Removal of partial agonism from parathyroid hormone (PTH)-related protein-(7-34)NH2 by substitution of PTH amino acids at positions 10 and 11

PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2 have previously been shown to be shown to be more potent antagonists than the corresponding PTH peptide, [Tyr34]bPTH(7-34)NH2. However, these peptides also display partial agonism for adenylate cyclase activity in ROS 17/2.8 cells. In this study, design of a pure potent antagonist of PTH and PTHrP by removal of agonism from PTHrP(7-34)NH2 with retention of antagonist potency was accomplished. Since [Tyr34]bPTH(7-34)NH2 lacks agonist activity, we introduced two amino acids native to the PTH sequence into their respective positions in PTHrP and the potent D-Trp12 analog. [Asn10Leu11]- and [Asn10,leu11,D-Trp12]-PTHrP(7-34)NH2 were found to be 23- and 26-fold more potent as antagonists in ROS cells than PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2, respectively. In addition, these peptides did not display partial agonism, even in an assay based on highly responsive cells pretreated with dexamethasone and pertussis toxin. In contrast, when the PTHrP sequence Asp10,Lys11 was inserted into [Tyr34]hPTH(7-34)NH2, antagonist potency declined by more than 6-fold and PTH-like agonist activity was installed. These results demonstrate that the activation domain of both PTH and PTHrP can be extended to include the 1-12 region and that the 10-12 region, in addition to the N-terminal hexapeptide, is important not only for receptor binding but also for hormonal signal transduction.     

A parathyroid hormone antagonist stimulates epidermal proliferation and hair growth in mice.

The biologic action of parathyroid hormone (PTH)-related peptide (PTHrP) in normal skin was investigated in cultured human keratinocytes and in SKH-1 hairless mice. The results indicate that the PTHrP agonists human PTHrP-(1-34) and PTH(1-34) are potent inhibitors of epidermal cell proliferation. [Nle8,18,Tyr34]bovine PTH-(7-34)-amide, an antagonist of the PTH/PTHrP receptor, blocked the inhibitory effect of PTH-(1-34) in cultured keratinocytes. In the SKH-1 mice, PTH-(7-34) caused a 244% increase of [3H]thymidine incorporation into isolated epidermal DNA and 246% and 180% increases in the number and length of hair shafts, respectively. Thus, PTH and PTHrP may play an important role in the normal physiology of skin, and their agonists and antagonists have potentially wide therapeutic applications in the treatment of hyperproliferative skin disorders and aging skin and could also be effective in stimulating and maintaining hair growth.     

Non-homologous sequences of parathyroid hormone and the parathyroid hormone related peptide bind to a common receptor on ROS 17/2.8 cells

We and others have recently shown that amino terminal sequences of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP), which share a 62% homology within the first 13 residues, bind to the same receptor on ROS 17/2.8 cells. The remaining PTHrP sequence is markedly different from PTH, suggesting that receptor binding may be dependent on the first 13 amino acids of either peptide. However, since the amino acid residues 14-34 have previously been recognized as an important binding domain for PTH, conformational similarity within this portion's secondary structure of both peptides could contribute to their capacity to bind to the same receptor. To test this hypothesis, we synthesized [Tyr36,Cys38]PTHrP-(14-38) and [Tyr34]bPTH(14-34)NH2, and studied binding of both peptides to the common PTH/PTHrP receptor on ROS 17/2.8 cells. Radioiodinated, HPLC-purified [Nle8,18, Tyr34]bPTH(1-34)NH2 (NlePTH) and [Tyr36]PTHrP-(1-36)NH2 were used to functionally define receptor binding requirements. [Tyr36,Cys38]PTHrP(14-38) and [Tyr34]bPTH(14-34)NH2 competed with 125I-NlePTH for binding sites on ROS 17/2.8 cells with apparent Kds of 10 microM and 50 microM respectively. Both peptides also competed with 125I-[Tyr36]PTHrP(1-36)NH2 with apparent Kds of 30 microM and 10 microM respectively. In the same assay system, NlePTH and [Tyr36,Cys38]PTHrP(1-38)inhibited binding of either radioiodinated ligand with apparent Kds of 0.3 and 1.0 nM. These studies indicate that although [Tyr34]bPTH(14-34)NH2 and [Tyr36,Cys38]PTHrP(14-38) share virtually no sequence homology, their secondary structures must be sufficiently similar to permit binding to a common PTH/PTHrP receptor.     

