• Molecules and Cells
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• v.25 no.2
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• pp.149-157
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• 2008

Dopamine is a major neurotransmitter in the mammalian central nervous system (CNS) that regulates neuroendocrine functions, locomotor activity, cognition and emotion. The dopamine system has been extensively studied because dysfunction of this system is linked to various pathological conditions including Parkinson's disease, schizophrenia, Tourette's syndrome, and drug addiction. Accordingly, intense efforts to delineate the full complement of signaling pathways mediated by individual receptor subtypes have been pursued. Dopamine D1-like receptors are of particular interest because they are the most abundant dopamine receptors in CNS. Recent work suggests that dopamine signaling could be regulated via dopamine receptor interacting proteins (DRIPs). Unraveling these DRIPs involved in the dopamine system may provide a better understanding of the mechanisms underlying CNS disorders related to dopamine system dysfunction and may help identify novel therapeutic targets.

• CELLMED
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• v.2 no.3
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• pp.27.1-27.6
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• 2012

Red ginseng, which has a variety of biological and pharmacological activities including antioxidant, anti-inflammatory, antimutagenic and anticarcinogenic effects, has been used for thousands of years as a general tonic in traditional oriental medicine. Here, we tested the immune regulatory activities of hydrolyzed red ginseng by malted barley (HRG) on the expressions of receptor interacting proteins (Rip) 2 and $I{\kappa}B$ kinase-beta (IKK-${\beta}$) in mouse peritoneal macrophages. We show that HRG increased the activations of Rip 2 and IKK-${\beta}$ for the first time. When HRG was used in combination with recombinant interferon-${\gamma}$ (rIFN-${\gamma}$), there was a marked cooperative induction of nitric oxide (NO) production. The increased expression of inducible NO synthase from rIFN-${\gamma}$ plus HRG-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor-${\kappa}B$ (NF-${\kappa}B$). In addition, the treatment of peritoneal macrophages with rIFN-${\gamma}$ plus HRG caused significant increases in tumor necrosis factor (TNF)-${\alpha}$ mRNA expression and production. Because NO and TNF-${\alpha}$ play an important role in the immune function and host defense, HRG treatment can modulate several aspects of the host defense mechanisms as a result of the stimulations of the inducible nitric oxide synthase and NF-${\kappa}B$. In conclusion, our findings demonstrate that HRG increases the productions of NO and TNF-${\alpha}$ from rIFN-${\gamma}$-primed macrophages and suggest that Rip2/IKK-${\beta}$ plays a critical role in mediating these immune regulatory effects of HRG.

• Molecules and Cells
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• v.27 no.6
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• pp.629-634
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• 2009

G protein-coupled receptors (GPCRs) are part of multi-protein networks called 'receptosomes'. These GPCR interacting proteins (GIPs) in the receptosomes control the targeting, trafficking and signaling of GPCRs. PDZ domain proteins constitute the largest protein family among the GIPs, and the predominant function of the PDZ domain proteins is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs. We present here a machine learning based approach for the identification of GPCR-binding PDZ domain proteins. In order to characterize the network of interactions between amino acid residues that contribute to the stability of the PDZ domain-ligand complex and to encode the complex into a feature vector, amino acid contact matrices and physicochemical distance matrix were constructed and adopted. This novel machine learning based method displayed high performance for the identification of PDZ domain-ligand interactions and allowed the identification of novel GPCR-PDZ domain protein interactions.

• BMB Reports
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• v.45 no.6
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• pp.331-336
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• 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.

• BMB Reports
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• v.44 no.6
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• pp.369-374
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• 2011

MHC-I molecules play a critical role in immune surveillance against viruses by presenting peptides to cytotoxic T lymphocytes. Although the mechanisms by which MHC-I molecules assemble and acquire peptides in the ER are well characterized, how MHC-I molecules traffic to the cell surface remains poorly understood. To identify novel proteins that regulate the intracellular transport of MHC-I molecules, MHC-I-interacting proteins were isolated by affinity purification, and their identity was determined by mass spectrometry. Among the identified MHC-I-associated proteins was Tmp21, the human ortholog of yeast Emp24p, which mediates the ER-Golgi trafficking of a subset of proteins. Here, we show that Tmp21 binds to human classical and non-classical MHC-I molecules. The Tmp21-MHC-I complex lacks ${\beta}_2$-microglobulin, and the number of the complexes is increased when free MHC-I heavy chains are more abundant. Taken together, these results suggest that Tmp21 is a novel protein that preferentially binds to ${\beta}_2$-microglobulin-free MHC-I heavy chains.

• Journal of Life Science
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• v.13 no.4
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• pp.541-550
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• 2003

E2 glycoprotein of hepatitis C virus (HCV) comprises a surface of viral particle together with E1 glycoprotein, and is thought to be involved in the attachment of HCV viral particle to receptor (s) on the permissible cells including hepatocytes, B cells, T cells, and monocytes. We constructed a phage library expressing cellular proteins of hepatocytes on the phage surface, which turned out to be 8.8${\times}$$10^5$ cfu of diversity and carried inserts in 95% of library. We screened both cDNA phage library and 12-mer peptide library to identify the cellular proteins binding to E2 protein. Some intracellular proteins including tensin and membrane band 4.1 which are involved in signal transduction of survival and cytoskeleton organization, were selected from cDNA phage library through several rounds of panning and screening. On the contrary, membrane proteins such as CCR7, CKR-L2, and insulin-like growth factor-1 receptor were identified through screening of peptide library. Phages expressing peptides corresponding to those membrane proteins were bound to E2 protein specifically as determined by neutralization of binding assay. Since it is well known that HCV can infect T cells as well as hepatocytes, we examined to see if E2 protein can bind to CCR7, a member of C-protein coupled receptor family expressed on T cells, using CCR7 transfected tells. Human CCR7 cDNA was cloned into pcDNA3.1(-) vector and transfected into human embryonic kidney cell, 293T, and expressed on the surface of the cell as shown by flow cytometer. Binding assay of E2 protein using CCR7 transfected cells indicated that E2 protein bound to CCR7 by dose-dependent mode, giving rise to the possibility that CCR7 might be a putative cellular receptor for HCV.

