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.

Functional analysis of chemically synthesized derivatives of the human CC chemokine CCL15/HCC-2, a high affinity CCR1 ligand.

The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N-terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino-terminal truncated and point-mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy-terminal alpha-helix were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (delta24-CCL15) converts the slightly active 92-residue delta0-CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell-based assays. The biological activity decreases from delta24-CCL15 to delta29-CCL15, and re-increases from delta29-CCL15 to delta30-CCL15. Thus, an exocyclic N-terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for delta24-CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy-terminal alpha-helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor-ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.

Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties.

The interferon (IFN)-inducible chemokines, specifically, IFN-gamma-inducible protein-10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), share a unique CXC chemokine receptor (CXCR3). Recently, the highly specific membrane-bound protease and lymphocyte surface marker CD26/dipeptidyl peptidase IV (DPP IV) was found to be responsible for posttranslational processing of chemokines. Removal of NH(2)-terminal dipeptides by CD26/DPP IV alters chemokine receptor binding and signaling, and hence inflammatory and anti-human immunodeficiency virus (HIV) activities. CD26/DPP IV and CXCR3 are both markers for Th1 lymphocytes and, moreover, CD26/DPP IV is present in a soluble, active form in human plasma. This study reports that at physiologic enzyme concentrations CD26/DPP IV cleaved 50% of I-TAC within 2 minutes, whereas for IP-10 and Mig the kinetics were 3- and 10-fold slower, respectively. Processing of IP-10 and I-TAC by CD26/DPP IV resulted in reduced CXCR3-binding properties, loss of calcium-signaling capacity through CXCR3, and more than 10-fold reduced chemotactic potency. Moreover, IP-10 and I-TAC cleaved by CD26/DPP IV acted as chemotaxis antagonists and CD26/DPP IV-truncated IP-10 and Mig retained their ability to inhibit the angiogenic activity of interleukin-8 in the rabbit cornea micropocket model. These data demonstrate a negative feedback regulation by CD26/DPP IV in CXCR3-mediated chemotaxis without affecting the angiostatic potential of the CXCR3 ligands IP-10 and Mig.

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.

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.

Identification of a novel human ADP receptor coupled to G(i).

We have cloned and expressed a novel human G-protein-coupled receptor closely related to the human P2Y(12) receptor. It corresponds to the orphan receptor called GPR86. GPR86 proved to be a G(i)-coupled receptor displaying a high affinity for ADP, similar to the P2Y(12) receptor and can therefore be tentatively called P2Y(13). In 1321N1 cells, the P2Y(13) receptor coupled to the phosphoinositide pathway only when coexpressed with Galpha(16). Inositol trisphosphate formation was stimulated equipotently by nanomolar concentrations of ADP and 2MeSADP, whereas 2MeSATP and ATP were inactive. In CHO-K1 cells expressing the P2Y(13) receptor, ADP and 2MeSADP had a biphasic effect on the forskolin-stimulated accumulation of cAMP: inhibition at nanomolar concentrations and potentiation at micromolar levels. In the same cells, ADP and 2MeSADP also stimulated the phosphorylation of Erk1 and Erk2, in a pertussis toxin-sensitive way. The tissue distribution of P2Y(13) was investigated by reverse transcriptase-polymerase chain reaction, and the predominant signals were obtained in spleen and brain. Although these can be discriminated by tissue distribution and some pharmacological features, the P2Y(12) and P2Y(13) receptors form a subgroup of related P2Y subtypes that is structurally different from the other P2Y subtypes but share coupling to G(i) and a high affinity for ADP.

The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54.

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.

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.

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.

The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C.

Ca(2+) elevations in Chinese hamster ovary cells stably expressing OX(1) receptors were measured using fluorescent Ca(2+) indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca(2+) elevations with an EC(50) around 1 nm. When the extracellular [Ca(2+)] was reduced to a submicromolar concentration, the EC(50) was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca(2+) was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca(2+). The shift in the potency was not caused by depletion of intracellular Ca(2+) but by a requirement of extracellular Ca(2+) for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the "Mn(2+)-quench technique" indicated a direct activation of a cation influx pathway by OX(1) receptor independent of Ca(2+) release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca(2+) entry, abolished the Ca(2+) response to low concentrations of orexin-A. The results thus suggest that OX(1) receptor activation leads to two responses, (i) a Ca(2+) influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.

CD26/dipeptidyl-peptidase IV down-regulates the eosinophil chemotactic potency, but not the anti-HIV activity of human eotaxin by affecting its interaction with CC chemokine receptor 3.

Chemokines attract and activate distinct sets of leukocytes. The CC chemokine eotaxin has been characterized as an important mediator in allergic reactions because it selectively attracts eosinophils, Th2 lymphocytes, and basophils. Human eotaxin has a penultimate proline, indicating that it might be a substrate for dipeptidyl-peptidase IV (CD26/DPP IV). In this study we demonstrate that eotaxin is efficiently cleaved by CD26/DPP IV and that the NH2-terminal truncation affects its biological activity. CD26/DPP IV-truncated eotaxin(3-74) showed reduced chemotactic activity for eosinophils and impaired binding and signaling properties through the CC chemokine receptor 3. Moreover, eotaxin(3-74) desensitized calcium signaling and inhibited chemotaxis toward intact eotaxin. In addition, HIV-2 infection of CC chemokine receptor 3-transfected cells was inhibited to a similar extent by eotaxin and eotaxin(3-74). Thus, CD26/DPP IV differently regulates the chemotactic and antiviral potencies of eotaxin by the removal of two NH2-terminal residues. This physiological processing may be an important down-regulatory mechanism, limiting eotaxin-mediated inflammatory responses.

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.

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.

Phosphoserine phosphatase deficiency in a patient with Williams syndrome.

Decreased serine levels were found in plasma and cerebrospinal fluid (CSF) of a boy with pre- and postnatal growth retardation, moderate psychomotor retardation, and facial dysmorphism suggestive of Williams syndrome. Fluorescence in situ hybridisation with an elastin gene probe indicated the presence of a submicroscopic 7q11.23 deletion, confirming this diagnosis. Further investigation showed that the phosphoserine phosphatase (EC 3.1.3.3.) activity in lymphoblasts and fibroblasts amounted to about 25% of normal values. Oral serine normalised the plasma and CSF levels of this amino acid and seemed to have some clinical effect. These data suggest that the elastin gene and the phosphoserine phosphatase gene might be closely linked. This seems to be the first report of phosphoserine phosphatase deficiency.

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.

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.

3-Phosphoglycerate dehydrogenase deficiency: an inborn error of serine biosynthesis.

Serine concentrations were markedly decreased in the cerebrospinal fluid of two brothers with congenital microcephaly, profound psychomotor retardation, hypertonia, epilepsy, growth retardation, and hypogonadism. The youngest boy also had congenital bilateral cataract. Magnetic resonance imaging of the brain showed evidence of dysmyelination. Plasma serine as well as plasma and cerebrospinal fluid glycine concentrations were also decreased but to a lesser extent. Treatment with oral serine in the youngest patient significantly increased cerebrospinal fluid serine and abolished the convulsions. In fibroblasts of both patients, a decreased activity was demonstrated of 3-phosphoglycerate dehydrogenase, the first step of serine biosynthesis (22% and 13% of the mean control value). This is an unusual disorder as the great majority of aminoacidopathies are catabolic defects. It is a severe but potentially treatable inborn error of metabolism that has not been previously reported in man.

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.