Ligand-supported purification of the urotensin-II receptor

Du, Ann T., Onan, Döne, Dinh, Diem T., Lew, Michael J., Ziogas, James, Aguilar, Marie-Isabel, Pattenden, Leonard K. and Thomas, Walter G. (2010) Ligand-supported purification of the urotensin-II receptor. Molecular Pharmacology, 78 4: 639-647. doi:10.1124/mol.110.065151


Author Du, Ann T.
Onan, Döne
Dinh, Diem T.
Lew, Michael J.
Ziogas, James
Aguilar, Marie-Isabel
Pattenden, Leonard K.
Thomas, Walter G.
Title Ligand-supported purification of the urotensin-II receptor
Journal name Molecular Pharmacology   Check publisher's open access policy
ISSN 0026-895X
1521-0111
Publication date 2010-10
Sub-type Article (original research)
DOI 10.1124/mol.110.065151
Volume 78
Issue 4
Start page 639
End page 647
Total pages 9
Place of publication Bethesda, MD, United States
Publisher American Society for Pharmacology and Experimental Therapeutics
Collection year 2011
Language eng
Formatted 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. Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics.
Keyword Protein-coupled receptor
Crystal-structure
Affinity-chromatography
Rhodopsin
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
School of Biomedical Sciences Publications
 
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Created: Sun, 03 Oct 2010, 00:03:16 EST