Nifedipine, but not verapamil, acutely elevates parathyroid hormone levels in premenopausal women

BACKGROUND Calcium channel antagonist therapy in humans has been associated with changes In anterior pitultary, thyroid and adrenal hormone secretion. Human studies assessing effects of calcium channel antagonists on calclotropic hormones have been few and typically involved small numbers of subjects studied for short periods of time. Few of these previously published studies have included women. The endocrine effects of calcium channel antagonists in women have become increasingly important as more women are taking these agents for diseases such as hypertension, angina, Raynaud's phenomenon and migraine. OBJECTIVE To assess both acute and chronic effects of calcium channel antagonists on calclotropic hormones in women. DESIGN A four-week prospective, randomized trial. SUBJECTS Twenty-nine premenopausal women, randomly assigned to receive either 240 mg of sustained release verapamil or 30 mg of sustained release nifedipine daily. LABORATORY END-POINTS Total and ionized serum calcium, phosphate, creatinine, parathyrold hormone (PTH), parathyroid hormone-related protein (PTHrP) and calcitonin, measured at baseline, after 24 hours, and 28 days of treatment. RESULTS Total and ionized calcium, phosphate, creatinine, PTHrP and calcitonin levels were not altered significantly after 24 hours or 28 days in any of the subjects, when compared to baseline. There were no significant differences in PTH levels after 24 hours or 28 days of verapamil treatment. There was a significant increase in serum PTH levels after 24 hours of nifedipine therapy; however, these differences were not evident after 28 days of therapy. CONCLUSIONS The short-term administration of nifedipine results in increased release of parathyroid hormone; however, long-term administration has no significant effect on the concentrations of calclotropic hormones.     

Bioactivity of PTH/PTHrP analogs lacking the 1-14 N-terminal domain

The N-terminal regions of 1-34 parathyroid hormone (PTH) and 1-34 parathyroid hormone related protein (PTHrP) are thought to be required for full agonist activity of these molecules and for signal transduction by cyclic AMP (cAMP). The C-terminal regions are thought to be involved in receptor binding and protein kinase C activation. In this study, two analogs of PTH/PTHrP lacking the segment 1-14 exhibited agonist activity in opossum kidney (OK) 3B2 cells. Analogs cPTHrP(15-34) and ANA NPY(13-36), an analog of neuropeptide Y, which both have amphipathic alpha helices, inhibited phosphate uptake and stimulated cAMP production in a dose-dependent manner, with half maximal activity in the microM range, compared to the nM range for hPTHrP(1-34) and hPTH(1-34). They also exhibited proportionately lower receptor binding affinities. cAMP production by these analogs was suppressed by the antagonist hPTHrP(7-34). Inhibition of phosphate uptake in response to the analogs was partially suppressed by H-89, but not by bisindolylmaleimide. The analogs also inhibited phosphate uptake and stimulated cAMP in parent OK cells and stimulated cAMP production in UMR-106 cells. These studies present the novel finding that in these cell types, a C-terminal region encompassing PTH/PTHrP(24-31), with the alpha-helical structure maintained, is sufficient for full activity at reduced potency.     

Production of parathyroid hormone and parathyroid-hormone-related protein by breast cancer cells in culture

Parathyroid-hormone-related protein (PTHrP) has been implicated in the origin of malignant hypercalcaemia. However, PTHrP production is not restricted to neoplastic cells, it is widespread among a variety of normal cell types and tissues. A physiological role for PTHrP has not been well defined. We describe a case of breast cancer with bone metastases and humoral hypercalcaemia of malignancy, with high levels of plasma C-terminal parathyroid hormone (PTH), mid-molecule PTH and PTHrP. Cells from breast cancer biopsies were cultured and medium samples assayed for the C-terminal and mid-molecule fragments, intact PTH and PTHrP. The data indicate a progressive increase in both PTH fragments and PTHrP levels, over a period of 30 days. No temporal parallelism exists between PTH fragments and PTHrP concentrations, the former being maximum at the 14th day, and the latter at the 30th day from the beginning of the culture. Our results indicate a coproduction of PTH and PTHrP by the breast cancer cells both in vivo and in vitro.Abbreviations PTH parathyroid hormone - PTHrP PTH-related protein - HHM humoral hypercalcaemia of malignancy This work was supported by a grant awarded by the Italian National Research Council (CNR): Clinical Applications of Oncology Research (ACRO) no. 92.02170.PF/39     

