共查询到20条相似文献,搜索用时 0 毫秒
1.
W P Sullivan B J Madden D J McCormick D O Toft 《The Journal of biological chemistry》1988,263(29):14717-14723
Progesterone receptors are phosphoproteins, in which phosphorylation has been proposed as a control mechanism for some stages of hormone action. Progesterone administration was shown to increase phosphorylation of the receptor from both cytosol and nuclear extracts of whole cells. We have analyzed the receptor phosphopeptides generated by chemical and proteolytic cleavage to assess the number of phosphorylation sites and their approximate location in the receptor. Progesterone receptor was labeled in situ in the presence or absence of hormone in medium containing [32P] orthophosphate, isolated by immunoprecipitation, and then digested with several proteases. The resulting 32P-labeled peptides were resolved by either two-dimensional electrophoresis:chromatography or by reverse-phase high performance liquid chromatography. Multiple phosphopeptides (3-6) were detected after cleavage with trypsin, chymotrypsin, or V8 protease. Major increases in phosphorylation occurred at existing sites since after hormone treatment no new phosphopeptides were found. Individual phosphopeptides showed variable increases in phosphorylation of 1.5-5-fold. The A and B receptor forms showed identical phosphorylation patterns, indicating similar processing in vivo. The phosphopeptide pattern for receptor in nuclear extracts resembled that of cytosol receptor. Chemical cleavage was used to assess the distribution of phosphorylation sites. Cyanogen bromide produced a large 40-kDa polypeptide which contained all of the phosphorylation sites and comprised the residues 129-449. Hydroxylamine was used to cleave a unique bond, Asn-372-Gly-373, in the 40-kDa polypeptide. All of the phosphorylation sites were located on the amino-terminal side of the cleavage. Thus, all of the phosphorylation sites were localized to a specific region (Met-129 to Asn-372) of the progesterone receptor that does not include either the DNA or steroid binding domains. 相似文献
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Kim Y Sun Y Chow C Pommier YG Simons SS 《The Journal of steroid biochemistry and molecular biology》2006,100(1-3):3-17
Varying the concentration of selected factors alters the induction properties of steroid receptors by changing the position of the dose-response curve (or the value for half-maximal induction=EC(50)) and the amount of partial agonist activity of antisteroids. We now describe a rudimentary mathematical model that predicts a simple Michaelis-Menten curve for the multi-step process of steroid-regulated gene induction. This model suggests that steps far downstream from receptor binding to steroid can influence the EC(50) of agonist-complexes and partial agonist activity of antagonist-complexes. We therefore asked whether inhibitors of three possible downstream steps can reverse the effects of increased concentrations of two factors: glucocorticoid receptors (GRs) and Ubc9. The downstream steps (with inhibitors in parentheses) are protein deacetylation (TSA and VPA), DNA unwinding (CPT), and CTD phosphorylation of RNA polymerase II (DRB and H8). None of the inhibitors mimic or prevent the effects of added GRs. However, inhibitors of DNA unwinding and CTD phosphorylation do reverse the effects of Ubc9 with high GR concentrations. These results support our earlier conclusion that different rate-limiting steps operate at low and high GR concentrations versus high GR with Ubc9. The present data also suggest that downstream steps can modulate the EC(50) of GR-mediated induction, thus both supporting the utility of our mathematical model and widening the field of biochemical processes that can modify the EC(50). 相似文献
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Hormone-dependent phosphorylation of the 1,25-dihydroxyvitamin D3 receptor in mouse fibroblasts 总被引:10,自引:0,他引:10
Experimental results, employing several immunologic techniques, suggest that the mouse receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) undergoes hormone-dependent phosphorylation in intact cells. Treatment of monolayer cultures of mouse 3T6 fibroblasts with 1,25(OH)2D3 reveals that the occupied 1,25(OH)2D3 receptor displays a minor reduction in electrophoretic mobility as compared to its unoccupied 54,500 dalton counterpart, a change consistent with covalent modification. Similar results were obtained by immunoprecipitation of metabolically-labeled receptors after incubation of 3T6 cells with [35S]methionine. This technique also provided greater insight into the precursor-product relationship between the two receptor forms. [32P]Orthophosphate-labeling of 3T6 cells, followed by immunoprecipitation indicated that only the form exhibiting covalent modification was phosphorylated. The temporal correspondence between the binding of 1,25(OH)2D3 to its cellular receptor and its phosphorylation suggests that the biochemical role of 1,25(OH)2D3 may be to induce a conformational change susceptible to phosphorylation and possibly functional activation. 相似文献
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Korkalainen M Tuomisto J Pohjanvirta R 《Biochemical and biophysical research communications》2000,273(1):272-281
Hamsters and Han/Wistar (Kuopio; H/W) rats show peculiarly selective responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). They are extremely resistant to its acute lethality but sensitive to, e.g. , enzyme induction. The biological effects of TCDD are mediated by the AH receptor (AHR). Recent studies on H/W rat AHR discovered a remodelled transactivation domain which appears to be critical for the TCDD resistance of these animals. Here, molecular cloning and sequencing of hamster AHR reveals another type of restructured transactivation domain. In hamsters, the functionally pivotal Q-rich region is substantially expanded and enriched in glutamine compared with all other AHRs cloned to date. By contrast, the amino-terminal end is highly conserved, which is in agreement with the H/W rat AHR. Because of the additional material in the transactivation domain, hamster AHR protein is larger than that in rats or mice, but the pattern of AHR mRNA expression in tissues is similar. 相似文献
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Phorbol ester-induced threonine phosphorylation of the human epidermal growth factor receptor occurs within the EGF binding domain 总被引:4,自引:0,他引:4
