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Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular receptor for muramyl dipeptide derived from the intestinal microbiota. Loss-of-function mutations in Nod2 are associated with the development of Crohn's disease, suggesting that NOD2 signaling plays critical roles in the maintenance of intestinal immune homeostasis. Although NOD2 activation prevents the development of short-term experimental colitis, it remains unknown whether the sensitivity to long-term experimental colitis is influenced by NOD2. In this study, we explored the roles played by NOD2 in the development of long-term adoptive transfer colitis. Unexpectedly, we found that Rag1-/-Nod2-/- mice were more resistant to adoptive transfer colitis than Rag1-/- mice and had reduced proinflammatory cytokine responses and enhanced accumulation of regulatory T cells (Tregs) expressing forkhead box P3 in the colonic mucosa. Prevention of colitis in Rag1-/-Nod2-/- mice was mediated by TGF-β1 because neutralization of TGF-β1 resulted in the development of more severe colitis due to reduced accumulation of Tregs. Such paradoxical Treg responses in the absence of NOD2 could explain why Nod2 mutations in humans are not sufficient to cause Crohn's disease.

Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such as Staphylococcus aureus and their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. S. aureus produce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show that S. aureus secretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-β response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.

Background Juvenile hormones (JHs) are a class of sesquiterpenoids that play a pivotal role in insect growth and reproduction. Synthetic JH agonists (JHAs), including pyriproxyfen, have been widely used as insecticides to control agricultural pests and disease vectors. The antimetamorphic action of JHAs is mediated by their intracellular receptor, the heterodimer of Methoprene-tolerant (Met) and Taiman (Tai) proteins. Although a range of bioassay systems has been developed to detect the activity of JHAs, each of these systems has its own drawback(s), such as poor reproducibility, the use of radioactive ligands or the effect of endogenous JH-signaling factors. Results To address these issues, we constructed a new luciferase reporter assay for JHAs in mammalian HEK293T cells transiently transfected with the Drosophila Met and Tai genes. This reporter system gave highly reproducible results and showed nanomolar sensitivity to natural JHs. We then applied this reporter system to a structure-activity relationship (SAR) analysis of 14 natural and synthetic JHAs, leading to identification of the ligand structural factors important for the transcription-inducing activity. Conclusion Because this reporter system is not affected by the signaling cascade downstream of the JH receptors, it is suitable for evaluating the intrinsic activity of JHAs. The SAR results obtained in this study therefore provide invaluable information on the rational design of novel JHA insecticides.

Brain inflammation generally accompanies and accelerates neurodegeneration. Here we report a microglial mechanism in which polyglutamine binding protein 1 (PQBP1) senses extrinsic tau 3R/4R proteins by direct interaction and triggers an innate immune response by activating a cyclic GMP-AMP synthase (cGAS)-Stimulator of interferon genes (STING) pathway. Tamoxifen-inducible and microglia-specific depletion of PQBP1 in primary culture in vitro and mouse brain in vivo shows that PQBP1 is essential for sensing-tau to induce nuclear translocation of nuclear factor κB (NFκB), NFκB-dependent transcription of inflammation genes, brain inflammation in vivo, and eventually mouse cognitive impairment. Collectively, PQBP1 is an intracellular receptor in the cGAS-STING pathway not only for cDNA of human immunodeficiency virus (HIV) but also for the transmissible neurodegenerative disease protein tau. This study characterises a mechanism of brain inflammation that is common to virus infection and neurodegenerative disorders., Brain inflammation generally accelerates neurodegeneration but the mechanisms of this are not fully characterised. Here the authors show that PQBP1 in microglia is important for sensing extrinsic Tau 3 R/4 R proteins and triggers an innate immune response through cGAS and STING resulting in cognitive impairment.

Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CARMERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3ζ did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CARMERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CARMERTK drives an ‘immunologically silent’ scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.

Lipopolysaccharide (LPS) sensing in the cytosol by the noncanonical inflammasome leads to pyroptosis and NLRP3 inflammasome activation. This mechanism may be more critical for sepsis development than recognition of LPS by Toll-like receptor 4. LPS is directly binding to its intracellular receptor caspase-4/5/11, mediated by outer membrane vesicles and guanylate-binding proteins that deliver LPS to the cytosol and mediate access of caspases to LPS. Caspase-11-dependent cleavage of gasdermin D is discussed as a link between LPS-induced activation of caspases and pyroptosis or NLRP3 inflammasome activation. Finally, we highlight recently described inhibitors of cytosolic LPS-triggered noncanonical inflammasome activation that might be considered as potential drugs for the treatment of sepsis.

The TSH receptor (TSHR) is the major autoantigen in Graves’ disease (GD). Bioinformatic analyses predict the existence of several human TSHR isoforms from alternative splicing, which can lead to the coexpression of multiple receptor forms. The most abundant of these is TSHRv1.3. In silico modeling of TSHRv1.3 demonstrated the structural integrity of this truncated receptor isoform and its potential binding of TSH. Tissue profiling revealed wide expression of TSHRv1.3, with a predominant presence in thyroid, bone marrow, thymus, and adipose tissue. To gain insight into the role of this v1.3 receptor isoform in thyroid pathophysiology, we cloned the entire open reading frame into a mammalian expression vector. Immunoprecipitation studies demonstrated that both TSHR-stimulating antibody and human TSH could bind v1.3. Furthermore, TSHRv1.3 inhibited the stimulatory effect of TSH and TSHR-Ab MS-1 antibody on TSHR-induced cAMP generation in a dose-dependent manner. To confirm the antigenicity of v1.3, we used a peptide ELISA against two different epitopes. Of 13 GD samples, 11 (84.6%) were positive for a carboxy terminal peptide and 10 (76.9%) were positive with a junction region peptide. To demonstrate that intracellular v1.3 could serve as an autoantigen and modulate disease, we used double-transfected Chinese hamster ovary cells that expressed both green fluorescent protein (GFP)-tagged TSHRv1.3 and full-length TSHR. We then induced cell stress and apoptosis using a TSHR monoclonal antibody and observed the culture supernatant contained v1.3-GFP protein, demonstrating the release of the intracellular receptor variant by this mechanism.

