scholarly journals ARH cooperates with AP-1B in the exocytosis of LDLR in polarized epithelial cells

2011 ◽  
Vol 193 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Richard S. Kang ◽  
Heike Fölsch
Keyword(s):  
Epithelial Cells ◽  
Autosomal Recessive ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lipoprotein Receptors ◽  
Recycling Endosomes ◽  
Sorting Motif ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor ◽  
Autosomal Recessive Hypercholesterolemia

The autosomal recessive hypercholesterolemia protein (ARH) is well known for its role in clathrin-mediated endocytosis of low-density lipoprotein receptors (LDLRs). During uptake, ARH directly binds to the FxNPxY signal in the cytoplasmic tail of LDLR. Interestingly, the same FxNPxY motif is used in basolateral exocytosis of LDLR from recycling endosomes (REs), which is facilitated by the epithelial-specific clathrin adaptor AP-1B. However, AP-1B directly interacts with neither the FxNPxY motif nor the second more distally located YxxØ sorting motif of LDLR. Here, we show that ARH colocalizes and cooperates with AP-1B in REs. Knockdown of ARH in polarized epithelial cells leads to specific apical missorting of truncated LDLR, which encodes only the FxNPxY motif (LDLR-CT27). Moreover, a mutation in ARH designed to disrupt the interaction of ARH with AP-1B specifically abrogates exocytosis of LDLR-CT27. We conclude that in addition to its role in endocytosis, ARH cooperates with AP-1B in basolateral exocytosis of LDLR from REs.

scholarly journals Differential Recognition of Tyrosine-based Basolateral Signals by AP-1B Subunit μ1B in Polarized Epithelial Cells

2002 ◽  
Vol 13 (7) ◽  
pp. 2374-2382 ◽  
Author(s):  
Hisashi Sugimoto ◽  
Masayuki Sugahara ◽  
Heike Fölsch ◽  
Yasuhiro Koide ◽  
Fubito Nakatsu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Mutant Form ◽  
Targeting Signals ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor ◽  
Basolateral Targeting

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit μ1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of μ1B. The mutant (M-μ1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit μ2 are critical for interacting with tyrosine-based endocytosis signals. We show M-μ1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-μ1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on μ1B or M-μ1B expression. Our results suggest that μ1B interacts with different classes of basolateral targeting signals in distinct ways.

scholarly journals Arf6 regulates AP-1B–dependent sorting in polarized epithelial cells

2011 ◽  
Vol 194 (6) ◽  
pp. 873-887 ◽  
Author(s):  
Elina Shteyn ◽  
Lucy Pigati ◽  
Heike Fölsch
Keyword(s):  
Epithelial Cells ◽  
Dominant Negative ◽  
Recycling Endosomes ◽  
Membrane Recruitment ◽  
Important Regulator ◽  
Basolateral Plasma Membrane ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor ◽  
Basolateral Targeting

The epithelial cell–specific clathrin adaptor complex AP-1B facilitates the sorting of various transmembrane proteins from recycling endosomes (REs) to the basolateral plasma membrane. Despite AP-1B’s clear importance in polarized epithelial cells, we still do not fully understand how AP-1B orchestrates basolateral targeting. Here we identify the ADP-ribosylation factor 6 (Arf6) as an important regulator of AP-1B. We show that activated Arf6 pulled down AP-1B in vitro. Furthermore, interfering with Arf6 function through overexpression of dominant-active Arf6Q67L or dominant-negative Arf6D125N, as well as depletion of Arf6 with short hairpin RNA (shRNA), led to apical missorting of AP-1B–dependent cargos. In agreement with these data, we found that Arf6 colocalized with AP-1B and transferrin receptor (TfnR) in REs. In addition, we observed specific recruitment of AP-1B into Arf6-induced membrane ruffles in nonpolarized cells. We conclude that activated Arf6 directs membrane recruitment of AP-1B, thus regulating AP-1B’s functions in polarized epithelial cells.

scholarly journals Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

2010 ◽  
Vol 21 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Ian C. Fields ◽  
Shelby M. King ◽  
Elina Shteyn ◽  
Richard S. Kang ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Amino Acid Residues ◽  
Recycling Endosomes ◽  
C Terminus ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P3 formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P3 formation in recycling endosomes is essential for AP-1B function.

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 The Adaptor Protein ARH Escorts Megalin to and through Endosomes

2003 ◽  
Vol 14 (12) ◽  
pp. 4984-4996 ◽  
Author(s):  
Masaaki Nagai ◽  
Timo Meerloo ◽  
Tetsuro Takeda ◽  
Marilyn Gist Farquhar
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Coated Pits ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Autosomal Recessive Hypercholesterolemia

Megalin is an endocytic receptor that binds multiple ligands and is essential for many physiological processes such as brain development and uptake of proteins by the kidney tubule, yolk sac, and thyroid. The cytoplasmic tail of megalin contains two FXNPXY motifs. Autosomal recessive hypercholesterolemia (ARH) is an adaptor protein that binds to the FXNPXY motif of the low-density lipoprotein receptor as well as clathrin and AP-2. We found that ARH also binds to the first FXNPXY motif of megalin in two-hybrid, pull-down and coimmunoprecipitation assays. ARH colocalizes with megalin in clathrin coated pits and in recycling endosomes in the Golgi region. When cells are treated with nocodazole, the recycling endosomes containing megalin and ARH disperse. On internalization of megalin, ARH and megalin are first seen in clathrin coated pits followed by sequential localization in early endosomes and tubular recycling endosomes in the pericentriolar region followed by their reappearance at the cell surface. Expression of ARH in Madin-Darby canine kidney cells expressing megalin mini-receptors enhances megalin-mediated uptake of125I-lactoferrin, a megalin ligand. These results show that ARH facilitates endocytosis of megalin, escorts megalin along its endocytic route and raise the possibility that transport through the endosomal system is selective and requires interaction with specific adaptor proteins.

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