Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PTHrP), which causes hypercalcemia in some cancer patients, stimulate multiple signals (cAMP, inositol phosphates, and calcium) probably by activating common receptors in bone and kidney. Using expression cloning, we have isolated a cDNA clone encoding rat bone PTH/PTHrP receptor from rat osteosarcoma (ROS 17/2.8) cells. The rat bone PTH/PTHrP receptor is 78% identical to the opossum kidney receptor; this identity indicates striking conservation of this receptor across distant mammalian species. Additionally, the rat bone PTH/PTHrP receptor has significant homology to the secretin and calcitonin receptors but not to any other G protein-linked receptor. When expressed in COS cells, a single cDNA clone, expressing either rat bone or opossum kidney PTH/PTHrP receptor, mediates PTH and PTHrP stimulation of both adenylate cyclase and phospholipase C. These properties could explain the diversity of PTH action without the need to postulate other receptor subtypes.
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PMID:Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. 131 66

The beta-adrenergic blocking agent propranolol was shown in previous studies to increase orthotopic bone formation in rats. To understand the cellular mechanisms underlying this observation, propranolol was tested for its effects on osteoblastic cells, which possess adenylate cyclase-coupled beta-adrenergic receptors. The ability of propranolol to modulate parathyroid hormone (PTH) and isoproterenol effects on adenylate cyclase activity and on alkaline phosphatase expression was studied in the osteoblast-like rat osteosarcoma cell line ROS 17/2.8. At concentrations between 0.1 and 10 microM, DL-propranolol specifically inhibited adenylate cyclase stimulation by the beta-adrenergic agonist isoproterenol, but did not alter either basal or PTH-stimulated activity. At these concentrations, propranolol also blunted the inhibition of alkaline phosphatase activity by isoproterenol but not PTH. Propranolol alone had minimal effects on ROS alkaline phosphatase activity at low concentrations (0.1-1 microM), but became inhibitory at high concentrations (10-100 microM). Thus, the direct effects of physiologically relevant propranolol concentrations on osteoblastic cells can be attributed principally to beta-adrenergic blockade. These findings further suggest that propranolol may enhance bone formation by preserving osteoblastic activity in the face of inhibition by beta-adrenergic agonists.
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PMID:Effects of beta-adrenergic blockade in an osteoblast-like cell line. 134 41

PTH receptors on osteoblasts and calcitonin receptors on osteoclasts are coupled to adenylate cyclase. Despite similar transduction mechanisms, these hormones have opposing physiological actions. We investigated the consequences of persistent protein phosphorylation on bone resorption in neonatal mouse calvariae using okadaic acid (OA) and calyculin-A, two inhibitors of protein phosphatase-1 and -2A. These two inhibitors caused different responses in bone at picomolar and low nanomolar concentrations. OA inhibited, in a dose-dependent manner, bone resorption stimulated by PTH, 1,25-Dihydroxyvitamin D3, phorbol ester, and prostaglandin E2 (PGE2). OA did not inhibit the generation of the second messengers cAMP or PGs and did not have nonspecific toxic effects, as measured by protein and RNA synthesis. Thus, OA appeared to mimic the global inhibitory action of calcitonin on bone resorption. Unlike OA, calyculin-A elicited a biphasic dose response. At concentrations of 3.3 nM and greater, calyculin-A inhibited, in a dose-dependent manner, stimulated bone resorption. However, calyculin-A alone, at 0.625 and 2.5 nM, stimulated bone resorption via a PG-independent pathway. In calvariae, OA and calyculin-A increased phosphorylation of a 58- to 60-kilodalton protein. A protein of similar molecular mass was hyperphosphorylated in OA-treated ROS 17/2.8 osteoblast-like cells. We conclude that in addition to hormonal regulation of protein kinase activity, protein dephosphorylation plays a functionally important role in the modulation of bone resorption.
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PMID:Protein phosphatase inhibitors and bone resorption: inhibition by okadaic acid and biphasic actions of calyculin-A. 137 1

We have previously shown that 1,25-dihydroxyvitamin D [1,25-(OH)2D3] and glucocorticoid modulate adenylate cyclase activation by PTH in osteoblast-like cells. Here we examine whether steroid effects on PTH receptor density explain the modulation of PTH action. Receptor assays were performed on late logarithmicphase monolayers of ROS 17/2.8 cells using human PTH-like peptide (hPLP) as radioligand. Kd and receptor density were computed from competition of tracer amounts of [125I-Tyr36] hPLP-(1-36) with unlabeled hPLP-(1-36) (0.1-30 nM). Steroid treatment had little or no effect on affinity for ligand. Pretreating cells with 10 nM 1,25-(OH)2D3 for 48 h decreased PTH receptor number to 17% of control values. Treating cells with 10 nM of the glucocorticoid triamcinolone acetonide (TRM) increased receptor number 10-fold, but simultaneous treatment with 1,25-(OH)2D3 (10 nM) completely prevented this receptor increase. Steroid effects required 13-18 h of treatment. Dose-response relationships for steroid modulation, determined from binding at 0.17 nM radioligand, indicated an EC50 of 0.3 nM for glucocorticoid augmentation of PTH receptor number and 0.02 nM for 1,25-(OH)2D3 reduction of receptor number in the presence of absence of the maximum TRM effect. The initial rate of cAMP production by receptor-saturating concentrations of PTH was 11,500 molecules per receptor per minute in untreated cells, comparable to reported turnover numbers for mammalian adenylate cyclase. Control experiments were validated measuring cAMP in intact cells as an indicator of adenylate cyclase activity. Cyclic AMP production was reduced 63% by 1,25-(OH)2D3 (10 nM) treatment. Glucocorticoid (10 nM) enhanced cAMP production twofold but reduced cAMP generation per receptor by 80%.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:1,25-dihydroxyvitamin D reduces parathyroid hormone receptor number in ROS 17/2.8 cells and prevents the glucocorticoid-induced increase in these receptors: relationship to adenylate cyclase activation. 165 16

