共查询到20条相似文献,搜索用时 0 毫秒
1.
M Ohmori T Endo M Ikeda T Onaya 《Biochemical and biophysical research communications》1991,178(2):733-738
To identify the site(s) on the thyrotropin (TSH) receptor that interacts with TSH or thyroid stimulating antibody (TSAb), we examined the effect of the synthetic TSH receptor peptide (termed N2 peptide, No. 35-50) on the cAMP accumulation induced by TSH or TSAb. Preincubation of bovine TSH with N2 peptide resulted in a significant and dose-dependent decrease in cAMP accumulation. This decrease was not observed when bovine TSH was preincubated with P1 peptide, which was used as a control (No. 398-417). In contrast, the N2 peptide did not affect TSAb activity in immunoglobulin fractions from three TSAb-positive patients with Graves' disease. P1 peptide also had no effect on TSAb activity. These results suggest that the N-terminal region of the TSH receptor is important for TSH action, and also that TSAb activity cannot be suppressed only by the application of the synthetic peptide corresponding to the N-terminal region. 相似文献
2.
3.
G D Chazenbalk Y Nagayama H Wadsworth D Russo B Rapoport 《Molecular endocrinology (Baltimore, Md.)》1991,5(10):1523-1526
We observed previously that the carboxyl-terminal region of the third loop of the TSH receptor (amino acid residues 617-625) is important in signal transduction. To analyze this region in more detail, in the present study we used site-directed mutagenesis to substitute, on an individual basis, the seven amino acids previously mutated as a group. These amino acids are either charged residues or potential phosphorylation sites. Six of the mutant TSH receptors with individual amino acid substitutions bound TSH with high affinity and displayed a cAMP response to TSH stimulation similar to the wild-type TSH receptor. The mutant receptor TSH-R-Gly625 (Arg----Gly) did not transduce a signal, but these results are noninformative because of the loss of high affinity TSH binding. The present data indicate that for each of the six informative amino acid substitutions, the individual residues are not critical for signal transduction. A corollary of this conclusion is that in the important carboxyl-terminal region of the third cytoplasmic loop of the TSH receptor multiple amino acid residues function as a unit. 相似文献
4.
Hm1 muscarinic cholinergic receptor internalization requires a domain in the third cytoplasmic loop.
J Lameh M Philip Y K Sharma O Moro J Ramachandran W Sadée 《The Journal of biological chemistry》1992,267(19):13406-13412
Selected regions of the Hm1 muscarinic cholinergic receptor were mutated to analyze the molecular mechanisms of agonist-induced receptor internalization (or sequestration). The wild-type and mutant Hm1 genes were expressed, using pSG5, in U293 human kidney cells. Whereas surface receptor density measured with the polar tracer N-[3H]methylscopolamine was rapidly reduced by carbachol exposure, total receptor content measured with [3H]quinuclidinyl benzilate did not decline for at least 24 h, indicating the absence of extensive receptor down-regulation in U293 cells. Carbachol stimulation of phosphatidylinositol turnover paralleled receptor internalization, both with EC50 values of 10-20 microM. Furthermore, a D71N point mutation that prevented receptor activation also abolished carbachol-induced receptor internalization, indicating that receptor activation (but not necessarily second messenger stimulation) was required for internalization. Truncation of the COOH-terminal tail (K447 trunc) and point mutations of several potential Ser and Thr phosphorylation sites to Ala failed to affect receptor activation and internalization. In contrast, partial deletions of the third intracellular loop (i3) (Tyr208-Thr366) resulted in receptor mutants deficient in agonist-induced receptor internalization/sequestration. Various deletions caused either complete loss of internalization (d 232-358) or impaired internalization, ranging from 10 to 30% over 2 h, whereas wild-type Hm1 internalized to approximately 50%. Whereas the reason for the observed differences among the deficient deletion mutants remains unclear, the initial rate of N-[3H]methylscopolamine binding loss from the cell surface was much slower than that of wild-type Hm1 in each case. The deletion of only one single domain, 284-292 (SMESLTSSE), in the middle of i3 was consistently associated with impaired internalization. Domain 284-292 is partially conserved among closely related muscarinic receptors, whereas most of the remainder of i3 is not (except for the i3 membrane junctions), and similar Ser- and Thr-rich regions are present in many other G protein-coupled receptors. We propose that a small receptor domain in the middle of the i3 loop of Hm1 is involved in agonist-induced receptor internalization. 相似文献
5.
