RANTES stimulates Ca2+ mobilization and inositol trisphosphate (IP3) formation in cells transfected with G protein-coupled receptor 75.

BACKGROUND AND PURPOSE: RANTES is an inflammatory chemokine with a critical role in T-lymphocyte activation and proliferation. Its effects are mediated through G protein-coupled heptahelical receptors (GPCRs). We show for the first time that RANTES activates the orphan G protein-coupled receptor 75 (GPR75). EXPERIMENTAL APPROACH: To identify a ligand for GPR75 we have used three different and independent methods, namely luciferase assay, bioluminescence assay and IP3 accumulation assay. KEY RESULTS: Treatment of cells expressing GPR75 with subnanomolar concentrations of RANTES led to stimulation of the luciferase activity in a reporter-gene assay, an increase in inositol trisphosphate, and intracellular Ca2+. The latter effect was blocked by the phospholipase-C inhibitor (PLC) U73122 indicating that Gq proteins mediate GPR75 signaling. RANTES enhanced the phosphorylation of AKT and mitogen-activated protein kinase (MAPK) in GPR75-transfected cells and this effect was blocked by the PLC inhibitor U73122 and the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. The hippocampal cell line HT22, which expresses GPR75 endogenously, but not the other known RANTES receptors, was used to study the effects of RANTES and GPR75 on neuronal survival. Treatment of HT22 cells with RANTES significantly reduced the neurotoxicity of amyloid-beta peptides, by activating PLC and PI3K. CONCLUSIONS AND IMPLICATIONS: This demonstrate clearly and undoubtedly the ability of RANTES to act on GPR75. Defects in the RANTES/GPR75-signaling pathway may contribute to neuroinflammatory and neurodegenerative processes as observed in Alzheimer's disease.

On-line adaptive algorithm with glucose prediction capacity for subcutaneous closed loop control of glucose: evaluation under fasting conditions in patients with Type 1 diabetes.

AIMS: To evaluate an algorithm with glucose prediction capacity and continuous adaptation of patient parameters-a model predictive control (MPC) algorithm-to control blood glucose concentration during fasting conditions in patients with Type 1 diabetes. In the subcutaneous (sc) route within a closed loop system. METHODS: Paired experiments were performed in six patients. Over 8 h the MPC algorithm was used to control glucose with s.c. insulin administration and two different glucose monitoring protocols: first, the algorithm was provided with intravenous (i.v.) glucose values for insulin dosage calculation directly (i.v.-s.c. route). Then, in the second experiment, i.v. glucose values were fed to the MPC with a delay of 30 min to simulate s.c. glucose measurements ('s.c.'-s.c. route). In both experiments plasma glucose, insulin dosage, and serum insulin levels were analysed. RESULTS: Glucose concentration was brought from hyper- to normoglycaemia and kept in the physiological range (6-7 mmol/l) with both routes in all subjects. Mean glucose concentration reached the threshold of 7 mmol/l approximately 2 (i.v.-s.c. route) and 3 ('s.c.'-s.c. route) hours after the start of glucose control with the MPC. During the last 2 h of automated glucose control, mean glucose concentration was 6.3 +/- 0.2 mmol/l and 6.6 +/- 0.3 mmol/l for i.v.-s.c. and 's.c.'-s.c. route, respectively. Glucose concentration, insulin doses, and serum insulin levels did not differ significantly between routes (P > 0.05). CONCLUSIONS: The MPC algorithm is suitable for glucose control during fasting within an extracorporeal artificial beta-cell in the subcutaneous route Type 1 diabetic patients.

The neuropeptide head activator induces activation and translocation of the growth-factor-regulated Ca(2+)-permeable channel GRC.

The neuropeptide head activator stimulates cell proliferation of neuronal precursor and neuroendocrine cells. The mitogenic signaling cascade requires Ca(2+) influx for which, as we show in this paper, the growth-factor-regulated Ca(2+)-permeable cation channel, GRC, is responsible. GRC is a member of the transient receptor potential channel family. In uninduced cells only low amounts of GRC are present on the plasma membrane but, upon stimulation with head activator, GRC translocates from an intracellular compartment to the cell surface. Head activator functions as an inducer of GRC translocation in neuronal and neuroendocrine cells, which express GRC endogenously, and also in COS-7 cells after transfection with GRC. Head activator is no direct ligand for GRC, but its action requires the presence of a receptor coupled to a pertussis-toxin inhibitable G-protein. Heterologously expressed GRC becomes activated by head activator, which results in opening of the channel and Ca(2+) influx. SK&F 96365, an inhibitor specific for TRP-like channels, blocks Ca(2+) entry and, consequently, translocation of GRC is prevented. Head activator-induced GRC activation and translocation are also inhibited by wortmannin and KN-93, blockers of the phosphatidylinositol 3-kinase and of the Ca(2+)/calmodulin-dependent kinase, respectively, which implies a role for both kinases in head-activator signaling to GRC.

