Etiological Mechanisms and Genetic/Biological Modulation Related to PTH1R in Primary Failure of Tooth Eruption.

Primary failure of eruption (PFE) is a rare disorder that is characterized by the inability of a molar tooth/teeth to erupt to the occlusal plane or to normally react to orthodontic force. This condition is related to hereditary factors and has been extensively researched over many years. However, the etiological mechanisms of pathogenesis are still not fully understood. Evidence from studies on PFE cases has shown that PFE patients may carry parathyroid hormone 1 receptor (PTH1R) gene mutations, and genetic detection can be used to diagnose PFE at an early stage. PTH1R variants can lead to altered protein structure, impaired protein function, and abnormal biological activities of the cells, which may ultimately impact the behavior of teeth, as observed in PFE. Dental follicle cells play a critical role in tooth eruption and root development and are regulated by parathyroid hormone-related peptide (PTHrP)-PTH1R signaling in their differentiation and other activities. PTHrP-PTH1R signaling also regulates the activity of osteoblasts, osteoclasts and odontoclasts during tooth development and eruption. When interference occurs in the PTHrP-PTH1R signaling pathway, the normal function of dental follicles and bone remodeling are impaired. This review provides an overview of PTH1R variants and their correlation with PFE, and highlights that a disruption of PTHrP-PTH1R signaling impairs the normal process of tooth development and eruption, thus providing insight into the underlying mechanisms related to PTH1R and its role in driving PFE.

Highly biased agonism for GPCR ligands via nanobody tethering.

Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present an approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.

Rare genetic disorders that impair parathyroid hormone synthesis, secretion, or bioactivity provide insights into the diagnostic utility of different parathyroid hormone assays.

PURPOSE OF REVIEW: Parathyroid hormone (PTH) is the major peptide hormone regulator of blood calcium homeostasis. Abnormal PTH levels can be observed in patients with various congenital and acquired disorders, including chronic kidney disease (CKD). This review will focus on rare human diseases caused by PTH mutations that have provided insights into the regulation of PTH synthesis and secretion as well as the diagnostic utility of different PTH assays. RECENT FINDINGS: Over the past years, numerous diseases affecting calcium and phosphate homeostasis have been defined at the molecular level that are responsible for reduced or increased serum PTH levels. The underlying genetic mutations impair parathyroid gland development, involve the PTH gene itself, or alter function of the calcium-sensing receptor (CaSR) or its downstream signaling partners that contribute to regulation of PTH synthesis or secretion. Mutations in the pre sequence of the mature PTH peptide can, for instance, impair hormone synthesis or intracellular processing, while amino acid substitutions affecting the secreted PTH(1-84) impair PTH receptor (PTH1R) activation, or cause defective cleavage of the pro-sequence and thus secretion of a pro- PTH with much reduced biological activity. Mutations affecting the secreted hormone can alter detection by different PTH assays, thus requiring detailed knowledge of the utilized diagnostic test. SUMMARY: Rare diseases affecting PTH synthesis and secretion have offered helpful insights into parathyroid biology and the diagnostic utility of commonly used PTH assays, which may have implications for the interpretation of PTH measurements in more common disorders such as CKD.

Parathyroid hormone-PTH1R signaling in cardiovascular disease and homeostasis.

Primary hyperparathyroidism (pHPT) afflicts our aging population with an incidence approaching 50 per 100 000 patient-years at a female:male ratio of ~3:1. Decisions surrounding surgical management are currently driven by age, hypercalcemia severity, presence of osteoporosis, renal insufficiency, or hypercalciuria with or without nephrolithiasis. Cardiovascular (CV) disease (CVD) is not systematically considered. This is notable since the parathyroid hormone (PTH) 1 receptor (PTH1R) is biologically active in the vasculature, and adjusted CV mortality risk is increased almost threefold in individuals with pHPT who do not meet contemporary recommendations for surgical cure. We provide an overview of epidemiology, pharmacology, and physiology that highlights the need to: (i) identify biomarkers that establish a healthy 'set point' for CV PTH1R signaling tone; (ii) better understand the pharmacokinetic-pharmacodynamic (PK-PD) relationships of PTH1R ligands in CV homeostasis; and (iii) incorporate CVD risk assessment into the management of hyperparathyroidism.

