Membrane Topology and Cell Surface Targeting of Microsomal Epoxide Hydrolase

Zhu, Qin-shi; Dippe, P von; Xing, Wenxue; Lévy, Daniel

doi:10.1074/jbc.274.39.27898

Cited by 52 publications

(34 citation statements)

References 49 publications

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“…Single transmembrane proteins with dual topology have been engineered by manipulating the charge near the membrane in proteins that normally have a single orientation (19). Cytochrome P450-2E1, with one transmembrane segment, has 2% inserted in the membrane in an inverted orientation (20), and a small fraction of epoxide hydrolase has been reported to have an inverted topology (21).…”

Section: Discussionmentioning

confidence: 99%

Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers

Sebag

Hinkle

2007

Proc. Natl. Acad. Sci. U.S.A.

155

200

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The melanocortin-2 (MC2) receptor accessory protein (MRAP) is required for trafficking of the G protein-coupled MC2 receptor to the plasma membrane. The mechanism of action and structure of MRAP, which has a single transmembrane domain, are unknown. Here, we show that MRAP displays a previously uncharacterized topology. Epitopes on both the N-and C-terminal ends of MRAP were localized on the external face of CHO cells at comparable levels. Using antibodies raised against N-and C-terminal MRAP peptides, we demonstrated that both ends of endogenous MRAP face the outside in adrenal cells. Nearly half of MRAP was glycosylated at the single endogenous N-terminal glycosylation site, and over half was glycosylated when the natural glycosylation site was replaced by one in the C-terminal domain. A mutant MRAP with potential glycosylation sites on both sides of the membrane was singly but not doubly glycosylated, suggesting that MRAP is not monotopic. Coimmunoprecipitation of differentially tagged MRAPs established that MRAP is a dimer. By selectively immunoprecipitating cell surface MRAP in one or the other orientation, we showed that MRAP homodimers are antiparallel and form a stable complex with MC2 receptor. In the absence of MRAP, MC2 receptor was trapped in the endoplasmic reticulum, but with MRAP, the MC2 receptor was glycosylated and localized on the plasma membrane, where it signaled in response to ACTH. MRAP acted specifically, because it did not increase surface expression of other melanocortin, ␤2-adrenergic, or TSH-releasing hormone receptors. MRAP is the first eukaryotic membrane protein identified with an antiparallel homodimeric structure.ACTH ͉ G protein-coupled receptor ͉ membrane ͉ orientation T he G protein-coupled receptor (GPCR) family is the largest group of membrane proteins in the human genome. GPCRs respond to a wide array of signals and regulate numerous intracellular signaling pathways. They are characterized by an extracellular amino terminus that is usually glycosylated, seven transmembrane domains, and a cytoplasmic carboxyl terminus. After synthesis in the endoplasmic reticulum, GPCRs must move to the plasma membrane before they can signal in response to extracellular ligands.In the adrenal gland, the melanocortin-2 (MC2) receptor, also referred to as the corticotropin (ACTH) receptor, is activated by the pituitary hormone ACTH and promotes glucocorticoid biosynthesis. The MC2 receptor is expressed primarily in the adrenal cortex and is positively coupled to adenylyl cyclase. The MC2 receptor is a member of the class A rhodopsin-like family and the smallest known GPCR. Individuals harboring inactivating mutations in the MC2 receptor suffer from familial glucocorticoid deficiency, or hereditary unresponsiveness to ACTH (1). Recently, it was discovered that familial glucocorticoid deficiency can also arise from mutations in an accessory protein required for ACTH signaling, the MC2 receptor accessory protein (MRAP) (2). Lack of functional MRAP, like the lack of an MC2 receptor, causes ACTH re...

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Section: Discussionmentioning

confidence: 99%

Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers

Sebag

Hinkle

2007

Proc. Natl. Acad. Sci. U.S.A.

155

200

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“…Biotinylated proteins were isolated from the cell extract with immobilized streptavidin, and the presence of His-tagged ⌬5-Des was detected in the pool of surface proteins by 10% SDS-PAGE and Western blotting using an anti-His tag polyclonal antibody (Santa Cruz Biotechnology, Inc.). To establish that the reagent labeled only cell surface proteins, the biotinylation of the well characterized cytoplasmic protein GroEL was also determined using a specific polyclonal antibody (23).…”