M Saji S Ikuyama T Akamizu L D Kohn 《Biochemical and biophysical research communications》1991,176(1):94-101
Thyrotropin (TSH) receptor mRNA levels in rat FRTL-5 thyroid cells are decreased by treatment with the calcium ionophores, A23187 or ionomycin, as well as with TSH, cholera toxin, forskolin, and 8-bromo-cAMP. Down regulation is, in each case, associated with a decrease in [125I]TSH binding and a decreased ability of TSH to increase cAMP levels. The ionophore does not alter cAMP levels and ethylene glycol-bis-(beta-aminoethyl ether) N, N'-tetraacetic acid (EGTA) in the medium prevents down regulation of TSH receptor mRNA levels by the ionophore, but not by TSH; the EGTA action is reversed by the simultaneous addition of Ca++. Whereas down regulation by TSH and its cAMP signal requires the presence of insulin and/or serum in the medium; down regulation by a calcium ionophore is still evident in their absence. Down regulation of TSH receptor mRNA levels and receptor desensitization by TSH/cAMP or an ionophore is lost in cells transfected with a full length TSH receptor cDNA devoid of regulatory elements, but able to reconstitute TSH receptor signal generation. 相似文献
6.
R Taub J C Hsu V M Garsky B L Hill B F Erlanger L D Kohn 《The Journal of biological chemistry》1992,267(9):5977-5984
Monoclonal antibodies, D2 and 4G11, selected by the autoantiidiotypic approach following injection of thyrotropin (TSH) into mice, mimic TSH in binding to receptors on thyroid membranes. Based on TSH receptor transfection studies, D2 and 4G11 show unequivocal specificity for the TSH receptor. To see if the complementary determining regions (CDRs) of these antibodies share any primary sequence similarities to regions of TSH critical for receptor binding, we deduced the primary structure of the variable regions of D2 and 4G11 by sequencing the immunoglobulin mRNA. We found that CDR1 of 4G11K and CDR2 of D2 mu show sequence similarity to regions of TSH alpha and TSH beta that had been previously implicated in the interaction of the hormone with its receptor. We tested the inhibitory effects of synthetic peptides from D2 mu-CDR2 and 4G11K-CDR1 on the binding of the corresponding antibodies to rat thyroid FRTL-5 cells and found an EC50 of 0.1 and 1 microM, respectively. TSH-derived peptides with similarity to D2 mu-CDR2 and 4G11K-CDR1 showed a significant but lesser effect on the binding of 4G11 or D2 to thyroid cells. Additionally, we tested the effects of the CDR peptides and TSH-derived peptides on TSH-stimulated cAMP production in FRTL-5 cells and found that D2 mu-CDR2 and 4G11K-CDR1 inhibited this activity, D2 mu-CDR2 most strongly (EC50 10 microM). Thus, linear sequences from the CDRs of these autoantiidiotypic antibodies with similarity to sequences from both subunits of TSH appear to interact with the TSH receptor. These data support previous studies indicating the complexity of the interaction between TSH and its receptor and advance earlier findings that such immunologic approaches are useful in dissecting receptor-ligand interactions. 相似文献
7.
