Characterisation of ligand binding to the parathyroid hormone/parathyroid hormone-related peptide receptor in MCF7 breast cancer cells and SaOS-2 osteosarcoma cells

Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP-receptor, PTH/PTHrP-R, are frequently expressed in mammary carcinomas as well as in bone cells. In this study we compared the ligand binding characteristics of the PTH/PTHrP-R in SaOS-2 human osteosarcoma cells with those in MCF7 breast cancer cells. We used both Scatchard analysis of saturation kinetics for iodinated ligand and the level of expressed receptor protein by visualising the single radio-labelled receptor-ligand complex from isolated membrane preparations from the two cell lines. In MCF7 cells, ligand binding, (receptor number) was increased by prior exposure of the cultured cells to epidermal growth factor (EGF), estradiol (E2), or dexamethasone (DEX), and decreased following calcitriol (1,25 DHCC). In contrast in the SaOS-2 cells, PTH/PTHrP-R number was increased by exposure to E2 and 1,25DHCC and decreased by DEX while EGF had no effect. These data were confirmed when the PTH/PTHrP-R was cross linked with (125)I-PTHrP-1-34(Tyr), and extended by visualising the intensity of the isolated radiolabelled receptor complex by autoradiography following SDS-PAGE at several time points during the treatment.     

Zebrafish express the common parathyroid hormone/parathyroid hormone-related peptide receptor (PTH1R) and a novel receptor (PTH3R) that is preferentially activated by mammalian and fugufish parathyroid hormone-related peptide.

To further explore the evolution of receptors for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), we searched for zebrafish (z) homologs of the PTH/PTHrP receptor (PTH1R). In mammalian genes encoding this receptor, exons M6/7 and M7 are highly conserved and separated by 81-84 intronic nucleotides. Genomic polymerase chain reaction using degenerate primers based on these exons led to two distinct DNA fragments comprising portions of genes encoding the zPTH1R and the novel zPTH3R. Sequence comparison of both full-length teleost receptors revealed 69% similarity (61% identity), but less homology with zPTH2R. When compared with hPTH1R, zPTH1R showed 76% and zPTH3R 67% amino acid sequence similarity; similarity with hPTH2R was only 59% for both teleost receptors. When expressed in COS-7 cells, zPTH1R bound [Tyr(34)]hPTH-(1-34)-amide (hPTH), [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), and [Ala(29),Glu(30), Ala(34),Glu(35), Tyr(36)]fugufish PTHrP-(1-36)-amide (fuguPTHrP) with a high apparent affinity (IC(50): 1.2-3.5 nM), and was efficiently activated by all three peptides (EC(50): 1.1-1.7 nM). In contrast, zPTH3R showed higher affinity for fuguPTHrP and hPTHrP (IC(50): 2.1-11.1 nM) than for hPTH (IC(50): 118.2-127.0 nM); cAMP accumulation was more efficiently stimulated by fugufish and human PTHrP (EC(50): 0.47 +/- 0.27 and 0.45 +/- 0.16, respectively) than by hPTH (EC(50): 9.95 +/- 1.5 nM). Agonist-stimulated total inositol phosphate accumulation was observed with zPTH1R, but not zPTH3R.     

Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney

A tumor-derived protein with a spectrum of biologic activities remarkably similar to that of parathyroid hormone (PTH) has recently been purified and its sequence deduced from cloned cDNA. This PTH-like protein (PLP) has substantial sequence homology with PTH only in the amino-terminal 1-13 region and shows little similarity to other regions of PTH thought to be important for binding to receptors. In the present study, we compared the actions of two synthetic PLP peptides, PLP-(1-34)amide and [Tyr36]PLP-(1-36)amide, with those of bovine parathyroid hormone (bPTH)-(1-34) on receptors and adenylate cyclase in bone cells and in renal membranes. Synthetic PLP peptides were potent activators of adenylate cyclase in canine renal membranes (EC50 = 3.0 nM) and in UMR-106 osteosarcoma cells (EC50 = 0.05 nM). Bovine PTH-(1-34) was 6-fold more potent than the PLP peptides in renal membranes, but was 2-fold less potent in UMR-106 cells. A competitive PTH receptor antagonist, [Tyr34]bPTH-(7-34)amide, rapidly and fully inhibited adenylate cyclase stimulation by the PLP peptides as well as bPTH-(1-34). Competitive binding experiments with 125I-labeled PLP peptides revealed the presence of high affinity PLP receptors in UMR-106 cells IC50 = 3-4 nM) and in renal membranes (IC50 = 0.3 nM). There was no evidence of heterogeneity of PLP receptors. Bovine PTH-(1-34) was equipotent with the PLP peptides in binding to PLP receptors. Likewise, PLP peptides and bPTH-(1-34) were equipotent in competing with 125I-bPTH-(1-34) for binding to PTH receptors in renal membranes. Photoaffinity cross-linking experiments revealed that PTH and PLP peptides both interact with a major 85-kDa and minor 55- and 130-kDa components of canine renal membranes. We conclude that PTH and PLP activate adenylate cyclase by binding to common receptors in bone and kidney. The results further imply that subtle differences exist between PTH and PLP peptides in their ability to induce receptor-adenylate cyclase coupling.     

A G protein-coupled receptor from zebrafish is activated by human parathyroid hormone and not by human or teleost parathyroid hormone-related peptide. Implications for the evolutionary conservation of calcium-regulating peptide hormones.

Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47-52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5' splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr(34)]hPTH(1-34)-amide (hPTH) (EC(50), 1.64 +/- 0. 95 nM) and [Ile(5),Trp(23),Tyr(36)]hPTHrP-(1-36)-amide ([Ile(5), Trp(23)]hPTHrP) (EC(50), 46.8 +/- 12.1 nM) but not when stimulated with [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), [Trp(23), Tyr(36)]hPTHrP-(1-36)-amide ([Trp(23)]hPTHrP), or [Ala(29),Glu(30), Ala(34),Glu(35),Tyr(36)]fugufish PTHrP-(1-36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.     

Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.     

Analogues of parathyroid hormone modified at positions 3 and 6. Effects on receptor binding and activation of adenylyl cyclase in kidney and bone.

Predictive and spectroscopic methods were used to develop a model of the structures of the 1-34 peptides of parathyroid hormone (PTH) and the PTH-related protein (PTHrP). Circular dichroism (CD) studies of bovine PTH-(1-34) and human PTHrP-(1-34)amide in the presence of trifluoroethanol suggest the presence of 24-26 alpha-helical residues. For both peptides, interactions between amino- and carboxyl-region alpha-helices are predicted to result in a hydrophobic core with externally facing hydrophilic residues that include probable determinants of receptor binding and activation. Two such residues, Ser3 and Gln6, are conserved in all known members of the PTH/PTHrP family. We have synthesized 13 novel analogues of bovine PTH-(1-34) monosubstituted at positions 3 and 6 and have determined their biological activities in renal and bone cell radioreceptor and adenylyl cyclase assays. Position 3 analogues displayed biological activity that was reduced in direct proportion to the volume of the substituent side-chain. Position 6 analogues also displayed reduced biological activity, but no simple correlation with side-chain volume or hydrophobicity was evident. The analogues fully displaced labeled PTH from binding sites in renal membranes and bone cells, but [Phe3]bPTH-(1-34), [Tyr3]bPTH-(1-34), [Phe6] bPTH-(1-34), and [Ser6]bPTH-(1-34) were only partial agonists in one or both adenylyl cyclase assays. Of these, [Phe3]bPTH-(1-34) and [Phe6]bPTH-(1-34) were tested for antagonist activity and were found to inhibit the activation of adenylyl cyclase in response to bPTH-(1-34) or hPTHrP-(1-34)amide. These results indicate that positions 3 and 6 contribute important determinants of PTH receptor binding and activation. Modification at these positions represents a novel approach to the development of antagonists of PTH action.     

