Structure-Encoded Location Biased Signaling in a Class B GPCR: Focus on the PTH Type 1 Receptor.

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein‐coupled receptor (GPCR) that regulates mineral‐ion, vitamin D, and bone homeostasis. PTH‐induced activation of the PTHR results in both transient and sustained cAMP production, from the plasma membrane and endosomes, respectively; however, it is not clear whether the spatial (location) or temporal (duration) components of cAMP signaling result in distinct biological outcomes. To answer this question, we generated a location biased ligand via epimerization of a single amino acid in PTH (PTH 7d ). Here we show that PTH 7d induces sustained cAMP responses exclusively from the plasma membrane that are very similar to those observed for PTH 1‐34 and a long‐acting PTH analog (LA‐PTH), two previously developed synthetic PTHR ligands that trigger sustained cAMP signaling from endosomes. PTH 7d location bias occurs as a result of a unique active PTHR conformation that triggers sustained cAMP signaling exclusively from the plasma membrane due to impaired β‐arrestin (βarr) coupling to the receptor. We further demonstrate the physiological importance of subcellular signaling location by the PTHR, as studies in mice show that sustained cAMP from endosomes is required for PTHR‐mediated elevations in serum Ca 2+ and active vitamin D levels. Additionally, assays in polarized epithelial cells reveal that endosomal cAMP signaling is a determinant for renal upregulation of the rate‐limiting hydroxylase that catalyzes the formation of active vitamin D. Together, these results advance our understanding of how location of GPCR signaling can regulate particular biological functions and add new insights into drug design based upon spatiotemporal manipulation of GPCR signaling.

Retromer in Osteoblasts Interacts With Protein Phosphatase 1 Regulator Subunit 14C, Terminates Parathyroid Hormone's Signaling, and Promotes Its Catabolic Response

Parathyroid hormone (PTH) type 1 receptor (PTH1R), as a typical class B1 G protein-coupled receptor (GPCR), is responsible for regulating bone turnover and maintaining calcium homeostasis, and its dysregulation has been implicated in the development of several diseases. The extracellular domain (ECD) of PTH1R is crucial for the recognition and binding of ligands, and the receptor may exhibit an autoinhibited state with the closure of the ECD in the absence of ligands. However, the correlation between ECD conformations and PTH1R activation remains unclear. Thus, this study combines enhanced sampling molecular dynamics (MD) simulations and Markov state models (MSMs) to reveal the possible relevance between the ECD conformations and the activation of PTH1R. First, 22 intermediate structures are generated from the autoinhibited state to the active state and conducted for 10 independent 200 ns simulations each. Then, the MSM is constructed based on the cumulative 44 μs simulations with six identified microstates. Finally, the potential interplay between ECD conformational changes and PTH1R activation as well as cryptic allosteric pockets in the intermediate states during receptor activation is revealed. Overall, our findings reveal that the activation of PTH1R has a specific correlation with ECD conformational changes and provide essential insights for GPCR biology and developing novel allosteric modulators targeting cryptic sites.

The classical paradigm of G protein-coupled receptor (GPCR) signaling via G proteins is grounded in a view that downstream responses are relatively transient and confined to the cell surface, but this notion has been revised in recent years following the identification of several receptors that engage in sustained signaling responses from subcellular compartments following internalization of the ligand-receptor complex. This phenomenon was initially discovered for the parathyroid hormone (PTH) type 1 receptor (PTH1R), a vital GPCR for maintaining normal calcium and phosphate levels in the body with the paradoxical ability to build or break down bone in response to PTH binding. The diverse biological processes regulated by this receptor are thought to depend on its capacity to mediate diverse modes of cyclic adenosine monophosphate (cAMP) signaling. These include transient signaling at the plasma membrane and sustained signaling from internalized PTH1R within early endosomes mediated by PTH. Here we discuss recent structural, cell signaling, and in vivo studies that unveil potential pharmacological outputs of the spatial versus temporal dimension of PTH1R signaling via cAMP. Notably, the combination of molecular dynamics simulations and elastic network model-based methods revealed how precise modulation of PTH signaling responses is achieved through structure-encoded allosteric coupling within the receptor and between the peptide hormone binding site and the G protein coupling interface. The implications of recent findings are now being explored for addressing key questions on how location bias in receptor signaling contributes to pharmacological functions, and how to drug a difficult target such as the PTH1R toward discovering nonpeptidic small molecule candidates for the treatment of metabolic bone and mineral diseases.