Production of parathyroid hormone-related protein by a rat parathyroid cell line

Parathyroid hormone-related protein (PTHrP), the peptide associated with humoral hypercalcemia of malignancy, has been identified in fetal and adult parathyroid glands. We here report a sub-clone of a rat parathyroid cell line which secretes a single peptide species corresponding in size to PTHrP(1-84). Biological activity of the secretion product was blocked by a specific antiserum against PTHrP, but not by parathyroid hormone (PTH) antiserum. Secretion of PTHrP by these cells was regulated by extracellular calcium in the physiological range. A single messenger RNA species for PTHrP was identified, though PTH mRNA could not be shown in these cells. Hybrid CAT genes containing 700-1000 bp of 5'-flanking DNA from the human PTH or PTHrP genes were transfected into these cells, and the PTHrP gene was expressed at 10-fold higher levels than the PTH gene. These cells thus provide a valuable model system for investigation expression of PTHrP in a non-transformed cell line.     

Parathyroid hormone(1-34) and parathyroid hormone-related protein(1-34) stimulate calcium release from human syncytiotrophoblast basal membranes via a common receptor

The placental syncytiotrophoblast is the site for mineral and nutrient exchange across the maternal-fetal interface. It has been proposed that parathyroid hormone-related protein (PTHrP) is a key factor in the maintenance of a maternal-fetal calcium gradient. Using simultaneously prepared microvillous (maternal facing) and basal (fetal facing) syncytiotrophoblast membranes from term human placentae (n=8), we determined the relative contribution of PTH(1-34), PTHrP(1-34) and PTHrP(67-94) to the regulation of syncytiotrophoblast calcium efflux. The vesicles had correct right-side-out membrane orientation and specific markers validated the fractionation of microvillous and basal membrane vesicles. Calcium efflux was studied by preloading vesicles with calcium-45 in the presence of calcium and magnesium and then incubating the vesicles at 37 degrees C for 15 min with the peptides. In basal membranes, PTHrP(1-! 34) significantly stimulated calcium efflux at a dose of 12.5 nmol/l, whereas PTH(1-34)-stimulated efflux was significant at 50 nmol/l (P<0.05, ANOVA). This efflux was significantly reduced in the presence of the PTH/PTHrP receptor antagonist (PTHrP(7-34)). Midmolecule PTHrP(67-94) had no significant effect on basal membrane calcium efflux. PTH(1-34), PTHrP(1-34) or PTHrP(67-94) had no significant effects on MVM calcium efflux. This study, using the human syncytiotrophoblast in vitro membrane system, demonstrated that PTHrP(1-34) and PTH(1-34) stimulate calcium transport across the basal, but not microvillous, syncytiotrophoblast membrane vesicles, mediated via the PTH/PTHrP receptor.     

Lung cancer associated with hypercalcemia induced by concurrently elevated parathyroid hormone and parathyroid hormone-related protein levels

In general, many cases of malignancy-associated hypercalcemia are due to HHM. In patients with humoral hypercalcemia of malignancy (HHM), it has been reported that plasma parathyroid hormone-related protein (PTHrP) and cyclic adenosine monophosphate (cAMP) levels were elevated, while plasma PTH and active vitamin D(3) levels were suppressed. Our patient showed hypercalcemia with a concurrent increase in plasma and tumor tissue PTHrP and PTH concentrations and also high cAMP and low 1-25(OH)(2)VD(3) levels in the plasma. These data suggest that the hypercalcemia exhibited by our patient was consistent with HHM due to lung cancer and its liver metastasis. Moreover, diagnostic imaging and autopsy findings showed no appreciable lesions of the parathyroid gland. In addition, histopathologic examination of the primary and metastatic tumors revealed the existence of PTH immunohistochemically stained with anti-PTH antibodies, suggesting an ectopic-PTH-producing lung tumor. From these data, our patient was diagnosed with a rare case of lung cancer, which produced both ectopic PTH and PTHrP.     