The hydrophobic residues phenylalanine 184 and leucine 187 in the type-1 parathyroid hormone (PTH) receptor functionally interact with the amino-terminal portion of PTH-(1-34).

Recent mutagenesis and cross-linking studies suggest that three regions of the PTH-1 receptor play important roles in ligand interaction: (i) the extreme NH(2)-terminal region, (ii) the juxtamembrane base of the amino-terminal extracellular domain, and (iii) the third extracellular loop. In this report, we analyzed the second of these segments in the rat PTH-1 receptor (residues 182-190) and its role in functional interaction with short PTH fragment analogs. Twenty-eight singly substituted PTH-1 receptors were transiently transfected into COS-7 cells and shown to be fully expressed by surface antibody binding analysis. Alanine-scanning analysis identified Phe(184), Arg(186), Leu(187), and Ile(190) as important determinants of maximum binding of (125)I-labeled bovine PTH-(1-34) and (125)I-labeled bovine PTH-(3-34) and determinants of responsiveness to the NH(2)-terminal analog, PTH-(1-14) in cAMP stimulation assays. Alanine mutations at these four sites augmented the ability of the COOH-terminal peptide [Glu(22), Trp(23)]PTHrP-(15-36) to inhibit the cAMP response induced by PTH-(1-34). At Phe(184) and Leu(187), hydrophobic substitutions (e.g. Ile, Met, or Leu) preserved PTH-(1-34)-mediated cAMP signaling potency, whereas hydrophilic substitutions (e.g. Asp, Glu, Lys, or Arg) weakened this response by 20-fold or more, as compared with the unsubstituted receptor's response. The results suggest that hydrophobicity at positions occupied by Phe(184) and Leu(187) in the PTH-1 receptor plays an important role in determining functional interaction with the 3-14 portion of PTH.     

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.     

Development of a photoreactive parathyroid hormone antagonist to probe antagonist-receptor bimolecular interaction.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert their calciotropic activities by binding to a specific seven-transmembrane-helix-containing G protein-coupled receptor mainly located in bone and kidney cells. In order to map in detail the nature of hormone-receptor interaction, we are employing 'photoaffinity scanning' of the bimolecular interface. To this end, we have developed photoreactive benzophenone (BP)-containing PTH analogs which can be specifically and efficiently cross-linked to the human (h) PTH/PTHrP receptor. In this report, we describe the photocross-linking of a BP-containing PTH antagonist, [Nle8,18,D-2-Nal12,Lys13(epsilon-BP),2-Nal23,Tyr34]bPT H(7-34)NH2 (ANT) to the recombinant hPTH/PTHrP receptor stably expressed in human embryonic kidney cells (HEK-293, clone C-21). This photoreactive antagonist has high affinity for the hPTH/PTHrP receptor and inhibits agonist-induced cyclase activity and intracellular calcium release. The photo-induced cross-linking of the radioiodinated antagonist (125I-ANT) to the recombinant hPTH/PTHrP receptor followed by SDS-PAGE analysis reveals a single radiolabeled band of approximately 85kDa, similar to that observed after cross-linking of a radioiodinated BP-containing agonist. The formation of this covalent 125I-ANT - hPTH/PTHrP receptor conjugate is competed dose-dependently by a variety of unlabelled PTH- and PTHrP-derived agonists and antagonists. This is the first report of a specific and efficient photocross-linking of a radioiodinated PTH antagonist to the hPTH/PTHrP receptor. Therefore, it provides the opportunity to study directly the nature of the bimolecular interaction of PTH antagonist with the hPTH/PTHrP receptor.     

Demonstration of receptors for parathyroid hormone on cultured rabbit costal chondrocytes

Parathyroid hormone (PTH) receptors on cultured rabbit costal chondrocytes were demonstrated using HPLC-purified, radioiodinated [Nle8,-Nle18, Tyr34] bovine PTH-(1-34)amide. PTH binding was found to be specific for PTH agonists and antagonists and dependent on the time and temperature of incubation. Both growth cartilage (GC) cells and resting cartilage (RC) cells were shown to have a single class of saturable, high affinity PTH binding sites with a dissociation constant of 0.6-0.7 nM. However, the numbers of receptors per cell were approximately 49,000 on GC cells and 19,000 on RC cells. After crosslinking the receptors on these cells with the radioligand, one, major 125I-labeled band of 76 kDa was separated by SDS-PAGE.     

