A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis ...elegans . The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein–coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein–coupled receptors such as DAF-37 with more promiscuous G-protein–coupled receptors such as DAF-38.
Abstract Among the neurotransmitter abnormalities that have been investigated in Alzheimer's disease (AD), deficits in the cholinergic system have been the most intensively studied. Another key ...neurotransmitter system involved with emotion and cognition is the dopaminergic system. Here we have investigated alterations in all five dopamine receptor subtypes in AD brain. Using antipeptide rabbit antibodies for each of the five dopamine receptors (D1–D5) we mapped the distribution of these receptors in postmortem AD and age-matched control brains in the frontal cortex, utilizing biotin–avidin immunocytochemistry. All five DR subtypes were expressed as cell surface and cytoplasmic proteins. Receptor-specific changes in control and AD brain were identified as follows: D4R and D3R were the predominant receptor subtypes in age-matched controls followed by D2R and D1R; D5R is the least expressed receptor subtype. In AD brain, D2R and D5R are well expressed in comparison to D1R, D3R and D4R. Expression of D1R, D3R and D4R was severely reduced in AD cortex. D2R expression is moderately reduced in the frontal cortex of AD brain. D5R is the only receptor subtype whose expression is increased in AD frontal cortex. Furthermore, in AD, we found comparable expression of D3R in astrocytes, whereas D5R-like immunoreactivity is significantly increased in astrocytes, in comparison to normal frontal cortex, where it was predominantly neuronal. These results demonstrate subtype-specific changes in dopamine receptors in AD that may be important in disease pathophysiology and that may also serve as potential targets for therapeutic intervention in AD.
During brain development, neurite formation plays a critical role in neuronal communication and cognitive function. In the present study, we compared developmental changes in the expression of ...crucial markers that govern the functional activity of neurons, including somatostatin (SST), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), brain nitric oxide synthase (bNOS), gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD-65) and synaptic vesicle protein synaptophysin (SYP) in non-differentiated and retinoic acid (RA)-induced differentiated SH-SY5Y cells. We further determined the role of SST in regulating subcellular distribution and expression of neurotransmitters. Our results indicate that SST potentiates RA-induced differentiation of SH-SY5Y cells and involves regulating the subcellular distribution and expression of neurotransmitter markers and synaptophysin translocation to neurites in a time-dependent manner, anticipating the therapeutic implication of SST in neurodegeneration.
The equilibrium potential for GABA-A receptor mediated currents (EGABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of ...K+/Cl- co-transporter (KCC2) but a relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in EGABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on EGABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus EGABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in EGABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in EGABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in EGABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In the present experimental investigation, biocomposites based on short banana fiber (20 wt%) and poly-lactic acid were fabricated using three different processing techniques, namely direct injection ...molding (DIM), extrusion injection molding (EIM) and extrusion compression molding (ECM). The thermal and mechanical characterization as well as dynamic mechanical analysis has been performed to understand and compare the performance of the developed biocomposites. FTIR analysis has been conducted to investigate the presence and type of interfacial interaction in the biocomposites. XRD analysis was conducted to investigate the structure and to measure the crystallinity of the biocomposites. A significant improvement in the mechanical (tensile and flexural properties), dynamic mechanical properties (storage modulus, loss modulus, and tan delta) and crystallinity of the biocomposites fabricated by EIM were observed. A novel approach was used to examine the orientation and distribution of the fibers within the developed biocomposites. The fiber damage in terms of breaking, bending, twisting and formation of the clusters have been observed. Scanning Electron Microscopy (SEM) analysis revealed that the fiber pull-out and fracture are dominating the failure of biocomposite under loading.
Calcium-sensing receptor (CASR), expressed in parathyroid gland and kidney, is a critical regulator of extracellular calcium homeostasis. This G protein-coupled receptor exists at the plasma membrane ...as a homodimer, although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H, R66C or N583X-inactivating mutations identified in familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroid patients, which were transiently expressed in kidney cells. All mutants were deficient in cell signaling responses to extracellular CASR ligands relative to wild-type. All mutants, although as well expressed as wild-type, lacked mature glycosylation, indicating impaired trafficking from the endoplasmic reticulum (ER). Dimerized forms of wild-type, R66H and R66C mutants were present, but not of the N583X mutant. By immunofluorescence confocal microscopy of non-permeabilized cells, although cell surface expression was observed for the wild-type, little or none was seen for the mutants. In permeabilized cells, perinuclear staining was observed for both wild-type and mutants. By colocalization fluorescence confocal microscopy, the mutant CASRs were localized within the ER but not within the Golgi apparatus. By the use of photobleaching fluorescence resonance energy transfer microscopy, it was demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER, whereas the N583X mutant was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface.