Treatment of bone-derived ROS 17/2.8 cells with dexamethasone and pertussis toxin enables detection of partial agonist activity for parathyroid hormone antagonists

In the design and biological evaluation of PTH antagonists, certain analogs, although antagonists in vitro, possess partial agonist properties in vivo that preclude their utility as antagonists. In an effort to identify weak agonism of PTH analogs, an attempt was made to enhance the responsiveness of the widely employed rat osteosarcoma (ROS 17/2.8) cell adenylate cyclase assay. Because responsiveness to PTH in these cells is enhanced upon treatment with dexamethasone (dex) or pertussis toxin (PT), we have evaluated their use to aid in detection of partial agonism for PTH and PTH-related protein (PTHrP) antagonist analogs. Treatment of cells with dex alone (30 nM for 3 days) or with PT alone (40 ng/ml for 1 day) increased basal adenylate cyclase activity by 27%. However, combination of the dex and PT treatments increased basal cAMP production 70%. The in vivo partial agonist [Nle8,18,Tyr34]bPTH(3-34)NH2 increased cAMP production 3-fold over basal levels in untreated cells, nearly 5-fold in PT-treated cells, 8-fold in cells treated with dex, and 10-fold in cells treated with dex plus PT. Similar results were obtained with PTHrP(7-34)NH2: the 6-fold stimulation observed in control cells was converted to 14-fold in cells treated with dex plus PT. Agonist activity undetectable in the conventional assay was observed in the dex plus PT system: [Tyr34]- and [D-Trp12,Tyr34]bPTH(7-34)NH2, which exhibit no agonist activity under control conditions, stimulated cAMP production 2.6- and 2.1-fold, respectively, under dex plus PT treatment. In contrast, the antagonist analogs [Asn10,Leu11]- and [Leu11,D-Trp12]PTHrP(7-34)NH2, hybrid peptides of PTH and PTHrP, had no agonist activity under any conditions. Because of increased responsiveness, this assay should occupy an important step in the pathway for evaluation of PTH antagonists and permit identification of weak partial agonist activity before extensive in vivo testing.     

Expression of parathyroid hormone-related protein in abnormal human parathyroids

The expression of parathyroid hormone-related protein (PTHrP) in abnormal human parathyroids was investigated. Northern blot analysis of RNA extracted from human benign parathyroid adenomata (n = 4) revealed multiple PTHrP mRNA species ranging in size from 1.8 to 4 kb. The relative abundance of PTHrP mRNA expressed in two of the adenomata was similar to that of a tumour (DAF) associated with humoral hypercalcaemia of malignancy, whereas PTHrP mRNA was of low abundance in a third and was undetectable in the fourth. PTHrP-like immunoreactivity was detected in extracts of abnormal parathyroid tissue (benign adenoma (n = 7), hyperplasia (n = 5) and parathyroid carcinoma (n = 2] using a sensitive specific two-site immunoradiometric assay for human (h) PTHrP(1-86) and a radioimmunoassay for hPTHrP(1-34). Ratios of hPTHrP(1-86)- and hPTHrP(1-34)-like immunoreactivities relative to hPTH(1-84)-like immunoreactivity in the parathyroid tissue extracts were, on average, less than 1%. PTHrP bioactivity in the extracts could not be distinguished from that of PTH, by an osteosarcoma cell bioassay. We conclude that, despite reports of over-expression of PTHrP mRNA in parathyroid adenomata, the potential contribution of PTHrP to the total PTH-like activity of adenomata and other abnormal parathyroid tissue may be insignificant relative to PTH.     