J R Arden O Nagata M S Shockley M Philip J Lameh W Sadée 《Biochemical and biophysical research communications》1992,188(3):1111-1115
We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling. Point mutations were constructed to test the role of charged amino acids in these junctions. A triple point mutation of Hm1 (E214 A/ E216K/ E221 K), which mimics the charge distribution in Hm2 (negatively coupled to cAMP) over the first 14 amino acids of i3, and a double point mutation in the N terminal junction, K359A/K361A, both failed to affect carbachol stimulated PI turnover. Therefore, charge distribution in the loop junctions appears to play a minor role in G protein coupling of Hm1 in HEK 293 cells. 相似文献
8.
A single gonadotropin treatment of newborn rats influenced the thyrotropin (TSH) provoked thyroxine (T4) production of 2 months old animals. On pretreatment with 10, 20, 50, 100 and 200 I.U. of gonadotropin, the T4 blood level of animals given 10 and 200 I.U. differed scarcely from the controls treated only by TSH, while intermediary doses had a damaging effect as the T4 values were well below the control values measured in animals treated by TSH in adulthood. 相似文献
9.
D Russo G D Chazenbalk Y Nagayama H L Wadsworth P Seto B Rapoport 《Molecular endocrinology (Baltimore, Md.)》1991,5(11):1607-1612
The most widely held model for the human TSH receptor is of holoreceptor of 80 kDa with two subunits of approximately 50 and 30 kDa linked by disulfide bridges, with the former subunit containing the major hormone-binding site. We reexamined this model by covalently cross-linking radiolabeled TSH to the recombinant human TSH receptor stably expressed in Chinese hamster ovary (CHO) cells. When cross-linking was performed after the preparation of CHO membranes, analysis of hormone-receptor complexes under reducing and nonreducing conditions provided results supporting the two-subunit TSH receptor model. In contrast, however, cross-linking of TSH to the TSH receptor in intact CHO cells before membrane preparation revealed, even under reducing conditions, an approximately 100-kDa receptor as well as an approximately 54-kDa hormone-binding subunit. The approximately 100-kDa holoreceptor size is consistent with the size of the TSH receptor, as predicted from its derived amino acid sequence. The proportions of the approximately 100-kDa TSH receptor and the 54-kDa fragment varied in different experiments, suggesting the occurrence of proteolytic cleavage. Cross-linking of radiolabeled TSH to intact cells expressing a mutant TSH receptor (TSHR-D1) lacking amino acids 317-366 localized the proteolytic cleavage site to just up-stream of amino acid residue 317. In summary, the present data obtained by cross-linking TSH to recombinant human TSH receptors in intact cells provides evidence that the receptor exists in vivo as an approximately 100-kDa glycoprotein with a single polypeptide chain with intramolecular disulfide bridges.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
Rat brain homogenate was incubated with various concentrations of ascorbate in a Tris-HC1 buffer. Thiobarbituric acid-reactive substances (TBARS) were measured and 3H-QNB (quinuclidinyl benzilate) binding was also assayed on the same homogenate. Good parallelism between TBARS formation and loss of 3H-QNB binding activity confirmed that loss of 3H-QNB binding resulted from ascorbate-induced lipid peroxidation. However, neither formation of TBARS nor loss of 3H-QNB binding occurred in phosphate buffer or in the Tris-HC1 system in the presence of metal-chelating reagents. This indicates that phosphate addition prevents the ascorbate effects due to complete chelation of intrinsic metal ions. 相似文献
11.
12.
MAP kinase activation by mu opioid receptor involves phosphatidylinositol 3-kinase but not the cAMP/PKA pathway. 总被引:2,自引:0,他引:2
The involvement of protein kinases was studied in mu opioid receptor activation of mitogen-activated protein (MAP) kinase using cells transfected with the receptor clone. The cAMP/protein kinase A (PKA) pathway is known to be the major biochemical pathway for mu opioid receptor signaling. However, our data showed that stimulating adenylyl cyclase or activating PKA had no effect on mu receptor enhancement of MAP kinase activity, suggesting that the cAMP/PKA pathway is not involved in mediating the mu receptor activation of MAP kinase. Inhibition of phosphatidylinositol (PI) 3-kinase reduced mu receptor enhancement of MAP kinase activity, suggesting PI 3-kinase involvement. Together, these results show that cross-talk between the mu opioid receptor and the MAP kinase cascade is not mediated by the cAMP/PKA pathway, but involves PI 3-kinase. 相似文献
13.
