Teriparatide prevented synovial inflammation and cartilage destruction in mice with DMM.

AIM: Emerging data have demonstrated that low-grade inflammation in osteoarthritis, a long-held degenerative disease. The inflamed synovium produces various cytokines that induce cartilage destruction and joint pain. A previous study showed that teriparatide, an FDA approved anti-osteoporotic drug, may enhance cartilage repair. Our study focuses on its role in OA synovitis. MATERIALS AND METHODS: Primary mouse articular chondrocytes were used to determine the most potent cytokines involved in OA inflammation and cartilage destruction. A destabilization of the medial meniscus mouse model was established to investigate the effect of teriparatide in OA, particularly, on synovial inflammation and cartilage degradation. RESULTS: In vitro experiments showed that TNF-α was the most potent inducer of cartilage matrix-degrading enzymes, and that teriparatide antagonized the TNF-α of effect. Consistently, articular cartilage samples from TNF-α transgenic mice contained more MMP-13 positive chondrocytes than those from wild type mice. In addition, more type II collagen was cleaved in human OA cartilage than in normal cartilage samples. CONCLUSIONS: Teriparatide can prevent synovitis and cartilage degradation by suppressing TNF-α mediated MMP-13 overexpression. Together with its chondroregenerative capability, teriparatide may be the first effective disease modifying osteoarthritis drug.

Solution design to extend the pH range of the pH-responsive precipitation of a CspB fusion protein.

Cell surface protein B (CspB) from Corynebacterium glutamicum has been developed as a reversible pH-responsive tag for protein purification. CspB fusion proteins precipitate at acidic pH, after that they completely dissolve at neutral pH. This property has been used in a non-chromatographic protein purification method named pH-responsive Precipitation-Redissolution of CspB tag Purification (pPRCP). However, it is difficult to apply pPRCP to proteins that are unstable under acidic conditions. In an effort to shift the precipitation pH to a milder range, we investigated the solution conditions of CspB-fused Teriparatide (CspB50TEV-Teriparatide) during the process of pH-responsive precipitation using pPRCP. The purified CspB50TEV-Teriparatide in buffer without additives precipitated at pH 5.3. By contrast, CspB50TEV-Teriparatide in buffer with 0.5 M Na2SO4 precipitated at pH 6.6 because of the kosmotropic effect. Interestingly, the pH at which precipitation occurred was independent of the protein concentration. The precipitated CspB50TEV-Teriparatide was fully redissolved at above pH 8.0 in the presence or absence of salt. The discovery that proteins can be precipitated at a mild pH will allow pPRCP to be applied to acid-sensitive proteins.

Effect of Clp protease from Corynebacterium glutamicum on heterologous protein expression.

The protease present in a host may reduce the yield and biological activity of heterologous proteins. In this study, we used protease overexpression and deletion strategies to examine the effect of the Clp protease system in Corynebacterium glutamicum on the recombinant protein and to produce a highly efficient heterologous protein expression host. In this study, we identified seven genes in the Clp protease family in Corynebacterium glutamicum ATCC 13032 through bioinformatics analysis, and studied their effects on the enhanced green fluorescent protein (EGFP) reporter protein. The fluorescence intensity of the knockout strain was significantly higher, and the effect of the clpS deletion strain was the most obvious. To verify the universal effect of the lack of clpS, the excellent industrial strain C. glutamicum 1.15647 was transformed to form recombinant 15647-ΔclpS. Based on the results, 15647-ΔclpS had a more significant effect on improving protein expression. Furthermore, recombinant human teriparatide (rhPTH) and variable domain of heavy chain of heavy-chain antibody (VHH) were selected to verify the universal applicability of the knockout strain for expressing heterologous proteins. Accordingly, we found that protease deficiency could increase the production of heterologous proteins. Finally, through a large-scale fermentation, the 15647-ΔclpS strain was used to produce VHH. Its yield was approximately 530 mg/L, which was 65% higher than that of WT-15647. In this study, a host that could effectively increase heterologous protein expression was successfully obtained.

Non-chromatographic purification of Teriparatide with a pH-responsive CspB tag.

