Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.

Quantifying degradation rates of transmembrane receptor kinases.

Transmembrane receptor-kinases are widespread throughout eukaryotes and their activities are known to regulate all kinds of cellular responses in diverse organs and cell types. In order to guarantee the correct amplitude and duration of signals, receptor levels at the cellular surface need to be tightly controlled. The regulation of receptor degradation is the most direct way to achieve this and elaborate mechanisms are in place to control this process. Therefore, the rate of receptor degradation is a parameter of central importance for understanding the dynamics of a signal transduction cascade. Unfortunately, degradation of transmembrane receptors is a complicated multistep process that involves internalization from the plasma membrane, invagination into the lumen of endosomal compartments, and finally fusion with the vacuole for degradation by vacuolar proteases. Therefore, degradation should be measured in an as noninvasive way as possible, such as not to interfere with the complicated transport processes. Here, a method for minimally invasive, in vivo turn-over measurements in intact organs is provided. This technique was used for quantifying the turn-over rates of the Brassinosteroid receptor kinase BRI1 (BRASSINOSTEROID INSENSITIVE 1) in Arabidopsis thaliana root meristems. Pulse-chase expression of a fluorescently labeled BRI1 variant was used and its turn-over rate was determined by quantitative confocal microscopy. This method is well suited to measure turn-over of transmembrane kinases, but can evidently be extended to measure turn-over of any types of transmembrane proteins.

Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the alpha 1B-adrenergic receptor.

The alpha 1B-adrenergic receptor (alpha 1BAR) and its truncated mutant T368 lacking the last 147 amino acids were stably expressed in Rat1 fibroblasts. The wild type alpha 1BAR was rapidly phosphorylated upon exposure to the agonist epinephrine as well as to phorbol ester as assessed by immunoprecipitation of the receptor with antiserum raised against its amino-terminal portion. Exposure of cells expressing the wild type alpha 1BAR to epinephrine resulted also in rapid homologous desensitization of receptor-mediated response on polyphosphoinositide hydrolysis. On the other hand, truncation of the serine- and threonine-rich carboxyl portion of the alpha 1BAR abolished agonist-induced phosphorylation and greatly impaired homologous desensitization of the receptor. The truncated receptor T368 could undergo agonist-induced decrease of cell surface receptors but to a lesser extent, as compared with the wild type alpha 1BAR. These results demonstrate that the carboxyl portion of the alpha 1BAR plays a crucial role in the regulation of receptor function. They also suggest a strong relationship between agonist-induced phosphorylation and desensitization of the alpha 1BAR, which were both insensitive to the inhibitor of protein kinase C RO-318220. Our findings support the emerging hypothesis that the biochemical mechanisms involved in rapid agonist-dependent regulation of G protein-coupled receptors, which activate polyphosphoinositide hydrolysis, do not primarily involve protein kinase C.

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.

Epineurial adipocytes are dispensable for Schwann cell myelination.

Previous clinical observations and data from mouse models with defects in lipid metabolism suggested that epineurial adipocytes may play a role in peripheral nervous system myelination. We have used adipocyte-specific Lpin1 knockout mice to characterize the consequences of the presence of impaired epineurial adipocytes on the myelinating peripheral nerve. Our data revealed that the capacity of Schwann cells to establish myelin, and the functional properties of peripheral nerves, were not affected by compromised epineurial adipocytes in adipocyte-specific Lpin1 knockout mice. To evaluate the possibility that Lpin1-negative adipocytes are still able to support endoneurial Schwann cells, we also characterized sciatic nerves from mice carrying epiblast-specific deletion of peroxisome proliferator-activated receptor gamma, which develop general lipoatrophy. Interestingly, even the complete loss of adipocytes in the epineurium of peroxisome proliferator-activated receptor gamma knockout mice did not lead to detectable defects in Schwann cell myelination. However, probably as a consequence of their hyperglycemia, these mice have reduced nerve conduction velocity, thus mimicking the phenotype observed under diabetic condition. Together, our data indicate that while adipocytes, as regulators of lipid and glucose homeostasis, play a role in nerve function, their presence in epineurium is not essential for establishment or maintenance of proper myelin.

Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909.

