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.

Expanding the role of PSMA PET in active surveillance.

INTRODUCTION: Accurate grading at the time of diagnosis is fundamental to risk stratification and treatment decision making, particularly for men being considered for Active Surveillance (AS). With the introduction of prostate-specific membrane antigen (PSMA) positron emission tomography (PET) there has been considerable improvement in sensitivity and specificity for the detection and staging of clinically significant prostate cancer. Our study aims to determine the role of PSMA PET/CT in men with newly diagnosed low or favourable intermediate risk prostate cancer to better select men for AS. METHOD: This is a retrospective single centre study performed from January 2019 and October 2022. This study includes men identified from electronic medical record system who had undergone a PSMA PET/CT following newly diagnosed low or favourable-intermediate risk prostate cancer. Primary outcome was to assess the change in management for men being considered for AS following PSMA PET/CT results on the basis of PSMA PET characteristics. RESULTS: In total, there were 11 of 30 men (36.67%) who were assigned management by AS and 19 of 30 men (63.33%) who had definitive treatment. 15 of the 19 men that needed treatment had concerning features on PSMA PET/CT results. Of the 15 men with concerning features on PSMA PET, 9 (60%) men were found to have adverse pathological features on final prostatectomy features. CONCLUSION: This retrospective study suggests that PSMA PET/CT has potential to influence the management of men with newly diagnosed prostate cancer that would otherwise be appropriate for active surveillance.

Isolated late testicular relapse of B-cell acute lymphoblastic leukemia treated with intensive systemic chemotherapy and response-based testicular radiation: A Children's Oncology Group study.

BACKGROUND: The incidence of isolated testicular relapse (ITR) of acute lymphoblastic leukemia (ALL) has decreased with contemporary treatment strategies, but outcomes are suboptimal with a 58% 5-year overall survival (OS). This study aimed to improve outcome in patients with ITR of B-cell ALL (B-ALL) occurring after 18 months of first clinical remission using intensive systemic chemotherapy and to decrease long-term sequelae by limiting use of testicular radiation. PROCEDURE: Forty patients in first ITR of B-ALL were enrolled. Induction (dexamethasone, vincristine, daunorubicin, and intrathecal triple therapy) was preceded by one dose of high-dose methotrexate (MTX, 5 g/m2 ). Following induction, 25 of 26 patients who had persistent testicular enlargement underwent testicular biopsy. Eleven had biopsy-proven disease and received bilateral testicular radiation (24 Gy), whereas twenty-nine did not. RESULTS: Overall 5-year event-free survival (EFS)/OS was 65.0 ± 8.8%/73.1 ± 8.3%, with 5-year EFS 62.1 ± 11.0% vs. 72.7 ± 14.4% for patients who did not receive radiation therapy (XRT) (n = 29) compared with those who did (n = 11), respectively (P = 0.64). There were six second bone marrow relapses and six second ITRs. The proportion of second relapses was similar in the patients that received testicular radiation and those who did not. However, the 5-year OS was similar for patients who did not receive XRT (72.6 ± 10.2%) compared with those who did (72.7 ± 14.4%) (P = 0.85). CONCLUSIONS: A 5-year OS rate of 73.1 ± 8.3% was obtained in children with first ITR of B-ALL occurring after 18 months of CR1 (length of first clinical remission) using intensive chemotherapy and limiting testicular radiation.

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH.

Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting "knock-in" mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1-34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.

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.

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.

Multiparametric Magnetic Resonance Imaging of the Prostate and Prostate-specific Membrane Positron Emission Tomography Prior to Prostate Biopsy (MP4 Study).

