Characterization of the interaction of parathyroid hormone with the mitochondrial ATPase

Parathyroid hormone (PTH) has been shown to bind specifically to the beta subunit of the mitochondrial ATPase on nitrocellulose blots. We have now examined this interaction further, using intact mitochondria, submitochondrial particles, and the purified F1 ATPase. With intact mitochondria, 1 microM concentrations of PTH and its biologically active 1-34 fragment activate the ATPase about 3-fold. This effect was reduced to a 1.4-fold activation with 3-34 and 7-34 fragments of the hormone, and oxidized PTH gave no detectable activity. Activation could only be observed below pH 7. PTH had no significant effect on the activity of the purified enzyme or on submitochondrial particles. However, specific binding of an iodinated PTH analog, [Nle 8,18-Tyr 34] bPTH (1-34) amide, was found with submitochondrial particles and the purified ATPase. Binding affinity with the purified enzyme was about 10(-3) that of the plasma membrane receptor, and the molar stoichiometry was close to 1:1 (PTH:intact enzyme). With submitochondrial particles the affinity was about 10-fold higher than with the purified enzyme. This binding was further examined with PTH derivatives and fragments, and compared to that seen in the plasma membrane receptor. Oxidation of methionine 18 in PTH reduced the affinity about 50%, oxidation of methionine 8 reduced the affinity 95%, and oxidation of both methionines further decreased affinity in both membranes and submitochondrial particles. However, when compared to the native hormone, the 3-34 and 7-34 PTH fragments had much higher affinity for the submitochondrial particles than for the plasma membranes. PTH also reduced chemical crosslinking of the ATP analog, p-fluorosulfonyl benzoyl 5'-adenosine, to the alpha subunit of this enzyme, but did not alter labeling of the enzyme with 3'-O-(4'-benzoyl) benzoyl ATP, suggesting that the hormone binds near a regulatory nucleotide binding site. Direct chemical crosslinking of PTH to the beta-subunit of the enzyme was attained with a cleavable, photoactivate crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3-dithiopropionate. The crosslinked protein was cleaved with cyanogen bromide and the labeled fragments were sequenced. The labeled fragments were found to be segments of the protein which have previously been implicated as being close to the noncatalytic ATP binding sites.     

The role of the methionine residues in the structure and function of parathyroid hormone

Forms of the biologically active N-terminal fragment of bovine parathyroid hormone oxidized at methionine 8, methionine 18, and both positions were prepared, separated from one another, and characterized as described earlier for the native hormone (A. L. Frelinger and J. E. Zull, (1984) J. Biol. Chem. 259, 5507). The biological properties of the oxidized forms were compared to those of the native hormone, using the renal membrane adenylyl cyclase assay. Oxidation at position 18 produced full agonists of the hormone with slightly reduced potency. Oxidation at position 8 produced partial agonists of greatly reduced potency. Oxidation at both positions produced partial agonists of even lower potency. Thus, methionine 8 is implicated both in binding and in activation of adenylyl cyclase, but methionine 18 is implicated only in binding. Further study showed that oxidation of both residues is dependent on the pH, ionic strength, and polarity of the solvent. However, methionine 8 is less easily oxidized than methionine 18. This difference is eliminated in 3 M guanidine-HCl with 1-34 and in 6 M guanidine-HCl with 1-84. On the other hand the difference in reactivity is greatly increased in high ionic strength, with methionine 8 becoming much less reactive. These results suggest that the methionine residues are important in the biologically active conformation of parathyroid hormone and that methionine 8 is less accessible than methionine 18 under certain conditions. These conclusions are discussed in the context of a specific model for the folding of parathyroid hormone.     

Synthesis and structural studies of new analogues of PTH(1–11) containing Cα-tetra-substituted amino acids in position 8

The N-terminal 1–34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses characteristic of the native intact PTH. Recently, analogues of PTH(1–11) fragments with helicity-enhancing substitutions have been demonstrated to yield potent analogues of PTH(1–34). The work describes the synthesis, biological activity and structure of analogues of the best modified PTH sequence H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH₂ (I). In particular, the effect of the Ala/Aib substitution at positions 1 and 3 as well as of the replacement of Nle in position 8 with d-Nle, l-(αMe)-Nle and d-(αMe)-Nle was studied. The resulting peptides were characterized structurally by CD spectroscopy, solution NMR and MD, and in vitro for activity with respect to the cognate receptor, parathyroid hormone receptor.     

