title: Calcium-dependent ligand binding and G-protein signaling of family B GPCR parathyroid hormone 1 receptor purified in nanodiscs.
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.
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Nivedita
authors.initials: N
authors.lastname: Mitra
authors.forename: Yuting
authors.initials: Y
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Eugene
authors.initials: E
authors.lastname: Serebryany
authors.forename: Victoria
authors.initials: V
authors.lastname: Mooney
authors.forename: Brian T
authors.initials: BT
authors.lastname: DeVree
authors.forename: Roger K
authors.initials: RK
authors.lastname: Sunahara
authors.forename: Elsa C Y
authors.initials: EC
authors.lastname: Yan
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D057809:HEK293 Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D049329:Nanostructures / Q000737:chemistry*; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000819:agonists / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; C473449:PTH1R protein, human; D044168:Receptor, Parathyroid Hormone, Type 1; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium