Eiken syndrome with parathyroid hormone resistance due to a novel parathyroid hormone receptor type 1 mutation: clinical features and functional analysis.

We report on 2 patients of East African ancestry with the same novel homozygous variant in the parathyroid hormone receptor type 1 (PTH1R). Both patients shared skeletal features, including brachydactyly, extensive metacarpal pseudo-epiphyses, elongated cone-shaped epiphyses, ischiopubic hypoplasia, and deficient sacral ossification, suggestive of Eiken syndrome. Strikingly, both patients exhibited clinically manifest parathyroid hormone (PTH) resistance with hypocalcemia and elevated serum phosphate levels. These laboratory and clinical abnormalities initially suggested pseudohypoparathyroidism, which is typically associated with GNAS abnormalities. In both patients, however, a homozygous novel PTH1R variant was identified (c.710 T > A; p.IIe237Asn, p.I237N) that is located in the second transmembrane helical domain. Previously, others have reported a patient with a nearby PTH1R mutation (D241E) who presented with similar clinical features (eg, delayed bone mineralization as well as clinical PTH resistance). Functional analysis of the effects of both novel PTH1R variants (I237N- and D241E-PTH1R) in HEK293 reporter cells transfected with plasmid DNA encoding the wild-type or mutant PTH1Rs demonstrated increased basal cAMP signaling for both variants, with relative blunting of responses to both PTH and PTH-related peptide (PTHrP) ligands. The clinical presentation of PTH resistance and delayed bone mineralization combined with the functional properties of the mutant PTH1Rs suggest that this form of Eiken syndrome results from alterations in PTH1R-mediated signaling in response to both canonical ligands, PTH and PTHrP.

Eiken syndrome is rare genetic disorder of skeletal development, 7 due to alterations in the gene for parathyroid hormone receptor type 1 (PTH1R). This receptor can bind 2 hormones: parathyroid hormone (PTH), the body's main regulator of the level of calcium in blood, and PTH-related peptide (PTHrP), that regulates bone development. We report 2 new cases of Eiken syndrome sharing the same not previously reported change in PTH1R. The patients had typical findings in the skeleton reported in previous cases, but with some variation in the features. Unlike previously reported cases, the 2 patients we describe had low blood calcium levels causing symptoms. We wanted to explore how this new mutation affects the function of the receptor, particularly how it might affect the signals generated when the receptor binds to its 2 t hormones, PTH and PTHrP. We genetically reprogrammed a cell line with the new mutation, and tested those cells' responses to stimulation by the hormones. We showed that the altered receptor appears unable to bind both hormones in a stable fashion, explaining why the patients showed changes both in the skeleton (due to altered PTHrP signaling) and in the blood level of calcium (due to altered PTH signaling).

Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A.

Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.

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.

Backbone Modification Provides a Long-Acting Inverse Agonist of Pathogenic, Constitutively Active PTH1R Variants.

Parathyroid hormone 1 receptor (PTH1R) plays a key role in mediating calcium homeostasis and bone development, and aberrant PTH1R activity underlies several human diseases. Peptidic PTH1R antagonists and inverse agonists have therapeutic potential in treating these diseases, but their poor pharmacokinetics and pharmacodynamics undermine their in vivo efficacy. Herein, we report the use of a backbone-modification strategy to design a peptidic PTH1R inhibitor that displays prolonged activity as an antagonist of wild-type PTH1R and an inverse agonist of the constitutively active PTH1R-H223R mutant both in vitro and in vivo. This peptide may be of interest for the future development of therapeutic agents that ameliorate PTH1R malfunction.

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 a new 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.

Substantially Delayed Maturation of Growth Plate Chondrocytes in "Humanized" PTH1R Mice with the H223R Mutation of Jansen's Disease.

Activating parathyroid hormone (PTH)/PTH-related Peptide (PTHrP) receptor (PTH1R) mutations causes Jansen's metaphyseal chondrodysplasia (JMC), a rare disease characterized by growth plate abnormalities, short stature, and PTH-independent hypercalcemia. Previously generated transgenic JMC mouse models, in which the human PTH1R allele with the H223R mutation (H223R-PTH1R) is expressed in osteoblasts via type Ia1 collagen or DMP1 promoters cause excess bone mass, while expression of the mutant allele via the type IIa1 collagen promoter results in only minor growth plate changes. Thus, neither transgenic JMC model adequately recapitulates the human disease. We therefore generated "humanized" JMC mice in which the H223R-PTH1R allele was expressed via the endogenous mouse Pth1r promoter and, thus, in all relevant target tissues. Founders with the H223R allele typically died within 2 months without reproducing; several mosaic male founders, however, lived longer and produced F1 H223R-PTH1R offspring, which were small and exhibited marked growth plate abnormalities. Serum calcium and phosphate levels of the mutant mice were not different from wild-type littermates, but serum PTH and P1NP were reduced significantly, while CTX-1 and CTX-2 were slightly increased. Histological and RNAscope analyses of the mutant tibial growth plates revealed markedly expanded zones of type II collagen-positive, proliferating/prehypertrophic chondrocytes, abundant apoptotic cells in the growth plate center and a progressive reduction of type X collagen-positive hypertrophic chondrocytes and primary spongiosa. The "humanized" H223R-PTH1R mice are likely to provide a more suitable model for defining the JMC phenotype and for assessing potential treatment options for this debilitating disease of skeletal development and mineral ion homeostasis. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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.