Proton NMR studies of the biologically active 1-34 fragment of bovine parathyroid hormone: examination of a structural model

Proton NMR spectra of the biologically active 1-34 fragment of bovine parathyroid hormone (bPTH) were studied as a function of pH over the range of pH 4 to 10, in buffer and in 6 M guanidine DC1. One of the histidine C-2 peaks titrated normally, with a pKa value of 6.8, but the other two histidines in this peptide had pKa values of 6.3. Denatured PTH showed only one histidine C-2 peak with a pKa of 6.7. An aliphatic peak identified as due to either a methionine or a glutamine residue also shifted with pH, and the pKa for this shift was 6.3. Finally, small but significant upfield shifts in the methyl and methylene resonances were observed as a function of pH, and when compared to the denatured peptide. These results indicate that the N-terminal domain of native PTH has considerable structure in solution, and are consistent with a theoretical model for the folding of this peptide.     

Parathyroid hormone (PTH) fragments relax the guinea-pig trachea in vitro

Synthetic bovine parathyroid hormone fragment containing the N-terminal 1-34 amino acids (bPTH-(1-34) ) relaxed the guinea-pig trachea constricted with histamine in vitro. Peptides with bovine and human sequences purchased from Peninsula Laboratories and Beckman Bioproducts produced similar effects. Substitution of methionine in positions 8 and 18 by norleucine did not affect this property of bPTH-(1-34). However, when the methionines were oxidized by treating the peptide with hydrogen peroxide, the peptide could no longer produce relaxation in the trachea. Oxidation of the methionine-replaced analog did not affect the action of the peptide on the trachea. It seems that the methionines per se are not necessary, but once oxidized the conformation of the molecule may be sufficiently altered to affect its ability to relax the trachea. While propranolol can block the relaxing action of isoproterenol, this blocking agent produces no inhibition of the bPTH-(1-34) effect. This action of PTH on the trachea may be related to cAMP because isobutyryl-methylxanthine, a phosphodiesterase inhibitor, potentiates and imidazole, a phosphodiesterase stimulator, inhibits the trachea relaxing action of bPTH-(1-34).     

Identification and purification of a kidney membrane protein which specifically binds the amino-terminal domain of native parathyroid hormone

Nitrocellulose blots of bovine kidney membrane proteins were prepared from denaturing polyacrylamide gels. Strips of the blots were incubated with parathyroid hormone (PTH), washed, and then incubated with antisera against the hormone. Exposure to horseradish peroxidase-linked second antibody led to staining of a 51-kDa protein. No staining was observed in blots not incubated with PTH. Fragments 35-84 and 19-84 of PTH reacted strongly with the antisera, but did not lead to staining of the 51-kDa protein on the blots. Staining was visible, but greatly reduced, when fragment 9-84 was used. Oxidation of the native hormone at positions 8 and 18 led to reductions in staining of the band which were quantitatively similar to the reductions in biological activity induced by such oxidations. These properties suggested that the 51-kDa protein recognizes the amino-terminal portions of PTH, which is the segment of the molecule required for its biological activities. Several micrograms of the 51-kDa protein were purified to homogeneity by selective extraction from the membranes with detergent and by elution from multiple two-dimensional gels. The purified protein retained its PTH-dependent staining and specificity. This protein may be a PTH receptor or a fragment of a PTH receptor from kidney.     

hPTH-fragments (53-84) and (28-48) antagonize the stimulation of calcium release and repression of alkaline phosphatase activity by hPTH-(1-34) in vitro

