scholarly journals Rapid Transport of Internalized P-Selectin to Late Endosomes and the Tgn

2000 ◽  
Vol 151 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Kimberly S. Straley ◽  
Samuel A. Green
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Endosomal Sorting ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Granule Membrane

Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20–25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.

scholarly journals Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

2000 ◽  
Vol 11 (8) ◽  
pp. 2643-2655 ◽  
Author(s):  
Lolita Zaliauskiene ◽  
Sunghyun Kang ◽  
Christie G. Brouillette ◽  
Jacob Lebowitz ◽  
Ramin B. Arani ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Domain ◽  
Gradient Centrifugation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Surface Proteins ◽  
Down Regulation ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Polar Residues

How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the “signal” within the TM necessary and sufficient for down-regulation is Thr11Gln17Thr19 (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well (∼79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.

LDL Receptor Mediates Effective Endocytosis of Activated Factor V.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1766-1766 ◽  
Author(s):  
Alexander B. Meijer ◽  
Sigrid D. Roosendaal ◽  
Vincent Limburg ◽  
Carmen van der Zwaan ◽  
Kees W. Rodenburg ◽  
...  
Keyword(s):  
Cho Cells ◽  
Fluorescent Protein ◽  
Low Density Lipoprotein ◽  
Yellow Fluorescent Protein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Functional Expression ◽  
Factor V ◽  
Early Endosomes ◽  
Density Lipoprotein Receptor

Abstract The platelet α-granules contain a unique pool of partially activated factor V, which has been suggested to originate form megakaryocyte endocytosis of factor V (FV) from plasma. The presence of activated FV (FVa) in plasma itself should be tightly controlled as dysfunction therein may predispose to thrombotic disorders. Previously, we have reported that FVa but not FV can bind the low-density lipoprotein receptor related protein (LRP). This multifunctional receptor can bind a multitude of ligands including FV’s homologue factor VIII (FVIII). We now investigated whether FV or FVa can, like FVIII, also bind the more restricted LDL receptor (LDLR). To this end, the endocytosis of FV and FVa by CHO cells expressing LDLR (CHO-LDLR+ cells) was assessed utilizing confocal microscopy. In the experimental setup, FV and FVa were visualized employing immuno-fluorescence staining techniques. The results showed that within 10 minutes after addition of FVa, fluorescent spots appeared inside the CHO-LDLR+ cells. In contrast, no fluorescent spots were observed after 10 minutes of incubation with FV. These observations suggest that FVa but not FV effectively interacts with the LDLR expressing CHO cells. We then assessed whether FVa can compete with FVIII for endocytosis by CHO-LDLR+ cells. In the presence of an equimolar amount of FVa and a FVIII derivative (FVIIIYFP) containing yellow fluorescent protein, both proteins were detected within the same vesicles inside the CHO-LDLR+ cells. Employing co-localization studies, we established that these vesicles represented early endosomes. In the presence of an access of FVa, however, the yellow fluorescence of FVIIIYFP was no longer observed. These results demonstrate that FVa can block the FVIII endocytosis by CHO-LDLR+ cells. The observations together suggest that LDLR can not only bind FVIII but also FVa. In line with this notion, we established that CHO-ldlA cells, which lack functional expression of LDLR, were unable to internalize neither FVa nor FVIIIYFP. In addition, the ligand-binding clusters II and cluster IV of LRP effectively inhibited the endocytic uptake of FVa by CHO-LDLR+ cells. This implies that structural elements of FVa involved in LRP binding may overlap with those required for LDLR dependent internalization. Our results suggest a so far unidentified role for members of the LDL receptor family in the regulation of FVa in plasma.

Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction

2009 ◽  
Vol 418 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Audrey Parent ◽  
Emilie Hamelin ◽  
Pascale Germain ◽  
Jean-Luc Parent
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glutathione Transferase ◽  
Direct Interaction ◽  
Small Gtpase ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Surface Receptors ◽  
Early Endosomes ◽  
Intracellular Domains

The β2ARs (β2-adrenergic receptors) undergo ligand-induced internalization into early endosomes, but then are rapidly and efficiently recycled back to the plasma membrane, restoring the numbers of functional cell-surface receptors. Gathering evidence suggests that, during prolonged exposure to agonist, some β2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment regulated by the small GTPase Rab11. In the present study, we demonstrate by co-immunoprecipitation studies that there is a β2AR–Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His6-tagged Rab11 protein and β2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of β2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the β2AR interacts preferentially with the GDP-bound form of Rab11. Arg333 and Lys348 in the C-terminal tail of the β2AR were identified as crucial determinants for Rab11 binding. A β2AR construct with these two residues mutated to alanine, β2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of β2AR RK/AA was drastically reduced when compared with wild-type β2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the β2AR RK/AA mutant failed to co-localize with Rab11 and recycle to the plasma membrane, in contrast with the wild-type receptor. To our knowledge, the present study is the first report of a direct interaction between the β2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.

scholarly journals Lipoprotein receptors – an evolutionarily ancient multifunctional receptor family

2010 ◽  
Vol 391 (11) ◽  
Author(s):  
Marco Dieckmann ◽  
Martin Frederik Dietrich ◽  
Joachim Herz
Keyword(s):  
Cell Surface ◽  
Receptor Gene ◽  
Ldl Receptor ◽  
Amyloid Peptide ◽  
Cell Surface Receptors ◽  
Lipoprotein Receptors ◽  
Surface Receptors ◽  
Wide Range ◽  
Extracellular Environment ◽  
Receptor Family

Abstract The evolutionarily ancient low-density lipoprotein (LDL) receptor gene family represents a class of widely expressed cell surface receptors. Since the dawn of the first primitive multicellular organisms, several structurally and functionally distinct families of lipoprotein receptors have evolved. In accordance with the now obsolete ‘one-gene-one-function’ hypothesis, these cell surface receptors were orginally perceived as mere transporters of lipoproteins, lipids, and nutrients or as scavenger receptors, which remove other kinds of macromolecules, such as proteases and protease inhibitors from the extracellular environment and the cell surface. This picture has since undergone a fundamental change. Experimental evidence has replaced the perception that these receptors serve merely as cargo transporters. Instead it is now clear that the transport of macromolecules is inseparably intertwined with the molecular machinery by which cells communicate with each other. Lipoprotein receptors are essentially sensors of the extracellular environment that participate in a wide range of physiological processes by physically interacting and coevolving with primary signal transducers as co-regulators. Furthermore, lipoprotein receptors modulate cellular trafficking and localization of the amyloid precursor protein (APP) and the β-amyloid peptide (Aβ), suggesting a role in the pathogenesis of Alzheimer's disease. Moreover, compelling evidence shows that LDL receptor family members are involved in tumor development and progression.

scholarly journals Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

2002 ◽  
Vol 13 (9) ◽  
pp. 3325-3335 ◽  
Author(s):  
Lora Melman ◽  
Hans J. Geuze ◽  
Yonghe Li ◽  
Lynn M. McCormick ◽  
Peter van Kerkhof ◽  
...  
Keyword(s):  
Cell Surface ◽  
Growth Hormone Receptor ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Related Protein ◽  
Density Lipoprotein Receptor ◽  
Cellular Turnover