In the present study, we describe the status of somatostatin receptor 2 and 5 (SSTR2 and SSTR5) as well as cannabinoid type 1 receptor (CB1R) in Huntingtin (Htt) knock-in striatal neuronal cells. In ...mutant Htt (mHtt) knock-in (STHdhQ111/111) and wild type (STHdhQ7/7) striatal neuronal cells, SSTRs and CB1R exhibit prominent cytoplasmic expression and respond to agonist in a receptor specific manner. In response to quinolinic acid (QUIN)-induced toxicity, STHdhQ111/111 cells are more vulnerable and display suppressed cell survival signaling pathways. Receptor-specific agonists protect cells from QUIN-induced toxicity and activate ERK1/2 in both STHdh cells. Co-activation of SSTRs and CB1R resulted in loss of protective effects, delayed ERK1/2 phosphorylation and altered receptor complex composition. These results provide firsthand evidence in support of the protective role of SSTRs in STHdh cells and the possible crosstalk between SSTRs and CB1R in the modulation of excitotoxicity in Huntington's disease.
•SSTR2/5 and CB1R are primarily expressed and colocalized intracellularly.•Activation of SSTR2, 5 and CB1R protected STHdh cells from QUIN-induced toxicity.•The protective effect of receptor activation is via ERK1/2.•Combined agonist treatment diminished the protective effect of single treatment.•Composition of receptor complex was altered upon ligand treatment.
Long term depression (LTD) in the CA1 region of the hippocampus, induced with a 20-Hz, 30 s tetanus to Schaffer collaterals, is enhanced in sleep-deprived (SD) rats. In the present study, we ...investigated the role of metabotropic glutamate receptors (mGluRs), γ-aminobutyric acid (GABA) B receptors (GABA(B)-Rs) and N-methyl-D-aspartic acid receptors (NMDARs) in the LTD of the population excitatory postsynaptic potential (pEPSP). The requirement of Ca(2+) from L- and T-type voltage-gated calcium channels (VGCCs) and intracellular stores was also studied. Results indicate that mGluRs, a release of Ca(2+) from intracellular stores and GABA(B)-Rs are required for LTD. Interestingly, while mGlu1Rs seem to be involved in both short-term depression and LTD, mGlu5Rs appear to participate mostly in LTD. CGP 55845, a GABA(B)-R antagonist, partially suppressed LTD in normally sleeping (NS) rats, while completely blocking LTD in SD rats. Moreover, GS-39783, a positive allosteric modulator for GABA(B)-R, suppressed the pEPSP in SD, but not NS rats. Since both mGluRs and GABA(B)-Rs seem to be involved in the LTD, especially in SD rats, we examined if the receptor expression pattern and/or dimerization changed, using immunohistochemical, co-localization and co-immunoprecipitation techniques. Sleep-deprivation induced an increase in the expression of GABA(B)-R1 and mGlu1αR in the CA1 region of the hippocampus. In addition, co-localization and heterodimerization between mGlu1αR/GABA(B)-R1 and mGlu1αR/GABA(B)-R2 is enhanced in SD rats. Taken together, our findings present a novel form of LTD sensitive to the activation of mGluRs and GABA(B)-Rs, and reveal, for the first time, that sleep-deprivation induces alterations in the expression and dimerization of these receptors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or ...modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.
1 Fraser Laboratories, Department of Medicine, and 3 Department of Surgery, McGill University, Montreal, Quebec H3A 1A1, Canada; and 2 Edison Biotechnology Institute and Department of Biomedical ...Sciences, College of Osteopapthic Medicine, Ohio University, Athens, Ohio 45701
Submitted 22 September 2003
; accepted in final form 5 May 2004
Growth hormone, acting through its receptor (GHR), plays an important role in carbohydrate metabolism and in promoting postnatal growth. GHR gene-deficient (GHR / ) mice exhibit severe growth retardation and proportionate dwarfism. To assess the physiological relevance of growth hormone actions, GHR / mice were used to investigate their phenotype in glucose metabolism and pancreatic islet function. Adult GHR / mice exhibited significant reductions in the levels of blood glucose and insulin, as well as insulin mRNA accumulation. Immunohistochemical analysis of pancreatic sections revealed normal distribution of the islets despite a significantly smaller size. The average size of the islets found in GHR / mice was only one-third of that in wild-type littermates. Total -cell mass was reduced 4.5-fold in GHR / mice, significantly more than their body size reduction. This reduction in pancreatic islet mass appears to be related to decreases in proliferation and cell growth. GHR / mice were different from the human Laron syndrome in serum insulin level, insulin responsiveness, and obesity. We conclude that growth hormone signaling is essential for maintaining pancreatic islet size, stimulating islet hormone production, and maintaining normal insulin sensitivity and glucose homeostasis.
glucose homeostasis; immunohistochemistry; Laron syndrome; insulin tolerance test
Address for reprint requests and other correspondence: J.-L. Liu, Fraser Laboratories, M315, Royal Victoria Hospital, 687 Pine Ave. West, Montreal, QC H3A 1A1, Canada (E-mail: jun-li.liu{at}mcgill.ca ).
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