LAUSR

Abstract The parathyroid hormone (PTH) type 1 receptor (PTHR) is a key regulator of bone turnover and calcium homeostasis. PTHR primarily activates the stimulatory G-protein, Gs, for adenylate cyclases that induce the production of cAMP. Our laboratory has demonstrated that PTH stimulates both transient and prolonged cAMP production at the plasma membrane and in endosomes, respectively. Activation of PKA through PTHR signaling in endosomes activates v-ATPases, which acidify the endosomal lumen. A decrease below pH 6.5 triggers the release of PTH from the receptor, thereby terminating Gs signaling. However, the structural mechanisms of PTH binding to PTHR and release from endosomal receptor are unclear. We studied the structural changes of PTH(1-34) upon binding to PTHR using nuclear magnetic resonance (NMR). We acquired 1H-15N HSQC, 2D 1H homonuclear NOESY, TOCSY, and 3D 15N-edited NOESY NMR spectra. The Hα chemical shifts of 15N-labeled PTH(1-34) residues, compared to “random coil” chemical shifts, demonstrate that at plasma membrane pH, unbound PTH consists of two halves: a helical C-terminal half (residues 18-34) and a less structured N-terminal half (1-13) connected by a random coil “hinge” (14-17). We hypothesize this hinge coordinates a two-step ligand binding mechanism, in which the helical C-terminal half of PTH binds to the extracellular domain of PTHR (ECD), followed by binding of the N-terminal half to the receptor transmembrane domain (TMD) and subsequent receptor activation. To investigate the first step of ligand binding, we compared the 1H-15N TROSY spectra of 15N-PTH(1-34) in the absence and presence of unlabeled ECD. HSQC peak intensities of PTH residues 21-34 are significantly reduced in the presence of ECD, suggesting that the flexibility of helical PTH(21-34) is compromised upon binding to ECD. Interestingly, peaks of L15 and S17, which participate in the hinge, disappear in the presence of ECD. In addition, new peaks for several PTH residues in both N- and C-terminal halves appear in the presence of ECD, indicating that ECD binding triggers new conformations of these residues. These data suggest that the increased rigidity of the hinge and new conformations of other N-terminal half residues position the N-terminal half for interaction with and binding to the TMD. To investigate the structural changes of PTH during endosomal acidification, we acquired HSQC spectra at pH 7.2, 6.5, and 5.9. These data reveal that the chemical shifts of PTH residues H9, H14, L15, and H32 are significantly affected by pH decrease, with H14 and L15 notably being most affected. Since H14 and L15 participate the hinge, we predict that conformational changes in the hinge as a result of pH decrease trigger ligand dissociation. Our work gives insight into the structural mechanisms of PTHR ligand binding and signaling regulation.