NOD1 is an intracellular receptor that, when activated, induces gene expression of pro-inflammatory factors promoting macrophages and neutrophils recruitment at the infection site. However, iE-DAP, the dipeptide agonist that promotes this receptor's activation, cannot permeate cell membranes. To develop a nanocarrier capable of achieving a high and prolonged activation over time, iE-DAP was encapsulated in nanoparticles (NPs) made of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The physicochemical properties, colloidal stability, encapsulation efficiency, and cellular uptake of iE-DAP-loaded PHVB NPs were analyzed. Results evidenced that physicochemical properties of iE-DAP-loaded NPs remained stable over time, and NPs were efficiently internalized into cells, a process that depends on time and concentration. Moreover, our results showed that NPs elicited a controlled cargo release in vitro, and the encapsulated agonist response was higher than its free form, suggesting the possibility of activating intracellular receptors triggering an immune response through the release of NOD1 agonist.

Endocannabinoid signaling depends upon the CB1 and CB2 cannabinoid receptors, their endogenous ligands anandamide and 2-arachidonoylglycerol, and intracellular proteins that mediate responses via the C-terminal and other intracellular receptor domains. The CB1 receptor regulates and is regulated by associated G proteins predominantly of the Gi/o subtypes, β-arrestins 1 and 2, and the cannabinoid receptor-interacting protein 1a (CRIP1a). Evidence for a physiological role for CRIP1a is emerging as data regarding the cellular localization and function of CRIP1a are generated. Here we summarize the neuronal distribution and role of CRIP1a in endocannabinoid signaling, as well as discuss investigations linking CRIP1a to development, vision and hearing sensory systems, hippocampus and seizure regulation, and psychiatric disorders including schizophrenia. We also examine the genetic and epigenetic association of CRIP1a within a variety of cancer subtypes. This review provides evidence upon which to base future investigations on the function of CRIP1a in health and disease.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an intracellular receptor for polysialic acid. MARCKS supports development, synaptic plasticity, and regeneration after injury. MARCKS binds with its functionally essential effector domain (ED) to polysialic acid. A 25-mer peptide comprising the ED of MARCKS stimulates neuritogenesis of primary hippocampal neurons after addition to the culture. This motivated us to investigate whether ED peptide has similar effects in spinal cord injury. ED peptide supported recovery and regrowth of monoaminergic axons in female, but not in male mice. Sex-specific differences in response to ED peptide application also occurred in cultured neurons. In female but not male neurons, the ED peptide enhanced neurite outgrowth that could be suppressed by inhibitors of the estrogen receptors α and β, fibroblast growth factor receptor-1, protein kinase C, and matrix metalloproteinase 2. In addition, we observed female-specific elevation of phosphorylated MARCKS levels after ED peptide treatment. In male neurons, the ED peptide enhanced neuritogenesis in the presence of an androgen receptor inhibitor to the extent seen in ED peptide-treated female neurons. However, inhibition of androgen receptor did not lead to increased phosphorylation of MARCKS. These results provide insights into the functions of a novel compound contributing to gender-dependent regeneration.

Ligand-dependent oligomerization of receptor tyrosine kinases (RTKs) results in their activation through highly specific conformational changes in the extracellular and intracellular receptor domains. These conformational changes are unique for each RTK sub-family, limiting cross-activation between unrelated RTKs. The proto-oncogene MET receptor tyrosine kinase overcomes these structural constraints and phosphorylates unrelated RTKs in numerous cancer cell lines. The molecular basis for these interactions is unknown. We investigated the mechanism by which MET phosphorylates the human epidermal growth factor receptor-3 (HER3 or ERBB3), a catalytically impaired RTK whose phosphorylation by MET has been described as an essential component of drug resistance to inhibitors targeting EGFR and HER2. We find that in untransformed cells, HER3 is not phosphorylated by MET in response to ligand stimulation, but rather to increasing levels of MET expression, which results in MET activation in a ligand-independent manner. Phosphorylation of HER3 by its canonical dimerization partners, EGFR and HER2, is achieved by engaging an allosteric site on the HER3 kinase domain, but this site is not required when HER3 is phosphorylated by MET. We also observe that HER3 preferentially interacts with MET during its maturation along the secretory pathway, before MET is post-translationally processed by cleavage within its extracellular domain. This results in accumulation of phosphorylated HER3 in the Golgi apparatus. We further show that in addition to HER3, MET phosphorylates other RTKs in the Golgi, suggesting that this mechanism is not limited to HER3 phosphorylation. These data demonstrate a link between MET overexpression and its aberrant activation in the Golgi endomembranes and suggest that non-canonical interactions between MET and unrelated RTKs occur during maturation of receptors. Our study highlights a novel aspect of MET signaling in cancer that would not be accessible to inhibition by therapeutic antibodies.

Since it was first postulated by Wigglesworth in 1934, juvenile hormone (JH) is considered a status quo hormone in insects because it prevents metamorphosis that is initiated by the molting hormone 20-hydroxyecdysone (20E). During the last decade, significant advances have been made regarding JH signaling. First, the bHLH-PAS transcription factor Met/Gce was identified as the JH intracellular receptor. In the presence of JH, with the assistance of Hsp83, and through physical association with a bHLH-PAS transcriptional co-activator, Met/Gce enters the nucleus and binds to E-box-like motifs in promoter regions of JH primary-response genes for inducing gene expression. Second, the zinc finger transcription factor Kr-h1 was identified as the anti-metamorphic factor which transduces JH signaling. Via Kr-h1 binding sites, Kr-h1 represses expression of 20E primary-response genes (i.e. Br, E93 and E75) to prevent 20E-induced metamorphosis. Third, through the intracellular signaling, JH promotes different aspects of female reproduction. Nevertheless, this action varies greatly from species to species. Last, a hypothetical JH membrane receptor has been predicted to be either a GPCR or a tyrosine kinase receptor. In future, it will be a great challenge to understand how the JH intracellular receptor Met/Gce and the yet unidentified JH membrane receptor coordinate to regulate metamorphosis and reproduction in insects.

