Modulation of NMDA Receptor Subunit mRNA in Butorphanol-Tolerant and —Withdrawing Rats

The NMDA receptor has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of butorphanol on the modulation of NMDA receptor subunit NR1, NR2A, NR2B, and NR2C gene expression were investigated by using in situ hybridization technique. Continuous intracerebroventricular (i.c.v.) infusion with butorphanol (26 nmol/l/h) resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels. The level of NR1 mRNA was significantly decreased in the cerebral cortex, thalamus, and CA1 area of hippocampus in butorphanol tolerant and withdrawal (7 h after stopping the infusion) rats. The NR2A mRNA was significantly decreased in the CA1 and CA3 of hippocampus in tolerant rats and increased in the cerebral cortex and dentate gyrus in butorphanol withdrawal rats. NR2B subunit mRNA was decreased in the cerebral cortex, caudate putamen, thalamus, CA3 of hippocampus in butorphanol withdrawal rats. No changes of NR1, NR2A, NR2C subunit mRNA in the cerebellar granule cell layer were observed in either butorphanol tolerant or withdrawal rats. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased significantly in all brain regions except in the thalamus and hippocampus, at the 7 hr after stopping the butorphanol infusion. These results suggest that region-specific changes of NMDA receptor subunit mRNA (NR 1 and NR2) as well as NMDA receptor binding ([³H]MK-801) are involved in the development of tolerance to and withdrawal from butorphanol.     

Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gα_s mutant proteins (from a screen of 66 Gα_s mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gα_s. This extended Linker 2 is the target site of a natural product inhibitor of G_q. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β₂/β₃ loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.     

Presenilin-1/γ-Secretase Controls Glutamate Release,Tyrosine Phosphorylation,and Surface Expression of N-Methyl-d-aspartate Receptor (NMDAR) Subunit GluN2B

Abnormally high concentrations of extracellular glutamate in the brain may cause neuronal damage via excitotoxicity. Thus, tight regulation of glutamate release is critical to neuronal function and survival. Excitotoxicity is caused mainly by overactivation of the extrasynaptic NMDA receptor (NMDAR) and results in specific cellular changes, including calcium-induced activation of calpain proteases. Here, we report that presenilin-1 (PS1) null mouse cortical neuronal cultures have increased amounts of calpain-dependent spectrin breakdown products (SBDPs) compared with WT cultures. NMDAR antagonists blocked accumulation of SBDPs, suggesting abnormal activation of this receptor in PS1 null cultures. Importantly, an increase in SBDPs was detected in cultures of at least 7 days in vitro but not in younger cultures. Conditioned medium from PS1 null neuronal cultures at 8 days in vitro contained higher levels of glutamate than medium from WT cultures and stimulated production of SBDPs when added to WT cultures. Use of glutamate reuptake inhibitors indicated that accumulation of this neurotransmitter in the media of PS1 null cultures was due to increased rates of release. PS1 null neurons showed decreased cell surface expression and phosphorylation of the GluN2B subunit of NMDAR, indicating decreased amounts of extrasynaptic NMDAR in the absence of PS1. Inhibition of γ-secretase activity in WT neurons caused changes similar to those observed in PS1 null neurons. Together, these data indicate that the PS1/γ-secretase system regulates release of glutamate, tyrosine phosphorylation, and surface expression of GluN2B-containing NMDARs.     

Expression of Set-α during Morphogenesis of Mouse Lower First Molar

The detailed in situ expression pattern of the Set-α gene has been studied. Previously we showed that Set-α is a differentially expressed gene in the embryonic mouse mandible at day 10.5 (E10.5) gestational age. Cells expressing Set-α were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5. At E12, they were slightly aggregated in an area where tooth germ of the lower first molar is estimated to be formed. At E13.5, Set-α was strongly expressed in the tooth germ. At the cap stage, Set-α was expressed in the enamel organ and dental papilla. At the bell stage, Set-α was distinctly expressed in the inner enamel epithelial and dental papilla cells facing the inner enamel epithelial layer, which were intended to differentiate into ameloblasts and odontoblasts, respectively. Interestingly, Set-α was also expressed in several embryonic craniofacial tissues derived from the ectoderm. This study is the first report that Set-α is distinctly expressed in the developing tooth germ, and suggests that Set-α plays an important role in both the initiation and the growth of the tooth germ, as well as in the differentiation of ameloblasts and odontoblasts.     

