孤儿核受体类固醇生成因子1

孤儿核受体是核受体超家族中较独特的成员,它参与了糖类、脂类及胆固醇和类固醇激素的代谢,可能是体内细胞基本功能的重要调节因子。孤儿核受体SF－1最初作为肾上腺和性腺中的P450羟化酶必需的调节子而被鉴定,在类固醇组织、垂体和下丘脑腹内侧核均有表达,SF01在基础结构上具有不同于其他核受体的特征结构域,并广泛参与许多基因如类固醇合成酶类、Muellerian抑制性物质、黄体生成素β亚基启动子等的表达调控,基因剔除实验证实SF－1是肾上腺类固醇合成和性别分化中的一个关键调节因子。     

核受体辅活化子PNRC与孤儿核受体SF1相互作用位点的鉴定

为了阐明核受体辅活化子 (proline richnuclearreceptorcoactivatorprotein ,PNRC)在孤儿核受体类固醇生成因子 1(steroidogenicfactor1,SF1)基因表达调控中的作用 ,采用酵母双杂合分析、缺失突变技术和瞬时转染等研究方法鉴定了PNRC与SF1的相互作用位点 .结果显示 ,PNRC中氨基酸 2 78～ 30 0区域是与SF1相互作用的位点 .该区域富含脯氨酸 ,其中有 1个SH3结合模体 (motif) ,单独的SH3模体不足以与SF1产生有效的相互作用 .瞬时转染分析表明 ,PNRC 2 70 32 7对野生型PNRC的辅激活功能具有负显性抑制效应 .研究结果表明 ,含SH3结合模体的PNRC 2 78 30 0区域是与SF1相互作用的位点     

Defining a Canonical Ligand-Binding Pocket in the Orphan Nuclear Receptor Nurr1

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核孤儿受体在胆固醇分解代谢过程中的调控作用

由胆固醇合成胆汁酸有两条途径。一条是经典途径或中性途径[1] ,也是合成胆汁酸最主要的途径 ,其限速反应为胆固醇 7α 羟化酶 (由Ｃｙｐ7ａ编码 )催化胆固醇羟化为 7α 羟胆固醇的反应。近几年发现另一条胆汁酸合成的酸性途径[2 ] ,这一途径开始于胆固醇转变为氧固醇 (ｏｘｙｓｔｅｒｏｌ) ,然后经氧固醇 7α 羟化酶 (由Ｃｙｐ7ｂ编码 )催化产生 7α 羟氧固醇化合物 ,再融入胆汁酸合成经典途径的下游步骤。由于胆固醇在体内有重要作用 ,有效调节胆固醇分解代谢以维持胆固醇水平的动态平衡十分重要。研究发现 ,胆汁酸以及合成过程中的中间产…     

Explosive Lineage-Specific Expansion of the Orphan Nuclear Receptor HNF4 in Nematodes

The nuclear receptor superfamily expanded in at least two episodes: one early in metazoan evolution, the second within the vertebrate lineage. An exception to this pattern is the genome of the nematode Caenorhabditis elegans, which encodes more than 270 nuclear receptors, most of them highly divergent. We generated 128 cDNA sequences for 76 C. elegans nuclear receptors, confirming that these are active genes. Among these numerous receptors are 13 orthologues of nuclear receptors found in arthropods and/or vertebrates. We show that the supplementary nuclear receptors (supnrs) originated from an explosive burst of duplications of a unique orphan receptor, HNF4. This origin has specific implications for the role of ligand binding in the function and evolution of the nematode supplementary nuclear receptors. Moreover, the supplementary nuclear receptors include a group of very rapidly evolving genes found primarily on chromosome V. We propose a model of lineage-specific duplications from a chromosome on which duplication and substitution rates are highly increased. Our results provide a framework to study nuclear receptors in nematodes, as well as to consider the functional and evolutionary consequences of lineage-specific duplications.Reviewing Editor: Dr. Nicolos Galtier     

