Role of mammalian vacuolar protein-sorting proteins in endocytic trafficking of a non-ubiquitinated G protein-coupled receptor to lysosomes

James N Hislop, Aaron Marley, Mark Von Zastrow

Research output: Contribution to journalArticle

98 Citations (Scopus)

Abstract

Many signaling receptors require covalent modification by ubiquitin for agonist-induced down-regulation via endocytic trafficking to lysosomes, a process that is mediated by a conserved set of endosome-associating proteins also required for vacuolar protein-sorting (VPS) in yeast. The delta opioid receptor (DOR) is a G protein-coupled receptor that can undergo agonist-induced proteolysis via endocytic trafficking to lysosomes but does not require covalent modification by ubiquitin to do so. This raises the question of whether lysosomal down-regulation of this "ubiquitination-independent" GPCR is mediated by a completely distinct biochemical mechanism or if similar VPS machinery is involved. Agonist-induced proteolysis of DOR was significantly inhibited by dominant negative mutant versions of Vps4/Skd1, an AAA-family ATPase required for a late step in lysosomal sorting of ubiquitinated membrane cargo. Furthermore, overexpression and interfering RNA-mediated knockdown indicated that lysosomal trafficking of opioid receptors is also dependent on Hrs, a VPS protein that mediates an early step in lysosomal sorting of ubiquitinated cargo. However, interfering RNA-mediated knockdown of Tsg101, a VPS protein that is essential for an intermediate step of the conserved lysosomal sorting mechanism, did not detectably affect agonist-induced proteolysis of DOR in the same cells in which (ubiquitination-dependent) lysosomal trafficking of epidermal growth factor receptors was clearly inhibited. These results indicate that opioid receptors, despite their ability to undergo efficient agonist-induced trafficking to lysosomes in the absence of covalent modification by ubiquitin, utilize some (Vps4 and Hrs) but perhaps not all (Tsg101) of the VPS machinery required for lysosomal sorting of ubiquitinated membrane cargo.
Original languageEnglish
Pages (from-to)22522-31
Number of pages10
JournalThe Journal of Biological Chemistry
Volume279
Issue number21
DOIs
Publication statusPublished - 21 May 2004

Fingerprint

Protein Transport
G-Protein-Coupled Receptors
Lysosomes
Sorting
delta Opioid Receptor
Ubiquitin
Proteolysis
Ubiquitination
Opioid Receptors
Proteins
Down-Regulation
RNA
Membranes
Endosomes
Machinery
Adenosine Triphosphatases
Yeasts
Yeast

Keywords

  • Adenosine Triphosphatases
  • Blotting, Western
  • Cell Line
  • DNA, Complementary
  • Densitometry
  • Down-Regulation
  • Endocytosis
  • Genes, Dominant
  • HeLa Cells
  • Humans
  • Lysosomes
  • Microscopy, Fluorescence
  • Mutation
  • Protein Transport
  • RNA Interference
  • RNA, Small Interfering
  • Receptors, G-Protein-Coupled
  • Signal Transduction
  • Time Factors
  • Transfection
  • Ubiquitin

Cite this

Role of mammalian vacuolar protein-sorting proteins in endocytic trafficking of a non-ubiquitinated G protein-coupled receptor to lysosomes. / Hislop, James N; Marley, Aaron; Von Zastrow, Mark.

In: The Journal of Biological Chemistry, Vol. 279, No. 21, 21.05.2004, p. 22522-31.

Research output: Contribution to journalArticle

@article{79debdd1a49a4f66abc024987bb48fc5,
title = "Role of mammalian vacuolar protein-sorting proteins in endocytic trafficking of a non-ubiquitinated G protein-coupled receptor to lysosomes",
abstract = "Many signaling receptors require covalent modification by ubiquitin for agonist-induced down-regulation via endocytic trafficking to lysosomes, a process that is mediated by a conserved set of endosome-associating proteins also required for vacuolar protein-sorting (VPS) in yeast. The delta opioid receptor (DOR) is a G protein-coupled receptor that can undergo agonist-induced proteolysis via endocytic trafficking to lysosomes but does not require covalent modification by ubiquitin to do so. This raises the question of whether lysosomal down-regulation of this {"}ubiquitination-independent{"} GPCR is mediated by a completely distinct biochemical mechanism or if similar VPS machinery is involved. Agonist-induced proteolysis of DOR was significantly inhibited by dominant negative mutant versions of Vps4/Skd1, an AAA-family ATPase required for a late step in lysosomal sorting of ubiquitinated membrane cargo. Furthermore, overexpression and interfering RNA-mediated knockdown indicated that lysosomal trafficking of opioid receptors is also dependent on Hrs, a VPS protein that mediates an early step in lysosomal sorting of ubiquitinated cargo. However, interfering RNA-mediated knockdown of Tsg101, a VPS protein that is essential for an intermediate step of the conserved lysosomal sorting mechanism, did not detectably affect agonist-induced proteolysis of DOR in the same cells in which (ubiquitination-dependent) lysosomal trafficking of epidermal growth factor receptors was clearly inhibited. These results indicate that opioid receptors, despite their ability to undergo efficient agonist-induced trafficking to lysosomes in the absence of covalent modification by ubiquitin, utilize some (Vps4 and Hrs) but perhaps not all (Tsg101) of the VPS machinery required for lysosomal sorting of ubiquitinated membrane cargo.",
keywords = "Adenosine Triphosphatases, Blotting, Western, Cell Line, DNA, Complementary, Densitometry, Down-Regulation, Endocytosis, Genes, Dominant, HeLa Cells, Humans, Lysosomes, Microscopy, Fluorescence, Mutation, Protein Transport, RNA Interference, RNA, Small Interfering, Receptors, G-Protein-Coupled, Signal Transduction, Time Factors, Transfection, Ubiquitin",
author = "Hislop, {James N} and Aaron Marley and {Von Zastrow}, Mark",
year = "2004",
month = "5",
day = "21",
doi = "10.1074/jbc.M311062200",
language = "English",
volume = "279",
pages = "22522--31",
journal = "The Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC",
number = "21",

