MON-747 Distinct Signaling Pathways of Dimeric R25CPTH(1-34) and Implications for Therapeutic Applications in Bone Metabolism

Abstract Disclosure: M. Noh: None. H.J. Kim: None. J. Yang: None. K. Kawakami: None. A. Inoue: None. D. Park: None. S. Lee: None. H. Lee: None. S. Lee: None. Our previous study reported that patients with hypoparathyroidism possessing a homozygous mutation in parathyroid hormone (PTH), which substitutes arginine with cysteine at position 25, exhibit increased bone density. The R25CPTH mutant forms homodimers with altered functionality compared to monomeric PTH. Typically, PTH binds strongly to the G protein-uncoupled (R0) conformation of the PTH type 1 receptor (PTH1R), whereas dimeric R25CPTH(1-34) selectively binds with the G protein-coupled (RG) conformation of PTH1R. Despite these changes, dimeric R25CPTH(1-34) demonstrates bone anabolic effects comparable to wild-type PTH(1-34) in vivo. This study aims to elucidate the specific downstream pathways activated by dimeric R25CPTH(1-34) and to understand its bone anabolic effects for therapeutic applications. Osteoblasts isolated from mouse calvaria were cultured and subjected to RNA-seq to observe transcriptomic changes in response to either chemically synthesized PTH(1-34) or dimeric R25CPTH(1-34) at 6, 12, and 24 hours post-injection. While early transcriptional responses to both ligands were similar, significant differences in gene expression emerged over time. To discern variations in downstream signaling pathways between the two ligands, we utilized a GPCR phospho array to detect changes in signaling pathways in GP-2.3 cells treated with either ligand, then substantial changes were observed in the number of phosphorylated proteins. Despite these alterations, dimeric R25CPTH(1-34) exhibited a comparable level of PTH1R binding ability as conventional PTH(1-34), as confirmed by the nanobit system. We subsequently verified the physiological effects of each ligand through acute injection in mice, revealing a significant difference in serum cAMP induction levels. Using atomic force microscopy, we observed changes in physical properties upon ligand binding to PTH1R, confirming notable differences in binding capabilities and strength and suggesting that such differences could lead to alterations in downstream signaling pathways following receptor binding. Lastly, qRT-PCR was conducted to confirm the variations in sub-signaling pathways between the ligands in specific tissues, showing significant divergences in the expression of genes associated with the PTH response in bone and kidney tissues. Our study suggests that dimeric R25CPTH(1-34) activates distinct signaling pathways, resulting in an alteration of bone metabolism compared to PTH(1-34). While dimeric R25CPTH(1-34) demonstrates comparable binding affinity to PTH1R as PTH(1-34), it substantially alters downstream signaling, leading to decreased expression of genes involved in bone metabolism. These findings suggest that dimeric R25CPTH(1-34) has the potential to serve as a novel therapeutic agent for the treatment of conditions associated with bone loss, through a unique mechanism of action. Presentation: Monday, July 14, 2025