Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Increased constitutive activity has been observed in the PTH receptor in association with naturally occurring mutations of two residues that are conserved between members of the glucagon/vasoactive intestinal peptide/calcitonin 7TM receptor family. Here, the corresponding residues of the glucagon receptor, His178 and Thr352, were probed by mutagenesis. An elevated level of basal cAMP production was observed after the exchange of His178 into Arg, but not for the exchange into Lys, Ala, or Glu. However, for all of these His178 substitutions, an increased binding affinity for glucagon was observed [dissociation constant (Kd) ranging from 1.1-6.4 nM, wild type: Kd = 12.0 nM]. A further increase in cAMP production was observed for the [H178R] construct upon stimulation with glucagon, albeit the EC50 surprisingly was increased approximately 10-fold relative to the wild-type receptor. Substitution of Thr352, located at the intracellular end of transmembrane segment VI, with Ala led to a slightly elevated basal cAMP level, while the introduction of Pro or Ser at this position affected rather the binding affinity of glucagon or the EC50 for stimulation of cAMP production. The large extracellular segment, which is essential for glucagon binding, was not required for constitutive activation of the glucagon receptor as the introduction of the [H178R] mutation into an N-terminally truncated construct exhibited an elevated basal level of cAMP production. The analog des-His1-[Glu9]glucagon amide, which in vivo is a glucagon antagonist, was an agonist on both the wild-type and the [H178R] receptor and did not display any activity as an inverse agonist. It is concluded that the various phenotypes displayed by the constitutively active glucagon receptor mutants reflect the existence of multiple agonist-preferring receptor conformers, which include functionally active as well as inactive states. This view agrees with a recent multi-state extension of the ternary complex model for 7TM receptor activation.
Mol Endocrinol 1998 Jan
PMID:Constitutive activity of glucagon receptor mutants. 944 Aug 12

The effect of parathyroid hormone-related protein (PTHrP) on proliferation was examined in Chinese hamster ovary (CHO) cells transfected with a PTH/PTHrP receptor encoding cDNA. Treatment with chicken PTHrP(1-36) (chPTHrP) lowered the cell number to 49 +/- 2% of untreated controls after 6 days with a half-maximal effect at 1 nM. The effect was mimicked by human (h) PTH(1-34), Br-cAMP and forskolin, but not by the receptor antagonist hPTH(3-34). Reduction of cell number was accompanied by increased PTH/PTHrP receptor expression and persistently activated adenylyl cyclase, together with altered cell morphology from epithelial to spindle-like forms, clustered growth and increased phosphate uptake. chPTHrP increased [Ca2+]i, but failed to activate membrane bound protein kinase C (PKC). Pretreatment with chPTHrP did not affect phorbol ester stimulated PKC activity, and chPTHrP or serum evoked increases in [Ca2+]i. In conclusion, PTHrP induced inhibition of proliferation and altered cell morphology is mediated by increased adenylyl cyclase rather than by PKC-dependent mechanisms in CHO cells.
Mol Cell Endocrinol 1997 Nov 30
PMID:Parathyroid hormone-related protein (PTHrP) inhibits proliferation of Chinese hamster ovary cells stably transfected with a PTH/PTHrP receptor cDNA. 945 37

PTH and PTH-related peptide (PTHrP) bind to the PTH/PTHrP receptor and stimulate cAMP accumulation with similar efficacy. Only PTH activates the PTH2 receptor. To examine the structural basis for this selectivity, we analyzed receptor chimeras in which the amino terminus and third extracellular domains of the two receptors were interchanged. All chimeric receptors bound radiolabeled PTH with high affinity. Transfer of the PTH2 receptor amino terminus to the PTH/PTHrP receptor eliminated high-affinity PTHrP binding and significantly decreased activation by PTHrP. A PTH/PTHrP receptor N terminus modified by deletion of the nonhomologous E2 domain transferred weak PTHrP interaction to the PTH2 receptor. Introduction of the PTH2 receptor third extracellular loop into the PTH/PTHrP receptor increased the EC50 for PTH and PTHrP, while preserving high-affinity PTH binding and eliminating high-affinity PTHrP binding. Similarly, transfer of the PTH/PTHrP receptor third extracellular loop preserved high-affinity PTH binding by the PTH2 receptor but decreased its activation. Return of Gln440 and Arg394, corresponding residues in the PTH/PTHrP and PTH2 receptor third extracellular loops, to the parent residue restored function of these receptors. Simultaneous interchange of wild-type amino termini and third extracellular loops eliminated agonist activation but not binding for both receptors. Function was restored by elimination of the E2 domain in the receptor with a PTH/PTHrP receptor N terminus and return of Gln440/Arg394 to the parent sequence in both receptors. These data suggest that the amino terminus and third extracellular loop of the PTH2 and PTH/PTHrP receptors interact similarly with PTH, and that both domains contribute to differential interaction with PTHrP.
Mol Endocrinol 1998 Feb
PMID:Multiple regions of ligand discrimination revealed by analysis of chimeric parathyroid hormone 2 (PTH2) and PTH/PTH-related peptide (PTHrP) receptors. 948 62