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators in PTHR signaling that could be extended broadly to GPCRs, in particular to class B peptide hormone receptors to expedite discoveries of small molecules toward the treatment of diverse hormonal and metabolic diseases.

The parathyroid hormone (PTH) type 1 receptor (PTHR) regulates a variety of key biological processes, including mineral ion and bone homeostasis, through the action of its two natural peptide ligands: PTH and PTH‐related peptide (PTHrP). Particularly, PTHrP is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling. Although mature forms of PTHR ligands consist of 84 and 141/173 amino acids for PTH and PTHrP, respectively, our current understanding on how endogenous PTHR ligands transduce signals through PTHR is largely derived from studies done with the N‐terminal fragments of both peptides, PTH1‐34 and PTHrP1‐36. While both peptides induce acute intracellular Ca2+ (iCa2+) release, they differ in the location and duration of cAMP signaling. PTH1‐34 induces sustained cAMP production from PTHR signaling complexes in endosomes, whereas PTHrP1–36 induces transient cAMP production derived from ligand–PTHR interactions at the plasma membrane. Although the transient nature of PTHrP1‐36 signaling has been regarded as a main PTHrP feature, here we demonstrate that PTHrP1‐36 cannot be considered as an analogue of the native PTHrP1‐141. We show that PTHrP1‐141 triggers sustained cAMP signaling from the plasma membrane without inducing b‐arrestin recruitment. Additionally, PTHrP1‐141 fails to stimulate iCa2+ release. We also show that the molecular basis for signaling differences between PTHrP1‐36 and native PTHrP1‐141 are caused by the stabilization of distinct PTHR conformations. These results indicate that native PTHrP1‐141 displays distinct signaling features from PTHrP1‐36 and can be considered an endogenous biased agonist for PTHR.

The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.

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 induces 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 H〈 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 in 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.Support or Funding InformationNIH R01 DK116780 and DK102495This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

EBP50 inhibits the anti-mitogenic action of the parathyroid hormone type 1 receptor in vascular smooth muscle cells

Evidence suggests that PTHrP [PTH (parathyroid hormone)-related protein] can act as an inflammatory mediator in several pathological settings including cardiovascular disease. The aim of the present study was to determine whether PTHrP might be involved in human platelet activation. We used a turbidimetric method to determine platelet aggregation. The expression of PTH1R (PTH type 1 receptor) in human platelets was analysed by Western blot and flow cytometry analyses. PTHrP-(1-36) (10(-7) mol/l) by itself failed to modify the activation of platelets. However, it significantly enhanced ADP-induced platelet activation, and also increased the ability of other agonists (thrombin, collagen and arachidonic acid) to induce platelet aggregation. H89 (10(-6) mol/l) and 25 x 10(-6) mol/l Rp-cAMPS (adenosine 3',5'-cyclic monophosphorothioate Rp-isomer), two protein kinase A inhibitors, and 25 x 10(-9) mol/l bisindolylmaleimide I, a protein kinase C inhibitor, partially decreased the enhancing effect of PTHrP-(1-36) on ADP-induced platelet activation. Meanwhile, 10(-6) mol/l PTHrP-(7-34), a PTH1R antagonist, as well as 10(-5) mol/l PD098059, a MAPK (mitogen-activated protein kinase) inhibitor, or a farnesyltransferase inhibitor abolished this effect of PTHrP-(1-36). Moreover, 10(-7) mol/l PTHrP-(1-36) increased (2-fold over control) MAPK activation in human platelets. PTH1R was detected in platelets, and the number of platelets expressing it on their surface in patients during AMI (acute myocardial infarction) was not different from that in a group of patients with similar cardiovascular risk factors without AMI. Western blot analysis showed that total PTH1R protein levels were markedly higher in platelets from control than those from AMI patients. PTH1R was found in plasma, where its levels were increased in AMI patients compared with controls. In conclusion, human platelets express the PTH1R. PTHrP can interact with this receptor to enhance human platelet activation induced by several agonists through a MAPK-dependent mechanism.

