The Trafficking Protein GABARAP Binds to and Enhances Plasma Membrane Expression and Function of the Angiotensin II Type 1 Receptor

Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin’s expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin’s plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.

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Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs

AJP Renal Physiology ◽

10.1152/ajprenal.00065.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. F411-F418

Author(s):

Casandra M. Monzon ◽

Jeffrey L. Garvin

Keyword(s):

Plasma Membrane ◽

Control Period ◽

Paracellular Pathway ◽

Membrane Expression ◽

No Donor ◽

Charge Selectivity ◽

Plasma Membrane Expression ◽

Cgmp Signaling ◽

Blocking Antibodies ◽

Dilution Potential

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was −18.2 ± 1.8 mV. After treatment with 200 μmol/l SPM, it was −14.7 ± 2.0 mV ( P < 0.04). In the presence of claudin-19 antibody, the dilution potential was −12.7 ± 2.1 mV. After SPM, it was −12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from −9.7 ± 1.0 to −6.3 ± 1.1 mV ( P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from −19.6 ± 2.6 to −17.2 ± 2.3 mV ( P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total ( P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

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Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

AJP Cell Physiology ◽

10.1152/ajpcell.00454.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. C857-C867 ◽

Cited By ~ 20

Author(s):

Silvia M. Uriarte ◽

Neelakshi R. Jog ◽

Gregory C. Luerman ◽

Samrath Bhimani ◽

Richard A. Ward ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Human Neutrophils ◽

Functional Responses ◽

Membrane Expression ◽

Granule Exocytosis ◽

Plasma Membrane Expression ◽

Microtubular Cytoskeleton ◽

Latrunculin A ◽

Azurophil Granule

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.

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Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells

The AAPS Journal ◽

10.1208/s12248-017-0121-5 ◽

2017 ◽

Vol 19 (5) ◽

pp. 1377-1386 ◽

Cited By ~ 13

Author(s):

Vineet Kumar ◽

Tot Bui Nguyen ◽

Beáta Tóth ◽

Viktoria Juhasz ◽

Jashvant D. Unadkat

Keyword(s):

Plasma Membrane ◽

Membrane Expression ◽

Plasma Membrane Expression ◽

In Cells

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Regulation of Plasma Membrane Expression of TRPV4 Channel by GSK1016790A and Hypotonic Stress in HeLa and M‐1 Cells

The FASEB Journal ◽

10.1096/fasebj.24.1_supplement.610.7 ◽

2010 ◽

Vol 24 (S1) ◽

Author(s):

Min Jin ◽

Jose James ◽

Roger G. O'Neil

Keyword(s):

Plasma Membrane ◽

Membrane Expression ◽

Hypotonic Stress ◽

Plasma Membrane Expression ◽

Trpv4 Channel

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Low Plasma Membrane Expression of the Miltefosine Transport Complex Renders Leishmania braziliensis Refractory to the Drug

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01694-08 ◽

2009 ◽

Vol 53 (4) ◽

pp. 1305-1313 ◽

Cited By ~ 54

Author(s):

María P. Sánchez-Cañete ◽

Luís Carvalho ◽

F. Javier Pérez-Victoria ◽

Francisco Gamarro ◽

Santiago Castanys

Keyword(s):

Plasma Membrane ◽

Cutaneous Leishmaniasis ◽

Oral Drug ◽

Leishmania Braziliensis ◽

Β Subunit ◽

Membrane Expression ◽

Plasma Membrane Expression ◽

Highly Sensitive ◽

P Type ◽

Transport Complex

ABSTRACT Miltefosine (hexadecylphosphocholine, MLF) is the first oral drug with recognized efficacy against both visceral and cutaneous leishmaniasis. However, some clinical studies have suggested that MLF shows significantly less efficiency against the cutaneous leishmaniasis caused by Leishmania braziliensis. In this work, we have determined the cellular and molecular basis for the natural MLF resistance observed in L. braziliensis. Four independent L. braziliensis clinical isolates showed a marked decrease in MLF sensitivity that was due to their inability to internalize the drug. MLF internalization in the highly sensitive L. donovani species requires at least two proteins in the plasma membrane, LdMT, a P-type ATPase involved in phospholipid translocation, and its β subunit, LdRos3. Strikingly, L. braziliensis parasites showed highly reduced levels of this MLF translocation machinery at the plasma membrane, mainly because of the low expression levels of the β subunit, LbRos3. Overexpression of LbRos3 induces increased MLF sensitivity not only in L. braziliensis promastigotes but also in intracellular amastigotes. These results further highlight the importance of the MLF translocation machinery in determining MLF potency and point toward the development of protocols to routinely monitor MLF susceptibility in geographic areas where L. braziliensis might be prevalent.

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Internalisation of the type I angiotensin II receptor (ATI) and angiotensin II function in the rat adrenal zona glomerulosa cell.

Journal of Endocrinology ◽

10.1677/joe.0.141r005 ◽

1994 ◽

Vol 141 (2) ◽

pp. R5-R9 ◽

Cited By ~ 10

Author(s):

G. P. Vinson ◽

M. M. Ho. ◽

J.R. Puddefoot ◽

R. Teja ◽

S. Barker

Keyword(s):

Monoclonal Antibody ◽

Plasma Membrane ◽

Angiotensin Ii ◽

Zona Glomerulosa ◽

Type I ◽

Specific Monoclonal Antibody ◽

Angiotensin Ii Receptor ◽

Cellular Localisation ◽

Glomerulosa Cells

ABSTRACT Little is known about the cellular localisation of the angiotensin II (AII) type 1 receptor (ATI) in the rat adrenal glomerulosa cell, but some studies have suggested that receptor internalisation and recycling may occur. Using a specific monoclonal antibody (6313/G2) to the first extracellular domain, we show here that most of the receptor is internalised in the unstimulated cell. When viable glomerulosa cells are incubated with 6313/G2, the receptor is transiently concentrated on the cell surface, and aldosterone output is stimulated. This stimulated output is enhanced by neither threshold nor maximal stimulatory concentrations of All amide, although the antibody does not inhibit All binding to the receptor. Conversely, the stimulatory actions of the antibody and those of ACTH are additive. The data suggest that recycling to the plasma membrane is constitutive, or regulated by unknown factors. Retention of the ATI receptor in the membrane is alone enough to allow sufficient G protein interaction to generate maximal stimulatory events.

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