Receptor signalling events including those initiated following activation of cytokine and growth factor receptors and the well-characterised death receptors (tumour necrosis factor receptor, type 1, FasR and TRAIL-R1/2) are initiated at the cell surface through the recruitment and formation of intracellular multiprotein signalling complexes that activate divergent signalling pathways. Over the past decade, research studies reveal that many of these receptor-initiated signalling events involve the sequential proteolysis of specific receptors by membrane-bound proteases and the γ-secretase protease complexes. Proteolysis enables the liberation of soluble receptor ectodomains and the generation of intracellular receptor cytoplasmic domain fragments. The combined and sequential enzymatic activity has been defined as regulated intramembrane proteolysis and is now a fundamental signal transduction process involved in the termination or propagation of receptor signalling events. In this review, we discuss emerging evidence for a role of the γ-secretase protease complexes and regulated intramembrane proteolysis in cell- and immune-signalling pathways.

Aims to investigate α2-AR subtype distribution and the relationship between receptor amounts and their functionality in normotensive and spontaneously hypertensive rats. Methods experiments were performed on left ventricular cardiomyocytes isolated from Wistar rats and SHR (2–2.5 months). Molecular routine tools (RT-PCR, Western blotting, immunocytochemistry) were used for semi-quantitative estimation of α2-AR subtypes. Fluorescence of both the Ca2+-dependent and NO-sensitive probes were used to define functionality of α2-AR, evaluated by changes in the dynamics of spontaneous Ca2+-transients and NO production in cardiomyocytes in response to the α2-AR agonist application. Results percentage of the three known α2-AR subtypes in Wistar and SHR cardiomyocytes is not principally different. Total amounts of α2A-AR subtype in SHR increases, for both the sarcolemmal and intracellular receptor pools. Total number of α2B-AR is also significantly higher in hypertensive rats with an increase in the sarcolemmal, but not the intracellular immunoreactivity. For α2C-AR subtype, no significant differences between Wistar and SHR were identified, despite the fact that its amounts in cardiomyocytes are somewhat higher than the other two subtypes. Notwithstanding the increased expression of α2-AR subtypes in SHR, α2-AR-agonist guanabenz was ineffective in suppression of spontaneous Ca2+-transients, as well as the lowering of free calcium levels in the cytosol. Guanabenz-induced NO synthesis is well correlated with the Ca2+-loading into sarcoplasmic reticulum and actually decreased in SHR cardiomyocytes. Conclusion data indicate α2-AR dysfunction and ineffectiveness of α2-AR-mediated signaling pathways in this model of cardiovascular pathologies. Results can be used for clinical practice for more effective control of cardiovascular functions in various disease states.

The aryl hydrocarbon receptor (AhR) was discovered as the intracellular receptor that bound with high affinity to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the AhR is required for mediating the toxicity induced by TCDD. Subsequent studies show that the AhR binds structurally diverse chemicals including plant-derived compounds that promote health and several AhR-active pharmaceuticals that exhibit anticancer activity. In this issue, there is a report that carbidopa, a drug used for treating Parkinson's disease, is also an AhR ligand, and this compound inhibits pancreatic cancer cell and tumor growth. These results are consistent with activities of other AhR-active compounds that inhibit carcinogenesis. Like carbidopa, these chemicals are selective AhR modulators with potential clinical applications that are AhR-dependent.

Circular RNAs are a subclass of noncoding RNAs in mammalian cells; however, whether these RNAs are involved in the regulation of astrocyte activation is largely unknown. Here, we have shown that the circular RNA HIPK2 (circHIPK2) functions as an endogenous microRNA-124 (MIR124-2HG) sponge to sequester MIR124-2HG and inhibit its activity, resulting in increased sigma non-opioid intracellular receptor 1 (SIGMAR1/OPRS1) expression. Knockdown of circHIPK2 expression significantly inhibited astrocyte activation via the regulation of autophagy and endoplasmic reticulum (ER) stress through the targeting of MIR124-2HG and SIGMAR1. These findings were confirmed in vivo in mouse models, as microinjection of a circHIPK2 siRNA lentivirus into mouse hippocampi inhibited astrocyte activation induced by methamphetamine or lipopolysaccharide (LPS). These findings provide novel insights regarding the specific contribution of circHIPK2 to astrocyte activation in the context of drug abuse as well as for the treatment of a broad range of neuroinflammatory disorders.

Steroid hormones classically mediate their actions by binding to intracellular receptor proteins that migrate to the nucleus and act as transcription factors to change gene expression. However, evidence is now accumulating for rapid, non-genomic effects of steroids. There is considerable controversy over the mechanisms underlying such effects. In a number of cases evidence has been presented for the direct activation of G-protein coupled receptors (GPCRs) by steroids, either at the plasma membrane, or at intracellular locations. Here, we will focus on the non-genomic actions of ecdysteroids on a Drosophila GPCR, DopEcR (CG18314), which can be activated by both ecdysone and the catecholamine, dopamine. We will also point out parallels between this system and the activation of the vertebrate GPCR, GPER1 (GPR30), which is thought to be activated by 17β-estradiol. We propose that the cellular localization and signalling properties of both DopEcR and GPER1 may be cell specific and depend upon their interactions with both accessory molecules and signalling pathways.

Cortisol modulates energy metabolism promoting the mobilization of glucose and increasing proteolysis to overcome stressful situations in teleost. The cortisol metabolic effects are attributed to genomic mechanisms that involve the interaction of cortisol with its glucocorticoid intracellular receptor. Furthermore, cortisol can also interact with plasma membrane glucocorticoid receptors activating a rapid nongenomic signaling; however, its contribution during the early acute phase stress response in fish is unknown. In the present work, we evaluated the effects of membrane-initiated cortisol actions in vivo in the proteome of rainbow trout (Oncorhynchus mykiss) skeletal muscle. Quantitative iTRAQ analyses were performed to examine proteomic changes in rainbow trout stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable cortisol conjugate. A total of 873 proteins were identified, among which 61 and 47 proteins were differentially expressed under cortisol and cortisol-BSA treatments, respectively. Functional clustering analysis revealed an upregulation of proteins associated with mitochondria and oxidative phosphorylation. These results were validated by Western blot analysis. Additionally, using rainbow trout myotubes, the participation of membrane glucocorticoid receptors in gene expression was evaluated. The results obtained suggest that cortisol acts through a membrane canonical glucocorticoid receptor and mediates the expression of proteins associated with mitochondrial oxidative phosphorylation.

