Characterization of fully active biotinylated parathyroid hormone analogs. Application to fluorescence-activated cell sorting of parathyroid hormone receptor bearing cells

[Nle8,18,Tyr34]bPTH-(1-34)amide (NlePTH) was biotinylated using sulfosuccinimidyl 6-(biotinamido)hexanoate, in dimethyl sulfoxide, and the multiple resulting peptides peaks were separated by reverse-phase high performance liquid chromatography. Their biological activities were compared with those of NlePTH, the parent compound, in radioreceptor and cAMP accumulation bioassays using rat osteosarcoma 17/2.8 cells; the earliest two eluting products, bioPTH 1 and 2, were equipotent, a third, bioPTH 3, was only 10% as potent, and the remaining, later eluting derivatives all were less than 0.1% as active. Competitive avidin binding assays using [3H]biotin suggested that bioPTH 1 and 2 had a single biotin congener per molecule, while bioPTH 3 contained two biotin residues. Upon Edman degradation, bioPTH 1 contained biotin on the lysine at position 13 of NlePTH; bioPTH 2's biotin was on the lysine at position 26 (or 27) and bioPTH 3 had biotins on lysines at both positions 13 and 26 (or 27). Avidin tagged with 125I, peroxidase, or fluorescein isothiocyanate was detected on bone-derived cells which had been incubated initially with bioPTH 2 (1, 10, and 100 nM) for 4 h, but not when NlePTH (1 microM) was added with bioPTH 2. A fluorescence-activated cell sorter detected a symmetrical shift in fluorescence of bone-derived cells incubated with 10 nM of bioPTH 2 and 10 micrograms/ml fluorescein isothiocyanate-avidin. Addition of a 30-fold molar excess of NlePTH, or omission of bioPTH 2, completely reversed this fluorescence shift, and no shift in fluorescence was seen with cells lacking PTH receptors. This fully active, high affinity biotinylated PTH-derivative should prove useful in the study of PTH receptor-bearing cells.     

Sequences of the cDNAs encoding canine parathyroid hormone-related protein and parathyroid hormone

The cDNA clones encoding canine parathyroid-hormone-related protein (cPTHrP) and parathyroid hormone (cPTH) have been isolated and sequenced. The predicted amino-acid sequences of the mature canine homologs have a high degree of homology to human PTHrP (hPTHrP) and PTH (hPTH), especially in the biologically active regions. The cPTHrP cDNA is unique, since it has homology to exon lA of hPTHrP which suggests that dogs utilize a promoter similar to PI of hPTHrP which has not been demonstrated in other species.     

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.     

Parathyroid hormone and parathyroid hormone-related peptide, and their receptors

Parathyroid hormone (PTH) has a central role in the regulation of serum calcium and phosphate, while parathyroid hormone-related peptide (PTHrP) has important developmental roles. Both peptides signal through the same receptor, the PTH/PTHrP receptor (a class B G-protein-coupled receptor). The different biological effects of these ligands result from their modes of regulation and secretion, endocrine vs. paracrine/autocrine. The importance of PTH and PTHrP is evident by the variety of clinical syndromes caused by deficiency or excess production of either peptide, and the demonstration that intermittent injection of PTH increases bone mass, and thus provides a means to treat osteoporosis. This, in turn, has triggered increased interest in understanding the mechanisms of PTH/PTHrP receptor action and the search for smaller peptide or non-peptide agonists that have efficacy at this receptor when administered non-parenterally.     

Synthesis and characterization of novel biotinylated carboxyl-terminal parathyroid hormone peptides that specifically crosslink to the CPTH-receptor

Parathyroid hormone (PTH) regulates calcium, phosphorous and skeletal homeostasis via interaction with the G protein-coupled PTH/PTHrP receptor, which is fully activated by the amino-terminal 34 amino-acid portion of the hormone. Recent evidence points to the existence of another class of receptors for PTH that recognize the carboxyl (C)-terminal region of intact PTH (1–84) (CPTHRs) and are highly expressed by osteocytes. Here we report the synthesis and characterization of two novel bifunctional CPTH ligands that include benzoylphenylalanine (Bpa) substitutions near their amino-termini and carboxyl-terminal biotin moieties, as well as a tyrosine³⁴ substitution to enable radioiodination. These peptides are shown to bind to CPTHRs with affinity similar to that of PTH (1–84) and to be specifically and covalently crosslinked to CPTHRs upon exposure to ultraviolet light. Crosslinking to osteocytes or osteoblastic cells generates complexes of 80 and 220 kDa, of which the larger form represents an aggregate that can be resolved into the 80 kDa. The crosslinked products can be further purified using immunoaffinity and avidin-based affinity procedures. While the molecular structure of the CPTHR(s) remains undefined, these bifunctional ligands represent powerful new tools for use in isolating and characterizing CPTHR protein(s).     