Different C-terminal fragments of parathyroid hormone (PTH)-(1-84) in blood participate in the regulation of calcium homeostasis by PTH-(1-84), and an antagonizing effect for the large carboxyl-terminal parathyroid hormone (C-PTH)-fragment (7-84) on calcium release has been described in vivo and in vitro. In this study the smaller C-PTH-fragment (53-84) and mid-regional PTH fragment (28-48), which represent discrete areas of activity in the PTH-(7-84) molecule, were assayed for their effects on calcium release and alkaline phosphatase (ALP) activity in a chick bone organ culture system. Neither PTH-(28-48) nor PTH-(53-84) had any effect on calcium release into the medium and both fragments stimulated ALP activity in the bone tissue, suggesting that the cAMP/PKA signalling pathway was not affected by these fragments. However they suppressed the calcium release induced by PTH-(1-34) and attenuated the down regulation of ALP activity caused by PTH-(1-34), suggesting that the effect on the cAMP/PKA signalling pathway may be indirectly. In conclusion, the study shows that the PTH-fragments (53-84) and (28-48) antagonize the PTH-(1-34) induced effects on calcium release and inhibition of ALP activity in a chick bone organ culture system.     

Chemical and biological properties of synthetic, sulfur-free analogues of parathyroid hormone.

Several analogues of the biologically active fragment of bovine parathyroid hormone (bPTH), based on the sequence of the NH2-terminal 34 amino acids, were prepared by solid phase synthesis and bioassayed in the in vitro adenylyl cyclase assay to provide further information concerning structure-activity relations in parathyroid hormone. In two analogues both methionines of the natural hormone were replaced with the sulfur-free and closely isosteric amino acid norleucine (Nle). The synthetic analogue [Nle-8, Nle-18]bPTH-(1-34) was highly active in the in vitro rat adenylyl cyclase bioassay, thus demonstrating that neither of the methionines, found in the native sequence, is indispensable for biological activity. Tyrosine was substituted for phenylalanine at position 34 in the synthesis of two other hormone analogues, [Try-34]bPTH-(1-34) and [Nle-8,Nle-18,Tyr-34]bPTH-(1-34). Both derivatives were exposed to conventional iodination procedures involving use of the oxidant chloramine T. Although iodination of [Try-34]bPTH-(1-34) resulted in virtually complete loss of biological activity, [Nle-8,Nle-18,Tyr-34]-bPTH-(1-34), which lacks methionine, could be exposed to oxidants and labeled efficiently with iodine with retention of nearly complete biological activity. These findings confirm that the loss of biological activity after oxidation of bPTH, as previously observed with the native hormone, is indeed attributable to the oxidation lability of methionine rather than to any other modifications. This sulfur-free, radioiodinated, biologically active analogue of parathyroid hormone may prove useful in studies of interaction of the hormone with the membrane receptors of target tissues and in studies of the metabolism of parathyroid hormone.     

Measuring Parathyroid Hormone (PTH) in Patients with Oxidative Stress – Do We Need a Fourth Generation Parathyroid Hormone Assay?

Oxidation of PTH at methionine residues results in loss of biological activity. PTH may be oxidized in patients with renal disease. The aim of this study was to develop an assay considering oxidation of PTH. Oxidized hPTH was analyzed by high resolution nano-liquid chromatography coupled to ESI-FTT tandem mass spectrometry (nanoLC-ESI-FT-MS/MS) directly and after proteolytic cleavage. The oxidized hPTH(1-84) sample shows TIC-peaks at 18-20 min and several mass peaks due to mass shifts caused by oxidations. No significant signal for oxidized hPTH(1-84) species after removal of oxidized PTH molecules by a specific column with monoclonal antibodies (MAB) raised against the oxidized hPTH was detectable. By using this column in samples from 18 patients on dialysis we could demonstrate that measured PTH concentrations were substantially lower when considering oxidized forms of PTH. The relationship between PTH concentrations determined directly and those concentrations measured after removal of the oxidized PTH forms varies substantially. In some patients only 7% of traditionally measured PTH was free of oxidation, whereas in other patients 34% of the traditionally measured PTH was real intact PTH. In conclusion, a huge but not constant proportion of PTH molecules are oxidized in patients requiring dialysis. Since oxidized PTH is biologically inactive, the currently used methods to detect PTH in daily clinical practice may not adequately reflect PTH-related bone and cardiovascular abnormalities in patients on dialysis.     

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.     

Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures.