In the past decades, glucocorticoid (GC) hormones and their cognate, intracellular receptor, the glucocorticoid receptor (GR), have been well established as critical checkpoints in mammalian energy homeostasis. Whereas many aspects in healthy nutrient metabolism require physiological levels and/or action of GC, aberrant GC/GR signalling has been linked to severe metabolic dysfunction, including obesity, insulin resistance and type 2 diabetes. Consequently, studies of the molecular mechanisms within the GC signalling axis have become a major focus in biomedical research, up-to-date particularly focusing on systemic glucose and lipid handling. However, with the availability of novel high throughput technologies and more sophisticated metabolic phenotyping capabilities, as-yet non-appreciated, metabolic functions of GC have been recently discovered, including regulatory roles of the GC/GR axis in protein and bile acid homeostasis as well as metabolic inter-organ communication. Therefore, this review summarises recent advances in GC/GR biology, and summarises findings relevant for basic and translational metabolic research.

Nucleotide-binding oligomerization domain-containing 2 (NOD2) is an intracellular receptor that plays an essential role in innate immunity as a sensor of a component of the bacterial cell wall, muramyl dipeptide (MDP). Crohn's disease (CD)-associated NOD2 variants lead to defective innate immune responses, including decreased NF-κB activation and cytokine production. We report herein that SAMP1/YitFc (SAMP) mice, which develop spontaneous CD-like ileitis in the absence of NOD2 genetic mutations, fail to respond to MDP administration by displaying decreased innate cytokine production and dysregulated NOD2 signaling compared with parental AKR control mice. We show that, unlike in other mouse strains, in vivo administration of MDP does not prevent dextran sodium sulfate-induced colitis in SAMP mice and that the abnormal NOD2 response is specific to the hematopoietic cellular component. Moreover, we demonstrate that MDP fails to enhance intracellular bacterial killing in SAMP mice. These findings shed important light on the initiating molecular events underlying CD-like ileitis.

It is well known that many of the actions of estrogens in the central nervous system are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. But there is also a compelling evidence for the involvement of membrane estrogen receptors in hypothalamic and other CNS functions. However, it is not well understood how estrogens signal via membrane receptors, and how these signals impact not only membrane excitability but also gene transcription in neurons. Indeed, it has been known for sometime that estrogens can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane delimited events. In addition, estrogens can affect second messenger systems including calcium mobilization and a plethora of kinases within neurons to alter cellular functions. Therefore, this brief review will summarize our current understanding of rapid membrane-initiated and intracellular signaling by estrogens in the hypothalamus, the nature of receptors involved and how these receptors contribute to maintenance of homeostatic functions, many of which go awry in menopausal states. This article is part of a Special Issue entitled Hormone Therapy .

Highlights: •Alkyl-LPA specifically interacts with PPARγ. •Alkyl-LPA treatments induces lipid accumulation in C2C12 cells. •Alkyl-LPA enhanced glucose uptake in C2C12 cells. •Alkyl-LPA-treated C2C12 cells express increased amounts of GLUT4 mRNA. •Alkyl-LPA is a novel therapeutic agent that can be used for the treatment of obesity and diabetes. -- Abstract: Studies on the effects of lipids on skeletal muscle cells rarely examine the effects of lysophospholipids. Through our recent studies, we identified select forms of phospholipids, such as alkyl-LPA, as ligands for the intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear hormone receptor implicated in many human diseases, including diabetes and obesity. We previously showed that alkyl-LPA is a specific agonist of PPARγ. However, the mechanism by which the alkyl-LPA–PPARγ axis affects skeletal muscle cells is poorly defined. Our objective in the present study was to determine whether alkyl-LPA and PPARγ activation promotes glucose uptake in skeletal muscle cells. Our findings indicate that PPARγ1 mRNA is more abundant than PPARγ2 mRNA in C2C12 cells. We showed that alkyl-LPA (3 μM) significantly activated PPARγ and increased intracellular glucose levels in skeletal muscle cells. We also showed that incubation of C2C12 cells with alkyl-LPA led to lipid accumulation in themore » cells. These findings suggest that alkyl-LPA activates PPARγ and stimulates glucose uptake in the absence of insulin in C2C12 cells. This may contribute to the plasma glucose-lowering effect in the treatment of insulin resistance.« less

International audience; Activation of the innate immune response in Metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor PGRP-LC and by the intracellular receptor PGRP-LE. Here, we show that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria.

Progesterone is a sexual steroid hormone that has a critical role in reproductive processes in males and females of several species, including humans. Furthermore, progesterone has been associated with pathological diseases such as breast, gynecological and brain cancer, regulating cell proliferation, apoptosis, and metastasis. In the past, progesterone actions were thought to be only mediated by its intracellular receptor (PR). However, recent evidence has demonstrated that membrane progesterone receptors (mPRs) mediate most of the non-classical progesterone actions. The role of the different mPRs subtypes in progesterone effects in reproduction and cancer is an emerging and exciting research area. Here we review studies to date regarding mPRs role in reproduction and cancer and discuss their functions and clinical relevance, suggesting mPRs as putative pharmacological targets and disease markers in cancer and diseases associated with reproduction.

The export of newly synthesized proteins, including G protein-coupled receptors (GPCR), from the endoplasmic reticulum (ER) and further transport to the plasma membrane is a tightly regulated process. ER export and subsequent cell surface targeting of GPCR is initially mediated through COPII-coated vesicles. It is governed by specific amino acid sequences located in extracellular as well as intracellular receptor domains, for example in the C-terminus (CT) of the receptor. Herein, we determined the role of the CT in the anterograde transport of the human neuropeptide Y receptor (hYR) type 2. We identified a short sequence motif in the membrane proximal CT: Y(x) 3 F(x) 3 F in the region of the putative 8th helix has a critical functional relevance for the anterograde transport of hY 2 R, since its deletion leads to accumulation of the receptor in the ER. It is sequence and position specific. Furthermore we identified a distinct role of C-terminal sequences in hY 1 R, hY 2 R, hY 4 R and hY 5 R. Regulation of hY 5 R export is regulated by a different mechanism as compared to hY 2 R. Different sequence elements with respect to function and localization are involved as demonstrated by the construction of a hY 2 /hY 5 receptor chimera and a noneffective deletion in the region of helix eight in the hY 5 R. In contrast to hY 2 R, deletion of the corresponding helical segment F(x) 3 L(x) 3 F has no influence on anterograde transport of hY 1 R, whereas deletion of F(x) 3 I(x) 3 V in hY 4 R restrains the receptor to the Golgi apparatus. Interestingly this pattern is not mirrored by repression of COPII vesicle transport by Sar1[H79G] overexpression. Whereas the 8th helix is involved before or at the level of Sar1 dependent export pathways in the ER for the hY 2 R, in hY 4 R helix eight is involved at later stages of anterograde transport.