Background: Prostate-specific membrane antigen (PSMA) positron emission tomography/computerised tomography (PET/CT) is increasingly being utilised in the diagnostic pathway for prostate cancer (PCa). Recent publications have suggested that this might help identify those who can avoid biopsy. Objective: The primary objective of this study was to determine whether PET magnetic resonance imaging (MRI) fusion could negate the need to biopsy prior to prostatectomy in a selected population of men. Design setting and participant: Multiparametric MRI (mpMRI) for PCa is our standard of care prior to prostate biopsy. Biopsy-naïve men with one or more Prostate Imaging Reporting and Data System (PI-RADS) 4 or 5 lesions ≥10 mm on mpMRI were invited to undergo PSMA PET/CT prior to biopsy. Following ethics approval, 60 men were recruited between September 2020 and March 2021. The key exclusion criteria included a previous history of PCa and previous prostate surgery or biopsy. Outcome measurements and statistical analysis: A positive PET MRI fusion scan was defined as "consistent with" as per the Memorial Sloan Kettering Cancer Center lexicon of certainty, and concordance with biopsy results was analysed. Clinically significant PCa (csPCa) was defined as grade group (GG) ≥2 on pathology. A chi-square analysis was performed with statistical significance defined at p < 0.05. Results and limitations: A total of 71 mpMRI lesions were positive on 61 (86%) PET MRI fusion scans. Fifty-nine of 61 lesions biopsied confirmed csPCa in 54 (92%). Of five of 59 lesions for which either biopsy was negative or low-grade cancer was found, three had rebiopsy of which two were confirmed to have csPCa corroborating with PET MRI fusion and one was reconfirmed to have GG1 only. For the remaining two, both had another lesion elsewhere in the gland confirming csPCa, and hence rebiopsy was not performed. Ultimately, 56 of 59 (95%) lesions with a positive PET MRI fusion scan were confirmed to have csPCa. All GG ≥3 cancers had a positive PET MRI fusion scan. Conclusions: This prospective study of PET MRI fusion assessment of men with PI-RADS 4 or 5 lesion ≥10 mm on mpMRI confirms that the majority of men (95%) with a positive PET MRI fusion scan will have csPCa. This supports recently published retrospective data suggesting that selected men might avoid prostate biopsy prior to radical prostatectomy. Patient summary: In this research, we have confirmed that prostate-specific membrane antigen positron emission tomography/computerised tomography in combination with magnetic resonance imaging could have an important role in enabling a diagnosis of prostate cancer. Using the combination of these scans, we could confidently predict the presence of aggressive prostate cancer in some men for which treatment is warranted. This means that there are some men who could possibility proceed directly to having prostate cancer surgery without the need for a confirmatory prostate biopsy.

Acute down-regulation of sodium-dependent phosphate transporter NPT2a involves predominantly the cAMP/PKA pathway as revealed by signaling-selective parathyroid hormone analogs.

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) in cells of the renal proximal tubule mediates the reduction in membrane expression of the sodium-dependent P(i) co-transporters, NPT2a and NPT2c, and thus suppresses the re-uptake of P(i) from the filtrate. In most cell types, the liganded PTHR1 activates Gα(S)/adenylyl cyclase/cAMP/PKA (cAMP/PKA) and Gα(q/11)/phospholipase C/phosphatidylinositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/PKC (IP(3)/PKC) signaling pathways, but the relative roles of each pathway in mediating renal regulation P(i) transport remain uncertain. We therefore explored the signaling mechanisms involved in PTH-dependent regulation of NPT2a function using potent, long-acting PTH analogs, M-PTH(1-28) (where M = Ala(1,12), Aib(3), Gln(10), Har(11), Trp(14), and Arg(19)) and its position 1-modified variant, Trp(1)-M-PTH(1-28), designed to be phospholipase C-deficient. In cell-based assays, both M-PTH(1-28) and Trp(1)-M-PTH(1-28) exhibited potent and prolonged cAMP responses, whereas only M-PTH(1-28) was effective in inducing IP(3) and intracellular calcium responses. In opossum kidney cells, a clonal cell line in which the PTHR1 and NPT2a are endogenously expressed, M-PTH(1-28) and Trp(1)-M-PTH(1-28) each induced reductions in (32)P uptake, and these responses persisted for more than 24 h after ligand wash-out, whereas that of PTH(1-34) was terminated by 4 h. When injected into wild-type mice, both M-modified PTH analogs induced prolonged reductions in blood P(i) levels and commensurate reductions in NPT2a expression in the renal brush border membrane. Our findings suggest that the acute down-regulation of NPT2a expression by PTH ligands involves mainly the cAMP/PKA signaling pathway and are thus consistent with the elevated blood P(i) levels seen in pseudohypoparathyroid patients, in whom Gα(s)-mediated signaling in renal proximal tubule cells is defective.

Engineering of therapeutic polypeptides through chemical synthesis: early lessons from human parathyroid hormone and analogues.

Application of chemical synthesis to gain access to high purity hPTH as well as more stable analogues was accomplished through a menu of extended NCL followed by metal free dethiylation.

Delay in discharge and its impact on unnecessary hospital bed occupancy.

