scholarly journals Distribution and Function of Ap-1 Clathrin Adaptor Complexes in Polarized Epithelial Cells

2001 ◽  
Vol 152 (3) ◽  
pp. 595-606 ◽  
Author(s):  
Heike Fölsch ◽  
Marc Pypaert ◽  
Peter Schu ◽  
Ira Mellman
Keyword(s):  
Membrane Proteins ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Apical Surface ◽  
Recycling Endosomes ◽  
Density Lipoprotein Receptor ◽  
Basolateral Plasma Membrane ◽  
Clathrin Adaptor ◽  
And Function ◽  
Golgi Network

Expression of the epithelial cell–specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD μ subunit, μ1A, being replaced by the closely related μ1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged μ1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.

scholarly journals Clathrin and clathrin adaptor AP-1 control apical trafficking of megalin in the biosynthetic and recycling routes

2019 ◽  
Vol 30 (14) ◽  
pp. 1716-1728 ◽  
Author(s):  
Diego Gravotta ◽  
Andres Perez Bay ◽  
Caspar T. H. Jonker ◽  
Patrick J. Zager ◽  
Ignacio Benedicto ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Transmembrane Protein ◽  
Low Density Lipoprotein ◽  
Basolateral Membrane ◽  
Density Lipoprotein ◽  
Apical Surface ◽  
Density Lipoprotein Receptor ◽  
Clathrin Adaptor ◽  
Apical Sorting ◽  
Apical Localization

Megalin (gp330, LRP-2) is a protein structurally related to the low-density lipoprotein receptor family that displays a large luminal domain with multiligand binding properties. Megalin localizes to the apical surface of multiple epithelia, where it participates in endocytosis of a variety of ligands performing roles important for development or homeostasis. We recently described the apical recycling pathway of megalin in Madin–Darby canine kidney (MDCK) cells and found that it is a long-lived, fast recycling receptor with a recycling turnover of 15 min and a half-life of 4.8 h. Previous work implicated clathrin and clathrin adaptors in the polarized trafficking of fast recycling basolateral receptors. Hence, here we study the role of clathrin and clathrin adaptors in megalin’s apical localization and trafficking. Targeted silencing of clathrin or the γ1 subunit of clathrin adaptor AP-1 by RNA interference in MDCK cells disrupted apical localization of megalin, causing its redistribution to the basolateral membrane. In contrast, silencing of the γ2 subunit of AP-1 had no effect on megalin polarity. Trafficking assays we developed using FM4-HA-miniMegalin-GFP, a reversible conditional endoplasmic reticulum–retained chimera, revealed that clathrin and AP-1 silencing disrupted apical sorting of megalin in both biosynthetic and recycling routes. Our experiments demonstrate that clathrin and AP-1 control the sorting of an apical transmembrane protein.

scholarly journals v-SNARE cellubrevin is required for basolateral sorting of AP-1B–dependent cargo in polarized epithelial cells

2007 ◽  
Vol 177 (3) ◽  
pp. 477-488 ◽  
Author(s):  
Ian C. Fields ◽  
Elina Shteyn ◽  
Marc Pypaert ◽  
Véronique Proux-Gillardeaux ◽  
Richard S. Kang ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Low Density Lipoprotein ◽  
Basolateral Membrane ◽  
Density Lipoprotein ◽  
Recycling Endosomes ◽  
Transport Vesicles ◽  
Density Lipoprotein Receptor ◽  
Target Membrane ◽  
Syntaxin 4 ◽  
Basolateral Plasma Membrane

The epithelial cell–specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B–dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.

scholarly journals The apoptotic engulfment protein Ced-6 participates in clathrin-mediated yolk uptake in Drosophila egg chambers

2012 ◽  
Vol 23 (9) ◽  
pp. 1742-1764 ◽  
Author(s):  
Anupma Jha ◽  
Simon C. Watkins ◽  
Linton M. Traub
Keyword(s):  
Ovarian Follicle ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Biochemical Properties ◽  
Ovarian Follicle Development ◽  
Density Lipoprotein Receptor ◽  
Egg Chambers ◽  
Clathrin Adaptor ◽  
Ptb Domain

Clathrin-mediated endocytosis and phagocytosis are both selective surface internalization processes but have little known mechanistic similarity or interdependence. Here we show that the phosphotyrosine-binding (PTB) domain protein Ced-6, a well-established phagocytosis component that operates as a transducer of so-called “eat-me” signals during engulfment of apoptotic cells and microorganisms, is expressed in the female Drosophila germline and that Ced-6 expression correlates with ovarian follicle development. Ced-6 exhibits all the known biochemical properties of a clathrin-associated sorting protein, yet ced-6–null flies are semifertile despite massive accumulation of soluble yolk precursors in the hemolymph. This is because redundant sorting signals within the cytosolic domain of the Drosophila vitellogenin receptor Yolkless, a low density lipoprotein receptor superfamily member, occur; a functional atypical dileucine signal binds to the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered.

