Rewiring PTH receptor signaling: Hormone dimerization restores endosomal signaling lost in hypocalcemia-linked PTH...

G protein-coupled receptors often form dimers and heterodimers at the plasma membrane to transduce signals from various ligands, including peptide hormones. However, the role of homodimerization in regulating signaling by the parathyroid hormone (PTH) type 1 receptor (PTH₁R) has remained ambiguous. Here, we show that PTH₁R exists as a monomer in live cells under both basal and ligand-bound conditions, even in the presence of a dimeric form of the PTH mutant, PTH^R25C (residue 25 of PTH), which is linked with hypoparathyroidism. Single-molecule fluorescence imaging and single-cell FRET assays support the monomeric behavior of PTH₁R, with molecular dynamics simulations using weighted ensemble sampling revealing that PTH^R25C destabilizes the active conformation of the receptor. In contrast, a synthetic dimeric PTH^R25C restores interactions near the receptor's N-terminal domain, maintains the active conformation, and rescues sustained cAMP signaling. These findings challenge previous assumptions about the homodimerization status of PTH₁R and highlight how ligand dimerization, rather than receptor dimerization, governs PTH₁R activation dynamics and location-biased cAMP signaling, offering mechanistic insights relevant to therapeutic strategies against hypoparathyroidism.