The mitogen-activated protein (MAP) kinases (p44mapk and p42mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44mapk/ERK1 and p42mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay using myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.
Mol Cell Biochem 1998 Jan
PMID:Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: attenuation of MAP kinase activation by cAMP, parathyroid hormone and forskolin. 954 82

We have initiated a mutational analysis of pathogenicity in the rice blast fungus, Magnaporthe grisea, in which hygromycin-resistant transformants, most generated by restriction enzyme-mediated integration (REMI), were screened for the ability to infect plants. A rapid primary infection assay facilitated screening of 5,538 transformants. Twenty-seven mutants were obtained that showed a reproducible pathogenicity defect, and 18 of these contained mutations that cosegregated with the hygromycin resistance marker. Analysis of eight mutants has resulted in the cloning of seven PTH genes that play a role in pathogenicity on barley, weeping lovegrass, and rice. Two independent mutants identified the same gene, PTH2, suggesting nonrandom insertion of the transforming DNA. These first 7 cloned PTH genes are described.
Mol Plant Microbe Interact 1998 May
PMID:Magnaporthe grisea pathogenicity genes obtained through insertional mutagenesis. 957 8

1,25-dihydroxyvitaminD3 [1,25-(OH)2D3] and PTH both act to increase serum calcium. In addition, 1,25-(OH)2D3 decreases PTH gene transcription, which is relevant both to the physiology of calcium homeostasis and to the management of the secondary hyperparathyroidism of patients with chronic renal failure. In chronic hypocalcemia there is secondary hyperparathyroidism with increased levels of PTH mRNA and serum PTH despite markedly increased levels of 1,25-(OH)2D3. We have studied the role of calreticulin in this resistance to 1,25-(OH)2D3. Weanling rats fed a low-calcium diet were hypocalcemic and had increased PTH mRNA levels despite high serum 1,25-(OH)2D3 levels. 1,25-(OH)2D3 given by continuous minipump infusion to normal rats led to the expected decrease in PTH mRNA. The hypocalcemic rats had an increased concentration of calreticulin in the nuclear fraction of their parathyroids, but not in other tissues. Gel shift assays showed that a purified vitamin D receptor and retinoid X receptor-beta bound to the PTH promoter's chicken and rat vitamin D response element (VDRE), and this binding was inhibited by added pure calreticulin. Transfection studies with a PTH VDRE-chloramphenicol acetyltransferase (CAT) construct showed that 1,25-(OH)2D3 decreased CAT transcription. Cotransfection of PTH VDRE-CAT with a calreticulin expression vector in the sense orientation prevented the transcriptional effect of 1,25-(OH)2D3, but a calreticulin vector in the antisense orientation had no effect. These results show that calreticulin prevents the binding of vitamin D receptor-retinoid X receptor-beta to the PTH VDRE in gel retardation assays and prevents the transcriptional effect of 1,25-(OH)2D3 on the PTH gene. This is the first report of calreticulin inhibiting a down-regulatory function of a sterol hormone and may help explain the refractoriness of the secondary hyperparathyroidism of many chronic renal failure patients to 1,25-(OH)2D3.
Mol Endocrinol 1998 Aug
PMID:Calreticulin inhibits vitamin D's action on the PTH gene in vitro and may prevent vitamin D's effect in vivo in hypocalcemic rats. 971 45

PTH maintains blood calcium concentrations within the physiological range by acting on a G protein-coupled heptahelical receptor (PTH1 Rc) located primarily in cells in bone and kidney. We have undertaken a photoaffinity cross-linking approach to elucidate the nature of the bimolecular interaction of PTH with the human (h) PTH1 Rc. Specifically, we have studied the region of the receptor that interacts with the midregion of PTH-(1-34), position 13, using a benzophenone-containing photoaffinity ligand, 125I-[Nle(8,18),Lys13(epsilon-pBz2),L-2-NaI23,Arg(26,2 7),Tyr34]bPTH-(1-34)NH2 (125I-K13). Using site-directed mutagenesis in combination with biochemical analysis, we have reduced our previously identified contact domain, 17 residues in the extracellular region of the receptor (173-189), to an 8-amino acid domain (182-189). Furthermore, we have found arginine 186 to be of critical importance to the interaction of the hPTH1 Rc with 125I-K13: modification of Arg186 to either lysine or alanine does not modify receptor avidity or signal transduction by the receptor, but eliminates cross-linking to 125I-K13.
Mol Endocrinol 1998 Nov
PMID:Arginine 186 in the extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for contact with position 13 of the hormone. 981 94