G Calapai G Mazzaglia M C Marciano F Squadrito D Altavilla B Zingarelli M Prosdocimi A P Caputi 《Life sciences》1992,51(26):2041-2048
Intravenous (640 micrograms/kg) or intracerebroventricular (0.5 and 1 microgram) injection of Escherichia coli endotoxin (LPS) causes inhibition of water intake induced by 24 hour period of water deprivation in the rat. Tumor necrosis factor alpha (TNF-alpha; 20 and 40 ng/rat) given into the lateral cerebral ventricle (i.c.v.) causes effects similar to those observed after LPS. Cloricromene, given either intravenously (1 and 2 mg/kg) or i.c.v. (250 and 500 ng), abolished the antidipsogenic effect induced by LPS (administered both i.v. and i.c.v.). Cloricromene (2 mg/kg, i.v. or 500 ng/rat, i.c.v.), on the contrary, did not modify the antidipsogenic effects induced by TNF-alpha. These data indicate that peripherally injected cloricromene (as well as that i.c.v. injected) antagonizes the effects of mediators of LPS on sites regulating thirst and suggest that cloricromene's action may be due to inhibition of brain TNF-alpha formation induced by LPS. 相似文献
14.
Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor. 总被引:12,自引:0,他引:12
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu-p55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4 beta-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct. 相似文献
15.
Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction. 总被引:32,自引:0,他引:32
D J Dripps B J Brandhuber R C Thompson S P Eisenberg 《The Journal of biological chemistry》1991,266(16):10331-10336
The interleukin-1 receptor antagonist (IL-1ra) is a protein capable of inhibiting receptor binding and biological activities of IL-1 without inducing an IL-1-like response. Equilibrium binding and kinetic experiments show that IL-1ra binds to the 80-kDa IL-1 receptor on the murine thymoma cell line EL4 with an affinity (KD = 150 pM) approximately equal to that of IL-1 alpha and IL-1 beta for this receptor. However, IL-1ra is unable to induce two early events associated with IL-1 activity. Surface-bound IL-1ra does not undergo receptor-mediated internalization, and IL-1ra does not activate the protein kinase activity responsible for down-modulation of the EGF receptor on the murine 3T3 fibroblast cell line. The failure to induce general, early responses characteristic of IL-1 indicates that IL-1ra is unlikely to act as an agonist on any cell expressing the 80-kDa receptor. 相似文献
16.
The effects and mechanisms of aging on corticosterone secretion in zona fasciculata-reticularis (ZFR) cells of ovariectomized (Ovx) rats were studied. Young (3-month) and old (24-month) female rats were Ovx for 4 days before decapitation. ZFR cells were isolated and incubated with different hormones or reagents at 37 degrees C for 30 min. Aging increased the basal secretion of corticosterone both in vivo and in vitro. The adrenocorticotropin (ACTH)-, forskolin-, 3-isobutyl-l-methylxanthine (IBMX)-, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP)-, and ovine prolactin (oPRL)-stimulated release of corticosterone by ZFR cells was greater in old than in young Ovx rats. H89, an inhibitor of protein kinase A (PKA), decreased the production of corticosterone in ZFR cells from young but not old Ovx rats. Forskolin-, or IBMX-induced production of cAMP was greater in old than in young Ovx animals, which correlated with the increase of corticosterone production by aging. The activity of 11 beta-hydroxylase that converts deoxycorticosterone (DOC, 10(-9) or 10(-8) M) to corticosterone in rat ZFR cells was decreased by age. However, the corticosterone production in response to high dose of DOC (10(-7) M) was indifferent between young and old groups. These results suggest that aging increases corticosterone production in Ovx rats via a mechanism in part associated with an increase of adenylyl cyclase activity and a decrease of phosphodiesterase activity, and then an increase of the generation of cAMP, but not related to either PKA activity or 11 beta-hydroxylase. 相似文献
17.