The genes for the human VPS10 domain-containing receptors are large and contain many small exons.

The two human proteins with a VPS10 domain, SorLA and sortilin, both bind neuropeptides. Searching for other VPS10-domain proteins in the database revealed three new putative human neuropeptide receptors. The new receptors were designated SorCS1, SorCS2 and SorCS3, due to their identical domain composition, which, except for the N-terminal VPS10 domain, differs from that of SorLA and sortilin. Using the databases of the human genome project we elucidated the exon-intron structures of the human VPS10-receptor genes. They contain many short exons, separated by introns, several of which extend over more than 50 kb. The three SorCS genes encompass more than 500 kb of genomic DNA and therefore represent some of the largest known human genes. All these genes map to chromosomal localisations of known genetic diseases, many of them neurological disorders, corresponding to the strong expression of these receptors in the brain. CpG islands are located in the first exon of each of the VPS10-receptor genes and might be involved in developmental or tissue-specific regulation of gene expression.

An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families.

Transient expression of SorCS in developing telencephalic and mesencephalic structures of the mouse.

Here we describe the expression of a third member of the VPS10 domain containing receptor family, SorCS, during mouse embryonal and early postnatal nervous system development. SorCS is expressed in a unique transient and dynamic pattern in regions where cells proliferate, as well as in areas where already differentiated cells reside, including the cerebral cortex, the ventral tegmental area, and the globus pallidus. Transcripts were absent from fiber tracts hinting at a neuronal expression. The only exception was hybridization signals on the developing optic nerve correlating with the appearance of astrocytes migrating into the retina.

Identification of SorCS2, a novel member of the VPS10 domain containing receptor family, prominently expressed in the developing mouse brain.

We report the identification of a fourth member of the VPS10 domain containing receptor family, SorCS2, highly expressed in the developing and mature murine central nervous system. During early central nervous system development its main site of expression is the floor plate. In addition, high transcript levels were detected transiently in a variety of brain regions including the dopaminergic midbrain nuclei and the dorsal thalamus. Outside the nervous system expression is detected in lung and heart and transiently in a variety of mesodermally derived tissues.

Gpr85, a novel member of the G-protein coupled receptor family, prominently expressed in the developing mouse cerebral cortex.

The G-protein coupled receptors (GPCRs) characterized by seven transmembrane domains represent the largest receptor superfamily to date and are implied in diverse cell signaling events, its members being present in a diversity of organs and tissues. Here we report the expression of Gpr85, a novel member of this gene family during mouse embryonal development and in the adult brain. Transcripts of Gpr85 were detected predominantly in tissues of neuroectodermal origin. In the central nervous system Gpr85 was expressed during phases of early neuronal differentiation. Highest transcript levels were observed in the developing cerebral cortex, pointing to a specific function of this gene for differentiation processes in the cerebral cortex. In addition, expression was also detected in derivatives of the neural crest and developing teeth.

Ectodomain shedding, translocation and synthesis of SorLA are stimulated by its ligand head activator.

The single transmembrane receptor SorLA is the mammalian orthologue of the head activator-binding protein, HAB, from hydra. The human neuronal precursor cell line NT2 and the neuroendocrine cell line BON produce head activator (HA) and respond to HA by entry into mitosis and cell proliferation. They express SorLA, and bind HA with nanomolar affinity. HA coupled to Sepharose is able to precipitate SorLA specifically proving that SorLA binds HA. Using antisera directed against extra- and intracellular epitopes we find SorLA as membrane receptor and as soluble protein released from cells into the culture medium. Cell lines differ strongly in processing of SorLA, with NT2 cells expressing SorLA mainly as membrane receptor, whereas release predominates in BON cells. Soluble SorLA lacks the intracellular domain and is shed from the transmembrane protein by a metalloprotease. Release from cells and brain slices is stimulated by HA and by phorbol ester, and it is blocked by a metalloprotease inhibitor and by lowering the temperature to 20 degrees C. Blockade of SorLA shedding and treatment of cells with SorLA antisense oligonucleotides lead to a decrease in the rate of cell proliferation. From this we conclude that SorLA is necessary to mediate the mitogenic effect of endogenous HA. HA enhances the translocation of SorLA from internal membranes to the cell surface and its internalization. In addition, HA stimulates SorLA synthesis hinting at an autocatalytic feedback loop in which the ligand activates production, processing, and translocation of its receptor.