Toll-like receptor (TLR) activation relies on biochemical recognition of microbial molecules and localization of the TLR within specific cellular compartments. Cell surface TLRs largely recognize bacterial membrane components, and intracellular TLRs are exclusively involved in sensing nucleic acids. Here we show that TLR11, an innate sensor for the Toxoplasma protein profilin, is an intracellular receptor that resides in the endoplasmic reticulum. The 12 membrane-spanning endoplasmic reticulum-resident protein UNC93B1 interacts directly with TLR11 and regulates the activation of dendritic cells in response to Toxoplasma gondii profilin and parasitic infection in vivo. A deficiency in functional UNC93B1 protein abolished TLR11-dependent IL-12 secretion by dendritic cells, attenuated Th1 responses against T. gondii, and dramatically enhanced susceptibility to the parasite. Our results reveal that the association with UNC93B1 and the intracellular localization of TLRs are not unique features of nucleic acid-sensing TLRs but is also essential for TLR11-dependent recognition of T. gondii profilin and for host protection against this parasite.

Dioxins are ubiquitous environmental contaminants that have attracted toxicological interest not only for the potential risk they pose to human health but also because of their unique mechanism of action. This mechanism involves a specific, phylogenetically old intracellular receptor (the aryl hydrocarbon receptor, AHR) which has recently proven to have an integral regulatory role in a number of physiological processes, but whose endogenous ligand is still elusive. A major acute impact of dioxins in laboratory animals is the wasting syndrome, which represents a puzzling and dramatic perturbation of the regulatory systems for energy balance. A single dose of the most potent dioxin, TCDD, can permanently readjust the defended body weight set-point level thus providing a potentially useful tool and model for physiological research. Recent evidence of response-selective modulation of AHR action by alternative ligands suggests further that even therapeutic implications might be possible in the future.

Since the discovery of the beneficial effects of adrenocortical extracts for treating adrenal insufficiency more than 80 years ago, glucocorticoids and their cognate, intracellular receptor, the glucocorticoid receptor have been characterized as critical checkpoints in the delicate hormonal control of energy homeostasis in mammals. Whereas physiological levels of glucocorticoids are required for proper metabolic control, aberrant glucocorticoid action has been linked to a variety of pandemic metabolic diseases, such as type II diabetes and obesity. Based on its importance for human health, studies of the molecular mechanisms of within the glucocorticoid signaling axis have become a major focus in biomedical research. In particular, the understanding of tissue-specific functions of the glucocorticoid receptor pathway has been proven to be of substantial value for the development of novel therapies in the treatment of chronic metabolic disorders. Therefore, this review focuses on the consequences of endogenous and experimental modulation of glucocorticoid receptor expression for metabolic homeostasis and dysregulation, particularly emphasizing tissue-specific contributions of the glucocorticoid pathway to the control of energy metabolism.

Seeing EPO The supply of red blood cells in mammals is controlled by the cytokine erythropoietin (EPO). In physiological situations, the concentration of EPO can change by 1000-fold. Becker et al. (p. 1404 , published online 20 May) used a combination of mathematical modeling and experimental analysis to discern how cells can maintain a linear response to such a broad range of EPO concentrations. Critical features included internalization of EPO-bound receptors and subsequent degradation of the EPO ligand. Replenishment of receptors at the cell surface required a large supply of EPO receptors maintained in reserve inside the cell. These mechanisms allow cells to experience large increases in EPO concentration without becoming refractory to further stimulation.

Contains fulltext : 87871.pdf (Publisher’s version ) (Closed access) Borrelia is the causative agent of Lyme disease, a widespread disease with important health consequences. Immune-mediated mechanisms are believed to play a major role in both host defense and in late complications of Lyme disease. Recognition of Borrelia and the initial activation of the innate immune system are important for host defense, as well as modulation of adaptive responses. Several classes of pattern recognition receptors (PRRs) have been suggested to be involved in the recognition of Borrelia: Toll-like receptors (TLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs). TLR2 has been found to be the most important receptor of the TLRs. The intracellular receptor NOD2, a member of the NLRs, might also play an important role in recognition. Mannose receptor is also involved in Borrelia recognition, but little is known about other CLRs such as dectin-1. After PRRs have recognized Borrelia, a signaling cascade is induced that leads to transcription of NF-kappaB, resulting in the production of pro-inflammatory cytokines. Understanding these pathways provides not only a better insight into the pathogenesis, but also provides potential, novel, therapeutic targets during active disease or post-infection complications. 01 maart 2010

The role of phospholipase D (PLD) in the regulation of the traffic of the PTH type 1 receptor (PTH1R) was studied in Chinese hamster ovary cells stably transfected with a human PTH1R (CHO-R3) and in rat osteosarcoma 17/2.8 (ROS) cells. PTH(1-34) increased total PLD activity by 3-fold in CHO-R3 cells and by 2-fold in ROS cells. Overexpression of wild-type (WT) PLD1 and WT-PLD2 increased basal PLD activity in CHO-R3 but not in ROS cells. Ligand-stimulated PLD activity greatly increased in CHO-R3 cells transfected with WT-PLD1 and WT-PLD2. However, only WT-PLD2 expression increased PTH-dependent PLD activity in ROS cells. Expression of the catalytically inactive mutants R898K-PLD1 (DN-PLD1) and R758K-PLD2 (DN-PLD2) inhibited ligand-dependent PLD activity in both cell lines. PTH(1-34) induced internalization of the PTH1R with a concomitant increase in the colocalization of the receptor with PLD1 in intracellular vesicles and in a perinuclear, ADP ribosylation factor-1-positive compartment. The distribution of PLD1 and PLD2 remained unaltered after PTH treatment. Expression of DN-PLD1 had a small effect on endocytosis of the PTH1R; however, DN-PLD1 prevented accumulation of the PTH1R in the perinuclear compartment. Expression of DN-PLD2 significantly retarded ligand-induced PTH1R internalization in both cell lines. The differential effects of PLD1 and PLD2 on receptor traffic were confirmed using isoform-specific short hairpin RNA constructs. We conclude that PLD1 and PLD2 play distinct roles in regulating PTH1R traffic; PLD2 primarily regulates endocytosis, whereas PLD1 regulates receptor internalization and intracellular receptor traffic.