• International Journal of Oral Biology
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• v.33 no.2
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• pp.71-76
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• 2008

The neurotrophin plays an important role in the development, differentiation and survival of the nervous system in vertebrates. It exerts its cellular effects through two different receptors, the Trk receptor tyrosine kinase neurotrophin receptor and the p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily. Trk and p75 neurotrophin receptors utilize specific target proteins to transmit signals into the cell. An ankyrin-rich membrane spanning protein (ARMS) was identified as a new p75 interacting protein and serves as a novel downstream target of p75 neurotrophin receptor. We sought to delineate the interaction between p75 and ARMS by deletion constructs of p75 and green fluorescent protein (GFP)-tagged ARMS. We examined the interaction between these two proteins after overexpressing them in HEK-293 cells. Using both Western blot analysis and immunocytochemistry followed by confocal laser scanning microscopy, we found out that the intracellular domain of the p75 neurotrophin receptor was important for the interaction with ARMS. The results from this study suggest that ARMS may play an important role for mediating the signals from p75 neurotrophin receptor into the cell.

• Animal cells and systems
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• v.5 no.2
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• pp.145-151
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• 2001

p62 is a phosphotyrosine-independent ligand of the SH2 domain of $p56^{Ick}$, a T-cell specific Src family tyrosine kinase. Recently p62 has been shown to interact with a number of proteins, such as $PKC\varsigma$ and ubiquitin, and implicated in important cellular functions such as cell proliferation. Since the two p62 interacting proteins, $p56^{Ick}$ and $PKC\varsigma$, have been reported to play roles in cell death, 1 have addressed the potential role of p62 during apoptosis in Jurkat cells in this study. Herein 1 show that p62 was specifically cleaved into two peptides by a caspase-3-like activity during Fas-receptor mediated apoptosis in Jurkat cells. This cleavage generated two fragments with molecular weights of about 35 kDa that differed in subcellular localizations. The N-terminal cleaved fragment was present in the detergent-insoluble fraction whereas the C-terminal fragment was found in the detergent-soluble fraction. In addition, the C-terminal fragment appeared to be subjected to further degradation as apoptosis prolonged. Moreover, overexpression of p62 in Jurkat cells attenuated the Fas receptor mediated apoptosis, suggesting that p62 is involved in apoptotic signal transduction pathway in lymphocytes.

• BMB Reports
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• v.49 no.2
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• pp.73-80
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• 2016

Autophagy is a process tightly regulated by various autophagy-related proteins. It is generally classified into non-selective and selective autophagy. Whereas non-selective autophagy is triggered when the cell is under starvation, selective autophagy is involved in eliminating dysfunctional organelles, misfolded and/or ubiquitylated proteins, and intracellular pathogens. These components are recognized by autophagy receptors and delivered to phagophores. Several selective autophagy receptors have been identified and characterized. They usually have some common domains, such as motif, a specific cargo interacting (ubiquitin-dependent or ubiquitin-independent) domain. Recently, structural data of these autophagy receptors has been described, which provides an insight of their function in the selective autophagic process. In this review, we summarize the most up-to-date findings about the structure-function of autophagy receptors that regulates selective autophagy.

• Archives of Pharmacal Research
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• v.26 no.10
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• pp.846-854
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• 2003

The oligomerization of G-proteincoupled receptors (GPCRs) has been shown to occur by various mechanisms, such as via disulfide covalent linkages, non covalent (ionic, hydrophobic) interactions of the N-terminal, and/or transmembrane and/or intracellular domains. Interactions between GPCRs could involve an association between identical proteins (homomers) or non-identical proteins (heteromers), or between two monomers (to form dimers) or multiple monomers (to form oligomers). It is believed that muscarinic receptors may also be arranged into dimeric or oigomeric complexes, but no systematic experimental evidence exists concerning the direct physical interaction between receptor proteins as its mechanism. We undertook this study to determine whether muscarinic receptors form homomers or a heteromers by direct protein-protein interaction within the same or within different subtypes using a yeast two-hybrid system. Intracellular loops (i1, i2 and i3) and the C-terminal cytoplasmic tails (C) of human muscarinic (Hm) receptor subtypes, Hm1, Hm2 and Hm3, were cloned into the vectors (pB42AD and pLexA) of a two-hybrid system and examined for heteromeric or homodimeric interactions between the cytoplasmic domains. No physical interaction was observed between the intracellular domains of any of the Hm/Hm receptor sets tested. The results of our study suggest that the Hm1, Hm2 and Hm3 receptors do not form dimers or oligomers by interacting directly through either the hydrophilic intracellular domains or the C-terminal tail domains. To further investigate extracellular domain interactions, the N-terminus (N) and extracellular loops (o1 and o2) were also cloned into the two-hybrid vectors. Interactions of Hm2N with Hm2N, Hm2o1, Hm2o2, Hm3N, Hm3o1 or Hm3o2 were examined. The N-terminal domain of Hm2 was found to have no direct interaction with any extracellular domain. From our results, we excluded the possibility of a direct interaction between the muscarinic receptor subtypes (Hm1, Hm2 and Hm3) as a mechanism for homo- or hetero-meric dimerization/oligomerization. On the other hand, it remains a possibility that interaction may occur indirectly or require proper conformation or subunit formation or hydrophobic region involvement.