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.

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.

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.

Structural details of a Class B GPCR-arrestin complex revealed by genetically encoded crosslinkers in living cells.

Understanding the molecular basis of arrestin-mediated regulation of GPCRs is critical for deciphering signaling mechanisms and designing functional selectivity. However, structural studies of GPCR-arrestin complexes are hampered by their highly dynamic nature. Here, we dissect the interaction of arrestin-2 (arr2) with the secretin-like parathyroid hormone 1 receptor PTH1R using genetically encoded crosslinking amino acids in live cells. We identify 136 intermolecular proximity points that guide the construction of energy-optimized molecular models for the PTH1R-arr2 complex. Our data reveal flexible receptor elements missing in existing structures, including intracellular loop 3 and the proximal C-tail, and suggest a functional role of a hitherto overlooked positively charged region at the arrestin N-edge. Unbiased MD simulations highlight the stability and dynamic nature of the complex. Our integrative approach yields structural insights into protein-protein complexes in a biologically relevant live-cell environment and provides information inaccessible to classical structural methods, while also revealing the dynamics of the system.

Modulation of PTH1R signaling by an extracellular binding antibody.

Parathyroid hormone receptor 1 (PTH1R) is a class B G-protein coupled receptor with key roles in bone development. The receptor signals through both the Gs and Gq G-proteins as well as through ß-arrestin in a G-protein independent manner. Current treatments for bone disorders, such as osteoporosis, target the PTH1R but are suboptimal in their efficacy. Monoclonal antibodies represent a major growth area in therapeutics as a result of their superior specificity and long serum half-life. Here, we discovered antibodies against the extracellular domain (ECD) of PTH1R from a phage display library. One of these antibodies, ECD-ScFvhFc, binds PTH1R with high affinity and although it has little or no effect on G-protein dependent receptor signaling, it does reduce PTH1R mediated ß-arrestin signaling. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the ECD-ScFvhFc binding site overlapped partially with that of the cognate ligand, PTH. The results of this study demonstrate the suitability of PTH1R as a target for therapeutic antibody development.

Biased GPCR signaling by the native parathyroid hormone-related protein 1 to 141 relative to its N-terminal fragment 1 to 36.

The parathyroid hormone (PTH)-related protein (PTHrP) is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling, and causes hypercalcemia in patients with malignancy. Although mature forms of PTHrP in the body consist of splice variants of 139, 141, and 173 amino acids, our current understanding on how endogenous PTHrP transduces signals through its cognate G-protein coupled receptor (GPCR), the PTH type 1 receptor (PTHR), is largely derived from studies done with its N-terminal fragment, PTHrP1-36. Here, we demonstrate using various fluorescence imaging approaches at the single cell level to measure kinetics of (i) receptor activation, (ii) receptor signaling via Gs and Gq, and (iii) receptor internalization and recycling that the native PTHrP1-141 displays biased agonist signaling properties that are not mimicked by PTHrP1-36. Although PTHrP1-36 induces transient cAMP production, acute intracellular Ca2+ (iCa2+) release and ß-arrestin recruitment mediated by ligand-PTHR interactions at the plasma membrane, PTHrP1-141 triggers sustained cAMP signaling from the plasma membrane and fails to stimulate iCa2+ release and recruit ß-arrestin. Furthermore, we show that the molecular basis for biased signaling differences between PTHrP1-36 and properties of native PTHrP1-141 are caused by the stabilization of a singular PTHR conformation and PTHrP1-141 sensitivity to heparin, a sulfated glycosaminoglycan. Taken together, our results contribute to a better understanding of the biased signaling process of a native protein hormone acting in conjunction with a GPCR.

Critical Extracellular Ca2+ Dependence of the Binding between PTH1R and a G-Protein Peptide Revealed by MD Simulations.

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.

Proteolytic Cleavage of the Extracellular Domain Affects Signaling of Parathyroid Hormone 1 Receptor.

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to Gs and increased activation of Gq compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.

IMPROvER: the Integral Membrane Protein Stability Selector.

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of [Formula: see text]-helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/ .

New views into class B GPCRs from the crystal structure of PTH1R.

The parathyroid hormone 1 receptor (PTH1R) is a major regulator of mineral ion homeostasis and bone metabolism and is thus considered an attractive drug target for the treatment of disorders in calcium metabolism and bone-related diseases such as osteoporosis. PTH1R is a member of the class B of GPCRs, which all share a dynamic multidomain binding mechanism to the peptide hormone. For a long time, these complexes have been recalcitrant to structural studies despite their great therapeutic relevance. Through extensive engineering of both the receptor and the peptide agonist ligand, we were able to determine the first high-resolution structure of a PTH1R-agonist complex. Comparisons of the PTH1R crystal structure with subsequently reported cryo-electron microscopy structures of the same receptor in complex with a G protein, as well as with other class B GPCR structures bound to antagonists, reveal new insights into the two-step activation mechanism of class B GPCRs and extend our understanding of the precise molecular rearrangements during receptor activation.

PTH/PTHrP Receptor Signaling, Allostery, and Structures.

The parathyroid hormone (PTH) type 1 receptor (PTHR) is the canonical G protein-coupled receptor (GPCR) for PTH and PTH-related protein (PTHrP) and the key regulator of calcium homeostasis and bone turnover. PTHR function is critical for human health to maintain homeostatic control of ionized serum Ca2+ levels and has several unusual signaling features, such as endosomal cAMP signaling, that are well-studied but not structurally understood. In this review, we discuss how recently solved high resolution near-atomic structures of hormone-bound PTHR in its inactive and active signaling states and discovery of extracellular Ca2+ allosterism shed light on the structural basis for PTHR signaling and function.

Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of ß-arrestin 2 coupling.

Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc's epitope which may partially overlap with the known PTH (1-34) binding site. However, PTH (1-34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits ß-arrestin-2 recruitment after PTH (1-34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism.

The type-1 parathyroid hormone receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH forms a complex with the PTHR1 that is rapidly internalized and induces prolonged cAMP production from endosomes. In contrast, PTHrP induces only transient cAMP production, which primarily arises from receptors on the cell surface. We show that backbone modification of PTH(1-34)-NH2 and abaloparatide (a PTHrP derivative) with a single homologous ß-amino acid residue can generate biased agonists that induce prolonged cAMP production from receptors at the cell surface. This unique spatiotemporal profile could be useful for distinguishing effects associated with the duration of cAMP production from effects associated with the site of cAMP production.

Characteristics of parathyroid hormone-1 receptor agonists and antagonists.

Aim: Parathyroid hormone-1 receptor (PTH1R) is a member of B G protein-coupled receptors. The agonistic activation of the PTH1R results in the production and secretion of osteoclast-stimulating cytokines while antagonists may be used to treat bone metastases, hypercalcemia, cachexia and hyperparathyroidism. Results: We built pharmacophore models and investigated the characteristics of PTH1R agonists and antagonists. The agonist model consists of three hydrophobic points, one hydrogen bond acceptor and one positive ionizable point. The antagonist model consists of one hydrogen bond donor and three hydrophobic points. Conclusion: The features of the two models are similar, but the hydrogen bond acceptor, which is the main difference between PTH1R agonists and antagonists, suggests it may be essential for the agonist.

Structure and dynamics of the active human parathyroid hormone receptor-1.

The parathyroid hormone receptor-1 (PTH1R) is a class B G protein-coupled receptor central to calcium homeostasis and a therapeutic target for osteoporosis and hypoparathyroidism. Here we report the cryo-electron microscopy structure of human PTH1R bound to a long-acting PTH analog and the stimulatory G protein. The bound peptide adopts an extended helix with its amino terminus inserted deeply into the receptor transmembrane domain (TMD), which leads to partial unwinding of the carboxyl terminus of transmembrane helix 6 and induces a sharp kink at the middle of this helix to allow the receptor to couple with G protein. In contrast to a single TMD structure state, the extracellular domain adopts multiple conformations. These results provide insights into the structural basis and dynamics of PTH binding and receptor activation.

High-resolution crystal structure of parathyroid hormone 1 receptor in complex with a peptide agonist.

Parathyroid hormone 1 receptor (PTH1R) is a class B multidomain G-protein-coupled receptor (GPCR) that controls calcium homeostasis. Two endogenous peptide ligands, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), activate the receptor, and their analogs teriparatide and abaloparatide are used in the clinic to increase bone formation as an effective yet costly treatment for osteoporosis. Activation of PTH1R involves binding of the peptide ligand to the receptor extracellular domain (ECD) and transmembrane domain (TMD), a hallmark of class B GPCRs. Here, we present the crystal structure of human PTH1R in complex with a peptide agonist at 2.5-Å resolution, allowing us to delineate the agonist binding mode for this receptor and revealing molecular details within conserved structural motifs that are critical for class B receptor function. Thus, this study provides structural insight into the function of PTH1R and extends our understanding of this therapeutically important class of GPCRs.

Report of second case and clinical and molecular characterization of Eiken syndrome.

We report a boy with Eiken syndrome caused by a homozygous missense variant in Parathyroid hormone 1 receptor (PTH1R) c.103G > A [p.(Glu35Lys)]. Eiken syndrome is a very rare skeletal dysplasia due to bi-allelic variants in PTH1R. Only one affected family has been known to-date. The hallmarks include delayed ossification of bone including the epiphyses, pubic symphysis, and primary ossification centers of the short tubular bones, coarse bone trabeculae, and modeling abnormalities. The phenotype being described here recapitulates the delayed ossification and modeling abnormalities of Eiken syndrome. In addition, supernumerary epiphyses of the tubular bones of the hands and primary failure of eruption of teeth were observed in our proband. This report characterizes Eiken syndrome and confirms that bi-allelic hypomorphic variants in PTH1R are probably to cause this condition.

Production and evaluation of parathyroid hormone receptor1 ligands with intrinsic or assembled peroxidase domains.

Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH1 receptor (PTHR1). We developed fusion protein ligands with enzymatic activity to probe PTHR1s at the cell surface. Two fusion proteins were generated by linking PTH to the N-terminus of either horseradish peroxidase (PTH-HRP) or the genetically modified soybean peroxidase APEX2 (PTH-APEX2). Alternatively, myc-tagged PTH (PTH-myc) was combined with antibodies, some of which HRP-conjugated, in the extracellular fluid. The three PTH-fusion proteins were produced as conditioned mediums (CM) by transfected producer HEK 293a cells. Binding of receptor-bound enzymatic ligands was revealed using widely available substrate/co-substrate systems. The stimulation of recipient HEK 293a expressing PTHR1s with the PTH-myc/antibodies combination or with PTH-APEX2 supported the histochemical or luminescent detection of recombinant PTHR1s (TrueBlueTM or luminol-based reagent). The PTH-HRP construction was the most sensitive and supported all tested peroxidase co-substrates (TrueBlueTM, tetramethylbenzidine (TMB), luminol, biotin-phenol with streptavidin-Qdots); the 3 latter schemes identified endogenous PTHR1 in the osteoblastic HOS cell line. The specificity of the fusion protein binding to PTHR1 was determined by its competition with an excess of PTH1-34. Bifunctional ligands possessing enzymatic activity detect intact receptors with various possible applications, including the screening of drugs that compete for receptor binding.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ß2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.