Objective The importance of signal transduction in cell activities has been generally accepted. The purpose of this study was to analyze the regulatory effect of intracellular signaling cascade-associated genes on rat liver regeneration (LR) at transcriptional level. Material and methods The associated genes were originally obtained through a search of the databases and related scientific publications; their expression profiles were then checked in rat LR using the Rat Genome 230 2.0 array. The LR-associated genes were identified by comparing the discrepancy in gene expression changes between the partial hepatectomy (PH) group and the sham operation (SO) group. Results A total of 566 genes associated with the intracellular signaling cascade were LR related. The genes involved in nine signaling pathways including intracellular receptor-, second messenger-, nitric oxide-, hormone-, carbohydrate-mediated, protein kinase, small GTPase, ER-nuclear and target of rapamycin (TOR) signaling pathways were detected to be enriched in a cluster characterized by up-regulated expression in LR. According to their expression similarity and time relevance, they were separately classified into 5 and 5 groups. Conclusions It is presumed that following PH, the second messenger-mediated signaling pathway inhibits the inflammatory response, while the protein kinase cascade and small GTPase-mediated signal transduction stimulate the immune response; the intracellular receptor-, second messenger-, small GTPase-mediated signal transduction and protein kinase cascade coordinately control cell replication; the intracellular receptor-, second messenger-mediated and ER-nuclear signaling pathways facilitate cell differentiation; the MAPK cascade and small GTPase-mediated signal transduction play a role in cytoskeletal reconstruction and cell migration; the second messenger-, small GTPase-mediated and IκB kinase/NFκB cascades take care of protein transport, etc., in LR.

It is well known that many of the actions of 17beta-estradiol (E2) in the central nervous system are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. However, there is compelling evidence for membrane steroid receptors for estrogen in hypothalamic and other brain neurons. But it is not well understood how estrogen signals via membrane receptors, and how these signals impact not only membrane excitability but also gene transcription in neurons. Indeed, it has been known for sometime that E2 can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane delimited events. In addition, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell signaling. Therefore, this review will consider our current knowledge of rapid membrane-initiated and intracellular signaling by E2 in the hypothalamus, the nature of receptors involved and how they contribute to homeostatic functions.

Human G-protein-coupled formyl pep- tide receptor-like 1 and its mouse homologue formyl peptide receptor 2 (mFPR2) mediate the chemotactic activity of a variety of pathogen and host-derived peptides, including amyloid 42 ,a key causative factor in Alzheimer's disease. In mouse microglia, mFPR2 is up-regulated by patho- gen-associated molecular patterns and proinflam- matory cytokines, as shown, for instance, in our previous study using peptidoglycan (PGN) of Gram bacteria. As PGN and its components have been reported to use TLR2 and an intracellular receptor nucleotide-binding oligomerization do- main 2 (NOD2), we investigated the capacity of palmitoyl-cys((RS)-2, 3-di(palmitoyloxy)-propyl)- Ala-Gly-OH (PamCAG), a specific TLR2 ligand, and muramyl dipeptide (MDP), a NOD2 ligand, to cooperatively regulate the expression and function of mFPR2 in microglia. We found that MDP and PamCAG as well as another TLR2-specific agonist palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), when used alone, each increased the expression of functional mFPR2 in microglial cells, and the com- bination of MDP and PamCAG or Pam3CSK4 ex- hibited an additive effect. Mechanistic studies re- vealed that MDP increased the levels of TLR2 ex- pression on the microglial cell surface and enhanced the levels of MAPKs p-38, ERK1/2, and NF-B activated by PamCAG. Our results suggest that TLR2 and NOD2 cooperate to up-regulate the expression of mFPR2 and therefore, may actively participate in the pathogenic processes of brain inflammation and neurodegenerative diseases. J. Leukoc. Biol. 83: 1467-1475; 2008.

S1P (sphingosine 1-phosphate) and SPC (sphingosylphosphorylcholine) have been recently recognized as important mediators of cell signalling, regulating basic cellular processes such as growth, differentiation, apoptosis, motility and Ca2+ homoeostasis. Interestingly, they can also act as first and second messengers. Although their activation of cell-surface G-protein-coupled receptors has been studied extensively, not much is known about their intracellular mechanism of action, and their target proteins are yet to be identified. We hypothesized that these sphingolipids might bind to CaM (calmodulin), the ubiquitous intracellular Ca2+ sensor. Binding assays utilizing intrinsic tyrosine fluorescence of the protein, dansyl-labelled CaM and surface plasmon resonance revealed that SPC binds to both apo- and Ca2+-saturated CaM selectively, when compared with the related lysophospholipid mediators S1P, LPA (lysophosphatidic acid) and LPC (lysophosphatidylcholine). Experiments carried out with the model CaM-binding domain melittin showed that SPC dissociates the CaM–target peptide complex, suggesting an inhibitory role. The functional effect of the interaction was examined on two target enzymes, phosphodiesterase and calcineurin, and SPC inhibited the Ca2+/CaM-dependent activity of both. Thus we propose that CaM might be an intracellular receptor for SPC, and raise the possibility of a novel endogenous regulation of CaM. Abbreviations: ANS, 8-anilinonaphthalene-1-sulfonic acid; CaM, calmodulin; CMC, critical micelle concentration; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LT-SPC, L-threo-sphingosylphosphorylcholine; mant-cGMP, 2′-O-(N-methylanthraniloyl)guanosine-3′,5′-cyclic monophosphate; PC, phosphatidylcholine; PDE, phosphodiesterase; PE, phosphatidylethanolamine; PNPP, p-nitrophenyl phosphate; PPase-2B, protein phosphatase 2B; PS, phosphatidylserine; RU, resonance units; S1P, sphingosine 1-phosphate; SM, sphingomyelin; SPC, sphingosylphosphorylcholine; SPR, surface plasmon resonance