Homozygous Ser-1 to Pro-1 mutation in parathyroid hormone identified in hypocalcemic patients results in secretion of a biologically inactive pro-hormone.

Like other secreted peptides, nascent parathyroid hormone (PTH) is synthesized with a pre- and a pro-sequence (25 and 6 amino acids, respectively). These precursor segments are sequentially removed in parathyroid cells before packaging into secretory granules. Three patients from two unrelated families who presented during infancy with symptomatic hypocalcemia were found to have a homozygous serine (S) to proline (P) change affecting the first amino acid of the mature PTH. Unexpectedly, biological activity of synthetic [P1]PTH(1-34) was indistinguishable from that of unmodified [S1]PTH(1-34). However, in contrast to conditioned medium from COS-7 cells expressing prepro[S1]PTH(1-84), medium from cells expressing prepro[P1]PTH(1-84) failed to stimulate cAMP production despite similar PTH levels when measured by an intact assay that detects PTH(1-84) and large amino-terminally truncated fragments thereof. Analysis of the secreted, but inactive PTH variant led to the identification of pro[P1]PTH(-6 to +84). Synthetic pro[P1]PTH(-6 to +34) and pro[S1]PTH(-6 to +34) had much less bioactivity than the corresponding PTH(1-34) analogs. Unlike pro[S1]PTH(-6 to +34), pro[P1]PTH(-6 to +34) was resistant to cleavage by furin suggesting that the amino acid variant impairs preproPTH processing. Consistent with this conclusion, plasma of patients with the homozygous P1 mutation had elevated proPTH levels, as determined with an in-house assay specific for pro[P1]PTH(-6 to +84). In fact, a large fraction of PTH detected by the commercial intact assay represented the secreted pro[P1]PTH. In contrast, two commercial biointact assays that use antibodies directed against the first few amino acid residues of PTH(1-84) for capture or detection failed to detect pro[P1]PTH.

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.

Kinetic and Thermodynamic Insights into Agonist Interactions with the Parathyroid Hormone Receptor-1 from a New NanoBRET Assay.

Polypeptides that activate the parathyroid hormone receptor-1 (PTHR1) are important in human physiology and medicine. Most previous studies of peptide binding to this receptor have involved the displacement of a radiolabeled ligand. We report a new assay format based on bioluminescence resonance energy transfer (BRET). Fusion of a NanoLuc luciferase (nLuc) unit to the N-terminus of the PTHR1 allows the direct detection of binding by an agonist peptide bearing a tetramethylrhodamine (TMR) unit. Affinity measurements from the BRET assay align well with results previously obtained via radioligand displacement. The BRET assay offers substantial operational benefits relative to affinity measurements involving radioactive compounds. The convenience of the new assay allowed us to explore several questions raised by earlier reports. For example, we show that although the first two residues of PTH(1-34) (the drug teriparatide) are critical for PTHR1 activation, these two residues contribute little or nothing to affinity. Comparisons among the well-studied agonists PTH(1-34), PTHrP(1-34), and "long-acting PTH" (LA-PTH) reveal that the high affinity of LA-PTH arises largely from a diminished rate constant for dissociation relative to the other two. A D-peptide recently reported to be comparable to PTH(1-34) as an agonist of the PTHR1 was found not to bind detectably to the receptor and to be a very weak agonist.

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.

Functional Properties of Two Distinct PTH1R Mutants Associated With Either Skeletal Defects or Pseudohypoparathyroidism.