Section: Methodsmentioning

confidence: 99%

Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

Díaz¹,

Mansilla²,

Vila³

et al. 2002

Journal of Biological Chemistry

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The Bacillus subtilis acyl-lipid desaturase (⌬5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of ⌬5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of ⌬5-Des with the reporter alkaline phosphatase (⌬5-Des-PhoA). The alkaline phosphatase activities of cells expressing the ⌬5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for ⌬5-Des as a polytopic membrane protein with six transmembrane-and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.The fatty acyl desaturases encompass a family of enzymes, representatives of which are found in all lower eukaryotes, plants and animals (for a review see Ref. 1), and some prokaryotes such as cyanobacteria (2) and bacilli (3), which have the function of introducing double bonds into fatty acyl chains.Although they all utilize molecular oxygen and reducing equivalents obtained from an electron transport chain, and the basic mechanism of the desaturation reaction may be very similar in all cases (4), fatty acid desaturases can be classified into three main subfamilies (1): (i) the soluble acyl carrier protein desaturases that introduce double bonds into fatty acids esterified to acyl carrier protein and are found in the stroma of plant plastids (4); (ii) the acyl-lipid desaturases, which are membranebound enzymes associated with the endoplasmic reticulum (1), the plant chloroplast membrane (5), the cytoplasmic membrane of some bacilli (6), and the plasmatic and thylacoid membranes of cyanobacteria (7) that desaturate fatty acids esterified in glycerolipids; (iii) the acyl-CoA desaturases, which introduce double bonds into fatty acids esterified to CoA (8) and are associated to the endoplasmic reticulum membrane of animals and fungi (1).The soluble and the membrane-bound desaturases show different consensus motifs (for a review, see 4). Database searching for these motifs reveals that they belong to two distinct multifunctional classes of iron-dependent enzymes, each of which includes desaturases, hydroxylases, and epoxidases that act on fatty acids ...

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“…1 is a bifunctional protein that is expressed with two distinct topological orientations on the hepatic endoplasmic reticulum (1,2), where it plays a central role in the metabolism of many carcinogenic xenobiotics (3). One of these topological forms is targeted to the plasma membrane, where it mediates the uptake of bile acids (4 -10).…”

Section: Microsomal Epoxide Hydrolase (Meh)mentioning

confidence: 99%

CCAAT/Enhancer-binding Protein α (C/EBPα) Activates Transcription of the Human Microsomal Epoxide Hydrolase Gene (EPHX1) through the Interaction with DNA-bound NF-Y

Zhu

Lévy

2004

Journal of Biological Chemistry

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Microsomal epoxide hydrolase (mEH) plays a central role in xenobiotic metabolism as well as mediating the sodium-dependent uptake of bile acids into the liver, where these compounds regulate numerous biological processes such as cholesterol metabolism and hepatocyte signaling pathways. Little is known, however, about the factors that control the constitutive and inducible expression of the mEH gene (EPHX1) that is altered during development and in response to numerous xenobiotics. In previous studies we have established that GATA-4 binding to the EPHX1 core promoter is critical for EPHX1 expression. The ؊80/؉25 bp core promoter also contained a reversed CCAAT box (؊5/؊1 bp), integrity of which was required for maximal basal EPHX1 transcription in HepG2 cells. Transient transfection of CCAAT/enhancer-binding protein ␣ (C/EBP␣) substantially stimulated EPHX1 promoter activity. Electrophoretic mobility shift assays, however, revealed that nuclear factor Y (NF-Y), but not C/EBP␣, directly bound to this site although increased expression of NF-Y had no effect on EPHX1 promoter activity. These results suggested that C/EBP␣ activated EPHX1 expression through its interaction with NF-Y bound to the CCAAT box. The existence of a C/EBP␣[NF-Y] complex was supported by electrophoretic mobility shift assays using antibodies against NF-Y and C/EBP␣ as well as by the ability of a dominant-negative NF-Y expression vector to inhibit promoter activity. The interaction between these transcription factors was established by co-immunoprecipitation analysis and glutathione S-transferase pull-down assays, whereas the association of the two factors and the interaction of NF-Y with the CCAAT box in vivo was confirmed by chromatin immunoprecipitation assays. C/EBP␣-dependent EPHX1 activation was also supported by reconstitution studies in HeLa cells that lack this protein. These results establish that EPHX1 expression is regulated by C/EBP␣ interacting with DNA-bound NF-Y.

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Membrane Topology and Cell Surface Targeting of Microsomal Epoxide Hydrolase

Cited by 52 publications

References 49 publications

Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers

Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers

Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

CCAAT/Enhancer-binding Protein α (C/EBPα) Activates Transcription of the Human Microsomal Epoxide Hydrolase Gene (EPHX1) through the Interaction with DNA-bound NF-Y

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