In some G protein-coupled receptors (GPCRs), agonist-dependent phosphorylation by specific GPCR kinases (GRKs) is an important mediator of receptor desensitization and endocytosis. Phosphorylation and the subsequent events that it triggers, such as arrestin binding, have been suggested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylation of the PTH receptor, a class II GPCR, also regulates receptor internalization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a number of receptor mutants in which individual serine residues had been replaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after agonist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response to PTH. The substitution of these serine residues by alanine affected neither the number of receptors expressed on the cell surface nor the ability of the receptor to signal via Gs. Overexpression of GRK2, but not GRK3, enhanced PTH-stimulated receptor phosphorylation, and this phosphorylation was abolished by alanine mutagenesis of residues 483, 485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor by the endogenous kinase in HEK-293 cells occurs on the same residues targeted by overexpressed GRK2. Strikingly, the rate and extent of PTH-stimulated internalization of mutated PTH receptors lacking phosphorylation sites were identical to that observed for the wild-type PTH receptor. Moreover, overexpressed GRK2, while enhancing the phosphorylation of the wild-type PTH receptor, had no affect on the rate or extent of receptor internalization in response to PTH. Thus, the agonist-occupied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.
Mol Endocrinol 1998 Dec
PMID:Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization. 984 59

1 ,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] negatively regulates expression of the avian PTH (aPTH) gene transcript, and a vitamin D response element (VDRE) near the promoter of the aPTH gene had previously been identified. The present report assessed whether the negative activity imparted by the aPTH VDRE could be converted to a positive transcriptional response through selective mutations introduced into the element. The tested sequences were derived from individual and combined mutations to 2 bp in the 3'-half of the direct repeat element, GGGTCAggaGGGTGT. Cold competition experiments using mutant and wild-type oligonucleotides in the mobility shift assay revealed minor differences in the ability of any of these sequences to compete for binding to a heterodimer complex comprised of recombinant proteins. Ethylation interference footprint analysis for each of the mutants produced unique patterns over the 3'-half-sites that were distinct from the weak, wild-type footprint. Transcriptional outcomes evaluated from a chloramphenicol acetyltransferase reporter construct utilizing the aPTH promoter found that the individual T-->A mutant produced an attenuated negative transcriptional response while the G-->C mutant resulted in a reproducibly weak positive transcriptional outcome. The double mutant, however, yielded a 4-fold increase in transcription, similar to the 7-fold increase observed from an analogous construct using the human osteocalcin VDRE. UV light crosslinking to gapped oligonucleotides assessed the polarity of heterodimer binding to the wild-type and double mutant sequences and was consistent with the vitamin D receptor preferentially binding to the 5'-half of both elements. Finally, DNA affinity chromatography was used to immobilize heterodimer complexes bound to the wild-type and double mutant sequences as bait to identify proteins that may preferentially interact with these DNA-bound heterodimers. This analysis revealed the presence of a p160 protein that specifically interacted with the heterodimer bound to the wild-type VDRE, but was absent from complexes bound to response elements associated with positive transcriptional activity. Thus, the sequence of the individual VDRE appears to play an active role in dictating transcriptional responses that may be mediated by altering the ability of a vitamin D receptor heterodimer to interact with accessory factor proteins.
Mol Endocrinol 1999 Mar
PMID:Turning a negative into a positive: vitamin D receptor interactions with the avian parathyroid hormone response element. 1007 2

Recent mutagenesis and cross-linking studies suggest that residues in the carboxyl-terminal portion of PTH(1-34) interact with the amino-terminal extracellular domain of the receptor and thereby contribute strongly to binding energy; and that residues in the amino-terminal portion of the ligand interact with the receptor region containing the transmembrane helices and extracellular loops and thereby induce second messenger signaling. We investigated the latter component of this hypothesis using the short amino-terminal fragment PTH(1-14) and a truncated rat PTH-1 receptor (r delta Nt) that lacks most of the amino-terminal extracellular domain. The binding of PTH(1-14) to LLC-PK1 or COS-7 cells transfected with the intact PTH-1 receptor was too weak to detect; however, PTH(1-14) dose-dependently stimulated cAMP formation in these cells over the dose range of 1-100 microM. PTH(1-14) also stimulated cAMP formation in COS-7 cells transiently transfected with r delta Nt, and its potency with this receptor was nearly equal to that seen with the intact receptor. In contrast, PTH(1-34) was approximately 100-fold weaker in potency with r delta Nt than it was with the intact receptor. Alanine scanning of PTH(1-14) revealed that for both the intact and truncated receptors, the 1-9 segment of PTH forms a critical receptor activation domain. Taken together, these results demonstrate that the amino-terminal portion of PTH(1-34) interacts with the juxtamembrane regions of the PTH-1 receptor and that these interactions are sufficient for initiating signal transduction.
Mol Endocrinol 1999 May
PMID:The (1-14) fragment of parathyroid hormone (PTH) activates intact and amino-terminally truncated PTH-1 receptors. 1031 18


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