Consistent with a vital role of parathyroid hormone (PTH) receptor type 1 (PTH1R) in skeletal development, homozygous loss-of-function PTH1R mutations in humans results in neonatal lethality (Blomstrand chondrodysplasia), whereas such heterozygous mutations cause a primary failure of tooth eruption (PFE). Despite a key role of PTH1R in calcium and phosphate homeostasis, blood mineral ion levels are not altered in such cases of PFE. Recently, two nonlethal homozygous PTH1R mutations were identified in two unrelated families in which affected members exhibit either dental and skeletal abnormalities (PTH1R-V204E) or hypocalcemia and hyperphosphatemia (PTH1R-R186H). Arg186 and Val204 map to the first transmembrane helix of the PTH1R, and thus to a critical region of this class B G protein-coupled receptor. We used cell-based assays and PTH and PTH-related protein (PTHrP) ligand analogs to assess the impact of the R186H and V204E mutations on PTH1R function in vitro. In transiently transfected HEK293 cells, PTH1R-R186H mediated cyclic adenosine monophosphate (cAMP) responses to PTH(1-34) and PTHrP(1-36) that were of comparable potency to those observed on wild-type PTH1R (PTH1R-WT) (half maximal effective concentrations [EC50s] = 0.4nM to 1.2nM), whereas the response-maxima were significantly reduced for the PTH1R-V204E mutant (maximum effect [Emax] = 81%-77% of PTH1R-WT, p ≤ 0.004). Antibody binding to an extracellular hemagglutinin (HA) tag was comparable for PTH1R-R186H and PTH1R-WT, but was significantly reduced for PTH1R-V204E (maximum binding level [Bmax] = 44% ± 11% of PTH1R-WT, p = 0.002). The potency of cAMP signaling induced by a PTH(1-11) analog was reduced by ninefold and threefold, respectively, for PTH1R-R186H and PTH1R-V204E, relative to PTH1R-WT, and a PTH(1-15) radioligand analog that bound adequately to PTH1R-WT exhibited little or no specific binding to either mutant receptor. The data support a general decrease in PTH1R surface expression and/or function as a mechanism for PFE and a selective impairment in PTH ligand affinity as a potential PTH1R-mutation-based mechanism for pseudohypoparathyroidism. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Activity-based, bioorthogonal imaging of phospholipase D reveals spatiotemporal dynamics of GPCR-Gq signaling.

Canonically, G-protein-coupled receptor (GPCR) signaling is transient and confined to the plasma membrane (PM). Deviating from this paradigm, the parathyroid hormone receptor (PTHR1) stimulates sustained Gs signaling at endosomes. In addition to Gs, PTHR1 activates Gq signaling; yet, in contrast to the PTHR1-Gs pathway, the spatiotemporal dynamics of the Gq branch of PTHR1 signaling and its relationship to Gs signaling remain largely ill defined. Recognizing that a downstream consequence of Gq signaling is the activation of phospholipase D (PLD) enzymes, we leverage activity-based, bioorthogonal imaging tools for PLD signaling to visualize and quantify the Gq branch of PTHR1 signaling. We establish that PTHR1-Gq signaling is short lived, exclusively at the PM, and antagonized by PTHR1 endocytosis. Our data support a model wherein Gq and Gs compete for ligand-bound receptors at the PM and more broadly highlight the utility of bioorthogonal tools for imaging PLDs as probes to visualize GPCR-Gq signaling.

Precise druggability of the PTH type 1 receptor.

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 of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.

Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein­coupled receptor (GPCR) that regulates mineral ion, vitamin D, and bone homeostasis. Activation of the PTHR by PTH induces both transient cell surface and sustained endosomal cAMP production. To address whether the spatial (location) or temporal (duration) dimension of PTHR-induced cAMP encodes distinct biological outcomes, we engineered a biased PTHR ligand (PTH7d) that elicits cAMP production at the plasma membrane but not at endosomes. PTH7d stabilized a unique active PTHR conformation that mediated sustained cAMP signaling at the plasma membrane due to impaired ß-arrestin coupling to the receptor. Experiments in cells and mice revealed that sustained cAMP production by cell surface PTHR failed to mimic the pharmacological effects of sustained endosomal cAMP production on the abundance of the rate-limiting hydroxylase catalyzing the formation of active vitamin D, as well as increases in circulating active vitamin D and Ca2+ and in bone formation in mice. Thus, similar amounts of cAMP generated by PTHR for similar lengths of time in different cellular locations, plasma membrane and endosomes, mediate distinct physiological responses. These results unveil subcellular signaling location as a means to achieve specificity in PTHR-mediated biological outcomes and raise the prospect of rational drug design based upon spatiotemporal manipulation of GPCR signaling.

Comparable Initial Engagement of Intracellular Signaling Pathways by Parathyroid Hormone Receptor Ligands Teriparatide, Abaloparatide, and Long-Acting PTH.