Cell surface protein B (CspB) from Corynebacterium glutamicum is used as a pH-responsive peptide tag to enable a simple solid-liquid separation method for isolating a CspB fusion protein. Here we demonstrate the first application of a CspB tag for the purification of Teriparatide, which is a biologic drug that is prescribed for osteoporosis. The Teriparatide was constructed as CspB50TEV-Teriparatide, comprising 50 amino acid residues of CspB, the cleavage site of TEV protease, and Teriparatide. CspB50TEV-Teriparatide was expressed in a culture supernatant by C. glutamicum secretion system at 3.0 g/L (equivalent to approximately 1.2 g/L Teriparatide). The CspB50TEV-Teriparatide was precipitated by reducing the pH of the culture supernatant, and the precipitate was then dissolved in a neutral buffer. A TEV protease treatment was applied to cleave the Teriparatide from the CspB50TEV-Teriparatide. Then, the remaining digested CspB50TEV, undigested CspB50TEV-Teriparatide, and TEV protease were precipitated in an acidic pH, whereas the soluble Teriparatide remained in the supernatant. The process had a yield of 96.5% and resulted in Teriparatide with a purity of 98.0% and productivity of 1.1 g/L of C. glutamicum culture. Thus, tag-free Teriparatide was successfully purified from the CspB fusion protein using only pH changes, centrifugation, and protease digestion without the need for chromatography. This versatile purification protocol is expected to be applicable to various proteins from laboratory to industrial scales.

[Pharmacological treatment for pregnancy and lactation associated osteoporosis.]

Pregnancy and lactation associated osteoporosis(PLO)is a rare disorder for women during pregnancy, the post-partum period, or while breastfeeding. It still remains unknown factors in its pathogenesis. That makes it there is no evident strategy for PLO now. In most cases, bone mineral density(BMD)of PLO patients spontaneously recovers after giving lactation up. On the other hand, patients with severe cases sometimes need bone specific therapy. There are some reports that show bisphosphonate, teriparatide and/or denosumab are effective on PLO cases. When the patients have wishes for having babies, we have to pay attention if the prescription effect on next pregnancy.

[ASBMR topics from clinical research(osteoporosis and sarcopenia).]

There are a lot of progressive topics about osteoporosis and sarcopenia in 2018 ASBMR Annual Meeting, involving an association between diabetes and bone microarchitecture, associations between atypical femoral fractures and bisphosphonate drug holidays as well as pre-treatment bone mineral density, an effect of combined denosumab and high-dose teriparatide on bone parameters, and relationships between muscle and deuterated creatine, a selective androgen receptor modulator, and high-dose vitamin D supplementation.

Potential effects of teriparatide (PTH (1-34)) on osteoarthritis: a systematic review.

Osteoarthritis (OA) is a common and prevalent degenerative joint disease characterized by degradation of the articular cartilage. However, none of disease-modifying OA drugs is approved currently. Teriparatide (PTH (1-34)) might stimulate chondrocyte proliferation and cartilage regeneration via some uncertain mechanisms. Relevant therapies of PTH (1-34) on OA with such effects have recently gained increasing interest, but have not become widespread practice. Thus, we launch this systematic review (SR) to update the latest evidence accordingly. A comprehensive literature search was conducted in PubMed, Web of Science, MEDLINE, the Cochrane Library, and Embase from their inception to February 2022. Studies investigating the effects of the PTH (1-34) on OA were obtained. The quality assessment and descriptive summary were made of all included studies. Overall, 307 records were identified, and 33 studies were included. In vivo studies (n = 22) concluded that PTH (1-34) slowed progression of OA by alleviating cartilage degeneration and aberrant remodeling of subchondral bone (SCB). Moreover, PTH (1-34) exhibited repair of cartilage and SCB, analgesic, and anti-inflammatory effects. In vitro studies (n = 11) concluded that PTH (1-34) was important for chondrocytes via increasing the proliferation and matrix synthesis but preventing apoptosis or hypertrophy. All included studies were assessed with low or unclear risk of bias in methodological quality. The SR demonstrated that PTH (1-34) could alleviate the progression of OA. Moreover, PTH (1-34) had beneficial effects on osteoporotic OA (OPOA) models, which might be a therapeutic option for OA and OPOA treatment.