Purification and characterization of recombinant human parathyroid hormone-related protein

Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.     

Preparation and characterization of a fully active biotinylated probe of cholecystokinin

In this study we describe the synthesis and purification of biotinylated cholecystokinin-8 (Bio-CCK-8) and characterize its use as a probe for the pancreatic cholecystokinin receptor. CCK-8 (0.1 umoles) was reacted with either radiolabeled d-[8,9(-3)H]biotin succinimide ester (0.5 umoles) or N-hydroxysuccinimidyl-biotin in dimethylformamide and triethylamine, and purified by anion exchange chromatography. Concentrations of Bio-CCK-8 and CCK-8 needed for half-maximal inhibition of [125]I-CCK-8 binding to pancreatic membranes were the same (1.0 and 1.3 nM). Bio-CCK-8 retained full biological activity as determined by stimulation of pancreatic protein secretion from rats, and the biotin group bound to CCK-8 retained its high sensitivity for avidin.     

Characterization of soluble and particulate parathyroid hormone receptors using a biotinylated bioactive hormone analog

A bioactive biotin-containing derivative of the synthetic bovine parathyroid hormone analog [Nle8,Nle18,Tyr34]bovine parathyroid hormone-(1-34) (bPTH-(1-34] amide was prepared by reacting the peptide with N-biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester. The derivative was incubated with particulate renal plasma membranes or with detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) extracts of renal cortical membranes, and two membrane components were identified. Labeling of these components was competitively inhibited by underivatized bPTH-(1-34) or bPTH-(3-34) but not by insulin, adrenocorticotropin, or oxidized rat PTH-(1-34). PTH-binding components that were immobilized on nitrocellulose could be detected by incubating the membrane with biotinyl-bPTH-(1-34). Binding components of apparent molecular mass 68, 70, and 150 kDa were specifically labeled in plasma membranes derived from canine, human, and porcine renal cortex, rat liver, and human fibroblasts. The 68-kDa binding protein was found to be consistently more acidic than the 70-kDa binding protein in human, porcine, and canine renal membranes analyzed by two-dimensional electrophoresis. The 68-70-kDa receptor doublet could be specifically isolated by streptavidin-agarose chromatography of solubilized membrane extracts that had first been incubated with biotinyl-BPTH-(1-34). Biotinyl-bPTH-(1-34) should be useful as a tool for further characterization and purification of the PTH receptor.     

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.     

Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.     

Parathyroid hormone and parathyroid hormone-related protein inhibit phasic contraction of pig duodenal smooth muscle

Parathyroid hormone (PTH) and a newly discovered PTH-related protein (PTHrP), which has amino-terminal homology with PTH, are potent relaxants of rat gastrointestinal tissues. Since their gastrointestinal relaxant effects have been described only in the rat, we examined their actions in another mammalian species in order to evaluate whether the relaxant property was more generally applicable. Longitudinal smooth muscle strips were obtained from the pig duodenum. The mucosa was removed, the strips were mounted in a tissue chamber, and changes in phasic contraction were detected with a force-displacement transducer and recorded using a polygraph. Acetylcholine-induced phasic contraction was inhibited rapidly in a dose-related manner by [Nle8,18,Tyr34]-bPTH-(1-34)-amide, or hPTHrP-(1-34). The IC50 values for these peptides were 2.6 nM and 6.1 nM, respectively. The maximal effect of both peptides was observed at 60 nM with an 84% decrease of the acetylcholine-induced contraction. At 400 nM, the PTH antagonist, [Nle8,18,Tyr34]-bPTH-(3-34)-amide, had no effect by itself. However, the same 400 nM concentration of this peptide totally blocked the decrease in phasic contraction induced by 10 nM of the bPTH-(1-34) analogue or hPTHrP-(1-34). Our results show that receptors for PTH or PTHrP are present in the muscular layer of the pig duodenum and that activation of these receptors inhibits the phasic contraction of the tissue. Furthermore, the ability of PTH-related peptides to relax gastrointestinal smooth muscle is not restricted to the rat.     

Characterization of the molecular motions of constitutively active G protein-coupled receptors for parathyroid hormone

The molecular mechanism of constitutive activity of the G protein-coupled receptor for human parathyroid hormone (PTH1) has been examined by molecular dynamics (MD) simulations. The single point mutations H223R, T410P, and I458R, of the PTH1 receptor result in ligand-independent receptor activation. Extensive MD simulations indicate that each of the mutations, through different mechanisms, lead to very similar conformational changes of the third intracellular loop. The structural changes, centered on K405 in the C-terminus of the third intracellular loop, can be traced back to the single-point mutations by calculation of the forces and torques responsible for the collective motions of the receptor. This analysis indicates a direct correlation between the conformational preferences of the cytoplasmic loop and the mutations in different locations of the receptor: TM2 (H223R), TM6 (T410P), and TM7 (1458R). Given the pivotal role of the third intracellular loop of PTH1 in coupling to the G proteins, the structural changes induced by these single-point mutations may be responsible for the ligand-free activation of the receptor. These results coupled with the high-resolution structure of the third cytoplasmic loop of PTH1, previously determined in our laboratory, provide unique insight into the mechanism of ligand free activation of the PTH1 receptor.     

