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Molecular Pharmacology

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Research ArticleArticle

Ligand-Supported Purification of the Urotensin-II Receptor

Ann T. Du, Döne Onan, Diem T. Dinh, Michael J. Lew, James Ziogas, Marie-Isabel Aguilar, Leonard K. Pattenden and Walter G. Thomas
Molecular Pharmacology October 2010, 78 (4) 639-647; DOI: https://doi.org/10.1124/mol.110.065151
Ann T. Du
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Döne Onan
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Diem T. Dinh
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Michael J. Lew
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James Ziogas
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Marie-Isabel Aguilar
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Leonard K. Pattenden
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Walter G. Thomas
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Abstract

A crucial limitation for structural and biophysical analysis of G protein-coupled receptors (GPCRs) is the inherent challenge of purifying and stabilizing these receptors in an active (agonist-bound) conformation. Peptide ligands, such as the vasoactive, cyclic hormone urotensin-II (U-II), may provide new purification tools, via high affinity, pseudo-irreversible binding suitable for ligand-based affinity purification. We show that the U-II receptor (UT) is resistant to desensitization as a result of low phosphorylation and diminished endocytosis. UT also displays an unusual proclivity to remain active with vasoconstriction sustained despite extensive washout of the ligand. To exploit these properties for ligand-supported purification, we modified the U-II ligand by attaching a biotin moiety and spacer arm to the N terminus, creating a novel affinity ligand (Bio-U-II) to interface with streptavidin media. Bio-U-II bound to UT with pharmacological properties analogous to those of the unmodified U-II ligand (high-affinity, pseudo-irreversible binding). The prebinding of Bio-U-II to UT (before exposure to detergent) facilitated specific capture of UT by stabilizing the receptor structure during solubilization with detergent. Solubilization of UT with the most compatible detergent, n-dodecyl β-d-maltoside, was dependent on the critical micelle concentration, and Gαq/11 protein was copurified with captured Bio-U-II-UT complexes. Furthermore, captured Bio-U-II-UT complexes were resistant to dissociation at elevated temperatures, suggesting that UT is relatively thermostable, making it an ideal candidate for future structural and biophysical studies. This work demonstrates the utility of pseudo-irreversible ligands to support the purification of a GPCR during detergent extraction, resulting in the first successful purification of the UT.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by National Health and Medical Research Council of Australia [Grants 268925, 251646, 334049] and by a Ph.D. scholarship from the Baker Heart Research Institute (to D.O.).

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.065151.

  • ABBREVIATIONS:

    GPCR
    G protein-coupled receptor
    U-II
    urotensin-II
    UT
    U-II receptor
    Bio-U-II
    biotinylated U-II
    HRP
    horseradish peroxidase
    Ang II
    angiotensin II
    HA
    hemagglutinin
    AT1A
    type 1A angiotensin receptor
    PDB
    Protein Data Bank
    HEK
    human embryonic kidney
    CHO
    Chinese hamster ovary
    DMEM
    Dulbecco's modified Eagle's medium
    PBS
    phosphate-buffered saline
    ERK
    extracellular signal-regulated kinase
    PAGE
    polyacrylamide gel electrophoresis
    DDM
    n-dodecyl β-d-maltoside
    CHAPS
    3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
    CMC
    critical micelle concentration.

  • Received March 30, 2010.
  • Accepted July 20, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (4)
Molecular Pharmacology
Vol. 78, Issue 4
1 Oct 2010
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Research ArticleArticle

Ligand-Supported Purification of the Urotensin-II Receptor

Ann T. Du, Döne Onan, Diem T. Dinh, Michael J. Lew, James Ziogas, Marie-Isabel Aguilar, Leonard K. Pattenden and Walter G. Thomas
Molecular Pharmacology October 1, 2010, 78 (4) 639-647; DOI: https://doi.org/10.1124/mol.110.065151

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Research ArticleArticle

Ligand-Supported Purification of the Urotensin-II Receptor

Ann T. Du, Döne Onan, Diem T. Dinh, Michael J. Lew, James Ziogas, Marie-Isabel Aguilar, Leonard K. Pattenden and Walter G. Thomas
Molecular Pharmacology October 1, 2010, 78 (4) 639-647; DOI: https://doi.org/10.1124/mol.110.065151
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