The brain-specific G-protein coupled receptor GPR85 with identical protein sequence in man and mouse maps to human chromosome 7q31.

A new G-protein coupled receptor, GPR85, was identified in man and mouse, which is completely conserved at the amino acid level. Transcripts of gpr85 were found in several human brain regions and, at lower levels, in spleen and placenta, whereas in mouse, gpr85 is confined to the brain. The hgpr85 gene was localized to human chromosome 7q31 by the polymerase chain reaction utilizing a radiation hybrid panel.

Alternative splicing of murine SorCS leads to two forms of the receptor that differ completely in their cytoplasmic tails.

We report the identification of a splice variant of SorCS, a member of the family of VPS10 domain containing receptors. These type I transmembrane proteins share the presence of internalization signals in their cytoplasmic tail as one common characteristic. We show that an alternatively spliced transcript of SorCS is generated by differential processing of a composite internal/terminal exon. This splice variant encodes a protein with an N-terminal VPS10 domain followed by a leucine-rich module and a transmembrane domain identical with the already described SorCS protein, but a divergent cytoplasmic tail. In contrast to the known intracellular regions of the related receptors, this splice variant contains no internalization or sorting signals.

Alternative splicing in the regulatory region of the human phosphatases CDC25A and CDC25C.

CDC25 phosphatases play key roles in cell proliferation by activating cell cycle-specific cyclin-dependent kinases (CDKs). We identified four new splice variants in the amino-terminal regulatory region of human cdc25C and one in cdc25A. All variants except one retain an intact catalytic domain. Alternative splicing results in loss of phosphorylation sites for kinases like CDK and the calcium/calmodulin-dependent kinase II (CaMKII), which influence CDC25 activity and compartmental localization. In NT2 teratocarcinoma cells, induced for nerve cell differentiation, the smaller sized variant of cdc25C was upregulated. At the protein level both phosphorylation state and isoform distribution differed between cell lines and cell cycle phases.

Identification and characterization of SorCS, a third member of a novel receptor family.

A novel receptor, SorCS, was isolated from murine brain. It shows homology to the mosaic receptor SorLA and the neurotensin receptor sortilin based on a common VPS10 domain which is the hallmark of this new receptor family. In the N-terminus of SorCS two putative cleavage sites for the convertase furin mark the beginning of the VPS10 domain, followed by a module of imperfect leucine-rich repeats and a transmembrane domain. The short intracellular C-terminus contains consensus signals for rapid internalization. The identified putative binding motifs for SH2 and SH3 domains are unique in the family of VPS10 domain receptors. SorCS is predominantly expressed in brain, but also in heart, liver, and kidney. SorCS transcripts detected by in situ hybridization in the murine central nervous system point to a neuronal expression.

A head-activator binding protein is present in hydra in a soluble and a membrane-anchored form.

The neuropeptide head activator plays an important role for proliferation and determination of stem cells in hydra. By affinity chromatography a 200 kDa head-activator binding protein, HAB, was isolated from the multiheaded mutant of Chlorohydra viridissima. Partial amino acid sequences were used to clone the HAB cDNA which coded for a receptor with a unique alignment of extracellular modules, a transmembrane domain, and a short carboxy-terminal cytoplasmic tail. A mammalian HAB homologue with identical alignment of these modules is expressed early in brain development. Specific antibodies revealed the presence of HAB in hydra as a transmembrane receptor, but also as secreted protein, both capable of binding head activator. Secretion of HAB during regeneration and expression in regions of high determination potential hint at a role for HAB in regulating the concentration and range of action of head activator.

Identification of a novel seven-transmembrane receptor with homology to glycoprotein receptors and its expression in the adult and developing mouse.