Unraveling the Interplay of Extracellular Domain Conformational Changes and Parathyroid Hormone Type 1 Receptor Activation in Class B1 G Protein-Coupled Receptors: Integrating Enhanced Sampling Molecular Dynamics Simulations and Markov State Models.

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.

ADAM19 cleaves the PTH receptor and associates with brachydactyly type E.

Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64-65. ADAM-19 cleavage increased Gq and decreased Gs activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.

Pth1r in Neural Crest Cells Regulates Nasal Cartilage Differentiation.

Neural crest cells (NCC) arise from the dorsal margin of the neural plate border and comprise a unique cell population that migrates to and creates the craniofacial region. Although factors including Shh, Fgf8, and bone morphogenetic proteins have been shown to regulate these biological events, the role of parathyroid hormone 1 receptor (Pth1r) has been less studied. We generated an NCC-specific mouse model for Pth1r and researched gene expression, function, and interaction focusing on nasal cartilage framework and midfacial development. Wnt1-Cre;Pth1rfl/fl;Tomatofl/+ mice had perinatal lethality, but we observed short snout and jaws, tongue protrusion, reduced NCC-derived cranial length, increased mineralization in nasal septum and hyoid bones, and less bone mineralization at interfrontal suture in mutants at E18.5. Importantly, the mutant nasal septum and turbinate cartilage histologically revealed gradual, premature accelerated hypertrophic differentiation. We then studied the underlying molecular mechanisms by performing RNA seq analysis and unexpectedly found that expression of Ihh and related signaling molecules was enhanced in mutant nasomaxillary tissues. To see if Pth1r and Ihh signaling are associated, we generated a Wnt1-Cre; Ihhfl/fl;Pth1rfl/fl;Tomatofl/+ (DKO) mouse and compared the phenotypes to those of each single knockout mouse: Wnt1-Cre; Ihhfl/fl;Pth1rfl/+;Tomatofl/+ (Ihh-CKO) and Wnt1-Cre;Ihhfl/+;Pth1rfl/fl;Tomatofl/+ (Pth1r-CKO). Ihh-CKO mice displayed a milder effect. Of note, the excessive hypertrophic conversion of the nasal cartilage framework observed in Pth1r-CKO was somewhat rescued DKO embryos. Further, a half cAMP responsive element and the 4 similar sequences containing 2 mismatches were identified from the promoter to the first intron in Ihh gene. Gli1-CreERT2;Pth1rfl/fl;Tomatofl/+, a Pth1r-deficient model targeted in hedgehog responsive cells, demonstrated the enlarged hypertrophic layer and significantly more Tomato-positive chondrocytes accumulated in the nasal septum and ethmoidal endochondral ossification. Collectively, the data suggest a relevant Pth1r/Ihh interaction. Our findings obtained from novel mouse models for Pth1r signaling illuminate previously unknown aspects in craniofacial biology and development.

Hedgehog activation promotes osteogenic fates of growth plate resting zone chondrocytes through transient clonal competency.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1-floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone ("patched roses") and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.

Endosomal signaling via cAMP in parathyroid hormone (PTH) type 1 receptor biology.

Compartmentalization of GPCR signaling is an emerging topic that highlights the physiological relevance of spatial bias in signaling. The parathyroid hormone (PTH) type 1 receptor (PTH1R) was the first GPCR described to signal via heterotrimeric G-protein and cAMP from endosomes after ß-arrestin mediated internalization, challenging the canonical GPCR signaling model which established that signaling is terminated by receptor internalization. More than a decade later, many other GPCRs have been shown to signal from endosomes via cAMP, and recent studies have proposed that location of cAMP generation impacts physiological outcomes of GPCR signaling. Here, we review the extensive literature regarding PTH1R endosomal signaling via cAMP, the mechanisms that regulate endosomal generation of cAMP, and the implications of spatial bias in PTH1R physiological functions.

Conserved class B GPCR activation by a biased intracellular agonist.

Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets1-5. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development6,7. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (Gs) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with Gs. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.

Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome.

The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor that plays key roles in regulating calcium homeostasis and skeletal development via binding the ligands, PTH and PTH-related protein (PTHrP), respectively. Eiken syndrome is a rare disease of delayed bone mineralization caused by homozygous PTH1R mutations. Of the three mutations identified so far, R485X, truncates the PTH1R C-terminal tail, while E35K and Y134S alter residues in the receptor's amino-terminal extracellular domain. Here, using a variety of cell-based assays, we show that R485X increases the receptor's basal rate of cAMP signaling and decreases its capacity to recruit ß-arrestin2 upon ligand stimulation. The E35K and Y134S mutations each weaken the binding of PTHrP leading to impaired ß-arrestin2 recruitment and desensitization of cAMP signaling response to PTHrP but not PTH. Our findings support a critical role for interaction with ß-arrestin in the mechanism by which the PTH1R regulates bone formation.

Scribble scrambles parathyroid hormone receptor interactions to regulate phosphate and vitamin D homeostasis.

G protein-coupled receptors, including PTHR, are pivotal for controlling metabolic processes ranging from serum phosphate and vitamin D levels to glucose uptake, and cytoplasmic interactors may modulate their signaling, trafficking, and function. We now show that direct interaction with Scribble, a cell polarity-regulating adaptor protein, modulates PTHR activity. Scribble is a crucial regulator for establishing and developing tissue architecture, and its dysregulation is involved in various disease conditions, including tumor expansion and viral infections. Scribble co-localizes with PTHR at basal and lateral surfaces in polarized cells. Using X-ray crystallography, we show that colocalization is mediated by engaging a short sequence motif at the PTHR C-terminus using Scribble PDZ1 and PDZ3 domain, with binding affinities of 31.7 and 13.4 µM, respectively. Since PTHR controls metabolic functions by actions on renal proximal tubules, we engineered mice to selectively knockout Scribble in proximal tubules. The loss of Scribble impacted serum phosphate and vitamin D levels and caused significant plasma phosphate elevation and increased aggregate vitamin D3 levels, whereas blood glucose levels remained unchanged. Collectively these results identify Scribble as a vital regulator of PTHR-mediated signaling and function. Our findings reveal an unexpected link between renal metabolism and cell polarity signaling.

PTH regulates osteogenesis and suppresses adipogenesis through Zfp467 in a feed-forward, PTH1R-cyclic AMP-dependent manner.

Conditional deletion of the PTH1R in mesenchymal progenitors reduces osteoblast differentiation, enhances marrow adipogenesis, and increases zinc finger protein 467 (Zfp467) expression. In contrast, genetic loss of Zfp467 increased Pth1r expression and shifts mesenchymal progenitor cell fate toward osteogenesis and higher bone mass. PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass in mice. Prrx1Cre; Zfp467fl/fl but not AdipoqCre; Zfp467fl/fl mice exhibit high bone mass and greater osteogenic differentiation similar to the Zfp467-/- mice. qPCR results revealed that PTH suppressed Zfp467 expression primarily via the cyclic AMP/PKA pathway. Not surprisingly, PKA activation inhibited the expression of Zfp467 and gene silencing of Pth1r caused an increase in Zfp467 mRNA transcription. Dual fluorescence reporter assays and confocal immunofluorescence demonstrated that genetic deletion of Zfp467 resulted in higher nuclear translocation of NFκB1 that binds to the P2 promoter of the Pth1r and increased its transcription. As expected, Zfp467-/- cells had enhanced production of cyclic AMP and increased glycolysis in response to exogenous PTH. Additionally, the osteogenic response to PTH was also enhanced in Zfp467-/- COBs, and the pro-osteogenic effect of Zfp467 deletion was blocked by gene silencing of Pth1r or a PKA inhibitor. In conclusion, our findings suggest that loss or PTH1R-mediated repression of Zfp467 results in a pathway that increases Pth1r transcription via NFκB1 and thus cellular responsiveness to PTH/PTHrP, ultimately leading to enhanced bone formation.

Molecular insights into peptide agonist engagement with the PTH receptor.