Teriparatide with Octacalcium Phosphate Collagen Composite Stimulates Osteogenic Factors.

Octacalcium phosphate and collagen composite (OCPcol) promotes osteogenic differentiation and angiogenesis, thereby enhancing bone regeneration. Although a newly developed freeze-dried composite of OCPcol and teriparatide (OCPcolTPTD) reinforced bone regeneration more than OCPcol, the mechanism of bone regeneration remains unresolved. In this study, disks containing OCPcolTPTD, OCPcol, or ß-tricalcium phosphate (ß-TCP) col were inserted into rodents with calvarial bone defects, before euthanasia 4 weeks later. Immunohistochemical and histochemical analyses were performed on bone samples to evaluate bone matrix development, angiogenesis, and osteoclast and osteoblast localization. In the OCPcolTPTD and OCPcol groups, bone regeneration was observed at the surface of calvarial dura mater and around acidophilic granular cells with abundant collagenous fiber-containing cells. Furthermore, the newly formed bone in the OCPcolTPTD group showed a larger total area and individual separated area than the other groups. Various osteogenic proteins were detected in the regenerated bone and peri-bone tissues by histochemistry and immunohistochemistry. Although the expression of several osteogenic biomarkers in the OCPcolTPTD group after 4 weeks of implantation was significantly lower than that in the OCPcol group, new bone formation by OCPcolTPTD in the center of the defect, where bone regeneration is difficult, tended to be superior to that by OCPcol. These results suggest that OCPcolTPTD enhanced bone regeneration more evenly and homogenously than OCPcol. Impact statement Our study suggests that octacalcium phosphate and collagen (OCPcol) together with a TPTD enhances bone regeneration in rodents with calvarial bone defects. Furthermore, we believe that composite of OCPcol and teriparatide (OCPcolTPTD) could be developed into novel clinical technique for the regeneration or repair of bone.

Parathyroid hormone and its related peptides in bone metabolism.

Parathyroid hormone (PTH) is an 84-amino-acid peptide hormone that is secreted by the parathyroid gland. It has different administration modes in bone tissue through which it promotes bone formation (intermittent administration) and bone resorption (continuous administration) and has great potential for application in sbone defect repair. PTH regulates bone metabolism by binding to PTH1R. PTH plays an osteogenic role by acting directly on mesenchymal stem cells, cells with an osteoblastic lineage, osteocytes, and T cells. It also participates as an osteoclast by indirectly acting on osteoclast precursor cells and osteoclasts and directly acting on T cells. In these cells, PTH activates the Wnt signaling, cAMP/PKA, cAMP/PKC, and RANKL/RANK/OPG pathways and other signaling pathways. Although PTH(1-34), also known as teriparatide, has been used clinically, it still has some disadvantages. Developing improved PTH-related peptides is a potential solution to teriparatide's shortcomings. The action mechanism of these PTH-related peptides is not exactly the same as that of PTH. Thus, the mechanisms of PTH and PTH-related peptides in bone metabolism were reviewed in this paper.

Pulsatile release of parathyroid hormone from an implantable delivery system.

Intermittent (pulsatile) administration of parathyroid hormone (PTH) is known to improve bone micro-architecture, mineral density and strength. Therefore, daily injection of PTH has been clinically used for the treatment of osteoporosis. However, this regimen of administration is not convenient and is not a favorable choice of patients. In this study, an implantable delivery system has been developed to achieve pulsatile release of PTH. A well-defined cylindrical device was first fabricated with a biodegradable polymer, poly(l-lactic acid) (PLLA), using a reverse solid-free form fabrication technique. Three-component polyanhydrides composed of sebacic acid, 1,3-bis(p-carboxyphenoxy) propane and poly(ethylene glycol) were synthesized and used as isolation layers. The polyanhydride isolation layers and PTH-loaded alginate layers were then stacked alternately within the delivery device. The gap between the stacked PTH-releasing core and the device frame was filled with PLLA to seal. Multi-pulse PTH release was achieved using the implantable device. The lag time between two adjacent pulses were modulated by the composition and the film thickness of the polyanhydride. The released PTH was demonstrated to be biologically active using an in vitro assay. Timed sequential release of multiple drugs has also been demonstrated. The implantable device holds promise for both systemic and local therapies.