Treatment of D2-receptor-expressing cells with specific drugs upregulates the receptor number at the cell surface independently of protein synthesis, leading to the concept of an intracellular receptor pool. However, how this pool is operating is still an enigma. Here, we report that a splice variant of the Gαi2 protein, protein sGαi2, plays a crucial role in the maintenance of this D2-receptor pool. Co-expression of sGi2 with D2 receptor reduced receptor localization to cell surface by one-third. This effect is associated with specific intracellular protein-protein interaction and the formation of a sGi2–D2-receptor complex. It has been suggested that the formation of this complex serves to prevent D2 receptors from reaching the cell membrane. Treatment of D2-receptor-expressing cells with agonists increased the number of cell surface D2 receptors and coincided with a reduction in these receptors from intracellular complexes, suggesting that agonist treatment released D2 receptors from the complex allowing them to localize to the cell membrane. Thus, in addition to elucidating how the intracellular pool of D2 receptor functions, our findings uncover a novel mechanism regulating the density of cell surface D2 receptors.

The nicotinic acetylcholine receptor (AChR) is the prototype ligand-gated ion channel, and its function is dependent on its lipid environment. In order to study the involvement of sphingolipids (SL) in AChR trafficking, we used pharmacological approaches to dissect the SL biosynthetic pathway in CHO-K1/A5 cells heterologously expressing the muscle-type AChR. When SL biosynthesis was impaired, the cell surface targeting of AChR diminished with a concomitant increase in the intracellular receptor pool. The SL-inhibiting drugs increased unassembled AChR forms, which were retained at the endoplasmic reticulum (ER). These effects on AChR biogenesis and trafficking could be reversed by the addition of exogenous SL, such as sphingomyelin. On the basis of these effects we propose a 'chaperone-like' SL intervention at early stages of the AChR biosynthetic pathway, affecting both the efficiency of the assembly process and subsequent receptor trafficking to the cell surface.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1., The structural basis of how G-protein coupled receptors respond to unique stimuli remains poorly understood. Here, Krumm et al. present new structures of the neurotensin receptor and reveal insights into how ligand binding is linked to structural rearrangements associated with receptor activation.

Neutrophil migration and activation are critical components of innate immunity and are mediated by a variety of inflammatory mediators, which include interleukin-8 (IL-8) and epithelial-derived neutrophil activating peptide-78 (ENA-78). Limited knowledge on the expression of receptors for these inflammatory mediators (CXCR1 and CXCR2) in bovine, in addition to the association of a polymorphism (G-->C) in position +777 of the CXCR1 gene with impaired neutrophil function, prompted evaluation of CXCR1 and CXCR2 mRNA and protein expression, ligand binding affinity, and intracellular receptor signaling in neutrophils from cows with different CXCR1 genotypes. Initial observations revealed that overall IL-8 receptor numbers appeared to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of SB225002, a CXCR2 inhibitor, CXCR1 affinity was about fivefold lower in cows with a CC genotype and may have resulted in an underestimation of receptor numbers in cows with this genotype. In addition, intracellular calcium ([Ca++]i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not ENA-78. Furthermore, when neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, [Ca++]i release was lower in cows with a CC genotype, suggesting differential CXCR1 signaling among genotypes. These findings offer knowledge of the role that each of these receptors plays in the inflammatory response in the bovine and provide insight into the potential mechanisms that may be affected in neutrophils of cows with different CXCR1 genotypes.

Steroid hormones are important endocrine signalling molecules controlling reproduction, development, metabolism, salt balance and specialized cellular responses, such as inflammation and immunity. They are lipophilic in character and act by binding to intracellular receptor proteins. These receptors function as ligand-activated transcription factors, switching on or off networks of genes in response to a specific hormone signal. The receptor proteins have a conserved domain organization, comprising a C-terminal LBD (ligand-binding domain), a hinge region, a central DBD (DNA-binding domain) and a highly variable NTD (N-terminal domain). The NTD is structurally flexible and contains surfaces for both activation and repression of gene transcription, and the strength of the transactivation response has been correlated with protein length. Recent evidence supports a structural and functional model for the NTD that involves induced folding, possibly involving α-helix structure, in response to protein–protein interactions and structure-stabilizing solutes.

Endocytosis represents an important mechanism regulating cell-surface expression of neurotransmitter receptors, including GABAA receptors, in neurons. Little is known, however, about trafficking of internalized receptors. Here, we used antibody tagging in living rat hippocampal neurons in culture to monitor GABAA receptor internalization. We show that cell-surface receptors have a homogeneous distribution reflecting their mobility in the membrane. Unexpectedly, internalized GABAA receptors were detected mainly in a subsynaptic pool associated with gephyrin at postsynaptic sites, whereas AMPA-type glutamate receptors were accumulated in the soma. This process was time-dependent and could be prevented by blocking clathrin-coated vesicle endocytosis. In control experiments, the existence of an intracellular pool of GABAA receptors associated with gephyrin was confirmed independently of internalization of surface receptors, and constitutive endocytosis, unrelated to antibody-tagging, could be demonstrated for both AMPA and GABAA receptors using a biotinylation assay. These results suggest that cycling of GABAA receptors between the cell surface and the subsynaptic pool provides a mechanism for the short-term regulation of GABAergic neurotransmission. Furthermore, the close association of gephyrin with internalized GABAA receptors suggests a role in intracellular receptor trafficking.

Sphingosine 1-phosphate and lysophosphatidic acid are two phospholipid growth factors whose importance in physiology and pathophysiology is becoming more and more apparent. Structure-activity relationships for agonism and antagonism at the thirteen known cell-surface and one intracellular receptor are described. Particular emphasis is placed on ligands having different selectivity than the parent molecules. Structural insights regarding agonist and antagonist recognition by the receptors from both computational modeling studies and crystallography are also discussed.