The antiviral drugs amantadine hydrochloride and rimantadine hydrochloride were tested as to their oncogenic potential using a serial line of Fischer rat embryo cells that previously had been shown to be an accurate indicator of chemicals known to be oncogenic in animal studies. Neither compound was found to have transforming activity. At slightly toxic levels, rimanbadine hydrochloride, but not amantadine hydrochloride, protected the same cell line from the transformation induced by the polycyclic hydrocarbons 3-methylcholanthrene and benzo(a)pyrene. 相似文献
18.
This experiment was performed to clarify the role of extracellular signal-regulated kinase, ERK1/2, in NADPH oxidase-dependent O2- production in rat peritoneal neutrophils. When neutrophils were exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) to stimulate an N-formyl peptide receptor, not only the production of Opact">- but also the activation of ERK1/2 was observed. The translocation of an NADPH oxidase component, p47phoxphox, from cytosol to membrane also occurred in neutrophils stimulated with fMLP. U0126, an ERK1/2 kinase inhibitor, inhibited both the production of Opact">- and the translocation of p47phoxphox elicited by fMLP. On the other hand, when complement receptor 3 of neutrophils was stimulated with opsonized zymosan (OZ), weaker activation of ERK1/2 than that by fMLP was observed. In this case, U0126 showed no inhibition against the production of Opact">- and slight inhibition against the translocation of p47phoxphox. Large inhibition against the OZ-induced production of Opact">- was only observed in neutrophils treated with GF109203X, a PKC inhibitor. The present study indicates that receptor dependence exists in the ERK1/2 signaling pathway leading to the activation of NADPH oxidase. 相似文献
19.
Lee KB Ptasienski JA Pals-Rylaarsdam R Gurevich VV Hosey MM 《The Journal of biological chemistry》2000,275(13):9284-9289
Desensitization of G protein-coupled receptors (GPCRs) involves the binding of members of the family of arrestins to the receptors. In the model system involving the visual GPCR rhodopsin, activation and phosphorylation of rhodopsin is thought to convert arrestin from a low to high affinity binding state. Phosphorylation of the M(2) muscarinic acetylcholine receptor (mAChR) has been shown to be required for binding of arrestins 2 and 3 in vitro and for arrestin-enhanced internalization in intact cells (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). For the M(2) mAChR, arrestin binding requires phosphorylation at multiple serine and threonine residues at amino acids 307-311 in the third intracellular (i3) loop. Here, we have investigated the molecular basis for the requirement of receptor phosphorylation for arrestin binding. Constructs of arrestin 2 that can bind to other GPCRs in a phosphorylation-independent manner were unable to interact with a mutant M(2) mAChR in which the Ser/Thr residues at 307-311 were mutated to alanines. However, although phosphorylation-deficient mutants of the M(2) mAChR that lacked 50-157 amino acids from the i3 loop were unable to undergo agonist-dependent internalization when expressed alone in tsA201 cells, co-expression of arrestin 2 or 3 restored agonist-dependent internalization. Furthermore, a deletion of only 15 amino acids (amino acids 304-319) was sufficient to allow for phosphorylation-independent arrestin-receptor interaction. These results indicate that phosphorylation at residues 307-311 does not appear to be required to activate arrestin into a high affinity binding state. Instead, phosphorylation at residues 307-311 appears to facilitate the removal of an inhibitory constraint that precludes receptor-arrestin association in the absence of receptor phosphorylation. 相似文献