Photoaffinity labeling of parathyroid hormone receptors in clonal rat osteosarcoma cells

A photoreactive derivative of a sulfur-free bovine parathyroid hormone (PTH) analogue, [Nle8,N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH), was purified from the products of the reaction of [Nle8,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NlePTH) with 4-fluoro-3-nitro-phenylazide and was used to identify binding components of the PTH receptor in clonal rat osteosarcoma cells (ROS 17/2.8). The purified analogue, NAP-NlePTH, is a fully active agonist in three different ROS 17/2.8 cell bioassays: 1) specific binding to saturable PTH receptors; 2) stimulation of cyclic AMP accumulation; and 3) inhibition of cellular alkaline phosphatase activity; this analogue gave dose response curves parallel to and 25-33% as potent as its parent molecule, NlePTH. Radioiodinated NAP-NlePTH (125I-labeled NAP-NlePTH) retained maximal receptor-binding potency. Radioligand saturation studies in intact cells showed that the Kd of PTH receptors for the photoligand was slightly less than that for 125I-labeled NlePTH (2.8 and 0.8 nM, respectively), but that the Bmax was essentially identical for both radioligands (8 fmol/10(5) cells). Photoaffinity labeling of ROS 17/2.8 cells revealed several 125I-labeled macromolecular components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One predominant 125I-labeled band, having an apparent Mr of 80,000 daltons (including Mr = 4,347 ligand; hereafter referred to as the Mr = 80,000 protein), was consistently demonstrated in both reducing and nonreducing conditions. Its labeling was completely inhibited by coincubation with NlePTH (10 nM) at 26-fold molar excess to the photoligand, but not by biologically inactive PTH fragments or unrelated hormone. Labeling of several other macromolecular components persisted in the presence of NlePTH (1 microM). Only the labeling of the Mr = 80,000 protein showed saturation kinetics for photoaffinity labeling; the dose of 125I-labeled NAP-NlePTH (0.8 nM) to half-saturate labeling of the Mr = 80,000 protein was close to the Kd (2.8 nM) of specific binding of the photoligand to receptors in intact ROS 17/2.8 cells. Pretreatment of the cells with NlePTH and dexamethasone led to the predicted proportional decrease or increase, respectively, in labeling of the Mr = 80,000 protein. Our data, using a highly purified photoactive derivative of PTH, having carefully defined chemical and biological properties, show a plasma membrane component of Mr = 80,000 in ROS 17/2.8 cells that possesses the affinity, binding capacity, and physiological characteristics of the PTH receptor.     

Parathyroid hormone receptor trafficking contributes to the activation of extracellular signal-regulated kinases but is not required for regulation of cAMP signaling

Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both G(s)- and G(q)-mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and beta-arrestin1 (beta-arrestin1-(319-418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signaling. PTH-(1-34) induced rapid and robust activation of ERK1/2. A PTHrP-based analog ([p-benzoylphenylalanine1, Ile5,Arg(11,13),Tyr36]PTHrP-(1-36)NH2), which selectively activates the G(s)/cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1-34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7-34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and G(q) (but not G(s)) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor.     