Alpha1a-adrenergic receptors (alpha1aARs) are present intracellularly and at the cell surface in cultured and natural cell models, where they are subject to agonist-mediated desensitization and internalization. To explore alpha1aAR trafficking, a hemagglutinin (HA)-tagged alpha1aAR/enhanced green fluorescent protein (EGFP) fusion protein was expressed in rat-1 fibroblasts and tracked by EGFP fluorescence and antibody labeling of surface receptors. Confocal analysis of antibody-labeled surface receptors revealed unexpected constitutive internalization in the absence of agonist stimulation. In partial agreement, the inverse agonist prazosin also caused a modest 20 +/- 2% increase in surface receptor levels, suggesting a partial block of constitutive internalization caused by decreased basal activation. However, prazosin was unable to prevent internalization of antibody-tagged surface receptors observed by confocal microscopy or cause obvious redistribution of intracellular receptor to the surface, suggesting that the alpha1aAR is internalizing even in a basal-inactive state. In contrast to the alpha1aAR, surface labeling of an HA-tagged alpha1b-EGFP fusion protein did not result in any apparent constitutive internalization. Constitutive internalization of the alpha1aAR seems to occur alongside reversible agonist-induced internalization, and both seem to involve clathrin-mediated endocytosis but not degradation in lysozymes. Surface receptor density must be maintained by recycling, because the protein synthesis inhibitor cycloheximide has no effect on total or surface receptor density in agonist-treated or untreated cells for 6 h. Constitutive agonist-independent trafficking of alpha1aARs may provide a novel mechanism by which an internal pool of alpha1aARs are maintained and recycled to allow continuous agonist-induced signaling.

The actions of glucocorticoid hormones are mediated by an intracellular receptor, the glucocorticoid receptor (GR). The mechanism of action of this ligand-inducible transcription factor is discussed, focusing on mechanisms of glucocorticoid resistance. Three mechanisms are highlighted: ligand-induced down-regulation of the receptor, the dominant-negative inhibition by the beta-isoform of the receptor, and repression by the transcription factor NF-kappa B. It has been shown that these mechanisms can significantly inhibit glucocorticoid signaling, and could therefore seriously decrease the efficacy of glucocorticoids used clinically.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of conserved subunits that each contains six transmembrane regions (TMRs) localized near the carboxyl terminus. Receptor subunit assembly into a tetramer appears to be a multideterminant process involving an additive contribution of membrane spanning helices and the short cytosolic carboxyl terminus (residues 2590-2749). Previous studies have shown that of the six membrane-spanning regions in each subunit, the 5th and 6th transmembrane regions, and the carboxyl terminus are strong determinants for assembly. The fifth and sixth TMRs contain numerous beta-branched amino acids that may participate in coiled/coil formation via putative leucine zipper motifs. InsP(3)R truncation mutants were expressed in COS-1 cells and analyzed by sucrose density gradient sedimentation and gel filtration for their ability to assemble. Chemical cross-linking with the homobifunctional reagents sDST or DMS of mammalian and bacterially expressed carboxyl-terminal containing receptor fragments reveals that sequences within the carboxyl terminus confer the formation of subunit dimers. A series of InsP(3) receptor carboxyl-terminal fragments and glutathione S-transferase (GST)/InsP(3)R chimeras were expressed in Escherichia coli and used in an in vitro assay to elucidate the minimal sequence responsible for association of the carboxyl termini into dimers. The results presented here indicate that this minimal sequence is approximately 30 residues in length and is localized between residues 2629 and 2654. These residues are highly conserved between the three InsP(3)R isoforms ( approximately 80% identity) as well as the ryanodine receptor ( approximately 40% identity) and suggest that a conserved assembly motif may exist between the two intracellular receptor families. We propose that assembly of the InsP(3) receptor to a tetramer involves intersubunit interactions mediated through both the membrane-spanning regions and residues 2629-2654 of the carboxyl terminus possibly through the formation of a dimer of dimers.

The ability of nonviral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications. In recent years, several efforts were focused on the elucidation of the gene transfer mechanisms and on the development of multicomponent systems in order to improve both targeted gene delivery and transfection efficiency. The transport of the therapeutic DNA from the cytoplasm into the nucleus is an inefficient process and is considered as the major limiting step in nondividing cells. One of the strategies to improve nuclear uptake of DNA is taking advantage of the cellular nuclear import machinery. Synthetic peptides containing a nuclear localization signal (NLS) are bound to the DNA so that the resulting DNA-NLS complex can be recognized as a nuclear import substrate by specific intracellular receptor proteins. In this review, we critically summarize recent studies applying this approach with a particular focus on NLS-sequence specificity. Implications of the observed results are also discussed in regards to future developments of this technology.

We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter s ignaling scenarios.

Glucocorticoids (GC) and their cognate intracellular receptor, the glucocorticoid receptor (GR), have been characterised as critical checkpoints in the endocrine control of energy homeostasis in mammals. Indeed, aberrant GC action has been linked to a variety of severe metabolic diseases, including obesity, insulin resistance and type 2 diabetes. As a steroid-binding member of the nuclear receptor superfamily of transcription factors, the GR translocates into the cell nucleus upon GC binding where it serves as a transcriptional regulator of distinct GC-responsive target genes that are – in many cases – associated with glucose and lipid regulatory pathways and thereby intricately control both physiological and pathophysiological systemic energy homeostasis. Here, we summarize the current knowledge of GC/GR function in energy metabolism and systemic metabolic dysfunction, particularly focusing on glucose and lipid metabolism.

Progesterone is synthesized in the nervous system by neurons and glial cells. Because of their simple structure, plasticity and capacity of regeneration, peripheral nerves are particularly well suited for studying the biosynthesis, mechanisms of action and effects of the hormone. Schwann cells, the myelinating glial cells in the peripheral nervous system, synthesize progesterone in response to a diffusible neuronal signal. In peripheral nerves, the local synthesis of progesterone plays an important role in the formation of myelin sheaths. This has been shown in vivo, after cryolesion of the mouse sciatic nerve, and in vitro, in cocultures of Schwann cells and sensory neurons. Schwann cells also express an intracellular receptor for progesterone, which thus functions as an autocrine signalling molecule. Progesterone may promote myelination by activating the expression of genes coding for transcription factors (Krox-20) and/or for myelin proteins (P0, PMP22). Recently, it has been proposed that progesterone may indirectly regulate myelin formation by influencing gene expression in neurons. Steroid hormones also influence the proliferation of Schwann cells: estradiol becomes a potent mitogen for Schwann cells when levels of cAMP are elevated and glucocorticosteroids have been shown to increase the mitogenic effects of peptide growth factors.