We have reported previously that parathyroid hormone (PTH) acts on cultured bone cells to stimulate creatine kinase (CK) activity and [3H]thymidine incorporation into DNA via phosphoinositide turnover, in addition to its other actions via increased cyclic AMP production. We also found that mid-region fragments of PTH stimulate [3H]thymidine incorporation into avian chondrocytes. In the present study of mammalian systems, we demonstrate differential effects of defined synthetic PTH fragments on CK activity and DNA synthesis, as compared with cyclic AMP production, in osteoblast-enriched embryonic rat calvaria cell cultures, in an osteoblast-like clone of rat osteosarcoma cells (ROS 17/2.8) and in chondroblasts from rat epiphysial cartilage cell cultures. Unlike full-length bovine (b)PTH-(1-84) or the fully effective shorter fragment human (h)PTH-(1-34), fragments lacking the N-terminal region of the hormone did not increase cyclic AMP formation, whereas they did stimulate increases in both DNA synthesis and CK activity. Moreover, the PTH fragment hPTH-(28-48) at 10 microM inhibited the increase in cyclic AMP caused by 10 nM-bPTH-(1-84). The increase of CK activity in ROS 17/2.8 cells caused by bPTH-(1-84) or hPTH-(28-48) was completely inhibited by either cycloheximide or actinomycin D, as was shown previously for rat calvaria cell cultures. These results indicated the presence of a functional domain of PTH in the central part of the molecule which exerts its mitogenic-related effects on osteoblast- and chondroblast-like cells in a cyclic AMP-independent manner. Since cyclic AMP formation by PTH leads to bone resorption, specific mid-region fragments of PTH might prove suitable for use in vivo to induce bone formation without concomitant resorption.     

Effects of bone in vitro of bovine parathyroid hormone and synthetic fragments representing residues 1-34, 2-34 and 3-34.

The biological activities of bovine parathyroid hormone (BPTH) and fragments comprising portions of its amino-terminal sequence have been compared in three different assay systems using embryonic rat bone in vitro. Whereas the 3-34 fragment was without significant activity the 1-34 fragment caused all the actions characteristic of BPTH 1-84, extending to bone previous evidence that the amino-terminal residues are sufficient for expression of the biological effects of intact parathyroid hormone. However, the relative potencies of the fragment and the intact hormone were different in the various systems. BPTH 1-34 showed relatively low osteolytic activity and induced anabolic effects in both osteoblasts and cartilage cells of cultivated embryonic mouse radii which were not evoked by the intact hormone. Further work is required to determine the mechanisms responsible for these interesting alterations in relative potency of fragment and native hormone.     

Characterization of parathyroid hormone fragments produced by cathepsin D

Cleavage of parathyroid hormone by cathepsin D was studied. Four primary products were detected and separated by high performance liquid chromatography. Two of the fragments are fluorescent and therefore contain residue 23 (tryptophan). These fragments are NH2-terminal in origin. The other two cross-react with antisera directed against COOH-terminal portions of the hormone; they are the complementary COOH-terminal fragments. Microsequencing and amino acid analysis showed that the two COOH-terminal fragments are 35-84 and 38-84 bovine parathyroid hormone. By CNBr cleavage and amino acid analysis, the two NH2-terminal fragments were shown to be the complementary 1-37 and 1-34 fragments. The 1-37 fragment is transitory and is rapidly hydrolyzed to 1-34, so that only relatively small amounts are detected at any one time. However, 34-84 was not converted to 38-84, although cleavage at other sites in the COOH-terminal fragments was observed with more exhaustive digestion. The 1-34 fragment appears to be the final product of the action of cathepsin D on parathyroid hormone. Both enzymatically produced NH2-terminal fragments were fully active in the renal membrane adenylyl cyclase assay system.     

