0.96 s(-1) for uPA and 6 +/- 3.6 s(-1) for plasmin) are fully compatible with a role in vivo. The activation of an abundant inactive precursor into a broad-spectrum chemokine by uPA and plasmin directly links the production of uPA by numerous tumors and their ability to recruit mononuclear leukocytes, without the need for the transcriptional activation of chemokine genes.

Gene expression of glucokinase regulatory protein in regenerating rat liver.

The activity and messenger RNA (mRNA) levels of glucokinase, and the concentration and mRNA levels of its regulatory protein, were analyzed during liver regeneration. The activity of glucokinase and the concentration of its regulatory protein decreased to 30% and 50%, respectively, after liver resection, remaining low after 1 week. No significant variations in the level of these proteins were found in sham-operated animals. The regulatory protein/glucokinase molar ratio increased during the replicative phase, to a maximum at 48 hours. The mRNA levels of glucokinase and of its regulatory protein decreased rapidly after partial hepatectomy to minimum values at 6 hours (15%) and at 12 hours (4%), respectively, returning to normal values at 24 hours and 168 hours, respectively. Sham-operated animals showed a similar decrease in mRNA levels during the prereplicative phase of liver regeneration, suggesting that the initial effects observed in the gene expression of these proteins were due to surgical stress. During the replicative phase, a specific inhibition of the regulatory protein's gene expression was observed in the regenerating liver. A decrease in the content of regulatory protein and the glucokinase activity, and an increase in the molar ratio of these two proteins correlate with the observed decrease in glycolytic flux, providing further evidence that the phosphorylation of glucose is a control point in the glycolytic/gluconeogenic flux during liver regeneration.

Cloning and expression of a Xenopus liver cDNA encoding a fructose-phosphate-insensitive regulatory protein of glucokinase.

Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein found in mammals.

The second extracellular loop of CCR5 is the major determinant of ligand specificity.

The chemokine receptor CCR5 binds macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation, normal T-cell expressed and secreted (RANTES), and constitutes the major co-receptor allowing infection of CD4(+) T lymphocytes, macrophages, and microglial cells by macrophage-tropic strains of human and simian immunodeficiency virus. CCR5 is most closely related to CCR2b, another chemokine receptor that responds to monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, and MCP-4. We have investigated by mutagenesis the regions of CCR5 and CCR2b involved in the specificity of binding and functional response to their respective ligands. We demonstrate that the key region of CCR5 involved in its specific interaction with MIP-1alpha, MIP-1beta, and RANTES, and its subsequent activation, lies within the second extracellular loop (and possibly the adjacent transmembrane segments). Conversely, the NH2-terminal domain of CCR2b is responsible for the high affinity binding of MCP-1, but is not sufficient to confer activation of the intracellular cascades. Extracellular loops of the receptor, among which the second loop plays a prominent role, are necessary to achieve efficient signaling of the receptor. These data complement our previous mapping of CCR5 domains functionally involved in the fusion process with the human immunodeficiency virus envelope, and will help in the development of agents able to interfere with the early steps of viral infection.