In the recent past, pharmacological and optical approaches have been employed to gain new insights on the basic mechanisms by which two different ligand systems—theparathyroid hormone (PTH) (endocrine and homeostatic) and the PTH-related peptide (PTHrP), autocrine or paracrine mediator of developmental tissues—achieve distinct biological effects by acting at the same cell surface receptor, the PTH type 1 receptor (PTHR). This chapter aims to present a comprehensive model to account for the molecular origin of signal differences mediated by the binding of PTH and PTHrP to the PTHR. It covers the kinetic properties of reactions engaged in the PTHR signalling system and endosomal G protein signalling as an emerging hypothesis for PTHR function.

The molecular mechanisms by which bone cells transduce mechanical stimuli into intracellular biochemical responses have yet to be established. There is evidence that mechanical stimulation acts synergistically with parathyroid hormone PTH(1-34) in mediating bone growth. Using picosecond time-resolved fluorescence microscopy and G protein-coupled receptor conformation-sensitive fluorescence resonance energy transfer (FRET), we investigated conformational transitions in parathyroid hormone type 1 receptor (PTH1R). 1) A genetically engineered PTH1R sensor containing an intramolecular FRET pair was constructed that enabled detection of conformational activity of PTH1R in single cells. 2) The nature of ligand-dependent conformational change of PTH1R depends on the type of ligand: stimulation with the PTH(1-34) leads to conformational transitions characterized by decrease in FRET efficiency while NH(2)-terminal truncated ligand PTH(3-34) stimulates conformational transitions characterized by higher FRET efficiencies. 3) Stimulation of murine preosteoblastic cells (MC3T3-E1) with fluid shear stress (FSS) leads to significant changes in conformational equilibrium of the PTH1R in MC3T3-E1 cells, suggesting that mechanical perturbation of the plasma membrane leads to ligand-independent response of the PTH1R. Conformational transitions induced by mechanical stress were characterized by an increase in FRET efficiency, similar to those induced by the NH(2)-terminal truncated ligand PTH(3-34). The response to the FSS stimulation was inhibited in the presence of PTH(1-34) in the flow medium. These results indicate that the FSS can modulate the action of the PTH(1-34) ligand. 4) Plasma membrane fluidization using benzyl alcohol or cholesterol extraction also leads to conformational transitions characterized by increased FRET levels. We therefore suggest that PTH1R is involved in mediating primary mechanochemical signal transduction in MC3T3-E1 cells.

The parathyroid hormone type 1 receptor (PTH1R), a canonical class B GPCR, is regulated by a positive allosteric modulator, extracellular Ca2+. Calcium ions prolong the residence time of PTH on the PTH1R, leading to increased receptor activation and duration of cAMP signaling. But the essential mechanism of the allosteric behavior of PTH1R is not fully understood. Here, extensive molecular dynamics (MD) simulations are performed for the PTH1R-G-protein combinations with and without Ca2+ to describe how calcium ions allosterically engage receptor-G-protein coupling. We find that the binding of Ca2+ stabilizes the conformation of the PTH1R-PTH-spep (the α5 helix of Gs protein) complex, especially the extracellular loop 1 (ECL1). Moreover, the MM-GBSA result indicates that Ca2+ allosterically promotes the interaction between PTH1R and spep, consistent with the observation of steered molecular dynamics (SMD) simulations. We further illuminate the possible allosteric signaling pathway from the stable Ca2+-coupling site to the intracellular G-protein binding site. These results unveil structural determinants for Ca2+ allosterism in the PTH1R-PTH-spep complex and give insights into pluridimensional GPCR signaling regulated by calcium ions.

The precise molecular mechanisms by which bone cells transduce mechanical stimulus into intracellular biochemical response have not been established yet. Here we show that mechanical perturbation of the plasma membrane leads to ligand‐independent conformational transitions in G protein coupled receptor (GPCR) such as parathyroid hormone type 1 receptor (PTH1r). By using picosecond time‐resolved fluorescence microscopy and GPCR conformation‐sensitive fluorescence energy transfer (FRET) we found that stimulation of MC3T3 cells with fluid shear stress or membrane fluidizing agent leads to a significant changes in conformational equilibrium of PTH1r in MC3T3 cells. The PTH1r conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single cell under fluid shear stress in real time using genetically engineered PTH1r containing intramolecular FRET pair. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of PTH1r. Therefore we suggest that PTH1r is involved in mediating primary mechanochemical signal transduction in MC3T3 cells. Furthermore we show that conformational transitions induced by mechanical stress are different from those induced by its ligand PTH(1‐34).This research was supported by the NIH grant HL086943.