Characterization of canine renal receptors for the parathyroid hormone-like protein associated with humoral hypercalcemia of malignancy

Parathyroid hormone-like proteins (PTHLP) display actions in the kidney which are similar to those of parathyroid hormone (PTH). We compared the binding properties of PTHLP and PTH in canine renal cortical membranes to determine if they interacted with the same or different receptors. Radioiodination to high specific activity (greater than 400 microCi/micrograms) of [Nle8,18,Tyr34]human PTH-(1-34)amide and [Tyr36]PTHLP-(1-36)amide was performed using the lactoperoxidase method. Complete enzymatic digestion of both radioligands demonstrated that the peptides were monoiodinated. Both radioligands retained full biological activity in the renal adenylate cyclase assay, and neither was significantly degraded during incubation with highly purified canine renal membranes under binding assays conditions. Specific binding reached equilibrium by 20 min at 20 degrees C. Competition binding studies using unlabeled [Nle8,18,Tyr34]human PTH-(1-34)amide, [Tyr36] PTHLP-(1-36)amide, and bovine PTH-(1-34) with either radioligand revealed similar binding affinities for all three peptides. Biologically inactive PTHLP fragments did not show significant displacement. In contrast to its similar binding affinity, [Tyr36]PTHLP-(1-36)amide was 6-15-fold less potent than bovine PTH-(1-34) in the renal adenylate cyclase assay, suggesting less efficient receptor-effector coupling. Photoaffinity cross-linking using either radioligand in canine renal membrane labeled indistinguishable 70,000-dalton proteins. In the presence of multiple protease inhibitors, binding to an 85-kDa component was observed. Labeling of both receptor forms was specifically abolished by an excess of either cold peptide and dose-response curves using affinity cross-linked membranes corroborated the apparent binding affinities determined by conventional radioligand binding assays. We conclude that PTHLP-(1-36) and amino-terminal PTH analogues bind to indistinguishable receptors in canine renal cortical membranes, but display differential coupling to post-receptor events.     

Phorbol ester induces desensitization of PTH-stimulated cyclic AMP production by decreasing the PTH receptor binding in UMR-106 cells

Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with the protein kinase C(PK-C) activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) results in a time dependent (1-12h) desensitization of PTH-stimulated cAMP production. Compared to controls, PMA-treated cells showed 50% decrease of PTH-stimulated cAMP production. PK-C inhibitor, H-7 significantly blocked this PMA-induced desensitization. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was decreased by about 20% in PMA-treated cells. H-7 treatment completely restored receptor binding in PMA-treated cells. These data suggest that PK-C might act directly on PTH receptor which is coupling to adenylate cyclase, and induce desensitization.     

Parathyroid hormone-related protein ameliorates death receptor-mediated apoptosis in lung cancer cells

Parathyroid hormone-related protein (PTHrP) is expressed in more advanced, aggressive tumors and may play an active role in cancer progression. This study investigated the effects of PTHrP on apoptosis after UV irradiation, Fas ligation, or staurosporine treatment in BEN human squamous lung carcinoma cells. Cells at 70% confluency were treated for 24 h with 100 nM PTHrP-(1-34), PTHrP-(38-64), PTHrP-(67-86), PTHrP-(107-139), or PTHrP-(140-173) in media with serum, exposed for 30 min to UV-B radiation (0.9 mJ/cm²), and maintained for another 24 h. Caspase-3, caspase-8, and caspase-9 activities increased fivefold. Pretreatment with PTHrP-(1-34) and PTHrP-(140-173) ameliorated apoptosis after UV irradiation, as indicated by reduced caspase activities, increased cell protein, decreased nuclear condensation, and increased clonal survival. Other peptides had no effect on measures of apoptosis. PTHrP-(140-173) also reduced caspase activities after Fas ligation by activating antibody, but neither peptide had effects on caspase-3 or caspase-9 activity after 1 µM staurosporine. These data indicate that PTHrP-(1-34) and PTHrP-(140-173) protect against death receptor-induced apoptosis in BEN lung cancer cells but are ineffective against mitochondrial pathways. PTHrP contributes to lung cancer cell survival in culture and could promote cancer progression in vivo. The mechanism for the protective effect against apoptosis remains to be determined. caspases; cell surface receptors; growth substances     

Specificity and stability of a new PTH1 receptor antagonist,mouse TIP(7-39)