The parathyroid hormone (PTH) 1 receptor (PTH1R) is a G protein-coupled receptor (GPCR) that regulates skeletal development and calcium homeostasis. Here, we describe cryo-EM structures of the PTH1R in complex with fragments of the two hormones, PTH and PTH-related protein, the drug abaloparatide, as well as the engineered tool compounds, long-acting PTH (LA-PTH) and the truncated peptide, M-PTH(1-14). We found that the critical N terminus of each agonist engages the transmembrane bundle in a topologically similar fashion, reflecting similarities in measures of Gαs activation. The full-length peptides induce subtly different extracellular domain (ECD) orientations relative to the transmembrane domain. In the structure bound to M-PTH, the ECD is unresolved, demonstrating that the ECD is highly dynamic when unconstrained by a peptide. High resolutions enabled identification of water molecules near peptide and G protein binding sites. Our results illuminate the action of orthosteric agonists of the PTH1R.

Parathyroid hormone receptor-1 signaling aggravates hepatic fibrosis through upregulating cAMP response element-binding protein-like 2.

BACKGROUND AND AIMS: Parathyroid hormone receptor-1 (PTH1R) is a class B G protein-coupled receptor central to skeletal development, bone turnover, and calcium homeostasis. However, the role of PTH1R signaling in liver fibrosis is largely unknown. Here, the role of PTH1R signaling in the activation of HSCs and hepatic fibrosis was examined. APPROACH AND RESULTS: PTH1R was highly expressed in activated HSCs and fibrotic liver by using human liver specimens or carbon tetrachloride (CCl 4 )-treated or methionine and choline-deficient diet (MCD)-fed C57/BL6 mice. The mRNA level of hepatic PTH1R was positively correlated to α-smooth muscle actin in patients with liver cirrhosis. Mice with HSCs-specific PTH1R deletion were protected from CCl 4 , MCD, or western diet, plus low-dose CCl 4 -induced liver fibrosis. Conversely, parathyroid hormone (PTH) aggravated liver fibrosis in CCl 4 -treated mice. Mouse primary HSCs and LX2 cell lines were used for in vitro experiments. Molecular analyses by luciferase reporter assays and chromatin immunoprecipitation assays in combination with mRNA sequencing in HSCs revealed that cAMP response element-binding protein-like 2 (Crebl2), a novel regulator in HSCs treated by PTH that interacted with mothers against decapentaplegic homolog 3 (SMAD3) and increased the transcription of TGFß in activating HSCs and collagen deposition. In agreement, HSCs-specific Crebl2 deletion ameliorated PTH-induced liver fibrosis in CCl 4 -treated mice. CONCLUSIONS: In both mouse and human models, we found that PTH1R was highly expressed in activated HSCs and fibrotic liver. PTH1R signaling regulated collagen production in the HSCs through Crebl2/SMAD3/TGFß regulatory circuits. Blockade of PTH1R signaling in HSCs might help mitigate the development of liver fibrosis.

MMP14 cleaves PTH1R in the chondrocyte-derived osteoblast lineage, curbing signaling intensity for proper bone anabolism.

Bone homeostasis is regulated by hormones such as parathyroid hormone (PTH). While PTH can stimulate osteo-progenitor expansion and bone synthesis, how the PTH-signaling intensity in progenitors is controlled is unclear. Endochondral bone osteoblasts arise from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). We found, via single-cell transcriptomics, that HC-descendent cells activate membrane-type 1 metalloproteinase 14 (MMP14) and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. Unlike Mmp14 global knockouts, postnatal day 10 (p10) HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. Mechanistically, MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling, and consistent with the implied regulatory role, in Mmp14ΔHC mutants, PTH signaling is enhanced. We found that HC-derived osteoblasts contribute ~50% of osteogenesis promoted by treatment with PTH 1-34, and this response was amplified in Mmp14ΔHC. MMP14 control of PTH signaling likely applies also to both HC- and non-HC-derived osteoblasts because their transcriptomes are highly similar. Our study identifies a novel paradigm of MMP14 activity-mediated modulation of PTH signaling in the osteoblast lineage, contributing new insights into bone metabolism with therapeutic significance for bone-wasting diseases.