Comparison of recombinant human PTH(1-34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(1-34) (RS-66271): In vitro activity and in vivo pharmacological effects in rats.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are believed to exert their biological actions through binding and activation of a common cell surface receptor. Recently, an analog of PTHrP (RS-66271), was described that demonstrated reduced binding affinity for the PTH/PTHrP receptor compared with bovine PTH(1-34) but retained equal biological activity. The present study investigated the receptor binding affinities of synthetic RS-66271 and recombinant human PTH(1-34) (LY333334) and compared their in vitro and in vivo pharmacological effects. RS-66271 had one hundredth the activity of PTH(1-34) in competing for the binding of [125I] [Nle8,18, Tyr34]human PTH(1-34) to the human PTH/PTHrP receptor stably expressed in a human kidney cell line. Despite this reduced binding affinity, RS-66271 had equivalent activity in increasing both cAMP production in osteoblast-like cells and bone resorption in neonatal mouse calvariae. However, RS-66271 was 7. 6-fold less active in stimulating inositol phosphate production. For in vivo studies, young, male Fisher rats received a daily subcutaneous dose of either 10 or 40 microg/kg of peptide for 1, 2, or 4 weeks. Volumetric bone mineral density and total bone mineral content of the proximal tibia were determined by peripheral quantitative computerized tomography. Trabecular and cortical bone of the distal femur were analyzed for calcium and dry weight. Lumbar vertebrae (L4-L6) were analyzed by histomorphometry. Trabecular and cortical bone mass showed a dose- and time-dependent increase in the treated animals compared with the controls. These increases were evident as early as 1 week after initiation of dosing. There were no consistent significant differences in the comparative effects of PTH(1-34) and RS-66271 on the measured bone parameters. In conclusion, despite the reduced binding affinity of RS-66271 for the PTH/PTHrP receptor compared with human PTH(1-34), both peptides displayed similar in vitro and in vivo pharmacological effects.

Down-regulation of the receptor for parathyroid hormone (PTH) and PTH-related peptide by PTH in primary fetal rat osteoblasts.

We studied the effects of parathyroid hormone (PTH) on PTH parathyroid hormone related peptide (PTHrP) receptor mRNA level, PTHrP binding and PTH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblasts, derived from fetal rat calvariae (ROB). Cells isolated during 10-70 minutes of collagenase treatment were seeded at a density of 25,000 cells/cm2 and cultured for 4 days. These cells show a fast increase in cAMP production after stimulation for 5 minutes with 20 nM bovine parathyroid hormone(1-34) (bPTH(1-34)). When ROB are incubated with bPTH(1-34) (0.04-40nM) for 24 h, a dose-dependent decrease of the PTH/PTHrP receptor mRNA level, PTHrP binding, and PTH-stimulated cAMP accumulation can be observed. Pretreatment of ROB with a high concentration of bPTH(1-34) (40 nM) leads within 15 minutes to a decrease in PTH-stimulated cAMP accumulation. However, it takes > or = 3 h before a significant decrease in PTH/PTHrP receptor mRNA level can be observed. Also a significant decrease in PTHrP binding is observed after only 4 h of incubation with bPTH(1-34). Compared with bPTH(1-34), pretreatment of ROB with bPTH(3-34) (40 and 100 nM) for 24 h causes smaller decreases in PTH-stimulated cAMP accumulation, PTHrP binding, and in the PTH/PTHrP receptor mRNA level. We investigated the possible involvement of the protein kinase A signaling pathway in the regulation of the PTH/PTHrP receptor mRNA expression. Both forskolin and (Bu)2cAMP decreased PTHrP binding and PTH/PTHrP mRNA levels. These observations suggest that chronic activation of the PKA signaling pathway may down-regulate PTH/PTHrP receptor expression and thus hormone responsiveness in "normal" osteoblasts. In short, we found that the decrease of the PTH-stimulated cAMP accumulation after long-term pretreatment with bPTH(1-34) is correlated with both PTH/PTHrP receptor mRNA level and PTHrP binding. These data also suggest that the initial desensitization (< 30 minutes) of PTH-stimulated cAMP responsiveness by pretreatment with a high concentration of bPTH(1-34) (40 nM) is not dependent on the number of available PTH/PTHrP receptors. The protein kinase A signaling pathway is involved in the regulation of the PTH/PTHrP receptor, but, regarding the effect of bPTH(3-34), other signaling systems are also involved.

