SGK1 increases Na,K-ATP cell-surface expression and function in Xenopus laevis oocytes

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

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Abrogation of cell surface expression of human class I transplantation antigens by an adenovirus protein in Xenopus laevis oocytes.

The Journal of Cell Biology ◽

10.1083/jcb.101.2.540 ◽

1985 ◽

Vol 101 (2) ◽

pp. 540-547 ◽

Cited By ~ 16

Author(s):

L Severinsson ◽

P A Peterson

Keyword(s):

Cell Surface ◽

Heavy Chain ◽

Viral Protein ◽

Surface Expression ◽

Class I ◽

Cell Surface Expression ◽

Xenopus Laevis Oocytes ◽

Human Class ◽

Heavy Chains ◽

Transplantation Antigens

Class I transplantation antigens form complexes with a virus protein encoded in the early region E3 of the adenovirus-2 genome. The interaction between this viral glycoprotein, E19, and nascent human class I antigens has been examined by microinjecting purified mRNA into Xenopus laevis oocytes. Both E19 and the two class I antigen subunits, the heavy chain and beta 2-microglobulin (beta 2M), were efficiently translated. The heavy chains did not become terminally glycosylated, as monitored by endoglycosidase H digestion, and were not expressed on the oocyte surface unless they were associated with beta 2M. The E19 protein did not become terminally glycosylated, and we failed to detect this viral protein on the surface of the oocytes. Co-translation of heavy chain and E19 mRNA demonstrated that the two proteins associate intracellularly. However, neither protein appeared to be transported to the trans-Golgi compartment. Similar observations were made in adenovirus-infected HeLa cells. Heavy chains bound to beta 2M became terminally glycosylated in oocytes in the presence of low concentrations of E19. At high concentrations of the viral protein, no carbohydrate modifications and no cell surface expression of class I antigens were apparent. Thus, beta 2M and E19 have opposite effects on the intracellular transport of the heavy chains. These data suggest that adenovirus-2 may impede the cell surface expression of class I antigens to escape immune surveillance.

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Regulation of the Cell Surface Expression and Function of Angiotensin II Type 1 Receptor by Rab1-mediated Endoplasmic Reticulum-to-Golgi Transport in Cardiac Myocytes

Journal of Biological Chemistry ◽

10.1074/jbc.m405988200 ◽

2004 ◽

Vol 279 (39) ◽

pp. 41077-41084 ◽

Cited By ~ 71

Author(s):

Catalin M. Filipeanu ◽

Fuguo Zhou ◽

William C. Claycomb ◽

Guangyu Wu

Keyword(s):

Endoplasmic Reticulum ◽

Angiotensin Ii ◽

Cell Surface ◽

Cardiac Myocytes ◽

Surface Expression ◽

Cell Surface Expression ◽

Golgi Transport ◽

And Function

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COMMD1 downregulates the epithelial sodium channel through Nedd4–2

AJP Renal Physiology ◽

10.1152/ajprenal.00257.2009 ◽

2010 ◽

Vol 298 (6) ◽

pp. F1445-F1456 ◽

Cited By ~ 32

Author(s):

Ying Ke ◽

A. Grant Butt ◽

Marianne Swart ◽

Yong Feng Liu ◽

Fiona J. McDonald

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Sodium Channel ◽

Rat Kidney ◽

Copper Metabolism ◽

Epithelial Sodium Channel ◽

Surface Expression ◽

Dominant Negative ◽

Cell Surface Expression ◽

Xenopus Laevis Oocytes

The epithelial sodium channel (ENaC) is important for the long-term control of Na+ homeostasis and blood pressure. Our previous studies demonstrated that Copper Metabolism Murr1 Domain-containing protein 1 (COMMD1; previously known as Murr1), a protein involved in copper metabolism, inhibited amiloride-sensitive current in Xenopus laevis oocytes expressing ENaC ( J Biol Chem 279: 5429, 2004). In this study, we report that COMMD1 inhibits amiloride-sensitive current in mammalian epithelial cells expressing ENaC, that the COMM domain of COMMD1 is sufficient for this effect, and that knockdown of COMMD1 increases amiloride-sensitive current. COMMD1 is coexpressed with ENaC in rat kidney medulla cells. COMMD1 increased ubiquitin modification of ENaC and decreased its cell surface expression. COMMD1 abolished insulin-stimulated amiloride-sensitive current and attenuated the stimulation of current by activated serum and glucocorticoid-regulated kinase (SGK1). COMMD1 was found to interact with both SGK1 and Akt1/protein kinase B, and knockdown of COMMD1 enhanced the stimulatory effect of both SGK1 and Akt1 on amiloride-sensitive current. COMMD1's effects were reduced in the presence of ENaC proteins containing PY motif mutations, abolished in the presence of a dominant negative form of Nedd4–2, and knockdown of COMMD1 reduced the inhibitory effect of Nedd4–2 on ENaC, but did not enhance current when Nedd4–2 was knocked down. These data suggest that COMMD1 modulates Na+ transport in epithelial cells through regulation of ENaC cell surface expression and this effect is likely mediated via Nedd4–2.