scholarly journals Carvedilol Ameliorates Experimental Atherosclerosis by Regulating Cholesterol Efflux and Exosome Functions

2019 ◽  
Vol 20 (20) ◽  
pp. 5202 ◽  
Author(s):  
Chen ◽  
Tsui ◽  
Chuang ◽  
Chiang ◽  
Chen ◽  
...  
Keyword(s):  
Cholesterol Efflux ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Nuclear Factor Κb ◽  
Experimental Atherosclerosis ◽  
Atherosclerotic Cardiovascular Disease ◽  
Beneficial Effects ◽  
Abca1 Expression ◽  
Density Lipoprotein Receptor ◽  
And Function

Carvedilol (Cav), a nonselective β-blocker with α1 adrenoceptor blocking effect, has been used as a standard therapy for coronary artery disease. This study investigated the effects of Cav on exosome expression and function, ATP-binding cassette transporter A1 (ABCA1) expression, and cholesterol efflux that are relevant to the process of atherosclerosis. Human monocytic (THP-1) cell line and human hepatic (Huh-7) cells were treated with Cav, and cholesterol efflux was measured. Exosomes from cell culture medium or mice serum were isolated using glycan-coated recognition beads. Low-density lipoprotein receptor knockout (ldlr−/−) mice were fed with high-fat diet and treated with Cav. Cav accentuated cholesterol efflux and enhanced the expressions of ABCA1 protein and mRNA in both THP-1 and Huh-7 cells. In addition, Cav increased expression and function of exosomal ABCA1 in THP-1 macrophage exosomes. The mechanisms were associated with inhibition of nuclear factor-κB (NF-κB) and protein kinase B (Akt). In hypercholesterolemic ldlr−/− mice, Cav enhanced serum exosomal ABCA1 expression and suppressed atherosclerosis by inhibiting lipid deposition and macrophage accumulation. Cav halts atherosclerosis by enhancing cholesterol efflux and increasing ABCA1 expression in macrophages and in exosomes, possibly through NF-κB and Akt signaling, which provides mechanistic insights regarding the beneficial effects of Cav on atherosclerotic cardiovascular disease.

scholarly journals Pathogenic effects of agrin V1727F mutation are isoform specific and decrease its expression and affinity for HSPGs and LRP4

2019 ◽  
Vol 28 (16) ◽  
pp. 2648-2658 ◽  
Author(s):  
John B Rudell ◽  
Ricardo A Maselli ◽  
Vladimir Yarov-Yarovoy ◽  
Michael J Ferns
Keyword(s):  
Matrix Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Extracellular Matrix Protein ◽  
Congenital Myasthenic Syndrome ◽  
Density Lipoprotein Receptor ◽  
Myasthenic Syndrome ◽  
And Function ◽  
Kinase Signaling Pathway ◽  
Pathogenic Effects

Abstract Agrin is a large extracellular matrix protein whose isoforms differ in their tissue distribution and function. Motoneuron-derived y+z+ agrin regulates the formation of the neuromuscular junction (NMJ), while y−z− agrin is widely expressed and has diverse functions. Previously we identified a missense mutation (V1727F) in the second laminin globular (LG2) domain of agrin that causes severe congenital myasthenic syndrome. Here, we define pathogenic effects of the agrin V1727F mutation that account for the profound dysfunction of the NMJ. First, by expressing agrin variants in heterologous cells, we show that the V1727F mutation reduces the secretion of y+z+ agrin compared to wild type, whereas it has no effect on the secretion of y−z− agrin. Second, we find that the V1727F mutation significantly impairs binding of y+z+ agrin to both heparin and the low-density lipoprotein receptor-related protein 4 (LRP4) coreceptor. Third, molecular modeling of the LG2 domain suggests that the V1727F mutation primarily disrupts the y splice insert, and consistent with this we find that it partially occludes the contribution of the y splice insert to agrin binding to heparin and LRP4. Together, these findings identify several pathogenic effects of the V1727F mutation that reduce its expression and ability to bind heparan sulfate proteoglycan and LRP4 coreceptors involved in the muscle-specific kinase signaling pathway. These defects primarily impair the function of neural y+z+ agrin and combine to cause a severe CMS phenotype, whereas y−z− agrin function in other tissues appears preserved.

A gp330/megalin-related protein is required in the major epidermis of Caenorhabditis elegans for completion of molting

Development ◽  
1999 ◽  
Vol 126 (3) ◽  
pp. 597-606 ◽  
Author(s):  
J. Yochem ◽  
S. Tuck ◽  
I. Greenwald ◽  
M. Han
Keyword(s):  
Caenorhabditis Elegans ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Apical Surface ◽  
Related Protein ◽  
Genetic Mosaic ◽  
Large Member ◽  
Fourth Larval Stage ◽  
Density Lipoprotein Receptor ◽  
Surface Staining