The induction of potent CD8+ T cell responses by vaccines to fight microbes or tumors remains a major challenge, as many candidates for human vaccines have proved to be poorly immunogenic. Deoxycytidyl-deoxyguanosin oligodeoxynucleotides (CpG ODNs) trigger Toll-like receptor 9, resulting in dendritic cell maturation that can enhance immunogenicity of peptide-based vaccines in mice. We tested whether a synthetic ODN, CpG 7909, could improve human tumor antigen-specific CD8+ T cell responses. Eight HLA-A2+ melanoma patients received 4 monthly vaccinations of low-dose CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund's adjuvant. All patients exhibited rapid and strong antigen-specific T cell responses: the frequency of Melan-A-specific T cells reached over 3% of circulating CD8+ T cells. This was one order of magnitude higher than the frequency seen in 8 control patients treated similarly but without CpG and 1-3 orders of magnitude higher than that seen in previous studies with synthetic vaccines. The enhanced T cell populations consisted primarily of effector memory cells, which in part secreted IFN- and expressed granzyme B and perforin ex vivo. In vitro, T cell clones recognized and killed melanoma cells in an antigen-specific manner. Thus, CpG 7909 is an efficient vaccine adjuvant that promotes strong antigen-specific CD8+ T cell responses in humans.

Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures.

During brain development, spontaneous neuronal activity has been shown to play a crucial role in the maturation of neuronal circuitries. Activity-related signals may cause selective neuronal cell death and/or rearrangement of neuronal connectivity. To study the effects of sustained inhibitory activity on developing inhibitory (GABAergic) neurons, three-dimensional primary cell cultures of fetal rat telencephalon were used. In relatively immature cultures, muscimol (10 microns), a GABAA receptor agonist, induced a transient increase in apoptotic cell death, as evidenced by a cycloheximide-sensitive increase of free nucleosomes and an increased frequency of DNA double strand breaks (TUNEL labeling). Furthermore, muscimol caused an irreversible reduction of glutamic acid decarboxylase activity, indicating a loss of GABAergic neurons. The muscimol-induced death of GABAergic neurons was attenuated by the GABAA receptor blockers bicuculline (100 microns) and picrotoxin (100 microns), by depolarizing potassium concentrations (30 mM KCl) and by the L-type calcium channel activator BAY K8644 (2 microns). As compared to the cholinergic marker (choline acetyltransferase activity), glutamic acid decarboxylase activity was significantly more affected by various agents known to inhibit neuronal activity, including tetrodotoxin (1 micron), flunarizine (5 microns), MK 801 (50 microns) and propofol (40 microns). The present results suggest that the survival of a subpopulation of immature GABAergic neurons is dependent on sustained neuronal activity and that these neurons may undergo apoptotic cell death in response to GABAA autoreceptor activation.

Transcriptional Activation and Receptor Dimerization is Affected by Mutations in the NR2E3 Ligand-Binding Domain

Purpose: Mutations in the ligand-binding domain (LBD) of NR2E3 cause recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS), Goldmann-Favre syndrome (GFS) and clumped pigmentary retinal degeneration (CPRD). In addition to ligand binding, the LBD contains also essential amino acid sequences for the oligomerization of nuclear receptors. The aim of our studies is to characterize the impact of mutations in the LBD on receptor oligomerization and transcriptional activity of NR2E3. Methods: The different NR2E3 mutants were generated by QuickChange mutagenesis and analyzed in 293T-based transactivation studies and BRET2 (bioluminescence resonance electron transfer) assays. In silico homology modeling of mutant proteins was also performed using available crystallographic data of related nuclear receptors. Results: The mutants p.W234S, p.A256V, p.A256E, p.L263P, p.R309G, p.R311Q, p.R334G, p.L336P, p.L353V, p.R385P and p.M407K, all located in the LBD, showed impaired receptor dimerization at various degrees. Impaired repressor dimerization as assessed by BRET2 assays did not always correlate with impaired repressor function of NR2E3 as assessed by cell-based reporter assays. There were minor differences of transcriptional activity of mutant proteins on mouse S-opsin (opn1sw), mouse cone arrestin (arr3) and human cone arrestin, suggesting that the effect of LBD mutations was independent of the promoter context. Conclusions: Mutational analysis and homology modeling allowed the characterization of potential oligomerization interfaces of the NR2E3 LBD. Additionally, mutations in NR2E3 LBD may cause recessive retinal degenerations by different molecular mechanisms.

Delayed mortality and attenuated thrombocytopenia associated with severe malaria in urokinase- and urokinase receptor-deficient mice.

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (-/-) mice. The mortality resulting from Plasmodium berghei ANKA infection was delayed in uPA(-/-) and uPAR(-/-) mice but was similar to that of the wild type (+/+) in tPA(-/-) mice. Parasitemia levels were similar in uPA(-/-), uPAR(-/-), and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR(-/-) mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR(-/-) mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA(-/-) and uPAR(-/-) mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR(-/-) mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR(-/-) mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR(-/-) mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.