}

TY - JOUR

T1 - Role of mammalian vacuolar protein-sorting proteins in endocytic trafficking of a non-ubiquitinated G protein-coupled receptor to lysosomes

AU - Hislop, James N

AU - Marley, Aaron

AU - Von Zastrow, Mark

PY - 2004/5/21

Y1 - 2004/5/21

N2 - Many signaling receptors require covalent modification by ubiquitin for agonist-induced down-regulation via endocytic trafficking to lysosomes, a process that is mediated by a conserved set of endosome-associating proteins also required for vacuolar protein-sorting (VPS) in yeast. The delta opioid receptor (DOR) is a G protein-coupled receptor that can undergo agonist-induced proteolysis via endocytic trafficking to lysosomes but does not require covalent modification by ubiquitin to do so. This raises the question of whether lysosomal down-regulation of this "ubiquitination-independent" GPCR is mediated by a completely distinct biochemical mechanism or if similar VPS machinery is involved. Agonist-induced proteolysis of DOR was significantly inhibited by dominant negative mutant versions of Vps4/Skd1, an AAA-family ATPase required for a late step in lysosomal sorting of ubiquitinated membrane cargo. Furthermore, overexpression and interfering RNA-mediated knockdown indicated that lysosomal trafficking of opioid receptors is also dependent on Hrs, a VPS protein that mediates an early step in lysosomal sorting of ubiquitinated cargo. However, interfering RNA-mediated knockdown of Tsg101, a VPS protein that is essential for an intermediate step of the conserved lysosomal sorting mechanism, did not detectably affect agonist-induced proteolysis of DOR in the same cells in which (ubiquitination-dependent) lysosomal trafficking of epidermal growth factor receptors was clearly inhibited. These results indicate that opioid receptors, despite their ability to undergo efficient agonist-induced trafficking to lysosomes in the absence of covalent modification by ubiquitin, utilize some (Vps4 and Hrs) but perhaps not all (Tsg101) of the VPS machinery required for lysosomal sorting of ubiquitinated membrane cargo.

AB - Many signaling receptors require covalent modification by ubiquitin for agonist-induced down-regulation via endocytic trafficking to lysosomes, a process that is mediated by a conserved set of endosome-associating proteins also required for vacuolar protein-sorting (VPS) in yeast. The delta opioid receptor (DOR) is a G protein-coupled receptor that can undergo agonist-induced proteolysis via endocytic trafficking to lysosomes but does not require covalent modification by ubiquitin to do so. This raises the question of whether lysosomal down-regulation of this "ubiquitination-independent" GPCR is mediated by a completely distinct biochemical mechanism or if similar VPS machinery is involved. Agonist-induced proteolysis of DOR was significantly inhibited by dominant negative mutant versions of Vps4/Skd1, an AAA-family ATPase required for a late step in lysosomal sorting of ubiquitinated membrane cargo. Furthermore, overexpression and interfering RNA-mediated knockdown indicated that lysosomal trafficking of opioid receptors is also dependent on Hrs, a VPS protein that mediates an early step in lysosomal sorting of ubiquitinated cargo. However, interfering RNA-mediated knockdown of Tsg101, a VPS protein that is essential for an intermediate step of the conserved lysosomal sorting mechanism, did not detectably affect agonist-induced proteolysis of DOR in the same cells in which (ubiquitination-dependent) lysosomal trafficking of epidermal growth factor receptors was clearly inhibited. These results indicate that opioid receptors, despite their ability to undergo efficient agonist-induced trafficking to lysosomes in the absence of covalent modification by ubiquitin, utilize some (Vps4 and Hrs) but perhaps not all (Tsg101) of the VPS machinery required for lysosomal sorting of ubiquitinated membrane cargo.

KW - Adenosine Triphosphatases

KW - Blotting, Western

KW - Cell Line

KW - DNA, Complementary

KW - Densitometry

KW - Down-Regulation

KW - Endocytosis

KW - Genes, Dominant

KW - HeLa Cells

KW - Humans

KW - Lysosomes

KW - Microscopy, Fluorescence

KW - Mutation

KW - Protein Transport

KW - RNA Interference

KW - RNA, Small Interfering

KW - Receptors, G-Protein-Coupled

KW - Signal Transduction

KW - Time Factors

KW - Transfection

KW - Ubiquitin

U2 - 10.1074/jbc.M311062200

DO - 10.1074/jbc.M311062200

M3 - Article

VL - 279

SP - 22522

EP - 22531

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

SN - 0021-9258

IS - 21

ER -