A genetic analysis of a gp330/megalin-related protein, LRP-1, has been undertaken in Caenorhabditis elegans. Consistent with megalin's being essential for development of mice, likely null mutations reveal that this large member of the low density lipoprotein receptor family is also essential for growth and development of this nematode. The mutations confer a striking defect, an inability to shed and degrade all of the old cuticle at each of the larval molts. The mutations also cause an arrest of growth usually at the molt from the third to the fourth larval stage. Genetic mosaic analysis suggests that the lrp-1 gene functions in the major epidermal syncytium hyp7, a polarized epithelium that secretes cuticle from its apical surface. Staining of whole mounts with specific monoclonal antibodies reveals that the protein is expressed on the apical surface of hyp7. Sterol starvation can phenocopy the lrp-1 mutations, suggesting that LRP-1 is a receptor for sterols that must be endocytosed by hyp7. These observations indicate that LRP-1 is related to megalin not only structurally but also functionally.

Lipoprotein assembly and function in an evolutionary perspective

2010 ◽  
Vol 1 (2) ◽  
pp. 165-183 ◽  
Author(s):  
Dick J. Van der Horst ◽  
Kees W. Rodenburg
Keyword(s):  
Lipid Transfer Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Microsomal Triglyceride Transfer Protein ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Density Lipoprotein Receptor ◽  
Exchangeable Basis ◽  
And Function

AbstractCirculatory fat transport in animals relies on members of the large lipid transfer protein (LLTP) superfamily, including mammalian apolipoprotein B (apoB) and insect apolipophorin II/I (apoLp-II/I). ApoB and apoLp-II/I, constituting the structural (non-exchangeable) basis for the assembly of various lipoproteins, acquire lipids through microsomal triglyceride-transfer protein, another LLTP family member, and bind them by means of amphipathic α-helical and β-sheet structural motifs. Comparative research reveals that LLTPs evolved from the earliest animals and highlights the structural adaptations in these lipid-binding proteins. Thus, in contrast to apoB, apoLp-II/I is cleaved post-translationally by a furin, resulting in the appearance of two non-exchangeable apolipoproteins in the single circulatory lipoprotein in insects, high-density lipophorin (HDLp). The remarkable structural similarities between mammalian and insect lipoproteins notwithstanding important functional differences relate to the mechanism of lipid delivery. Whereas in mammals, partial delipidation of apoB-containing lipoproteins eventually results in endocytic uptake of their remnants, mediated by members of the low-density lipoprotein receptor (LDLR) family, and degradation in lysosomes, insect HDLp functions as a reusable lipid shuttle capable of alternate unloading and reloading of lipid. Also, during muscular efforts (flight activity), an HDLp-based lipoprotein shuttle provides for the transport of lipid for energy generation. Although a lipophorin receptor – a homolog of LDLR – was identified that mediates endocytic uptake of HDLp during specific developmental periods, the endocytosed lipoprotein appears to be recycled in a transferrin-like manner. These data highlight that the functional adaptations in the lipoprotein lipid carriers in mammals and insects also emerge with regard to the functioning of their cognate receptors.

scholarly journals Peroxisome proliferator-activated receptor-γ regulates the expression and function of very-low-density lipoprotein receptor

2010 ◽  
Vol 298 (1) ◽  
pp. E68-E79 ◽  
Author(s):  
Huan Tao ◽  
Srikanth Aakula ◽  
Naji N. Abumrad ◽  
Tahar Hajri
Keyword(s):  
Adipose Tissue ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Low Density Lipoprotein Receptor ◽  
Density Lipoprotein Receptor ◽  
And Function

Very-low-density lipoprotein receptor (VLDLR) is a member of the low-density receptor family, highly expressed in adipose tissue, heart, and skeletal muscle. It binds apolipoprotein E-triglyceride-rich lipoproteins and plays a significant role in triglyceride metabolism. PPARγ is a primary regulator of lipid metabolism in adipocytes and controls the expression of an array of genes involved in lipid trafficking in adipocytes. However, it is not known whether VLDLR is also under the control of PPARγ. In this study, we investigated the role of PPARγ in the regulation of VLDLR expression and function in vivo and in vitro. During the differentiation of 3T3-L1 preadipocytes, the levels of VLDLR protein and mRNA increased in parallel with the induction of PPARγ expression and reached maximum in mature adipocytes. Treatment of differentiated adipocytes with PPARγ agonist pioglitazone upregulated VLDLR expression in dose- and time-dependent manners. In contrast, specific inhibition of PPARγ significantly downregulated the protein level of VLDLR. Induction of VLDLR is also demonstrated in vivo in adipose tissue of wild-type (WT) mice treated with pioglitazone. In addition, pioglitazone increased plasma triglyceride-rich lipoprotein clearance and increased epididymal fat mass in WT mice but failed to induce similar effects in vldlr−/−mice. These results were further corroborated by the finding that pioglitazone treatment enhanced adipogenesis and lipid deposition in preadipocytes of WT mice, while its effect in VLDLR-null preadipocytes was significantly blunted. These findings provide direct evidence that VLDLR expression is regulated by PPARγ and contributes in lipid uptake and adipogenesis.

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