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Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants

Journal of Clinical Investigation ◽

10.1172/jci8688 ◽

2000 ◽

Vol 105 (7) ◽

pp. 887-895 ◽

Cited By ~ 387

Author(s):

Jean-Pierre Morello ◽

Ali Salahpour ◽

André Laperrière ◽

Virginie Bernier ◽

Marie-Françoise Arthus ◽

...

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Vasopressin Receptor ◽

Pharmacological Chaperones ◽

V2 Vasopressin Receptor ◽

And Function

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Actin-binding Protein α-Actinin-1 Interacts with the Metabotropic Glutamate Receptor Type 5b and Modulates the Cell Surface Expression and Function of the Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m608880200 ◽

2007 ◽

Vol 282 (16) ◽

pp. 12143-12153 ◽

Cited By ~ 27

Author(s):

Nuria Cabello ◽

Rosaria Remelli ◽

Laia Canela ◽

Ana Soriguera ◽

Josefa Mallol ◽

...

Keyword(s):

Cell Surface ◽

Glutamate Receptor ◽

Metabotropic Glutamate Receptor ◽

Surface Expression ◽

Receptor Type ◽

Actin Binding ◽

Cell Surface Expression ◽

Actin Binding Protein ◽

Metabotropic Glutamate ◽

And Function

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Nedd4-2 isoforms ubiquitinate individual epithelial sodium channel subunits and reduce surface expression and function of the epithelial sodium channel

AJP Renal Physiology ◽

10.1152/ajprenal.00339.2007 ◽

2008 ◽

Vol 294 (5) ◽

pp. F1157-F1165 ◽

Cited By ~ 22

Author(s):

Nandita S. Raikwar ◽

Christie P. Thomas

Keyword(s):

Cell Surface ◽

Sodium Channel ◽

Collecting Duct ◽

Epithelial Sodium Channel ◽

Surface Expression ◽

Cell Surface Expression ◽

Membrane Expression ◽

Ww Domains ◽

Ubiquitin Proteasome ◽

And Function

We previously reported the existence of multiple isoforms of human Nedd4-2 ( Am J Physiol Renal Physiol 285: F916–F929, 2003). When overexpressed in M-1 collecting duct epithelia, full-length Nedd4-2 (Nedd4-2), Nedd4-2 lacking the NH2-terminal C2 domain (Nedd4-2ΔC2), and Nedd4-2 lacking WW domains 2 and 3 (Nedd4-2ΔWW2,3) variably reduce benzamil-sensitive Na+ transport. We investigated the effect of each of the Nedd4-2 isoforms on cell surface expression and ubiquitination of ENaC subunits. We find that αENaC when transfected alone or with β and γENaC is expressed at the cell surface and this membrane expression is variably reduced by coexpression with each of the Nedd4-2 isoforms. Nedd4-2 reduces the half-life of ENaC subunits and enhances the ubiquitination of α, β, and γENaC subunits when expressed alone or together suggesting that each subunit is a target for Nedd4-2-mediated ubiquitination. As has been reported recently, we confirm that the surface-expressed pool of ENaC is multi-ubiquitinated. Inhibitors of the proteasome increase ubiquitination of ENaC subunits and stimulate Na+ transport in M-1 cells consistent with a role for the ubiquitin-proteasome pathway in regulating Na+ transport in the collecting duct.

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Molecular pathogenesis of human CD59 deficiency

Neurology Genetics ◽

10.1212/nxg.0000000000000280 ◽

2018 ◽

Vol 4 (6) ◽

pp. e280 ◽

Cited By ~ 5

Author(s):

Netanel Karbian ◽

Yael Eshed-Eisenbach ◽

Adi Tabib ◽

Hila Hoizman ◽

B. Paul Morgan ◽

...

Keyword(s):

Cell Membrane ◽

Cell Surface ◽

Chronic Inflammatory Demyelinating Polyneuropathy ◽

Membrane Attack Complex ◽

Surface Expression ◽

Surface Proteins ◽

Cell Surface Expression ◽

Congenital Deficiency ◽

Recurrent Strokes ◽

And Function

ObjectiveTo characterize all 4 mutations described for CD59 congenital deficiency.MethodsThe 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality.ResultsImmunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation.ConclusionsAll 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non–MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis.

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