Expressions of parathyroid hormone‐related protein (PTHrP) and PTH/PTHrP‐receptor (PTH/PTHrP‐R) in gastrointestinal stromal tumours (GISTs), leiomyomas and schwannomas

Background: Gastrointestinal stromal tumours (GISTs) are rare, c‐kit and CD34 positive, and different from other mesenchymal tumours of the gastrointestinal tract (e.g. leiomyomas and schwannomas). The purpose of this study was to investigate the roles of parathyroid hormone‐related protein (PTHrP) and parathyroid hormone/parathyroid hormone‐related protein‐receptor (PTH/PTHrP‐R) in the growth and differentiation of GISTs. Methods: Nineteen GISTs, six leiomyomas and five schwannomas were examined in this study. Results: All of the GISTs and leiomyomas, and four of the schwannomas (80.0%) were positive for PTHrP. Immunohistochemical staining revealed that all of the leiomyomas, 90% of the GISTs and 80% of the schwannomas expressed PTH/PTHrP‐R. Furthermore, both PTHrP and PTH/PTHrP‐R were expressed in the cytoplasm of identical cells in all of these tumours. Conclusion: Our results suggest that both PTHrP and PTH/PTHrP‐R play an important role in the growth and differentiation of GISTs, leiomyomas and schwannomas.     

Expressions of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP-receptor (PTH/PTHrP-R) in gastrointestinal stromal tumours (GISTs), leiomyomas and schwannomas

BACKGROUND: Gastrointestinal stromal tumours (GISTs) are rare, c-kit and CD34 positive, and different from other mesenchymal tumours of the gastrointestinal tract (e.g. leiomyomas and schwannomas). The purpose of this study was to investigate the roles of parathyroid hormone-related protein (PTHrP) and parathyroid hormone/parathyroid hormone-related protein-receptor (PTH/PTHrP-R) in the growth and differentiation of GISTs. METHODS: Nineteen GISTs, six leiomyomas and five schwannomas were examined in this study. RESULTS: All of the GISTs and leiomyomas, and four of the schwannomas (80.0%) were positive for PTHrP. Immunohistochemical staining revealed that all of the leiomyomas, 90% of the GISTs and 80% of the schwannomas expressed PTH/PTHrP-R. Furthermore, both PTHrP and PTH/ PTHrP-R were expressed in the cytoplasm of identical cells in all of these tumours. CONCLUSION: Our results suggest that both PTHrP and PTH/PTHrP-R play an important role in the growth and differentiation of GISTs, leiomyomas and schwannomas.     

Regulation of PTH/PTH-related protein receptor expression by endogenous PTH-related protein in the rat osteosarcoma cell line ROS 17/2.8

We have utilized clonal lines of the rat osteoblastic cell line ROS 17/2.8 stably transfected with full-length parathyroid hormone-related protein (PTHrP) cDNA in a sense or an antisense orientation to examine the effects of alteration in the production of endogenous PTHrP on expression of the PTH/PTHrP receptor. In the stably transfected clonal cell lines, changes in PTH/PTHrP receptor expression were evaluated by Northern blot analysis, whole-cell ligand binding of ¹²⁵I-[Tyr³⁶] PTHrP (1–36), and exogenous PTHrP (1–34)-stimulated cyclic adenosine monophosphate (cAMP) accumulation. Compared to control (vector-transfected) cells, PTHP-overproducing (sense-transfected) cells exhibited a marked decrease in the expression of PTH/PTHrP receptor mRNA and PTHrP ligand binding, as well as a corresponding decrease in the PTHrP (1–34)-stimulated cAMP response. By contrast, the antisense-transfected cells showed a marked increase in expression of PTH/PTHrP receptor mRNA and PTHrP (1–34) ligand binding, but a significant increase in the PTHrP (1–34)-stimulated cAMP response was not detected. Using antisense-transfected ROS cells, PTH/PTHrP receptor mRNA expression and ¹²⁵I-[Tyr³⁶] PTHrP (1–36) binding were downregulated by treatment for 24 h with exogenous PTHrP (1–36), forskolin, or dibutyryl cAMP. The findings extend those of earlier studies showing receptor downregulation by exogenous PTH by indicating that endogenous PTHrP, as well as circulating PTH, may help regulate receptor production; and suggesting that even very low concentrations of the peptide may influence receptor production.     

The role of protein kinase-A activity in the evaluation of agonist/antagonist properties of analogs of parathyroid hormone-related protein in opossum kidney cells.