核受体辅活化子PNRC与孤儿核受体SF1的相互作用有赖于SF1的AF-2功能结构域

核受体辅活化子PNRC(proline richnuclearreceptorcoregulatoryprotein ,富含脯氨酸的核受体辅调节蛋白 )可通过含SH3结合模体的PNRC2 78 30 0区域与孤儿核受体类固醇生成因子 1(steroido genicfactor 1,SF1)相互作用 .激活功能 2 (activationfunction 2 ,AF 2 )结构域在核受体配体依赖性转录激活中发挥了重要作用 ,为探讨AF 2结构域在SF1转录激活中的作用机制 ,采用酵母双杂合分析、缺失突变技术和瞬时转染等研究方法考察了AF 2结构域对SF1反式激活功能及SF1与PNRC相互作用的影响 .SF1的反式激活功能有赖于AF 2结构域 ,其机制是SF1AF 2结构域的突变严重影响了SF1与PNRC的有效相互作用 ,并消除了PNRC对SF1反式激活功能的辅激活作用 .结果表明 ,SF1与PNRC的相互作用有赖于AF 2的功能结构域     

Regulation of Platelet-derived Growth Factor Receptor Function by Integrin-associated Cell Surface Transglutaminase

A functional collaboration between growth factor receptors such as platelet derived growth factor receptor (PDGFR) and integrins is required for effective signal transduction in response to soluble growth factors. However, the mechanisms of synergistic PDGFR/integrin signaling remain poorly understood. Our previous work showed that cell surface tissue transglutaminase (tTG) induces clustering of integrins and amplifies integrin signaling by acting as an integrin binding adhesion co-receptor for fibronectin. Here we report that in fibroblasts tTG enhances PDGFR-integrin association by interacting with PDGFR and bridging the two receptors on the cell surface. The interaction between tTG and PDGFR reduces cellular levels of the receptor by accelerating its turnover. Moreover, the association of PDGFR with tTG causes receptor clustering, increases PDGF binding, promotes adhesion-mediated and growth factor-induced PDGFR activation, and up-regulates downstream signaling. Importantly, tTG is required for efficient PDGF-dependent proliferation and migration of fibroblasts. These results reveal a previously unrecognized role for cell surface tTG in the regulation of the joint PDGFR/integrin signaling and PDGFR-dependent cell responses.Adhesion of cells to the extracellular matrix (ECM)² regulates a wide range of cellular processes, including cell survival, growth, migration, and differentiation. A central paradigm in the field entails both physical association and functional collaboration between integrins and growth factor receptors (GFRs) in the regulation of cell responses to the ECM and soluble growth factors (1). In particular, the engagement of β1 and αvβ3 integrins with ECM ligands transiently activates platelet-derived growth factor (PDGF) receptor-tyrosine kinase even in the absence of its soluble ligands and promotes and sustains growth factor-initiated signaling by PDGFR (2). Despite a significance of this synergistic signaling, the molecular mechanisms underlying the cross-talk between the two receptor systems remain unknown. A direct or indirect association between these two types of signaling receptors may be enhanced by their co-sequestering in cholesterol-enriched membrane microdomains (3). Because integrins and receptor-tyrosine kinases share many downstream signaling targets, integrin-ECM interaction may also increase availability of signal relay enzymes and adapter proteins to receptor-tyrosine kinases by promoting their recruitment from cytosol to the plasma membrane (4).PDGF is a major survival factor, mitogen, and motogen for mesenchymal cells (5). This ligand-receptor pair is implicated in tumor-associated processes, including autocrine growth stimulation of tumor cells, tumor angiogenesis, and regulation of stromal fibroblasts (6). Atherosclerosis in the vessel wall and restenosis after angioplasty also involve hyperactivation of the PDGF-PDGFR signaling axis in vascular smooth muscle cells (7). Likewise, skin wound healing and liver, lung, and kidney fibrosis depend on PDGF-mediated signaling and cell responses (8). Importantly, ECM composition and cell-matrix interactions modulate cell responsiveness to PDGF (9).Upon binding a dimeric PDGF molecule, PDGFR undergoes dimerization and autophosphorylation of tyrosine residues in trans because of the juxtaposition of cytoplasmic tails of the receptor. Phosphorylation of the conserved tyrosine residue in the kinase domain (Tyr-849 of PDGFRα and Tyr-857 of PDGFRβ) increases catalytic activity of the kinases, whereas autophosphorylation of tyrosine residues outside the kinase domain creates docking sites for signal transduction proteins containing Src homology 2 domains. The latter include various enzymes such as phosphatidylinositol 3-kinase, phospholipase Cγ, the Src family tyrosine kinases, the tyrosine phosphatase Shp-2, and the GTPase activating protein for Ras, RasGAP. Other PDGFR binding partners including Grb2, Grb7, Nck, Shc, and Crk lacking enzymatic activity but serve adapter functions in the downstream signaling pathways (10).Previous studies revealed a transient PDGF-independent tyrosine phosphorylation of PDGFRβ in human fibroblasts during adhesion on fibronectin or collagen type I, whereas similar PDGFRβ activation response was reproduced by application of external strain to quiescent cells (2). Clustering of integrins with fibronectin-coated beads was shown to stimulate PDGFR phosphorylation in fibroblasts (11). Furthermore, fibronectin was found to promote PDGF-mediated signaling in fibroblasts by increasing association of phosphatase Shp-2 with PDGFR and limiting the time that the negative signaling regulator, RasGAP, interacts with the receptor (4). Whereas these results implicate cell-ECM interactions and integrin function in the regulation of PDGFR activity, many details of this functional cross-talk remain unknown.Tissue transglutaminase (tTG) is a multifunctional protein that possesses Ca²⁺-dependent transamidating and GTPase activities (12). On the surface of various cells, all the tTG forms stable non-covalent complexes with β1 and β3 integrins and functionally collaborates with these receptors by acting as a co-receptor for fibronectin (13). This adhesive function of tTG is involved in the assembly of fibronectin matrices and cell migration on fibronectin (14–16). tTG broadly affects integrin signaling by promoting their clustering and increasing activation of focal adhesion kinase and RhoA (13, 17). Thus, we set to examine whether signaling mediated by GFRs, which depends on the integrin function, is altered by tTG.Here we present a novel mechanistic insight into the cross-talk between integrin and PDGFR signaling pathways. We provide evidence that tTG interacts with PDGFR on the cell surface and mediates its physical association with integrins. In turn, the formation of stable integrin-tTG-PDGFR ternary complexes promotes PDGFR activation and downstream signaling, regulates the receptor turnover, and amplifies PDGFR-mediated cellular responses. These studies reveal a novel function of tTG in coupling the adhesion-mediated and growth factor-dependent signaling pathways. They suggest that this tTG activity might be involved in pro-inflammatory function of this protein in normal wound healing and tissue fibrosis (18), vascular remodeling (19), and tumor metastasis (20).     