Characterization of parathyroid hormone-related protein in the human term placenta.

To characterize parathyroid hormone-related protein (PTHrP) in the human placenta, we measured PTHrP-like immunoreactivity (PRP-LI) in the term placenta and studied the elution profiles of placental tissue extracts on Sephadex G-75 chromatography with a specific RIA. We also examined the gene expression of PTHrP mRNA by Northern blot analysis and the localization of PRP-LI in the placenta by immunohistochemistry. The amount of PRP-LI in placental extracts (n = 7) was 20.9 +/- 2.2 pg/g wet tissue (mean +/- SE). Dilution curves of placental tissue ran parallel to those of synthetic PTHrP (1-34) standards. Sephadex G-75 gel chromatography demonstrated two major PRP-LI peaks; the first peak was eluted around the molecular size between 10 kilodaltons (Kda) and 20 Kda and the other around 5 Kda. Northern blot analysis of PTHrP mRNA extracted from placental tissues showed a major hybridization signal around 18S. PTHrP immunohistochemistry showed PRP-LI staining in the cytoplasm of syncytiotrophoblasts and stroma cells (Hofbauer cells) in the term placenta. These results suggest that syncytiotrophoblasts and stroma cells in the term placenta synthesize PTHrP in two major molecular forms, 10 Kda-20 Kda and around 5 Kda.     

Nuclear and nucleolar localization of parathyroid hormone-related protein

Parathyroid hormone-related protein (PTHrP) was first discovered as the factor causing hypercalcaemia produced by solid tumours frequently associated with the head and neck, breast, lung and kidney. The homology of its amino-terminus to parathyroid hormone (PTH; eight of the first 13 residues are identical), enables it to share the same receptor and perform similar biological functions to PTH. The sequences of PTHrP C-terminal to its PTH-like region confer functions such as transplacental calcium transport, renal bicarbonate excretion and in vitro osteoclast inhibition. Recent findings have shown that PTHrP is a nuclear/nucleolar protein in certain tissues and that this localization is cell cycle-regulated, mediated by the middle portion of the molecule, and involves the nuclear import receptor importin beta1. The present review discusses what is known about the pathway by which PTHrP localizes to the nucleus/nucleolus and the putative roles it may have there.     

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.     

Parathyroid hormone and parathyroid hormone-related protein exert both pro- and anti-apoptotic effects in mesenchymal cells

During bone formation, multipotential mesenchymal cells proliferate and differentiate into osteoblasts, and subsequently many die because of apoptosis. Evidence suggests that the receptor for parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), the PTH-1 receptor (PTH-1R), plays an important role in this process. Multipotential mesenchymal cells (C3H10T1/2) transfected with normal or mutant PTH-1Rs and MC3T3-E1 osteoblastic cells were used to explore the roles of PTH, PTHrP, and the PTH-1R in cell viability relative to osteoblastic differentiation. Overexpression of wild-type PTH-1R increased cell numbers and promoted osteocalcin gene expression versus inactivated mutant receptors. Furthermore, the effects of PTH and PTHrP on apoptosis were dramatically dependent on cell status. In preconfluent C3H10T1/2 and MC3T3-E1 cells, PTH and PTHrP protected against dexamethasone-induced reduction in cell viability, which was dependent on cAMP activation. Conversely, PTH and PTHrP resulted in reduced cell viability in postconfluent cells, which was also dependent on cAMP activation. Further, the proapoptotic-like effects were associated with an inhibition of Akt phosphorylation. These data suggest that parathyroid hormones accelerate turnover of osteoblasts by promoting cell viability early and promoting cell departure from the differentiation program later in their developmental scheme. Both of these actions occur at least in part via the protein kinase A pathway.     

Molecular characterization of the receptor-ligand complex for parathyroid hormone.

