N- and C-terminal degradation of ecdysteroid receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases

S Schauer, T Burster, M Spindler-Barth

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action.

Original languageEnglish
Pages (from-to)383-94
Number of pages12
JournalInsect Molecular Biology
Volume21
Issue number3
DOIs
Publication statusPublished - Jun 2012

Fingerprint

Cysteine Proteases
CHO Cells
proteasome endopeptidase complex
Proteasome Endopeptidase Complex
Threonine
threonine
cysteine
Protein Isoforms
proteinases
degradation
receptors
cathepsin S
Ubiquitination
ubiquitin
Ubiquitin
cells
dipeptidases
cathepsin B
Endopeptidases
Cathepsin B

Keywords

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • CHO Cells
  • Calpain
  • Cathepsins
  • Cricetinae
  • Cysteine Proteases
  • Drosophila Proteins
  • Drosophila melanogaster
  • Leupeptins
  • Mammals
  • Molecular Sequence Data
  • Oligopeptides
  • Protease Inhibitors
  • Proteasome Endopeptidase Complex
  • Proteasome Inhibitors
  • Protein Isoforms
  • Protein Multimerization
  • Protein Stability
  • Protein Structure, Tertiary
  • Proteolysis
  • Receptors, Steroid
  • Threonine
  • Transcription, Genetic
  • Ubiquitination
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

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title = "N- and C-terminal degradation of ecdysteroid receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases",
abstract = "Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action.",
keywords = "Amino Acid Sequence, Animals, Blotting, Western, CHO Cells, Calpain, Cathepsins, Cricetinae, Cysteine Proteases, Drosophila Proteins, Drosophila melanogaster, Leupeptins, Mammals, Molecular Sequence Data, Oligopeptides, Protease Inhibitors, Proteasome Endopeptidase Complex, Proteasome Inhibitors, Protein Isoforms, Protein Multimerization, Protein Stability, Protein Structure, Tertiary, Proteolysis, Receptors, Steroid, Threonine, Transcription, Genetic, Ubiquitination, Journal Article, Research Support, Non-U.S. Gov't",
author = "S Schauer and T Burster and M Spindler-Barth",
note = "{\circledC} 2012 The Authors. Insect Molecular Biology {\circledC} 2012 The Royal Entomological Society.",
year = "2012",
month = "6",
doi = "10.1111/j.1365-2583.2012.01144.x",
language = "English",
volume = "21",
pages = "383--94",
journal = "Insect Molecular Biology",
issn = "0962-1075",
publisher = "Wiley Blackwell",
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}

TY - JOUR

T1 - N- and C-terminal degradation of ecdysteroid receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases

AU - Schauer, S

AU - Burster, T

AU - Spindler-Barth, M

N1 - © 2012 The Authors. Insect Molecular Biology © 2012 The Royal Entomological Society.

PY - 2012/6

Y1 - 2012/6

N2 - Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action.

AB - Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action.

KW - Amino Acid Sequence

KW - Animals

KW - Blotting, Western

KW - CHO Cells

KW - Calpain

KW - Cathepsins

KW - Cricetinae

KW - Cysteine Proteases

KW - Drosophila Proteins

KW - Drosophila melanogaster

KW - Leupeptins

KW - Mammals

KW - Molecular Sequence Data

KW - Oligopeptides

KW - Protease Inhibitors

KW - Proteasome Endopeptidase Complex

KW - Proteasome Inhibitors

KW - Protein Isoforms

KW - Protein Multimerization

KW - Protein Stability

KW - Protein Structure, Tertiary

KW - Proteolysis

KW - Receptors, Steroid

KW - Threonine

KW - Transcription, Genetic

KW - Ubiquitination

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1111/j.1365-2583.2012.01144.x

DO - 10.1111/j.1365-2583.2012.01144.x

M3 - Article

VL - 21

SP - 383

EP - 394

JO - Insect Molecular Biology

JF - Insect Molecular Biology

SN - 0962-1075

IS - 3

ER -