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The neuropeptide head activator stimulates cell proliferation of neuronal precursor and neuroendocrine cells. The mitogenic signaling cascade requires Ca2+ influx for which, as we show in this paper, the growth-factor-regulated Ca2+-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 Ca2+ influx. SK&F 96365, an inhibitor specific for TRP-like channels, blocks Ca2+ 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 Ca2+/calmodulin-dependent kinase, respectively, which implies a role for both kinases in head-activator signaling to GRC.

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.

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°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.

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.

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.

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.

A human cDNA fragment bearing the complete coding region for the β 2-adrenergic receptor was introduced into the genome of Autographa californianuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [ 125I]iodocynnopindolol showed that Sf9 insect cells infected with the recombinant virus expressed ≈ 1 × 10 4β 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membraines revealed a molecular weight of ≈ 46000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.

A human cDNA fragment bearing the complete coding region for the β2‐adrenergic receptor was introduced into the genome of Autographa californianuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocynnopindolol showed that Sf9 insect cells infected with the recombinant virus expressed ≈ 1 × 104β2‐adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membraines revealed a molecular weight of ≈ 46000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs‐protein.

Using the ‘fusogen’ polycthyleneglycol (PEG), Dawson et al. [1] have concluded that both guanosine triphosphate (GTP)-mduced calcium efflux and the enhancement of IP 3-promoted calcium release from rat liver microsomal vesicles could be attributed to a GTP-dependent vesicle fusion. We have studied GTP-induced fusion of microsomal vesicles from rat exocrine pancreas using light scatter and fluorescence dequenching methods. In the presence of PEG (3%), GTP (10 μM) induced a decrease in light scatter and an increase in fluorescence in the fluorescence dequenching assay (GTP-effect) indicating fusion of the vesicles. Guanosine 5'- O-(3-thiotriphosphate) (10 μM) had no effect on its own and inhibited the GTP-induced signals. Preincubation of the vesicles with adenosine triphosphate (ATP) (4 mM) increased the GTP-effect by 80%. whereas bafilomycin B 1, a specific inhibitor of vacuolar type H +-ATPases, and the protonophore CCCP (10 μM) inhibited only the ATP-dependent part of the GTP-effect. Inhibitors of the vacuolar type H +-ATPase, which are also SH-alkylating reagents such as N-ethylmaleimid (100 μM) and the tyrosine-,cysteinc- and lysine-reactive reagent 7-chloro-4-nitrobenz-2-exa-1,3-diazole (10 μM) [2,3j. abolished the GTP-effect in the absence or presence of ATP. We conclude that GTP induces fusion of pancreatic microsomes which is increased by an H +gradient established by a vacuolar type H +-ATPase.

Using the ‘fusogen’ polycthyleneglycol (PEG), Dawson et al. [1] have concluded that both guanosine triphosphate (GTP)‐mduced calcium efflux and the enhancement of IP3‐promoted calcium release from rat liver microsomal vesicles could be attributed to a GTP‐dependent vesicle fusion. We have studied GTP‐induced fusion of microsomal vesicles from rat exocrine pancreas using light scatter and fluorescence dequenching methods. In the presence of PEG (3%), GTP (10 μM) induced a decrease in light scatter and an increase in fluorescence in the fluorescence dequenching assay (GTP‐effect) indicating fusion of the vesicles. Guanosine 5'‐O‐(3‐thiotriphosphate) (10 μM) had no effect on its own and inhibited the GTP‐induced signals. Preincubation of the vesicles with adenosine triphosphate (ATP) (4 mM) increased the GTP‐effect by 80%. whereas bafilomycin B1, a specific inhibitor of vacuolar type H+‐ATPases, and the protonophore CCCP (10 μM) inhibited only the ATP‐dependent part of the GTP‐effect. Inhibitors of the vacuolar type H+‐ATPase, which are also SH‐alkylating reagents such as N‐ethylmaleimid (100 μM) and the tyrosine‐,cysteinc‐ and lysine‐reactive reagent 7‐chloro‐4‐nitrobenz‐2‐exa‐1,3‐diazole (10 μM) [2,3j. abolished the GTP‐effect in the absence or presence of ATP. We conclude that GTP induces fusion of pancreatic microsomes which is increased by an H+gradient established by a vacuolar type H+‐ATPase.

Das Rahmenprogramm des Gastlandes der Chemie‐Olympiade ist zwar wichtig und notwendig, aber letztlich bestimmen die Aufgaben den Erfolg des Wettbewerbs‐nach innen und nach außen.