Parathyroid hormone 1 receptor (PTH1R) is a family-B GPCR that plays a crucial role in bone remodeling. Previous studies show extracellular Ca2+ is a positive allosteric modulator for one PTH1R ligand, parathyroid hormone (PTH), which is approved by US FDA to treat severe osteoporosis. Moreover, PTH residues E19 & E22 have shown to be involved in Ca2+ sensing. However, the effects of PTH Ca2+ sensing on intracellular G-protein binding are unknown. Here, we used FRET-based SPASM sensors to study the interaction between PTH1R and different Ga peptides. SPASM sensors, which are isolated in native HEK293T membranes through optimized protocol, contain PTH1R followed by the acceptor fluorophore, a flexible linker, the donor fluorophore, and a peptide from a Ga subunit that mimics the interaction of the full G-protein heterotrimer. In the current study, two SPASM sensor preparation methods, Giant Plasma Membrane Vessiculation and native membrane preparation, were employed. The quality and integrality of the SPASM sensors isolated through each method were evaluated and compared to one another. We performed FRET experiments to quantify the activation of different Ga isoforms by PTH and its Ca2+ sensing mutant, PTHE19AE22A. PTH binding to PTH1R SPASM sensors causes differential interactions between PTH1R and the Gs, Gq and Gi peptides. PTHE19AE22A activation of PTH1R-SPASM sensors leads to distinct interaction profiles between each G-peptide isoforms, which were further modulated by the presence of extracellular Ca2+. Quantifying the differential activation of the specific Ga isoforms by PTH and PTHE19AE22A in the presence and absence of Ca2+ will delineate mechanistic details of PTH1R activation and its role in bone-related diseases. Further, understanding the extracellular Ca2+ modulation of PTH signaling will provide insight for developing treatments for chronic hypocalcemia associated with hypoparathyroidism, while uncovering PTH1R novel regulation in bone remodeling.
Level of Honors
magna cum laude
Jiang, Difei, "Mechanism of PTH CA2+ Sensing on G-Protein Interactions with PTH1R" (2023). Lawrence University Honors Projects. 180.