Ligand-supported purification of the urotensin-II receptor.
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 Gα(q/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.
Du
Ann T
AT
Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.
Onan
Döne
D
Dinh
Diem T
DT
Lew
Michael J
MJ
Ziogas
James
J
Aguilar
Marie-Isabel
MI
Pattenden
Leonard K
LK
Thomas
Walter G
WG
D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008024:Ligands; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000302:isolation & purification* / Q000378:metabolism*; D014579:Urotensins / Q000378:metabolism
D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D014579:Urotensins; C473142:Uts2r protein, rat; C029677:urotensin II