Structural modifications of the amino-terminal region of PTH resulted in the generation of PTH analogs with potent antagonist activities and markedly reduced agonist activities. Further development of these structure-function relationships to modifications of the sequence of the PTH-related protein (PTHrP) resulted in PTH/PTHrP antagonist analogs with increased antagonist activity and little if any agonist effects. Since studies from our laboratory have shown that measurement of protein kinase-A activity appears to be a more sensitive index of the actions of PTH than measurements of total cAMP, the present studies were designed to examine the effects of a series of PTHrP-based peptides for agonist/antagonist effects in opossum kidney (OK) cells. The results show that PTHrP-(7-34)NH2, which does not increase cAMP, displays agonist activity in terms of increasing protein kinase-A activity. Furthermore, [Leu11,D-Trp12]PTHrP-(7-34)NH2 and [D-Trp12]PTHrP-(7-34)NH2, which appear to be effective antagonists of rat PTH-(1-34)-stimulated cAMP generation, were less effective in antagonizing the effects on protein kinase-A and only [Leu11,D-Trp12] PTHrP-(7-34)NH2 appeared to exhibit any antagonist activity. The Ki for [Leu11,D-Trp1/]PTHrP-(7-34)NH2 to antagonize the stimulatory effect of 1 nM rat PTH-(1-34) was easily demonstrable by measurements of cAMP, but could not be demonstrated using the assay of protein kinase-A activity. These data underscore the observation that measurement of protein kinase-A is a more sensitive index of the effects of PTH than measurements of cAMP and that significant agonist activity on the cAMP/protein kinase pathway cannot be excluded without measurements of protein kinase-A activity.     

Concurrent primary hyperparathyroidism and humoral hypercalcemia of malignancy in a patient with multiple endocrine neoplasia type 1

We report a patient with multiple endocrine neoplasia type 1 presenting with elevation of parathyroid hormone-related protein (PTHrP) from a metastatic pancreatic neuroendocrine tumor (PNET), and parathyroid hormone (PTH) from primary hyperparathyroidism, resulting in severe hypercalcemia. Parathyroid hormone-related protein production by the PNET was confirmed by immunohistochemical analysis. Hypercalcemia and elevated PTHrP improved markedly with hepatic artery chemoembolization of liver metastasis. Thus, in multiple endocrine neoplasia type 1, correct identification of the cause of hypercalcemia as PTHrP production from a PNET or PTH production from a parathyroid tumor has important therapeutic implications.     

Historical Perspective and Evolutionary Origins of Parathyroid Hormone-Related Protein

After the discovery of parathyroid hormone-related protein (PTHrP) as the cause of the hypercalcemia of malignancy, it was found to be distributed widely in tissues, with its actions driving many physiologic and pathologic conditions. Its involvement in cancer extended to a contribution to the ability of cancer cells to promote bone resorption and establish as metastases. It was found to have multiple activities within the sequence, including a nuclear localizing sequence and a specific nuclear transport system. PTHrP and parathyroid hormone (PTH) appear to have arisen from a common ancestral gene and the comparative endocrinology and genomics studies focused on finding where the two genes appeared. PTHrP has been identified in bony and cartilaginous fish in the same tissues as in humans, indicating that PTHrP has fundamental and basic physiological roles in all vertebrates. PTHrP has been localized in fish neoplasms suggesting that PTHrP’s role in tumor formation is a conserved role from at least fish to humans. Interestingly, PTH has been identified in both bony and cartilaginous fish even though they lack a parathyroid gland and indicate that PTH’s evolutionary history is much longer too. So the point where PTHrP and PTH were duplicated is still unknown. A comparison among the analogous human, mouse, chicken, xenopus, and fugu sequences of the PTHrP gene demonstrates elements of conservation. When coupled with human Encode data and knowledge of interspecies gene structure, it offers rudimentary insights into function and underwrites hypotheses on physiology.     

