• BMB Reports
• /
• 제45권6호
• /
• pp.331-336
• /
• 2012

The retinoid-related orphan nuclear receptor gamma ($ROR{\gamma}$) plays critical roles in regulation of development, immunity and metabolism. As transcription factor usually forms a protein complex to function, thus capturing and dissecting of the $ROR{\gamma}$ protein complex will be helpful for exploring the mechanisms underlying those functions. After construction of the recombinant tandem affinity purification (TAP) plasmid, pMSCVpuro $ROR{\gamma}$-CTAP(SG), the nuclear localization of $ROR{\gamma}$-CTAP(SG) fusion protein was verified. Following isolation of $ROR{\gamma}$ protein complex by TAP strategy, seven candidate interacting proteins were identified. Finally, the heat shock protein 90 (HSP90) and receptor-interacting protein 140 (RIP140) were confirmed to interplay with $ROR{\gamma}$ by co-immunoprecipitation. Interference of HSP90 or/and RIP140 genes resulted in dramatically decreased expression of CYP2C8 gene, the $ROR{\gamma}$ target gene. Data from this study demonstrate that HSP90 and RIP140 proteins interact with $ROR{\gamma}$ protein in a complex format and function as co-activators in the $ROR{\gamma}$-mediated regulatory processes of HepG2 cells.

• Molecules and Cells
• /
• 제44권1호
• /
• pp.1-12
• /
• 2021

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator in adipogenesis. PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPARγ-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPARγ in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPARγ. The heterodimer formation of PPARγ-RXRα was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPARγ. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPARγ by blocking RXRα recruitment to the PPARγ-RXRα heterodimer complex in adipogenesis.

• IMMUNE NETWORK
• /
• 제5권3호
• /
• pp.144-149
• /
• 2005

Background: Fc receptor-mediated phagocytosis is a complex process involving the activation of kinases and phosphatases. FcgammaRIIB has been known to transduces inhibitory signals through an immunoreceptor tyrosine-based inhibitory motif (ITIM) in cytoplasmic domains. In this study, we examined the involvement of inositol-phosphatase in the Fc receptor-mediated phagocytosis. Methods: J774 cells were infected using vaccinia viral vector containing SH2 domain-containing inositol-phosphatase (SHIP) cDNA and stimulated with the sensitized sheep red blood cells. Results: Stimulation of J774 cells induced the tyrosine phosphorylation of SHIP which was maximal at 5 minutes. Phosphatidylinositol-3 (PI-3) kinase inhibitor (wortmannin) inhibits J774 cell phagocytosis of sensitized sheep red blood cells in a dose-dependent manner. Heterologious expression of SHIP in J774 cells inhibits phagocytosis of sensitized sheep red blood cells in a dose-dependency manner, but catalytically dead mutants of SHIP has no effect on phagocytosis. Conclusion: These results strongly suggest that the active signals mediated by PI-3 kinase are opposed by inhibitory signals through SHIP in the regulation of Fc receptor-mediated phagocytosis.

• 약학회지
• /
• 제47권3호
• /
• pp.180-183
• /
• 2003

Crystal structure of TRAF6 in complex with TRAF6-binding sites from CD40 was previously determined. The structure revealed a distinct TRAF6-binding groove of CD40, the key structural determinant of interaction. The structural information leads to a proposed TRAF6-binding motif. This allows the identification of TRAF6-binding sequences in the hIRAK protein, whose functional requirement in IL-1/Toll-like receptor superfamilies-mediated signal transduction is further demonstrated using site-directed mutagenesis. The mutational effects of hIRAK on the down-stream NF-kB signaling shows the importance of the TRAF6 interface for signaling by IL-1/Toll-like receptor superfamilies.

• 통합자연과학논문집
• /
• 제10권3호
• /
• pp.125-130
• /
• 2017

Glucagon like pepide-2, one of the GLPs, is involved in various metabolic functions in the gastrointestinal tract. It plays a major role in the regulation of mucosal epithelium and the intestinal crypt cell proliferation. Because of their therapeutic importance towards the diseases in the gastrointestinal tract, it becomes necessary to study their interaction with its receptor, GLP-2R. In this study, we have developed protein-protein docking complexes of GLP-2 - GLP-2 receptor. Homology models of GLP-2 are developed, and a reliable model out of the predicted models was selected after model validation. The model was bound with the receptor, to study the important interactions of the complex. This study could be useful in developing novel and potent drugs for the diseases related with GLP-2.

• Biomolecules & Therapeutics
• /
• 제18권2호
• /
• pp.152-158
• /
• 2010

Melanin concentrating hormone is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G-protein coupled receptor family, especially plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome. Although fluorescence-based calcium mobilization assay platform has been one of the most widely accepted tools for receptor research and drug discovery, fluorescence interference and shallow assay window limit their application in high throughput screening and have led to a growing interest in alternative, luminescence-based technologies. Herein, a luminescence-based functional assay system for the MCH1 receptor was developed and validated with the mitochondrial targeted aequorin. Aequorin based functional assay system for MCH1 presented excellent Z' factor (0.8983) and high signal-to-noise ratio (141.9). The nonpeptide MCH1 receptor antagonist, SNAP 7941 and GSK 803430, exhibited $IC_{50}$ values of 0.62 ${\pm}$ 0.11 and 12.29 ${\pm}$ 2.31 nM with excellent correlation coefficient. These results suggest that the aequorin based assay system for MCH1 is a strong alternative to the traditional GPCR related tools such as radioligand binding experiments and fluorescence functional determinations for the compound screening and receptor research.