Histidine at position 5 is the specificity "switch" between two parathyroid hormone receptor subtypes.

The PTH and PTH-related protein (PTHrP) system consists of two hormones, at least two G protein-coupled seven-transmembrane domain receptors, and at least two intracellular signal transduction pathways for each receptor. The PTH/PTHrP receptor is present in the conventional target tissues of PTH action, namely kidney and bone. Both PTH and PTHrP bind to and activate the PTH/PTHrP receptor with equal affinity and efficacy. The newly discovered receptor, termed the human (h) PTH2 receptor, has 70% homology with the PTH/PTHrP receptor, but is found predominantly in brain and pancreas. It interacts selectively with PTH and not with PTHrP. PTH and PTHrP differ in several positions, including position 5 (Ile in PTH; His in PTHrP). To define the role of position 5 in receptor selectivity, we designed and synthesized a series of hybrid analogs containing specific elements of both the PTH and PTHrP sequences. Using human cell lines stably expressing either human receptor subtype, we evaluated the biological profile of the hybrids in assays of receptor binding and action. Both point-mutated hybrids, [Ile5]PTH-(1-34) and [His5]PTH-(1-34), bind to and stimulate cAMP accumulation and the release of cytosolic free calcium in HEK293/C-21, a clonal human embryonic kidney cell line stably expressing the recombinant hPTH/PTHrP receptor. However, only [Ile5]PTHrP-(1-34), and not [His5]PTH-(1-34), binds to and stimulates cAMP accumulation and the release of cytosolic free calcium in HEK293/BP-16, a clonal human embryonic kidney cell line stably expressing the recombinant hPTH2 receptor. The segmental hybrid PTHrP-(1-14)-PTH-(15-34) binds to and activates the hPTH/PTHrP receptor, but not the hPTH2 receptor, similar to the biological profile of His5-containing ligands: PTHrP-(1-34) and [His5]PTH-(1-34). Exchanging Ile5 for His5 in the segmental hybrid produces the analog [Ile5]PTHrP-(1-14)-PTH-(15-34), which interacts with both receptor subtypes. We conclude that His5 in PTHrP is the major structural determinant of receptor subtype specificity in the hPTH/PTHrP and hPTH2 two-receptor system. The mechanism of the specificity "switch" remains to be elucidated, but may result from a subtle perturbation of the bioactive conformation and/or from a direct steric hindrance at the hPTH2 receptor-ligand interface created by histidine at position 5. The hPTH2, but not the hPTH/PTHrP, receptor can discriminate between the two hormones based on the structural differences generated at position 5.

Effects of human parathyroid hormone (1-34), LY333334, on bone mass, remodeling, and mechanical properties of cortical bone during the first remodeling cycle in rabbits.

We have previously shown that parathyroid hormone (PTH) increases cortical bone mass and mechanical strength of female rabbits after 140 days of treatment. However, cortical porosity was also shown to increase. If cortical porosity increases prior to the change in geometry, there may be a transient decrease in cortical bone strength that could make the bone more susceptible to fracture in the early phase of treatment. The purpose of this study is to examine the effects of PTH on the remodeling dynamics and mechanical properties of cortical bone in rabbits, which exhibit haversian remodeling, during the first remodeling cycle after the initiation of treatment. Fifty 9-month-old intact female New Zealand white rabbits were randomized into five groups. A baseline control group was killed at the start of the experiment. The two PTH-treated groups were given human PTH(1-34) at 10 microg/kg daily subcutaneously for 35 (P35) or 70 (P70) days. Two respective age-matched control groups (V35, V70) were injected with vehicle. Histomorphometry of the cortical bone in the tibial midshaft showed that, although intracortical activation frequency was significantly increased by PTH at 35 days, there was no significant increase of cortical porosity in the first remodeling cycle (70 days). Moreover, stimulation of cortical surface bone formation in the treated animals led to significantly greater cortical area and greater bone strength in both P35 and P70. We conclude that, although intracortical remodeling increases within the first remodeling period (70 days) in animals treated with 10 microg/kg PTH, the greater cortical area due to acceleration of bone formation on cortical surfaces increases cortical bone strength. There is no mechanical risk during the first remodeling cycle associated with intermittent PTH treatment in animals with normal bone mass.