The PTH activates both adenylate cyclase and a mechanism that increases membrane-associated protein kinase-C (PKC) activity. To define the hormone's PKC activation domain we have used a panel of PTH fragments and ROS 17/2 rat osteosarcoma cells as the target cells. PTH equally and maximally increased PKC activity in ROS 17/2 cell membranes at physiological concentrations between 1-50 pM and 5-50 nM, but not at intermediate concentrations or concentrations above 50 nM. The PKC-stimulating picomolar concentrations of PTH did not stimulate adenylate cyclase in ROS 17/2 cells, while the PKC-stimulating nanomolar concentrations of the hormone did stimulate adenylate cyclase, with an EC50 of 1-2 nM. Very high concentrations of PTH, such as 100 nM, that did not increase membrane PKC activity were still able to maximally stimulate adenylate cyclase. PTH fragments lacking the N-terminal amino acids needed for adenylate cyclase activation increased membrane PKC activity, and the PKC activation domain was found to lie within the 28-34 region of the PTH molecule. This was confirmed by showing that optimally effective picomolar concentrations of the human PTH-(28-34) fragment itself were able to increase membrane-associated PKC activity to the same extent as the optimally effective picomolar concentrations of the intact PTH-(1-84) or the larger PTH-(1-34) or PTH-(3-34) fragments.
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PMID:The protein kinase-C activation domain of the parathyroid hormone. 172 20

The effects of transforming growth factor-alpha (TGF alpha) were determined on the ability of parathyroid hormone (PTH) or parathyroid hormone-related protein (PTHrP) to stimulate bone resorption and adenylate cyclase in vitro. Bovine PTH-(1-34) and human PTHrP-(1-34) were equipotent in their ability to stimulate bone resorption in neonatal mouse calvaria with maximal stimulation (2.9 and 2.8-fold increases in 45Ca release, respectively) at a concentration of 10 nM. Combinations of TGF alpha with bPTH-(1-34) or hPTHrP-(1-34) had additive effects on their ability to stimulate bone resorption when submaximal concentrations of the agonists were used. There was no evidence of synergism between TGF alpha bPTH-(1-34) or hPTHrP-(1-34) in their ability to stimulate bone resorption in vitro, nor was TGF alpha able to increase bone resorption induced by maximal concentrations of bPTH-(1-34) or hPTHrP-(1-34). TGF alpha potentiated the effects of either bPTH-(1-34) or hPTHrP-(1-34) on the stimulation of adenylate cyclase in osteoblast-like ROS 17/2.8 cells. These data indicate that TGF alpha has additive effects with submaximal concentrations of PTH or PTHrP on their ability to stimulate bone resorption which may be important in the pathogenesis of humoral hypercalcemia of malignancy.
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PMID:Effects of transforming growth factor-alpha on parathyroid hormone- and parathyroid hormone-related protein-mediated bone resorption and adenylate cyclase stimulation in vitro. 178 99

We present a new human osteosarcoma cell line designated OHS-4. These cells showed a high alkaline phosphatase activity that is not regulated by 1,25 dihydroxyvitamin D3. They exhibited a sensitive adenylate cyclase response to parathyroid hormone but not to prostaglandin E2 or human calcitonin. By Northern blot analysis we could detect type I collagen mRNA but none for type III collagen. The cells were able to produce human osteocalcin at a maximum level of 35 ng per million cells when exposed to 2.4 nM 1,25-dihydroxyvitamin D3 for 96 h. We purified this protein from conditioned media using successive chromatography and assessed its identity by partial amino acid sequencing. When injected into nude mice, the cells retained their osteogenic activity and developed calcified tumors. After Von Kossa staining, we observed nonmineralized osteoid deposits and mineralized deposits with a structure similar to that of trabecular bone by light microscopy. On the basis of its osteoblastic characteristics, this new osteosarcoma cell line may represent the human counterpart of the ROS 17/2 cell line. This cell line represents a valuable model for the isolation and characterization of human bone specific proteins.
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PMID:Characterization of a new human osteosarcoma cell line OHS-4. 186 Aug 86

PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2 have previously been shown to be shown to be more potent antagonists than the corresponding PTH peptide, [Tyr34]bPTH(7-34)NH2. However, these peptides also display partial agonism for adenylate cyclase activity in ROS 17/2.8 cells. In this study, design of a pure potent antagonist of PTH and PTHrP by removal of agonism from PTHrP(7-34)NH2 with retention of antagonist potency was accomplished. Since [Tyr34]bPTH(7-34)NH2 lacks agonist activity, we introduced two amino acids native to the PTH sequence into their respective positions in PTHrP and the potent D-Trp12 analog. [Asn10Leu11]- and [Asn10,leu11,D-Trp12]-PTHrP(7-34)NH2 were found to be 23- and 26-fold more potent as antagonists in ROS cells than PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2, respectively. In addition, these peptides did not display partial agonism, even in an assay based on highly responsive cells pretreated with dexamethasone and pertussis toxin. In contrast, when the PTHrP sequence Asp10,Lys11 was inserted into [Tyr34]hPTH(7-34)NH2, antagonist potency declined by more than 6-fold and PTH-like agonist activity was installed. These results demonstrate that the activation domain of both PTH and PTHrP can be extended to include the 1-12 region and that the 10-12 region, in addition to the N-terminal hexapeptide, is important not only for receptor binding but also for hormonal signal transduction.
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PMID:Removal of partial agonism from parathyroid hormone (PTH)-related protein-(7-34)NH2 by substitution of PTH amino acids at positions 10 and 11. 216 25

In the design and biological evaluation of PTH antagonists, certain analogs, although antagonists in vitro, possess partial agonist properties in vivo that preclude their utility as antagonists. In an effort to identify weak agonism of PTH analogs, an attempt was made to enhance the responsiveness of the widely employed rat osteosarcoma (ROS 17/2.8) cell adenylate cyclase assay. Because responsiveness to PTH in these cells is enhanced upon treatment with dexamethasone (dex) or pertussis toxin (PT), we have evaluated their use to aid in detection of partial agonism for PTH and PTH-related protein (PTHrP) antagonist analogs. Treatment of cells with dex alone (30 nM for 3 days) or with PT alone (40 ng/ml for 1 day) increased basal adenylate cyclase activity by 27%. However, combination of the dex and PT treatments increased basal cAMP production 70%. The in vivo partial agonist [Nle8,18,Tyr34]bPTH(3-34)NH2 increased cAMP production 3-fold over basal levels in untreated cells, nearly 5-fold in PT-treated cells, 8-fold in cells treated with dex, and 10-fold in cells treated with dex plus PT. Similar results were obtained with PTHrP(7-34)NH2: the 6-fold stimulation observed in control cells was converted to 14-fold in cells treated with dex plus PT. Agonist activity undetectable in the conventional assay was observed in the dex plus PT system: [Tyr34]- and [D-Trp12,Tyr34]bPTH(7-34)NH2, which exhibit no agonist activity under control conditions, stimulated cAMP production 2.6- and 2.1-fold, respectively, under dex plus PT treatment. In contrast, the antagonist analogs [Asn10,Leu11]- and [Leu11,D-Trp12]PTHrP(7-34)NH2, hybrid peptides of PTH and PTHrP, had no agonist activity under any conditions. Because of increased responsiveness, this assay should occupy an important step in the pathway for evaluation of PTH antagonists and permit identification of weak partial agonist activity before extensive in vivo testing.
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PMID:Treatment of bone-derived ROS 17/2.8 cells with dexamethasone and pertussis toxin enables detection of partial agonist activity for parathyroid hormone antagonists. 216 26

Previous studies examining the interaction of PTH and PTH-related protein (PTHrP) with target tissue have for the most part emphasized the similarity between the two hormones in binding to and activating receptors. This observation that two peptides with limited homology have equal affinities for the same receptor is unusual. In this report we investigated two aspects of PTH/PTHrP-receptor interactions. First, the nonhomologous 14-34 regions of PTH and PTHrP were synthesized and evaluated. Second, hybrid peptides containing the 7-18 fragment of one hormone combined with the 19-34 region of the other hormone were studied to determine whether interactions between these two regions are required for receptor recognition. All four peptides were examined in bovine renal cortical membrane and rat osteosarcoma (ROS 17/2.8) cell PTH-binding and PTH-stimulated adenylate cyclase assays. The results indicate that the receptor-binding domains of PTH and PTHrP lie outside of the 1-13 region, the region containing sequence homology shared by the two hormones, and that two peptides of different amino acid sequence bind with equal affinity to the bovine renal PTH receptor. However, in the absence of the N-terminal region, the rat bone PTH receptor displays a preference for the C-terminal (19-34 sequence) region of PTHrP.
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PMID:The bovine renal parathyroid hormone (PTH) receptor has equal affinity for two different amino acid sequences: the receptor binding domains of PTH and PTH-related protein are located within the 14-34 region. 216 27


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