Parathyroid hormone 1 receptor signaling mediates breast cancer metastasis to bone in mice.

Bone metastases are a common complication of breast cancer. We have demonstrated that intermittent administration of parathyroid hormone (PTH[1-34]) reduces the incidence of bone metastases in murine models of breast cancer by acting on osteoblasts to alter the bone microenvironment. Here, we examined the role of signaling mediated by PTH 1 receptor (PTH1R) in both osteoblasts and breast cancer cells in influencing bone metastases. In mice with impaired PTH1R signaling in osteoblasts, intermittent PTH did not reduce bone metastasis. Intermittent PTH also did not reduce bone metastasis when expression of PTH1R was knocked down in 4T1 murine breast cancer cells by shRNA. In 4T1 breast cancer cells, PTH decreased expression of PTH-related protein (PTHrP), implicated in the vicious cycle of bone metastases. Knockdown of PTHrP in 4T1 cells significantly reduced migration toward MC3T3-E1 osteoblasts, and migration was further inhibited by treatment with intermittent PTH. Conversely, overexpression of PTHrP in 4T1 cells increased migration toward MC3T3-E1 osteoblasts, and this was not inhibited by PTH. In conclusion, PTH1R expression is crucial in both osteoblasts and breast cancer cells for PTH to reduce bone metastases, and in breast cancer cells, this may be mediated in part by suppression of PTHrP.

Molecular Mechanisms of PTH/PTHrP Class B GPCR Signaling and Pharmacological Implications.

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.

Altered signaling at the PTH receptor via modified agonist contacts with the extracellular domain provides a path to prolonged agonism in vivo.

The parathyroid hormone type 1 receptor (PTHR1), a Class B GPCR, is activated by long polypeptides, including drugs for osteoporosis and hypoparathyroidism. The PTHR1 engages peptide agonists via a two-step mechanism. Initial contact involves the extracellular domain (ECD), which has been thought to contribute primarily to receptor-peptide binding, and then the N terminus of the peptide engages the receptor transmembrane domain (TMD), which is thought to control the message conveyed to intracellular partners. This mechanism has been suggested to apply to other Class B GPCRs as well. Here, we show that modification of a PTHR1 agonist at ECD-contact sites can alter the signaling profile, an outcome that is not accommodated by the current two-step binding model. Our data support a modified two-step binding model in which agonist orientation on the ECD surface can influence the geometry of agonist-TMD engagement. This expanded binding model offers a mechanism by which altering ECD-contact residues can affect signaling profile. Our discoveries provide a rationale for exploring agonist modifications distal from the TMD-contact region in future efforts to optimize therapeutic performance of peptide hormone analogs.

Dynamic Balance between PTH1R-Dependent Signal Cascades Determines Its Pro- or Anti-Osteogenic Effects on MSC.

Parathyroid hormone (PTH) is one of the key regulators of calcium and phosphate metabolism in the body, controlling bone metabolism and ion excretion by the kidneys. At present, attempts to use PTH as a therapeutic agent have been associated with side-effects, the nature of which is not always clear and predictable. In addition, it is known that in vivo impairment of PTH post-receptor signaling is associated with atypical differentiation behavior not only of bone cells, but also of connective tissues, including adipose tissue. In this work, we studied the functional responses of multipotent mesenchymal stromal cells (MSCs) to the action of PTH at the level of single cells. We used MSCs isolated from the periosteum and subcutaneous adipose tissue to compare characteristics of cell responses to PTH. We found that the hormone can activate three key responses via its receptor located on the surface of MSCs: single transients of calcium, calcium oscillations, and hormone-activated smooth increase in intracellular calcium. These types of calcium responses led to principally different cellular responses of MSCs. The cAMP-dependent smooth increase of intracellular calcium was associated with pro-osteogenic action of PTH, whereas phospholipase C dependent calcium oscillations led to a decrease in osteogenic differentiation intensity. Different variants of calcium responses are in dynamic equilibrium. Suppression of one type of response leads to increased activation of another type and, accordingly, to a change in the effect of PTH on cell differentiation.