核受体转录辅激活蛋白：结构与功能

核受体超家族大体可分为 3个亚类 :(1 )由雌激素 (ｅｓｔｒｏｇｅｎ ,ＥＲ)、孕激素 (ｐｒｏ ｇｅｓｔｉｎ ,ＰＲ)和糖皮质激素 (ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ ,ＧＲ)等类固醇激素受体构成的Ｉ类受体 ;(2 )由甲状腺素 (ｔｈｙｒｏｉｄｈｏｒｍｏｎｅ ,ＴＲ)、维生素Ｄ(ｖｉｔａ ｍｉｎＤ ,ＶＤＲ)、9 顺 /反式视黄酸 (9 ｃｉｓ/ｔｒａｎｓ ｒｅｔｉｎｏｉｃａｃｉｄ ,ＲＸＲ ,ＲＡＲ)等构成的ＩＩ类受体 ;(3)天然配体未知或不需要的孤儿受体。三类核受体的作用方式虽然不同 ,但在结构上却有共同的特点 ,它们的典型结构分为6个部分 ,即Ａ、…     

An Evolutionarily Conserved Arginine Is Essential for Tre1 G Protein-Coupled Receptor Function During Germ Cell Migration in Drosophila melanogaster

Background

G protein-coupled receptors (GPCRs) play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1) is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration.

Methodology/Principal Findings

First, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo.

Conclusions/Significance

These structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors.     

Receptor Tyrosine Kinase Signaling and Primordial Germ Cell Development

Primordial germ cells (PGCs) give rise to sperms and eggs. Their development is crucial to species propagation and has to be precisely controlled. Studies in several model organisms have identified many genes involved in the specification and guided migration of PGCs. However, the mechanisms governing the behaviors of this unique type of cells remain to be investigated. Interestingly, PGCs share certain cellular properties with metastasizing cancer cells including proliferation, invasion of other tissues, survival, and migration. Recently we have shown that in Drosophila the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development. In later stages, activation of both STAT and Ras, likely by other molecules, is required continuously for PGC migration. The requirement for RTK suggests molecular conservation between flies and mice in PGC development and also suggests that germ cells and cancer cells share certain intracellular signaling strategies.