Production of biologically active fragments of parathyroid hormone by isolated Kupffer cells

Cleavage of parathyroid hormone (PTH) by isolated Kupffer cells from rat liver was examined. Iodinated PTH labeled at position 43 was converted into two radioactive fragments which were shown by Edman degradation to have residues 35 and 38 as their NH2 termini. Cleavage at these positions is characteristic of cathepsin D. Amino-terminal fragments were detected by bioassay of fractions obtained by high performance liquid chromatography. These fragments eluted in positions characteristic of the 1-34 and 1-37 peptides also previously shown to be produced by purified cathepsin D. The putative 1-37 fragment was rapidly converted to 1-34 upon digestion with cathepsin D, whereas the putative 1-34 fragment was not further digested by this enzyme, behavior previously shown to be characteristic of 1-37 and 1-34 bovine PTH. Fragmentation of PTH as measured by generation of fragments soluble in trichloroacetic acid was inhibited by methylamine, monensin, and ammonium chloride. In addition, monensin significantly inhibited production of both carboxyl- and amino-terminal fragments. Finally, active PTH fragments were also produced by elicited peritoneal macrophages. It is concluded that Kupffer cells, and other macrophages, can produce active fragments of PTH which appear in the medium. These fragments may be generated by cathepsin D within the cells.     

The central part of parathyroid hormone stimulates thymidine incorporation of chondrocytes

The stimulation of DNA synthesis in primary cell cultures of chicken chondrocytes by parathyroid hormone was studied by assaying [3H]thymidine incorporation into DNA. Optimal assay conditions were determined by varying cell age, plating density, and incubation time. Under these conditions DNA synthesis was significantly stimulated by parathyroid hormone (PTH) and some of its fragments: cells treated with human (h)PTH(1-84), bovine (b)PTH(1-34) and [Nle8,18,Tyr34]bPTH(3-34)amide and hPTH(13-34) displayed 2.6-fold enhanced [3H]thymidine incorporation in a dose-dependent manner. The fragment hPTH(28-48) led to a similar stimulation, whereas [Tyr43]hPTH(43-68) and [Tyr52,Asp76]hPTH(52-84) had no effect. Using a series of synthetic hPTH peptides covering the central region of the hormone molecule (residues 25-47), we could delimitate further this putative mitogenic functional domain to a core region between amino acid residues 30 and 34. The effect of PTH on [3H]thymidine incorporation could not be mimicked by forskolin, indicating that the corresponding signal is not mediated by cAMP. It is, however, inhibited by EGTA and cannot be provoked in the absence of calcium ions in the medium. Therefore, the results presented indicate a hitherto unidentified functional domain of PTH in the central part of the molecule which exerts its mitogenic effect on chondrocytes in a cAMP-independent manner but seems to involve calcium ions for signal transduction.     

Methionine oxidation in human growth hormone and human chorionic somatomammotropin. Effects on receptor binding and biological activities

The oxidation of the methionine residues of human growth hormone (hGH) and human chorionic somatomammotropin (hCS) to methionine sulfoxide by hydrogen peroxide has been studied. The kinetics of oxidation of individual methionine residues has been measured by reverse-phase high pressure liquid chromatography tryptic peptide mapping. Met-170 is completely resistant to oxidation in both hormones. The other 3 methionine residues in hCS (Met-64, Met-96, and Met-179) have markedly different reaction rates. Oxidation of the methionine residues does not appear to cause gross conformational changes in either hGH or hCS, as judged by CD and 1H NMR spectroscopy. Oxidation of Met-14 and Met-125 in hGH has little effect on affinity of the hormone for lactogenic receptors or on its potency in the Nb2 rat lymphoma in vitro bioassay for lactogenic hormones. The oxidation of Met-64 and/or Met-179 in hCS reduces profoundly both its affinity for lactogenic receptors and its in vitro biological potency. It is inferred by induction that residues 64 and/or 179 are critical for the binding of both hGH and hCS to lactogenic receptors and the expression of lactogenic biological activity.     