Cell populations in lesions of cutaneous leishmaniasis of leishmania (L.) amazonensis- infected rhesus macaques, Macaca mulatta

The cellular nature of the infiltrate in cutaneous lesion of rhesus monkeys experimentally infected with Leishmania (L.) amazonensis was characterized by immunohistochemistry. Skin biopsies from infected animals with active or healing lesions were compared to non-infected controls (three of each type) to quantitate inflammatory cell types. Inflammatory cells (composed of a mixture of T lymphocyte subpopulations, macrophages and a small number of natural killer cells and granulocytes) were more numerous in active lesions than in healing ones. T-cells accounted for 44.7 ± 13.1% of the infiltrate in active lesions (versus CD2+= 40.3 ± 5.7% in healing lesions) and T-cell ratios favor CD8+ cells in both lesion types. The percentage of cells expressing class II antigen (HLA-DR+) in active lesions (95 ± 7.1%) was significantly higher (P < 0.005) from the healing lesions (42.7 ± 12.7%). Moreover, the expression of the activation molecules CD25 (@ 16%), the receptor for interleukin-2, suggests that many T cells are primed and proliferating in active lesions. Distinct histopathological patterns were observed in lesions at biopsy, but healing lesions contained more organized epithelioid granulomas and activated macrophages, followed by fibrotic substitution. The progression and resolution of skin lesions appears to be very similar to that observed in humans, confirming the potential for this to be used as a viable model to study the immune response in human cutaneous leishmaniasis.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

In vitro neuroendocrine effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the AhR-expressing hypothalamic rat GnV-3 cell line.

The aryl hydrocarbon receptor (AhR) is involved in a wide variety of biological and toxicological responses, including neuroendocrine signaling. Due to the complexity of neuroendocrine pathways in e.g. the hypothalamus and pituitary, there are limited in vitro models available despite the strong demand for such systems to study and predict neuroendocrine effects of chemicals. In this study, the applicability of the AhR-expressing rat hypothalamic GnV-3 cell line was investigated as a novel model to screen for neuroendocrine effects of AhR ligands using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as reference compound. The qRT-PCR analyses demonstrated the presence of several sets of neurotransmitter receptors in the GnV-3 cells. TCDD (10nM) altered neurotransmitter signaling by up-regulation of glutamate (Grik2), gamma-amino butyric acid (Gabra2) and serotonin (Ht2C) receptor mRNA levels. However, no significant changes in basal and serotonin-evoked intracellular Ca(2+) concentration ([Ca(2+)]i) or serotonin release were observed. On the other hand, TCDD de-regulated period circadian protein homolog 1 (Per1) and gonadotropin releasing hormone (Gnrh) mRNA levels within a 24-h time period. Both Per1 and Gnrh genes displayed a similar mRNA expression pattern in GnV-3 cells. Moreover, the involvement of AhR in TCDD-induced alteration of Neuropeptide Y (Npy) gene expression was found and confirmed by using siRNA targeted against Ahr in GnV-3 cells. Overall, the combined results demonstrate that GnV-3 cells may be a suitable model to predict some mechanisms of action and effects of AhR ligands in the hypothalamus.

Deficient T cell fate specification in mice with an induced inactivation of Notch1.

Notch proteins are cell surface receptors that mediate developmental cell specification events. To explore the function of murine Notch1, an essential portion of the gene was flanked with loxP sites and inactivation induced via interferon-regulated Cre recombinase. Mice with a neonatally induced loss of Notch1 function were transiently growth retarded and had a severe deficiency in thymocyte development. Competitive repopulation of lethally irradiated wild-type hosts with wild-type- and Notch1-deficient bone marrow revealed a cell autonomous blockage in T cell development at an early stage, before expression of T cell lineage markers. Notch1-deficient bone marrow did, however, contribute normally to all other hematopoietic lineages. These findings suggest that Notch1 plays an obligatory and selective role in T cell lineage induction.

Consequences of sleep deprivation on neurotransmitter receptor expression and function.

Several pieces of evidence suggest that sleep deprivation causes marked alterations in neurotransmitter receptor function in diverse neuronal cell types. To date, this has been studied mainly in wake- and sleep-promoting areas of the brain and in the hippocampus, which is implicated in learning and memory. This article reviews findings linking sleep deprivation to modifications in neurotransmitter receptor function, including changes in receptor subunit expression, ligand affinity and signal transduction mechanisms. We focus on studies using sleep deprivation procedures that control for side-effects such as stress. We classify the changes with respect to their functional consequences on the activity of wake-promoting and/or sleep-promoting systems. We suggest that elucidation of how sleep deprivation affects neurotransmitter receptor function will provide functional insight into the detrimental effects of sleep loss.

Malignant transformation of DMBA/TPA-induced papillomas and nevi in the skin of mice selectively lacking retinoid-X-receptor alpha in epidermal keratinocytes.

Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.