Multiple analogs of parathyroid hormone, all of which bind to the PTH/PTHrP receptor PTH1R, are used for patients with osteoporosis and hypoparathyroidism. Although ligands such as abaloparatide, teriparatide (hPTH 1-34 [TPTD]), and long-acting PTH (LA-PTH) show distinct biologic effects with respect to skeletal and mineral metabolism endpoints, the mechanistic basis for these clinically-important differences remains incompletely understood. Previous work has revealed that differential signaling kinetics and receptor conformation engagement between different PTH1R peptide ligands. However, whether such acute membrane proximal differences translate into differences in downstream signaling output remains to be determined. Here, we directly compared short-term effects of hPTH (1-34), abaloparatide, and LA-PTH in multiple cell-based PTH1R signaling assays. At the time points and ligand concentrations utilized, no significant differences were observed between these three ligands at the level of receptor internalization, ß-arrestin recruitment, intracellular calcium stimulation, and cAMP generation. However, abaloparatide showed significantly quicker PTH1R recycling in washout studies. Downstream of PTH1R-stimulated cAMP generation, protein kinase A regulates gene expression via effects on salt inducible kinases (SIKs) and their substrates. Consistent with no differences between these ligands on cAMP generation, we observed that hPTH (1-34), abaloparatide, and LA-PTH showed comparable effects on SIK2 phosphorylation, SIK substrate dephosphorylation, and downstream gene expression changes. Taken together, these results indicate that these PTH1R peptide agonists engage downstream intracellular signaling pathways to a comparable degree. It is possible that differences observed in vivo in preclinical and clinical models may be related to pharmacokinetic factors. It is also possible that our current in vitro systems are insufficient to perfectly match the complexities of PTH1R signaling in bona fide target cells in bone in vivo. © 2020 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands.

Antibodies raised against many cell surface proteins, including G protein-coupled receptors, remain important tools for their functional characterization. By linking antibodies to ligands for cell surface proteins, such adducts can be targeted to the surface of a cell type of choice. Site-specific functionalization of full-size antibodies with synthetic moieties remains challenging. Here we present new approaches in which single domain antibodies (known as VHHs or nanobodies) that target either cell surface proteins or conventional antibodies are used to indirectly deliver ligands for GPCRs to their sites of action. The combination of high yield production of nanobodies, facile site-specific functionalization, and compatibility with commercially available mouse and rabbit antibodies should enable wide application of this approach.

Ligand-Dependent Effects of Methionine-8 Oxidation in Parathyroid Hormone Peptide Analogues.

LA-PTH is a long-acting parathyroid hormone (PTH) peptide analogue in preclinical development for hypoparathyroidism (HP). Like native PTH, LA-PTH contains a methionine at position 8 (Met8) that is predicted to be critical for function. We assessed the impact of Met oxidation on the functional properties of LA-PTH and control PTH ligands. Oxidation of PTH(1-34) resulted in marked (~20-fold) reductions in binding affinity on the PTH receptor-1 (PTHR1) in cell membranes, similarly diminished potency for 3',5'-cyclic AMP signaling in osteoblastic cell lines (SaOS-2 and UMR106), and impaired efficacy for raising blood calcium in mice. Surprisingly, oxidation of LA-PTH resulted in little or no change in these functional responses. The signaling potency of oxidized-LA-PTH was, however, reduced approximately 40-fold compared to LA-PTH in cells expressing a PTHR1 construct that lacks the N-terminal extracellular domain (ECD). Molecular modeling revealed that while Met8 of both LA-PTH and PTH(1-34) is situated within the orthosteric ligand-binding pocket of the receptor's transmembrane domain bundle (TMD), the Met8 sidechain position is shifted for the 2 ligands so that on Met8 oxidation of PTH(1-34), steric clashes occur that are not seen with oxidized LA-PTH. The findings suggest that LA-PTH and PTH(1-34) engage the receptor differently in the Met8-interaction environment of the TMD bundle, and that this interaction environment can be allosterically influenced by the ECD component of the ligand-receptor complex. The findings should be useful for the future development of novel PTH-based peptide therapeutics for diseases of bone and mineral ion metabolism.

Allosteric interactions in the parathyroid hormone GPCR-arrestin complex formation.

Peptide ligands of class B G-protein-coupled receptors act via a two-step binding process, but the essential mechanisms that link their extracellular binding to intracellular receptor-arrestin interactions are not fully understood. Using NMR, crosslinking coupled to mass spectrometry, signaling experiments and computational approaches on the parathyroid hormone (PTH) type 1 receptor (PTHR), we show that initial binding of the PTH C-terminal part constrains the conformation of the flexible PTH N-terminal signaling epitope before a second binding event occurs. A 'hot-spot' PTH residue, His9, that inserts into the PTHR transmembrane domain at this second step allosterically engages receptor-arrestin coupling. A conformational change in PTHR intracellular loop 3 permits favorable interactions with ß-arrestin's finger loop. These results unveil structural determinants for PTHR-arrestin complex formation and reveal that the two-step binding mechanism proceeds via cooperative fluctuations between ligand and receptor, which extend to other class B G-protein-coupled receptors.