BACKGROUND: Elderly patients are potentially more vulnerable to prolonged hospital stay as they frequently require additional resources to facilitate their discharge. In an acute hospital setting, we aimed to quantify and compare length of stay (LOS) for all patients over and under the age of 65, and identify the number and cause of days lost under the care of a single surgical unit. METHODS: Over a 4 month period from January to April 2010, data on the management and source of potential delay was collected daily on consecutive patients admitted and discharged under the care of one consultant surgeon at a district general hospital. Statistical analysis was then performed with particular focus on actual delays affecting elderly patients. RESULTS: A total of 99 complete inpatients episodes were recorded. There were 30 elective and 69 acute admissions. 10 (33%) elective vs. 42 (61%) acute patients encountered delays, losing 39 and 232 days respectively (χ2 [1, N = 99] = 6.36, p = .012). 23 of a total 39 elderly patients admitted acutely required specialist care of the elderly opinion and placement in community hospitals resulting in delays of 188 days. vs. 36 days for the 16 discharged home and 8 days for 30 patients under 65 (χ2 (2, N = 69) = 26.54, p = <.001). CONCLUSIONS: Elderly patients experiencing acute surgical admission and discharge to community hospitals had prolonged LOS due to significant delays associated with care of the elderly provision. The financial considerations behind bed capacity in primary and secondary care and the provision of care of elderly services need to be balanced against unnecessary occupancy of acute hospital beds with its associated health and economic implications.

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.

Projecting climate change effects on forest net primary productivity in subtropical Louisiana, USA.

This study projected responses of forest net primary productivity (NPP) to three climate change scenarios at a resolution of 5 km x 5 km across the state of Louisiana, USA. In addition, we assessed uncertainties associated with the NPP projection at the grid and state levels. Climate data of the scenarios were derived from Community Climate System Model outputs. Changes in annual NPP between 2000 and 2050 were projected with the forest ecosystem model PnET-II. Results showed that forest productivity would increase under climate change scenarios A1B and A2, but with scenario B1, it would peak during 2011-2020 and then decline. The projected average NPP under B1 over the years from 2000 to 2050 was significantly different from those under A1B and A2. Forest NPP appeared to be primarily a function of temperature, not precipitation. Uncertainties of the NPP projection were due to large spatial resolution of the climate variables. Overall, this study suggested that in order to project effects of climate change on forest ecosystem at regional level, modeling uncertainties could be reduced by increasing the spatial resolution of the climate projections.

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.

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.

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia.

Jansen's metaphyseal chondrodysplasia (JMC) is a rare disease of bone and mineral ion physiology that is caused by activating mutations in PTHR1. Ligand-independent signaling by the mutant receptors in cells of bone and kidney results in abnormal skeletal growth, excessive bone turnover, and chronic hypercalcemia and hyperphosphaturia. Clinical features further include short stature, limb deformities, nephrocalcinosis, and progressive losses in kidney function. There is no effective treatment option available for JMC. In previous cell-based assays, we found that certain N-terminally truncated PTH and PTHrP antagonist peptides function as inverse agonists and thus can reduce the high rates of basal cAMP signaling exhibited by the mutant PTHR1s of JMC in vitro. Here we explored whether one such inverse agonist ligand, [Leu11 ,dTrp12 ,Trp23 ,Tyr36 ]-PTHrP(7-36)NH2 (IA), can be effective in vivo and thus ameliorate the skeletal abnormalities that occur in transgenic mice expressing the PTHR1-H223R allele of JMC in osteoblastic cells via the collagen-1α1 promoter (C1HR mice). We observed that after 2 weeks of twice-daily injection and relative to vehicle controls, the IA analog resulted in significant improvements in key skeletal parameters that characterize the C1HR mice, because it reduced the excess trabecular bone mass, bone marrow fibrosis, and levels of bone turnover markers in blood and urine. The overall findings provide proof-of-concept support for the notion that inverse agonist ligands targeted to the mutant PTHR1 variants of JMC can have efficacy in vivo. Further studies of such PTHR1 ligand analogs could help open paths toward the first treatment option for this debilitating skeletal disorder. © 2019 American Society for Bone and Mineral Research.

Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor.

PTH and PTHrP use the same G protein-coupled receptor, the PTH/PTHrP receptor (PTHR), to mediate their distinct biological actions. The extent to which the mechanisms by which the two ligands bind to the PTHR differ is unclear. We examined this question using several pharmacological and biophysical approaches. Kinetic dissociation and equilibrium binding assays revealed that the binding of [(125)I]PTHrP(1-36) to the PTHR was more sensitive to GTPgammaS (added to functionally uncouple PTHR-G protein complexes) than was the binding of [(125)I]PTH(1-34) ( approximately 75% maximal inhibition vs. approximately 20%). Fluorescence resonance energy transfer-based kinetic analyses revealed that PTHrP(1-36) bound to the PTHR more slowly and dissociated from it more rapidly than did PTH(1-34). The cAMP signaling response capacity of PTHrP(1-36) in cells decayed more rapidly than did that of PTH(1-34) (t(1/2) = approximately 1 vs. approximately 2 h). Divergent residue 5 in the ligand, Ile in PTH and His in PTHrP, was identified as a key determinant of the altered receptor-interaction responses exhibited by the two peptides. We conclude that whereas PTH and PTHrP bind similarly to the G protein-coupled PTHR conformation (RG), PTH has a greater capacity to bind to the G protein-uncoupled conformation (R(0)) and, hence, can produce cumulatively greater signaling responses (via R(0)-->RG isomerization) than can PTHrP. Such conformational selectivity may relate to the distinct modes by which PTH and PTHrP act biologically, endocrine vs. paracrine, and may help explain reported differences in the effects that the ligands have on calcium and bone metabolism when administered to humans.

Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein receptor: selectivity of a modified PTH(1-15) radioligand for GalphaS-coupled receptor conformations.

Mechanisms of ligand binding to the PTH/PTHrP receptor (PTHR) were explored using PTH fragment analogs as radioligands in binding assays. In particular, the modified amino-terminal fragment analog, (125)I-[Aib(1,3),Nle8,Gln10,homoarginine11,Ala12,Trp14,Tyr15]rPTH(1-15)NH2, (125)I-[Aib(1,3),M]PTH(1-15), was used as a radioligand that we hypothesized to bind solely to the juxtamembrane (J) portion of the PTHR containing the extracellular loops and transmembrane helices. We also employed (125)I-PTH(1-34) as a radioligand that binds to both the amino-terminal extracellular (N) and J domains of the PTHR. Binding was examined in membranes derived from cells expressing either wild-type or mutant PTHRs. We found that the binding of (125)I-[Aib(1,3),M]PTH(1-15) to the wild-type PTHR was strongly (approximately 90%) inhibited by guanosine 5'-O-(3-thio)triphosphate (GTPgammaS), whereas the binding of (125)I-PTH(1-34) was only mildly (approximately 25%) inhibited by GTPgammaS. Of these two radioligands, only (125)I-[Aib(1,3),M]PTH(1-15) bound to PTHR-delNt, which lacks most of the receptor's N domain, and again this binding was strongly inhibited by GTPgammaS. Binding of (125)I-[Aib(1,3),M]PTH(1-15) to the constitutively active receptor, PTHR-H223R, was only mildly (approximately 20%) inhibited by GTPgammaS, as was the binding of (125)I-PTH(1-34). In membranes prepared from cells lacking Galpha(S) via knockout mutation of Gnas, no binding of (125)I-[Aib(1,3),M]PTH(1-15) was observed, but binding of (125)I-[Aib(1,3),M]PTH(1-15) was recovered by virally transducing the cells to heterologously express Galpha(S). (125)I-PTH(1-34) bound to the membranes with or without Galpha(S). The overall findings confirm the hypothesis that (125)I-[Aib(1,3),M]PTH(1-15) binds solely to the J domain of the PTHR. They further show that this binding is strongly dependent on coupling of the receptor to Galpha(S)-containing heterotrimeric G proteins, whereas the binding of (125)I-PTH(1-34) can occur in the absence of such coupling. Thus, (125)I-[Aib(1,3),M]PTH(1-15) appears to function as a selective probe of Galpha(S)-coupled, active-state PTHR conformations.

Binding Selectivity of Abaloparatide for PTH-Type-1-Receptor Conformations and Effects on Downstream Signaling.

The PTH receptor type 1 (PTHR1) mediates the actions of two endogenous polypeptide ligands, PTH and PTHrP, and thereby plays key roles in bone biology. Based on its capacity to stimulate bone formation, the peptide fragment PTH (1-34) is currently in use as therapy for osteoporosis. Abaloparatide (ABL) is a novel synthetic analog of human PTHrP (1-34) that holds promise as a new osteoporosis therapy, as studies in animals suggest that it can stimulate bone formation with less of the accompanying bone resorption and hypercalcemic effects that can occur with PTH (1-34). Recent studies in vitro suggest that certain PTH or PTHrP ligand analogs can distinguish between two high-affinity PTHR1 conformations, R(0) and RG, and that efficient binding to R(0) results in prolonged signaling responses in cells and prolonged calcemic responses in animals, whereas selective binding to RG results in more transient responses. As intermittent PTH ligand action is known to favor the bone-formation response, whereas continuous ligand action favors the net bone-resorption/calcemic response, we hypothesized that ABL binds more selectively to the RG vs the R(0) PTHR1 conformation than does PTH (1-34), and thus induces more transient signaling responses in cells. We show that ABL indeed binds with greater selectivity to the RG conformation than does PTH (1-34), and as a result of this RG bias, ABL mediates more transient cAMP responses in PTHR1-expressing cells. The findings provide a plausible mechanism (ie, transient signaling via RG-selective binding) that can help account for the favorable anabolic effects that ABL has on bone.