Molecular models for the interaction of parathyroid hormone (PTH) with its G-protein-coupled receptors (PTH1 and PTH2) have been developed. The proposed ligand-receptor complex is based on experimental data from spectroscopic investigations of the hormone and receptor fragments as well as theoretical structure predictions based on homology analysis with proteins of known structure. From the insight afforded by the models, biochemical and pharmacological observations can be correlated with specific molecular or atomic interactions. The ligand selectivity of PTH2, specifically the lack of binding of His5-containing analogues, can be ascribed to unfavorable steric interactions (the binding pocket is markedly smaller in PTH2 than PTH1) as well as repulsive Coulombic forces between amino acids of like-charge (a positively charged H384 is located in the binding pocket in PTH2). The model of PTH1 suggests that the constitutive activity observed from the incorporation of a positively charged amino acid at position 223, found at the cytoplasmic end of TM2, is caused by a Coulombic attraction to E465, at the cytoplasmic end of TM7, leading to an association of TM2 and TM7 and thereby ligand-free activation. Additionally, a number of important interactions in the ligand-receptor complex are described along with predictions of the pharmacological profile which will result from specific modifications at these sites. In this regard, the models described here allow for atomic insight into the biochemical data currently available and allow targeting of future mutations to probe specific ligand/receptor interactions and thereby further our understanding of the functioning of this important hormone system.     

Synthesis and characterization of CN-modified protein analogues as potential vibrational contrast agents

A recombinant VH single-domain antibody recognizing staphylococcal protein A was functionalized on reactive lysine residues with N-hydroxysuccimidyl-activated 4-cyanobenzoate. Surface plasmon resonance analysis of antibody-antigen binding revealed that modified and unmodified antibodies bound protein A with similar affinities. Raman imaging of the modified antibodies indicated that the benzonitrile group provides vibrational contrast enhancement in a region of the electromagnetic spectrum that is transparent to cellular materials. Thus, the modified single-domain antibody may be amenable to detecting protein A from samples of the human pathogen Staphylococcus aureus using vibronic detection schemes such as Raman and coherent anti-Stokes Raman scattering. The generality of this labeling strategy should make it applicable to modifying an array of proteins with varied structure and function.     

Photoaffinity cross-linking identifies differences in the interactions of an agonist and an antagonist with the parathyroid hormone/parathyroid hormone-related protein receptor

Analogs of parathyroid hormone (PTH)-related protein (PTHrP), singularly substituted with a photoreactive L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in human embryonic kidney cells stably expressing the recombinant human PTH/PTHrP receptor. Two of these analogs, in which the photoreactive residue is either in position 1 or 2 (Bpa(1)- and Bpa(2)-PTHrP, respectively) displayed high affinity binding. Bpa(1)-PTHrP also displayed high efficacy for the stimulation of increased cAMP levels. Surprisingly, Bpa(2)-PTHrP was found to be a potent antagonist, despite the presence of the principal activation domain (sequence 1-6). Analysis of the digestion profiles of the ligand-receptor photoconjugates revealed that both the agonist and the antagonist cross-link to the S-CH(3) group of Met(425) in transmembrane domain 6 of the human PTH/PTHrP receptor. However, the antagonist Bpa(2)-PTHrP also cross-links to a proximal site within the receptor domain Pro(415)-Met(425). Unlike the antagonist Bpa(2)-PTHrP, the potent agonist Bpa(2)-PTH, also bearing the Bpa residue in position 2, cross-links only to the S-CH(3) group of Met(425) (similar to Bpa(1)-PTHrP and Bpa(1)-PTH). Taken together, these results suggest that the antagonist Bpa(2)-PTHrP is able to distinguish between two distinct conformations of the receptor. The comparison between PTHrP analogs substituted by Bpa at two consecutive positions and across PTH and PTHrP reveals insights into the PTH/PTHrP ligand-receptor bimolecular interaction at the level of a single amino acid.     

Establishment and characterization of a novel cell line derived from a human small cell lung carcinoma that secretes parathyroid hormone, parathyroid hormone-related protein, and pro-opiomelanocortin

There are few case reports describing small cell lung carcinoma (SCLC), which secrete parathyroid hormone (PTH)-related protein (PTHrP) and result in hypercalcemia. We have established a novel cell line, derived from a 37-year-old woman with SCLC, which produced PTH, PTH-rP, and a part of proopiomelanocortin (POMC), and led to hypercalcemia. The cell line, named SS-1, was grown as floating cell clusters in DMEM/F12 medium supplemented with 10% fetal bovine serum and had a population doubling time of 72 h. The modal chromosome number was 47 (88%); marker chromosomes were not observed. The SS-1 cell line secreted not only PTHrP but also PTH, and both were decreased by CaCl₂ administration. Decreasing the concentration of Ca⁺⁺ in the growth medium stimulated the secretion of both PTHrP and PTH. The cell line had calcium sensing receptor (Cas-R). Since PTHrP and PTH secretion from the SS-1 cells was related to Ca⁺⁺ concentration in the growth medium, the cell line might be useful for the study of PTH-rP and PTH regulation as well as for SCLC analysis. In addition, the cells secreted N terminal POMC, the precursor of adrenocorticotropic hormone, in response to stimulation with corticotropin releasing hormone. In summary, we established a novel cell line, SS-1 from SCLC, which produced PTHrP, PTH and N terminal POMC.