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca²⁺ channel complexes. Ca_Vα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca_V1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca_Vα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca_Vα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca_Vα2δ1-mediated increase in the peak current density and voltage-dependent gating of Ca_V1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca_Vα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca_Vα2δ1 as well as the Ca_Vα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca_Vα2δ1, and furthermore that N-glycosylation of Ca_Vα2δ1 is essential to produce functional L-type Ca²⁺ channels.     

Pharmacophore-directed Homology Modeling and Molecular Dynamics Simulation of G Protein-coupled Receptor： Study of Possible Binding Modes of 5-HT2c Receptor Agonists

A new pharmacophore-based modeling procedure, including homology modeling, pharmacophore study, flexible molecular docking, and long-time molecular dynamics (MD) simulations, was employed to construct the structure of the human 5-HT_(2C) receptor and determine the characteristics of binding modes of 5-HT_(2C) receptor agonists. An agonist-receptor complex has been constructed based on homology modeling and a pharmacophore hypothesis model based on some high active compounds. Then MD simulations of the ligand-receptor complex in an explicit membrane environment were carried out. The conformation of the 5- HT_(2C) receptor during MD simulation was explored, and the stable binding modes of the studied agonist were determined. Flexible molecular docking of several structurally diverse agonists of the human 5-HT_(2C) receptor was carried out, and the general binding modes of these agonists were investigated. According to the models presented in this work and the results of Flexi-Dock, the involvement of the amino acid residues Asp134, Ser138, Ash210, Asn331, Tyr358, Ile131, Ser132, Val135, Thr139, Ile189, Val202, Val208, Leu209, Phe214, Val215, Gly218, Ser219, Phe223, Trp324, Phe327, and Phe328 in agonist recognition was studied. The obtained binding modes of the human 5-HT_(2C) receptor agonists have good agreement with the site-directed mutagenesis data and other studies.     

Differential Modulation of ��- and ��-Opioid Receptor Agonists by Endogenous RGS4 Protein in SH-SY5Y Cells

Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins have been reported to modulate opioid receptor signaling. Here we show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express μ- and δ-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by ∼90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of δ-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was increased compared with control cells. This effect was reversed by transient transfection of a stable RGS4 mutant (HA-RGS4C2S). Furthermore, MAPK activation by SNC80 was increased in cells with knockdown of RGS4. In contrast, there was no change in the μ-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the δ-opioid receptor but not the μ-opioid receptor could be precipitated together with the stable RGS4. Using chimeras of the δ- and μ-opioid receptors, the C-tail and third intracellular domain of the δ-opioid receptor were suggested to be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating δ-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4.μ- and δ-opioid receptors are members of the G protein-coupled receptor family and interact with Gα_i/o proteins (1, 2). This results in signaling to a variety of downstream effectors, including adenylyl cyclase and the mitogen-activated protein kinase (MAPK)² cascade. Signaling of opioid receptors is regulated negatively by regulator of G protein signaling (RGS) proteins (3, 4). These are a family of molecules containing a “RGS consensus” domain that bind to Gα subunits and act as GTPase-accelerating proteins to increase the rate of GTP hydrolysis. This results in a decrease in the lifetime of the active Gα-GTP and free Gβγ subunits and limits signaling to downstream effectors (5–8). The mechanisms by which RGS proteins selectively modulate G protein-mediated receptor signal transduction pathways, especially opioid receptor signaling, are beginning to unfold (9–12). The foundation for the function and selectivity of RGS proteins in regulating opioid signaling lies in their ability to interact with opioid receptors and their cognate G proteins. In general, the selectivity or the preference of an RGS protein for a particular receptor is determined by a variety of factors, including tissue-specific expression and precise interaction with the intracellular domains of receptor proteins, G protein subunits, and effectors as well as other pathway-specific components (13).The effects of RGS proteins on opioid receptor signaling have been examined in several systems. The findings are not always consistent, probably due to the different methodologies used. It has been shown that members of the RZ, R4, and R7 subfamilies (7) of RGS proteins play crucial roles not only in terminating acute opioid agonist action but also in opioid receptor desensitization, internalization, recycling, and degradation (3, 14), thereby affecting opioid tolerance and dependence (15–18). Much work has been performed with RGS4, because it is a smaller RGS protein with a structure consisting of the RGS consensus (box) sequence and a small N terminus (19, 20). It also has a wide distribution in the brain, especially in brain regions important for opioid actions, including the striatum, locus coeruleus, dorsal horn of the spinal cord, and cerebral cortex (21). In vitro RGS4 has been shown to reverse δ-opioid receptor agonist-induced inhibition of cAMP synthesis in membranes prepared from NG108-15 cells (6). Overexpression of RGS4 in HEK293 cells also attenuated morphine-, [d-Ala²,N-Me-Phe⁴,Gly-ol⁵]enkephalin (DAMGO)-, and [d-Pen²,d-Pen⁵]enkephalin (DPDPE)-induced inhibition of adenylyl cyclase (22, 23). Co-expression of RGS4 with GIRK1/GIRK2 channels in Xenopus oocytes reduced the basal K⁺ current and accelerated the deactivation of GIRK channels activated by κ-opioid receptor agonist {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (24). Although these previous studies have provided evidence that RGS4 can negatively regulate opioid receptor signaling, they do not confirm a functional role for endogenous RGS4 in endogenous, nontransfected systems.Human neuroblastoma SH-SY5Y cells endogenously express μ- and δ-opioid receptors and a variety of Gα_i/o proteins (25–27). Here we show that RGS4 is abundantly found at both the mRNA and protein levels in these cells. Consequently, we used SH-SY5Y cells to examine the hypothesis that RGS4 negatively modulates opioid receptor signaling under physiological conditions. The endogenously expressed RGS4 level in SH-SY5Y cells was reduced using lentiviral delivery of short hairpin RNA (shRNA) targeting the RGS4 gene. This resulted in changes in δ- but not μ-opioid receptor-mediated signaling to adenylyl cyclase and the MAPK pathway. These findings argue for a selective interaction of RGS4 with the δ-opioid receptor. To test this, we expressed FLAG-tagged μ- and δ-opioid receptors together with a construct for a stable, proteosome-resistant RGS4 protein in HEK293T cells. Co-immunoprecipitation indicated that the δ-opioid but not the μ-opioid receptor was closely associated with RGS4, providing further evidence for a selective interaction between RGS4 and δ-opioid receptor signaling.     

Determinants in the β and δ Subunit Cytoplasmic Loop Regulate Golgi Trafficking and Surface Expression of the Muscle Acetylcholine Receptor

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25–28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.     

Expression,Purification, and Evaluation of Anti-IL-2Rα Antibody Secreted by Leishmania tarentolae

International Journal of Peptide Research and Therapeutics - Producing a functional anti-IL-2Rα antibody in Leishmania tarentolae, a trypanosomatid protozoan non-pathogenic to human, is a...     

Citronellol and Geraniol,Components of Rose Oil,Activate Peroxisome Proliferator-Activated Receptor α and γ and Suppress Cyclooxygenase-2 Expression

We evaluated the effects of rose oil on the peroxisome proliferator-activated receptor (PPAR) and cyclooxygenase-2 (COX-2). Citronellol and geraniol, the major components of rose oil, activated PPARα and γ, and suppressed LPS-induced COX-2 expression in cell culture assays, although the PPARγ-dependent suppression of COX-2 promoter activity was evident only with citronellol, indicating that citronellol and geraniol were the active components of rose oil.     

Estrogen Receptor β Signaling Induces Autophagy and Downregulates Glut9 Expression

Glut9 is highly expressed in the human kidney proximal convoluted tubular and plays a crucial role in the regulation of plasma urate levels. The gene effects were stronger among women. Our results show that 17-β-estradiol (E2) through ER (estrogen receptor) β downregulates Glut9 protein expression on human renal tubular epithelial cell line (HK2). Intriguingly, E2 does not affect the expression of Glut9 mRNA. ERβ is linked to PTEN, the PTEN gene negatively regulates the PI3K/AKT pathway, and the PI3K/AKT pathway inhibition may lead to autophagy. Further study indicates that ERβ may affect the expression of Glut9 though autophagy.