Abstract：
:GPCRs mediate intracellular signaling upon external stimuli, making them ideal drug targets. However, little is known about their activation mechanisms due to the difficulty in purification. Here, we introduce a method to purify GPCRs in nanodiscs, which incorporates GPCRs into lipid bilayers immediately after membrane solubilization, followed by single-step purification. Using this approach, we purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R), which regulates calcium and phosphate homeostasis and is a drug target for osteoporosis. We demonstrated that the purified PTH1R in nanodiscs can bind to PTH(1-34) and activate G protein. We also observed that Ca(2+) is a weak agonist of PTH1R, and Ca(2+) in millimolar concentration can switch PTH(1-34) from an inverse agonist to an agonist. Hence, our results show that nanodiscs are a viable vehicle for GPCR purification, enabling studies of GPCRs under precise experimental conditions without interference from other cellular or membrane components.
journal_name
ACS Chem Bioljournal_title
ACS chemical biologyauthors
Mitra N,Liu Y,Liu J,Serebryany E,Mooney V,DeVree BT,Sunahara RK,Yan ECdoi
10.1021/cb300466nsubject
Has Abstractpub_date
2013-03-15 00:00:00pages
617-25issue
3eissn
1554-8929issn
1554-8937journal_volume
8pub_type
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