Conformational studies of RS-66271, an analog of parathyroid hormone-related protein with pronounced bone anabolic activity.

Both the parathyroid hormone (PTH) and the functionally similar parathyroid hormone-related protein (PTHrP) have served as templates for the development of novel bone anabolic agents for the treatment of osteoporosis. The PTHrP analog RS-66271 (Vickery, B. H.; Avnur, Z.; Cheng Y.; Chiou, S.-S.; Leaffer, D.; Caulfield, J. P.; Kimmel, D. B.; Ho, T.; Krstenansky, J. L. J. Bone Miner. Res. 1996, 11, 1943-1951), in which the amino acids 22-31 have been substituted by the sequence E22-L-L-E-K-L-L-E-K-L31 (a model amphiphilic peptide), is a potent bone anabolic agent in vivo. Therefore, RS-66271 is a good candidate for structural analysis with the aim of developing a structure-activity relationship. The structural characterization described here was carried out in aqueous solution employing circular dichroism and nuclear magnetic resonance spectroscopy. We find that the incorporated amphiphilic decapeptide is indeed helical. In addition, it induces the adjacent residues, up to residue 16, to adopt the helical conformation. The helical domain, including residues 16-32, incorporates most of the previously identified principal receptor binding domain PTHrP(25-34). We discuss the relevance of the distinct and extensive helicity in light of the reduced in vitro receptor affinity/ activity and the enhanced in vivo bone anabolic efficacy of RS-66271.

Parathyroid hormone-related protein (PTHrP) action in rat articular chondrocytes: comparison of PTH(1-34), PTHrP(1-34), PTHrP(1-141), PTHrP(100-114) and antisense oligonucleotides against PTHrP.

Parathyroid hormone-related protein (PTHrP) is thought to be an important autocrine/paracrine factor for chondrocyte metabolism since mice lacking the PTHrP gene exhibit abnormal cartilage development. To determine the biological role of PTHrP in chondrocytes, we first compared the agonist potency of human (h) PTHrP(1-34) with hPTH(1-34) in cultured rat articular chondrocytes. Neither hPTHrP(1-34) nor hPTH(1-34) altered basal DNA synthesis, but attenuated the stimulatory effect of transforming growth factor beta (TGF-beta). Both agents suppressed the expression of alpha(1) type II collagen mRNA in a dose-response fashion with the same potency. In addition, the action of exogenously added hPTHrP(1-34) and hPTH(1-34) on intracellular cAMP and [Ca2+]i levels was similar. We next compared the effect of PTHrP within its entire amino acid sequence (1-141). With regard to thymidine incorporation, alpha(1) type II collagen gene expression and accumulation of cAMP and [Ca2+]i level, there was no significant difference between hPTHrP(1-34) and hPTHrP(1-141). PTHrP C-terminal (100-114) did not show any function. To further investigate PTHrP function, intracellular PTHrP translation was inhibited by a transgene of antisense oligonucleotides against PTHrP. Antisense oligonucleotides decreased PTHrP mRNA translation, specifically inhibited DNA synthesis in control as well as TGF-beta-treated chondrocytes and enhanced alpha(1) type II collagen mRNA expression in TGF-beta-treated chondrocytes. These results suggest that there is no significant difference between exogenously added hPTH(1-34), hPTHrP(1-34) and PTHrP(1-141) with regard to the biological action of these agents, including cell growth, differentiation and second messenger pathway. However, the result of DNA synthesis in the antisense PTHrP-inhibition study suggests that intracellular PTHrP may have an as yet unknown biological role, in addition to a classical PTH/PTHrP receptor-mediated function in the rat articular chondrocyte.

Quantitative cell membrane-based radioligand binding assays for parathyroid hormone receptors.