• Journal of Plant Biotechnology
• /
• 제42권4호
• /
• pp.277-283
• /
• 2015

Plant genome analyses, including Arabidopsis thaliana showed a large gene family of plant receptor kinases with various extracellular ligand-binding domain. Now intensively studies to understand physiological and cellular functions for higher plant receptor kinases in diverse and complex biological processes including plant growth, development, ligands perception including steroid hormone and plant-microbe interactions. Brassinosteroids (BRs) as a one of well know steroid hormone are plant growth hormones that control biomass accumulation and also tolerance to many biotic and abiotic stress conditions and hence are of relevance to agriculture. BRI1 receptor kinase, which is localized in plasma membrane in the cell sense BRs and it bind to a receptor protein known as BRASSINOSTEROID INSENSITIVE 1 (BRI1). Recently, we reported that BRI1 and its co-receptor, BRI1-ASSOCIATED KINASE (BAK1) autophosphorylated on tyrosine residue (s) in vitro and in vivo and thus are dual-specificity kinases. Other plant receptor kinases are also phosphorylated on tyrosine residue (s). Post-translational modifications (PTMs) can be studied by altering the residue modified by directed mutagenesis to mimic the modified state or to prevent the modification. These approaches are useful to not only characterize the regulatory role of a given modification, but may also provide opportunities for plant improvement.

• BMB Reports
• /
• 제31권5호
• /
• pp.468-474
• /
• 1998

Membrane depolarization in PC12 cells induces calcium influx via an L-type voltage-sensitive calcium channel (L-VSCC) and increases intracellular free calcium, which leads to tyrosine phosphorylation of epidermal growth factor (EGF) receptor and the associated adaptor protein, She. This activated EGF receptor complex then can activate mitogen-activated protein (MAP) kinase, as in nerve growth factor (NGF) receptor activation. In the present study, we investigated the role of EGF receptor in the signaling pathway initiated by membrane depolarization of PC12 cells. Prolonged membrane depolarization induced phosphorylation of extracellular signal-regulated kinase (ERK) within 1 min in undifferentiated PC12 cells. Pretreatment of PC12 cells with the calcium chelator EGTA abolished depolarization-stimulated ERK phosphorylation, but NGF-induced phosphorylation of ERK was not affected. The chronic treatment of phorbol ester, which down-regulated the activity of protein kinase C (PKC), did not affect the phosphorylation of ERK upon depolarization. In the presence of an inhibitor of EGF receptor, neither depolarization nor calcium ionophore increased the level of ERK phosphorylation. These data imply that the EGF receptor is functionally necessary to activate ERK and neurite outgrowth in response to the prolonged depolarization in PC12 cells, and also that PKC is apparently not involved in this signaling pathway.

• Clinical Psychopharmacology and Neuroscience
• /
• 제16권4호
• /
• pp.383-390
• /
• 2018

Objective: Autism spectrum disorder (ASD) is a complex neurodevelopmental syndrome with an increasingly prevalent etiology, yet not fully understood. It has been thought that vitamin D, complex B vitamin levels and homocysteine are associated with environmental factors and are important in ASD. The aim of this study was to examine serum vitamin D, vitamin D receptor (VDR), homocysteine, vitamin B6, vitamin B12 and folate levels in ASD. Methods: In this study, serum vitamin D and VDR, homocysteine, vitamins B6, B12 and folate levels were determined in 60 patients with ASD (aged 3 to 12 years) and in 45 age-gender matched healthy controls. In addition, calcium, phosphorus and alkaline phosphatase, which are associated with vitamin D metabolism, were measured from serum in both groups. ASD severity was evaluted by the Childhood Autism Rating Scale (CARS). Results: Serum vitamin D and VDR were substantially reduced in patients with ASD in comparision to control group. However, homocysteine level was significantly higher and vitamin B6, vitamin B12 and folate were also reduced in patients with ASD. Total CARS score showed a positive association with homocysteine and a negative correlation with vitamins D,B6, B12, folate and VDR. Conclusion: This comprehensive study, which examines many parameters has shown that low serum levels of vitamins D, B6, B12, folate and VDR as well as high homocysteine are important in the etiopathogenesis of ASD. However, further studies are required to define the precise mechanism(s) of these parameters and their contributions to the etiology and treatment of ASD.

• Archives of Pharmacal Research
• /
• 제21권4호
• /
• pp.423-428
• /
• 1998

The ligand binding signals to a wide variety of seven transmembrane cell surface receptors are transduced into intracellular signals through heterotrimeric G-proteins. Recently, there have been reports which show diverse coupling patterns of ligand-activated receptors to the members of Gq family $\alpha$ subunits. In order to shed some light on these complex signal processing networks, interactions between G$\alpha$q family of G protein and neurokinin-2 receptor as well as muscarinic M$_{1}$ receptor, which are considered to be new thearpeutic targets in asthma, were studied. Using washed membranes from Cos-7 cells co-transfected with different G.alpha.q and receptor cDNAs, the receptors were stimulated with various concentrations of carbachol and neurokinin A and the agonist-dependent release of [$^3H$]inositol phosphates through phospholipase C beta-1 activation was measured. Differential coupling of Gaq family of G-protein to muscarinic M$_{1}$ receptor and neurokinin-2 receptor was observed. The neurokinin-2 receptor shows a ligand-mediated response in membranes co-transfected with G$\alpha$q, G$\alpha$11 and G$\alpha$14 but not G$\alpha$16 and the ability of the muscarinic $M_1$ receptor to activate phospholipase C through G$\alpha$/11 but not G$\alpha$14 and G$\alpha$16 was demonstrated. Clearly G$\alpha$/11 can couple $\M_1$ and neurokinin-2 receptor to activate phospholipase C. But, there are differences in the relative coupling of the G$\alpha$14 and G$\alpha$16 subunits to these receptors.