A two-receptor model for the action of parathyroid hormone on osteoblasts: a role for intracellular free calcium and cAMP

It has been suggested that intracellular Ca2+, in addition to cAMP, plays an important role in PTH-stimulated bone resorption. There is now strong evidence indicating that the osteoblast is the main target cell for PTH action, regulating indirectly, via cell-cell communication, osteoclastic bone resorption. In order to investigate the possible role of free cytosolic calcium in stimulated bone resorption, we studied the effects of the intact hormone (bPTH 1-84) and some of its fragments (bPTH (1-34), bPTH(3-34,) (Nle-8, Nle-18,Tyr-34) bPTH (3-34) amide) on their capacity to modify the cytosolic Ca2+ concentration in rat osteoblast-like cells. The experiments were performed using Quin-2, a fluorescent indicator of free calcium. We found an excellent correlation between the ability of PTH and PTH fragments to transiently increase cytosolic Ca2+ concentration in rat osteoblast-like cells and their ability to stimulate bone resorption in embryonic rat calvaria in vitro. On the other hand, no direct correlation was found for the cAMP and bone-resorbing responses. On the ground of these data we propose a two-receptor model for PTH action in osteoblasts, in which one receptor is coupled to the production of cAMP, whereas the other is involved in the increase of cytosolic Ca2+. Activation of both receptors by PTH (1-84) or PTH (1-34) leads to the full physiological response in osteoblasts, most probably the release of one or more factors which stimulate the activity of existing osteoclasts and others which stimulate the recruitment of additional osteoclasts.     

New aspects of radioimmunochemical measurement of human parathyroid hormone using the labelled antibody technique.

Two forms of heterogeneity of parathyroid hormone (PTH) have given rise to conflicting results: one due to the heterogeneity of the secreted species from the gland and their peripheral metabolism and the other representing the immunochemical heterogeneity of the available antibodies. We have developed sequence specific assays using the technique of labelled antibodies. Therefore, results of assays measuring the C-terminal part and the (1-34)-N-terminal part of the molecule could be compared to those of an assay for hormone bearing both N- and C-terminal antigenic determinants. This assay is supposed to detect predominantly (1-84)-intact hormone. The immunoradiometric assay of (1-34)-PTH has a sensitivity of 0.04 ng/ml. This technique avoids the critical iodination of the hormone fragment containing no tyrosine. There is the expected overlap between normal subjects and patients with primary and secondary hyperparathyroidism. The most important finding are results from patients undergoing neck catheterization. We demonstrated nonuniform secretion of different species of PTH by parathyroid adenomata and normal glands. This supports the hypothesis of cleavage of the (1-84)-molecule in the gland.     

Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50)

Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes beta-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1-34), and PTH-(1-84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7-34) and naturally circulating PTH-(7-84) induce internalization of PTH1R in a beta-arrestin-independent but dynamin-dependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7-34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7-34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.     

Characterization of the rabbit renal receptor for native parathyroid hormone employing a radioligand purified by reversed-phase liquid chromatography

Iodinated native bovine parathyroid hormone (bPTH(1-84)) was separated from uniodinated hormone by reversed-phase liquid chromatography techniques after lactoperoxidase labeling. Analysis of iodinated residues after enzymatic digestion indicated that the major labeled product was largely monoiodinated on the sole tyrosine residue. This material retained full bioactivity in an in vitro renal adenylate cyclase assay. Binding of 125I-bPTH(1-84) to rabbit renal membranes at 4 degrees C was proportional to membrane protein concentration and was saturable and dissociable. Radioligand binding was inhibited by concentrations of unlabeled bPTH(1-84) required to stimulate adenylate cyclase in the same membrane preparation but was not inhibited by non-PTH peptides other than adrenocorticotropin at high concentrations (greater than 10 microM). Synthetic NH2-terminal analogues of bPTH(1-84) all elicited approximately equivalent inhibition of radioligand binding which was, however, less potent than unlabeled bPTH(1-84), suggesting a role for the carboxyl region of the molecule in the interaction of bPTH(1-84) with its receptor. Activity of the NH2-terminal agonists was similar to bPTH(1-84) in stimulating adenylate cyclase. Although substitution in sequence position one, of serine in human PTH(1-34) for alanine in bPTH(1-34), reduced activity in the adenylate cyclase assay, inhibition of 125I-bPTH(1-84) binding by both peptides and by an analogue of bPTH(3-34) was equivalent, consistent with a minimal contribution of the first 2 residues for receptor binding of the NH2-terminal region of PTH. The results illustrate the utility of the radiolabeled preparation of native bPTH we have developed and emphasize the importance of probing the PTH receptor with an intact hormone to maximize information concerning the mechanism of PTH action.