Most current assays of PTH receptor ligand binding employ whole cells as the vehicle for receptor. Whole cell binding does not easily permit the estimation of physically meaningful binding parameters, the detection of multiple receptor states, or the evaluation of the effects of receptor modulators such as guanine nucleotides. We have developed quantitative methods for the measurement of equilibrium ligand binding parameters at cloned parathyroid hormone (PTH) receptors in cell membrane preparations. Centrifugation is used to separate bound and free [125I]-labeled peptide radioligands, and nonfat dried milk is used as a blocking agent to reduce nonspecific binding. This method is useful for measurement of agonist and antagonist radioligand binding at the PTH-1 receptor and binding of [125I]PTH(1-34) at the PTH-2 receptor. Less than 25% of [125I]PTH(1-34) or [125I]PTHrP(1-36) is degraded during the assay incubation. We demonstrated the utility of the assay using measurements of ligand binding properties at the PTH-1 receptor. (1) Homologous displacement experiments provided estimates of Kd and Bmax for the radioligands. (2) Displacement of radiolabeled antagonist binding ([125I]PTH(3-34)) by an unlabeled agonist (RS-66271) revealed multiple affinity states of agonist-receptor interaction. (3) Comparison of RS-66271 displacement in the presence and absence of GTP-gammaS demonstrated that the highest affinity state is guanine nucleotide-sensitive, suggesting that this state requires stabilization by G-protein. This assay thus allows more mechanistic interpretation of binding data than PTH binding assays in current use. A more convenient rapid-filtration method was also developed for measurement of radioligand binding at PTH-1 and PTH-2 receptors.

Time-resolved fluorescence ligand binding for G protein-coupled receptors.

G protein-coupled receptors (GPCRs) and their ligands are traditionally characterized by radioligand-binding experiments. These experiments yield excellent quantitative data, but have low temporal and spatial resolution. In addition, the use of radioligands presents safety concerns. Here we provide a general procedure for an alternative approach with high temporal and spatial resolution, based on Tb(+)-labeled fluorescent receptor ligands and time-resolved fluorescence resonance energy transfer (TR-FRET). This protocol and its design are detailed here for the parathyroid hormone receptor, a class B GPCR, and its fluorescently labeled 34-amino acid peptide ligand, but it can be easily modified for other receptors and their appropriately labeled ligands. We discuss three protocol options that use Tb(+)-labeled fluorescent ligands: a time-resolved fluorescence separation option that works on native receptors but requires separation of bound and unbound ligand; a TR-FRET option using SNAP-tag-labeled receptors for high-throughput screening; and a TR-FRET option that uses fluorescently labeled antibodies directed against an epitope engineered into the Flag-labeled receptors' N terminus. These protocol options can be used as standard procedures with very high signal-to-background ratios in order to characterize ligands and their receptors in living cells and in cell membranes via straightforward plate-reader measurements.

ß-arrestin-selective G protein-coupled receptor agonists engender unique biological efficacy in vivo.

Biased G protein-coupled receptor agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, D-Trp(12),Tyr(34)-bPTH(7-34) [bPTH(7-34)], a biased agonist for the type 1 PTH receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both bPTH(7-34) and the conventional agonist hPTH(1-34) stimulate anabolic bone formation. To understand how two PTH receptor ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 wk with vehicle, bPTH(7-34) or hPTH(1-34). Treatment of wild-type mice with bPTH(7-34) primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival, and migration. These responses were absent in ß-arrestin2-null mice, identifying them as downstream targets of ß-arrestin2-mediated signaling. In contrast, hPTH(1-34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. hPTH(1-34) actions were less dependent on ß-arrestin2, as might be expected of a ligand capable of G protein activation. In vitro, bPTH(7-34) slowed the rate of preosteoblast proliferation, enhanced osteoblast survival when exposed to an apoptotic stimulus, and stimulated cell migration in wild-type, but not ß-arrestin2-null, calvarial osteoblasts. These results suggest that bPTH(7-34) and hPTH(1-34) affect bone mass in vivo through predominantly separate genomic mechanisms created by largely distinct receptor-signaling networks and demonstrate that functional selectivity can be exploited to change the quality of G protein-coupled receptor efficacy.