The low-density lipoprotein receptor (LDLR)-related protein (LRP) is a multiligand endocytic receptor that has broad cellular and physiological functions. Previous studies have shown that both tyrosine-based and di-leucine motifs within the LRP cytoplasmic tail are responsible for mediating its rapid endocytosis. Little is known, however, about the mechanism by which LRP is targeted for degradation. By examining both endogenous full-length and a minireceptor form of LRP, we found that proteasomal inhibitors, MG132 and lactacystin, prolong the cellular half-life of LRP. The presence of proteasomal inhibitors also significantly increased the level of LRP at the cell surface, suggesting that the delivery of LRP to the degradation pathway was blocked at a compartment from which recycling of the receptor to the cell surface still occurred. Immunoelectron microscopy analyses demonstrated a proteasomal inhibitor-dependent reduction in LRP minireceptor within both limiting membrane and internal vesicles of the multivesicular bodies, which are compartments that lead to receptor degradation. In contrast to the growth hormone receptor, we found that the initial endocytosis of LRP minireceptor does not require a functional ubiquitin–proteasome system. Finally, using truncated cytoplasmic mutants of LRP minireceptors, we found that a region of 19 amino acids within the LRP tail is required for proteasomal regulation. Taken together our results provide strong evidence that the cellular turnover of a cargo receptor, i.e., LRP, is regulated by the proteasomal system, suggesting a broader function of the proteasome in regulating the trafficking of receptors into the degradation pathway.

scholarly journals Low Density Lipoprotein (LDL) Receptor-related Protein 1B Impairs Urokinase Receptor Regeneration on the Cell Surface and Inhibits Cell Migration

2002 ◽  
Vol 277 (44) ◽  
pp. 42366-42371 ◽  
Author(s):  
Yonghe Li ◽  
Jane M. Knisely ◽  
Wenyan Lu ◽  
Lynn M. McCormick ◽  
Jieyi Wang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Urokinase Receptor ◽  
Low Density ◽  
Related Protein

Recycling of Cell-surface Receptors: Observations from the LDL Receptor System

1982 ◽  
Vol 46 (0) ◽  
pp. 713-721 ◽  
Author(s):  
M. S. Brown ◽  
R. G. W. Anderson ◽  
S. K. Basu ◽  
J. L. Goldstein
Keyword(s):  
Cell Surface ◽  
Ldl Receptor ◽  
Cell Surface Receptors ◽  
Receptor System ◽  
Surface Receptors

scholarly journals A mutation and an antibody that affect chemical cross-linking of low-density lipoprotein receptors on human fibroblasts

1993 ◽  
Vol 289 (2) ◽  
pp. 569-573 ◽  
Author(s):  
D D Patel ◽  
A K Soutar ◽  
B L Knight
Keyword(s):  
Tertiary Structure ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Surface Membrane ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cross Linking ◽  
Low Density ◽  
Ldl Receptors ◽  
Surface Receptors

Treatment of normal fibroblasts with the bifunctional cross-linking reagent DTSSP [3,3′-dithiobis(sulphosuccinimidylpropionate)] at 4 degrees C converted approximately 40% of the cell-surface low-density lipoprotein (LDL) receptors into a high-M(r) form, thought to represent receptor dimers. Preincubation of the cells with anti-(LDL receptor) monoclonal antibody 10A2 increased the proportion of surface receptors in the high-M(r) form after treatment with DTSSP at 4 degrees C to over 70%. Preincubation with LDL did not affect the proportion cross-linked, but prevented the increase produced by antibody 10A2. Cross-linking at 37 degrees C was less efficient than at 4 degrees C and was not affected by preincubation with antibody 10A2. Surface LDL receptors on fibroblasts from the homozygous familial hypercholesterolaemic subject MM were not cross-linked by DTSSP, confirming that the mutation had produced a change in the conformation of the receptor molecule. Taken together, the results suggest that normal LDL receptors on at least one region of the surface membrane may be loosely associated in some form of multimeric array which alters its alignment differently in response to antibody 10A2 and to cooling. Mutations that alter the tertiary structure of the receptors could affect LDL binding by disturbing the arrangement of the array.

scholarly journals Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes.

1997 ◽  
Vol 8 (3) ◽  
pp. 517-532 ◽  
Author(s):  
R P Czekay ◽  
R A Orlando ◽  
L Woodward ◽  
M Lundstrom ◽  
M G Farquhar
Keyword(s):  
Gene Family ◽  
Receptor Gene ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Density Lipoprotein Receptor ◽  
Receptor Gene Family

Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (approximately 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.

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