Using a PCR-based cloning strategy we have isolated a cDNA from mouse brain and named it fex, because it codes for a novel putative G protein-coupled receptor expressed in follicles. The deduced amino acid sequence shows a higher degree of homology to the family of glycoprotein receptors, namely those for FSH, LH, and TSH, than to other G protein-coupled receptors. With 18 leucine-rich repeats FEX exhibits features in its N-terminal portion characterizing it as unique within the glycoprotein receptor family. In the adult mouse fex expression was detected in the male and female gonads, the adrenal medulla, and the olfactory bulb of the brain. During embryonic development fex transcripts were detected transiently in various tissues, particularly in selected regions of the central nervous system, the developing face, the intervertebral discs anlagen, and the limb buds. Because fex was expressed during periods of active morphogenesis, it may be an important receptor for signals controlling growth and differentiation of specific embryonic tissues.

Involvement of a Gardos-type potassium channel in head activator-induced mitosis of BON cells.

The human neuroendocrine cell line BON was used to study second messengers involved in signal transduction for entry into mitosis. BON cells produce the neuropeptide head activator (HA) and use it as autocrine growth factor. HA stimulates BON cell proliferation by triggering entry into mitosis. HA-induced mitosis is mediated by an inhibitory G protein, the action of which is blocked by pertussis toxin. HA signaling requires inhibition of the cAMP pathway, calcium influx, and hyperpolarization of cells. The latter is a very important and sensitive step involving a calcium-activated potassium channel. Cell cycle progression and proliferation of BON cells are most efficiently inhibited with specific inhibitors of this potassium channel. Pharmacology and RNA analysis suggest identity with the recently cloned Gardos-type potassium channel.

Unique expression pattern of a novel mosaic receptor in the developing cerebral cortex.

Recently, a new type of transmembrane protein with a unique combination of protein domains was characterized from human, rabbit and chicken. This protein exhibits features of the low-density lipoprotein receptor family and shows homology to the receptor of the neuropeptide head activator isolated from hydra. To study the temporal and spatial pattern of expression of this unusual new receptor we have isolated a murine homolog and, in accordance with its human counterpart, named it mSorLA. Northern blot analysis revealed the highest abundance of mSorLA transcripts in the adult brain, lower levels in a variety of other organs and expression during embryogenesis. In situ hybridization showed predominant localization in neurons of the cortex, the hippocampus and the cerebellum. During embryonic development mSorLA displayed a unique pattern of expression in the cerebral cortex, where a subpopulation of neurons was labeled before final differentiation. Transcripts of mSorLA were also detected outside the central nervous system in regions active in morphogenesis.

Expression and characterization of the "laminin binding protein" in hydra.

Recently, a cDNA was isolated from hydra with extensive homology to a mammalian and invertebrate gene which codes for a protein called laminin binding protein (LBP). In this paper we describe the protein expression of the hydra LBP in Escherichia coli. On SDS gels the recombinant hydra LBP displayed an apparent molecular mass of 43 kDa, although the calculated mass, including six additional histidines, is 33.7 kDa. Polyclonal antibodies were produced against the hydra recombinant LBP. The antiserum reacted with a 42-kDa and a 43-kDa protein from Hydra vulgaris and from a multiheaded mutant of Chlorohydra viridissima, respectively. In hydra, LBP RNA and protein were highly expressed in cells with short cell cycles, such as all cells of the interstitial cell lineage, less in slowly cycling epithelial cells, and at very reduced levels or not at all in differentiated cells. Higher expression in the multiheaded mutant of C. viridissima than in H. vulgaris, the cells of which differ in doubling time, hint at a function in cell proliferation. This is supported by the finding that in vitro hydra LBP is a substrate for the cell-cycle-specific kinase CDC2.

Isolation of a marker for head-specific cell differentiation in hydra.

A cDNA was isolated from Hydra vulgaris coding for a 360 amino acid long polypeptide with no significant similarity to any known protein. In situ hybridization showed that the corresponding transcript is expressed exclusively in endodermal cells of the hypostome, hence its name hyp 1. Hyp 1 therefore represents a highly specific marker for the head region of hydra. In accordance with this, we found that hyp 1 expression was strong in tissues regenerating a head. Hyp 1 reappeared early during head regeneration, namely 6-8 h after initiation of regeneration by cutting. These findings imply that hyp 1 is an excellent marker for monitoring early events in head-specific differentiation processes in hydra.