Ligand-supported purification of the urotensin-II receptor

Ann Thi Du, Done Onan, Diem Thi Dinh, Michael J Lew, James Ziogas, Marie -Isabel Aguilar, Leonard K Pattenden, Walter G Thomas

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4 Citations (Scopus)

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 due to 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 analogous pharmacological properties to the unmodified U-II ligand (high affinity, pseudo-irreversible binding). The pre-binding 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 beta-D-maltoside, was dependent on the critical micelle concentration and Galpha(q/11) protein was co-purified 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.
Original languageEnglish
Pages (from-to)639 - 647
Number of pages9
JournalMolecular Pharmacology
Volume78
Issue number4
DOIs
Publication statusPublished - 2010

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