Parathyroid hormone 1 (PTH1) receptor antagonists might be of benefit in hypercalcemia of malignancy (HHM) and hyperparathyroidism. We previously identified bovine tuberoinfundibular peptide (7-39) (bTIP(7-39)) as a high-affinity PTH1 receptor antagonist. Mouse TIP(7-39) is an antagonist (rPTH1 K(B)=44 nM, rPTH2=940 nM) that is more potent than other known PTH1 receptor antagonists: bTIP(7-39) (210 nM), PTH-related protein (PTHrP)(7-34) (640 nM), and bPTH(7-34) (>3000 nM). Plasma proteases slowly (t(1/2)=81 min) inactivated [125I] mTIP(7-39). Intravenous plasma [125I]mTIP(7-39) was bi-phasically cleared (radioactivity t(1/2)=2.9 min (70%) and 120 min (30%), binding activity t(1/2)=3.6 min (92%), and t(1/2)=21 min (8%)). Loss of unlabeled mTIP(7-39) (250 microg/kg i.v.) receptor binding was similar. mTIP(7-39)'s high-affinity should facilitate animal evaluation of effects of PTH1 receptor antagonism.     

Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis.

We have investigated receptor structural components responsible for ligand-dependent inverse agonism in a constitutively active mutant of the human parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (hP1R). This mutant receptor, hP1R-H223R (hP1R(CAM-HR)), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa(2),Ile(5),Trp(23),Tyr(36)]PTHrP-(1-36)-amide (Bpa(2)-PTHrP-(1-36)), which has valine 2 replaced by p-benzoyl-l-phenylalanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1R(CAM-HR). This analog cross-linked to hP1R(CAM-HR) at two contiguous receptor regions as follows: the principal cross-link site (site A) was between receptor residues Pro(415)-Met(441), spanning the TM6/extracellular loop three boundary; the second cross-link site (site B) was within the TM4/TM5 region. Within the site A interval, substitution of Met(425) to Leu converted Bpa(2)-PTHrP-(1-36) from an inverse agonist to a weak partial agonist; this conversion was accompanied by a relative shift of cross-linking from site A to site B. The functional effect of the M425L mutation was specific for Bpa(2)-containing analogs, as inverse agonism of Bpa(2)-PTH-(1-34) was similarly eliminated, whereas inverse agonism of [Leu(11),d-Trp(12)]PTHrP-(5-36) was not affected. Overall, our data indicate that interactions between residue 2 of the ligand and the extracellular end of TM6 of the hP1R play an important role in modulating the conversion between active and inactive receptor states.     

Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells

Synthetic peptides corresponding to the amino-terminal region of the human parathyroid hormone-related peptide (hPTHrp) were used to characterize the interaction of hPTHrp with parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8). Both hPTHrp-(1-34) and [Tyr40]hPTHrp-(1-40) showed full agonist activity in stimulating cyclic AMP accumulation in ROS cells; human PTHrp-(1-34) was approximately 2.5-fold as potent as hPTH-(1-34). Both [Tyr-40]hPTHrp-(3-40) and hPTH-(3-34) inhibited the cyclic AMP increase induced by either hPTHrp or PTH with parallel dose-inhibition curves. Binding to intact ROS cells of a 125I-labeled [Tyr40]hPTHrp-(1-40) (125I-[Tyr40]hPTHrp-(1-40)) which retains full biological activity was time- and temperature-dependent and reversible. Binding of 125I-[Tyr40]hPTHrp-(1-40) and 125I-labeled [Nle8, Nle18, Tyr34]bovine PTH-(1-34)NH2 to ROS cells was competed for, to the same extent and with the comparable potency, by either unlabeled hPTHrp or PTH peptides. The binding capacity and affinity of receptors in ROS cells were strikingly similar for hPTHrp and PTH. Affinity cross-linking with either radioligand resulted in high affinity, specific labeling of an apparently identical macromolecule centering at Mr = 80,000, which was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both reducing and nonreducing conditions. The data indicate that hPTHrp and PTH, their amino-terminal fragments at least, interact with the identical receptors with regard to affinity, capacity, specificity, and physicochemical characteristics in osteoblastic ROS 17/2.8 cells.