Evidence for a novel parathyroid hormone-related protein in fetal lamb parathyroid glands and sheep placenta: comparisons with a similar protein implicated in humoral hypercalcaemia of malignancy

Parathyroid hormone (PTH)-like bioactivity, assayed as adenylate cyclase response in UMR 106-01 osteogenic sarcoma cells, was present in extracts of sheep fetal and maternal parathyroid glands and placenta. Preincubation of extracts with PTH(1-34) antiserum inhibited approximately 40% of the bioactivity in fetal parathyroid extracts, 50% in maternal parathyroid extracts, but only 10% of the bioactivity in the placental extract. Partial purification of placental extracts by chromatography yielded fractions containing PTH-like bioactivity which were similar in behaviour to that of PTH-related protein (PTHrP) from a human lung cancer cell line (BEN). An antiserum against synthetic PTHrP(1-16) partially inhibited the bioactivity of the placental extract and synthetic PTHrP(1-34), but had no effect on the bioactivity of bovine PTH(1-34) or bovine PTH(1-84). The placental PTH-like bioactivity was higher in mid- than in late gestation. Fetal parathyroid glands contained the highest PTH-like bioactivity. Thyroparathyroidectomy of one fetal twin lamb in each of 16 ewes between 110 and 125 days of gestation resulted in decreases of the plasma calcium concentration and reversal of the placental calcium gradient that existed between the ewe and the intact fetus. Perfusion of the placenta of each twin in anaesthetized ewes was carried out sequentially with autologous fetal blood in the absence of the exsanguinated fetus. The plasma calcium concentration in the blood perfusing the placenta of each twin increased, but reached a plateau at a lower concentration in the perfusing blood of thyroparathyroidectomized fetuses than in that of the intact fetuses. Addition of extracts of fetal parathyroid glands or of partially purified PTHrP resulted in further increases in plasma calcium in the autologous blood perfusing the placentae of thyroparathyroidectomized fetuses, but addition of bovine PTH(1-84) or rat PTH(1-34) had no effect. The presence of this PTH-like protein in the fetal parathyroid gland and placenta may contribute to the relative hypercalcaemia of the fetal lamb. This protein, which is similar to PTHrP associated with humoral hypercalcaemia of malignancy, stimulates the placental calcium pump responsible for maintaining a relative fetal hypercalcaemia during gestation.     

Mutation of phenylalanine-34 of parathyroid hormone disrupts NHERF1 regulation of PTH type I receptor signaling

Internalization of the PTH type I receptor (PTH1R) is regulated in a cell- and ligand-specific manner. We previously demonstrated that the sodium/proton exchanger regulatory factor type 1 (NHERF1; EBP50) is pivotal in determining the range of peptides that internalize the PTH1R. Antagonist PTH fragments can internalize the PTH1R in some kidney and bone cell models. PTH(7-34), which binds to, but does not activate, the PTH1R, internalizes the PTH1R in kidney distal tubule (DT) cells, where NHERF1 is not expressed. The effect of antagonist PTHrP peptides has not, to this point, been assessed. PTH1R internalization was measured by real-time confocal fluorescence microscopy of DT cells stably expressing 10⁵ EGFP-tagged PTH1R/cell (1). PTHrP(7-34) internalized the PTH1R in a manner indistinguishable from PTH(7-34). Introduction of NHERF1 into DT cells, however, blocked PTH(7-34)—, but not PTHrP(7-34)—, induced PTH1R internalization. To delineate the sequences within PTHrP that determine whether PTH1R internalization is affected by NHERF1, chimeric PTH/PTHrP fragments were tested for their ability to induce PTH1R internalization. PTH(7-21)/PTHrP (22-34), PTH(7-32)/PTHrP(33-34), and PTH(7-33)/PTHrP(34) at 1 μM each internalized the PTH1R 50–70% in a NHERF1-independent manner. When the C terminus of PTHrP was replaced with homologous amino acids from PTH, NHERF1 inhibited PTH1R internalization. It was determined that simply mutating F34 to A in PTH induced PTH1R internalization in a NHERF1-independent manner. None of the chimeric peptides activated the PTH1R but all effectively competed for 1 nM PTH(1-34) in cyclic AMP assays. In addition, all chimeric peptides competed for radiolabeled PTH(1-34) in binding assays in DT cells. PTH(1-34) and PTHrP(7-34), but not PTH(7-34), efficiently recruited β-arrestin1 to plasma membrane PTH1Rs. We, therefore, conclude that PTH(1-34) and PTHrP(7-34) induce a conformational change in the PTH1R that promotes arrestin binding and dissociates NHERF1 from PTH1R internalization.