Partial proteolysis with trypsin has been used to map the sites of phorbol ester-induced phosphorylation of the epidermal growth factor (EGF) receptor. Both 12-O-tetradecanoylphorbol 13-acetate (TPA) and EGF stimulate phosphorylation of the EGF receptor in intact human carcinoma cells. Under the conditions examined, EGF is more effective than TPA in stimulating phosphorylation of a 45 kDa intracellular receptor domain, while TPA is more effective than EGF in inducing phosphorylation of a 120 kDa transmembrane EGF-binding domain. The phosphorylation of the 120 kDa peptide occurs primarily on threonine residues. Two-dimensional peptide mapping indicates that the two major phosphopeptides found in the 120 kDa receptor fragment correspond to the major new phosphopeptides found in intact EGF receptor following treatment with TPA. Thus, the major sites of TPA-induced threonine phosphorylation reside in the 120 kDa binding domain of the EGF receptor. 相似文献
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Patel PD Lyons DM Zhang Z Ngo H Schatzberg AF 《The Journal of steroid biochemistry and molecular biology》2000,72(3-4):115-123
Squirrel monkeys are among a diverse group of New World primates that demonstrate unusually high levels of circulating corticosteroids and glucocorticoid receptor (GR) insensitivity. Recent evidence suggests that overexpression of an immunophilin impairs dexamethasone binding to GR in the Bolivian squirrel monkey (Saimiri boliviensis). Here we describe the cloning, expression, and functional characterization of GR from the closely related Guyanese squirrel monkey (S. sciureus). The cloned Guyanese squirrel monkey GR (gsmGR) cDNA closely resembles human GR (hGR) cDNA, and yields a high affinity dexamethasone binding receptor when expressed in COS-1 cells. Transactivation analysis of hGR and gsmGR expressed in CV-1 cells and cultured squirrel monkey kidney (SMK) cells indicates that: (1) SMK cells elaborate a functional high activity GR from human GR cDNA; (2) gsmGR is an order of magnitude less efficient than hGR at transactivation in CV-1 and SMK cells; and (3) maximal transactivation by gsmGR is attenuated in both cell lines. Glucocorticoid resistance in S. sciureus is at least partly attributable to a naturally occurring mutation in the GR gene that results in impaired GR transactivation. 相似文献
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Montuori N Pacifico F Mellone S Liguoro D Di Jeso B Formisano S Gentile F Consiglio E 《Biochemical and biophysical research communications》2000,268(1):42-46
We have previously reported that the rat hepatic lectin-1 (RHL-1) subunit of rat asialoglycoprotein receptor (ASGPr), the endocytic receptor found on the basolateral surface of hepatocytes, was expressed in rat thyroid tissue and localized on the apical surface of polarized rat thyroid FRT cells. Here we show that PC Cl3 cells, a differentiated rat thyroid cell line, bound thyroglobulin (Tg) via ASGPr. In fact, both the bacterial recombinant carbohydrate recognition domain of RHL-1 (rCRD(RHL-1)) and the anti-rCRD(RHL-1) antibody markedly inhibited (125)I-Tg binding to the cell surface of PC Cl3 cells. Ligand blot assays with deglycosylated Tg show that the rCRD(RHL-1) was able to interact with Tg even after remotion of sugars. The region of Tg involved in the binding to RHL-1 was investigated by ligand blot assays with biotinylated rCRD(RHL-1) on thermolysin-digested native and desialated rat thyroglobulin. It is shown that the rCRD(RHL-1) specifically recognized a thyroglobulin fragment with an apparent M(r) of 68,000, corresponding to the amino-terminal part of the molecule. To our knowledge, this is the first report that attributes to the amino-terminal portion of Tg molecule, containing its earliest and major hormonogenic site, the function of binding to a cell surface receptor of the thyroid. Moreover, we show that oligosaccharides are not the only molecular signals for binding to RHL-1, but amino acidic determinants could also play a role. 相似文献
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David B. Haslam Thomas Borén Per Falk Dag Liver Amy Chou Zheng Xu Staffan Normark † 《Molecular microbiology》1994,14(3):399-409
Pyelonephritic isolates of Escherichia coli commonly express P-pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P-pili have been defined, based on varying specificity for galabiose-containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the ammo-terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the ammo-terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino-terminus of the PapG adhesin and is independent of the complex pilus architecture. 相似文献
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J H van Laar C A Berrevoets J Trapman N D Zegers A O Brinkmann 《The Journal of biological chemistry》1991,266(6):3734-3738
Phosphorylation of the androgen receptor was investigated in the absence of hormone as well as during and after transformation of the receptor to the tight nuclear binding form. Human prostate tumor cells (LNCaP) were labeled for 4 h with [32P]orthophosphate in the presence or absence of steroid. Subsequently, androgen receptors were immunoprecipitated either from total cell lysates or from nuclear extracts using a specific monoclonal antibody. The immunoprecipitated receptor preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, using a polyclonal antiserum, and autoradiography. It was observed that the androgen receptor is already phosphorylated in the absence of hormone, but undergoes a hormone-induced additional phosphorylation. After administration of 10 nM R1881, a 1.8-fold increase in phosphorylation over nonstimulated control cells was reached. Moreover, the amount of nuclear extractable androgen receptor was increased; the acquisition of tight nuclear binding capacity was accompanied by hormone-induced receptor phosphorylation. 相似文献
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Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain 总被引:3,自引:0,他引:3
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The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo. 相似文献
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Tajia L. Green Kelly Tung Debora Lim Stacey M. Leventhal Kiho Cho David G. Greenhalgh 《Biochemistry and Biophysics Reports》2017
Glucocorticoids are one of the most widely used therapeutics in the treatment of a variety of inflammatory disorders. However, it is known that there are variable patient responses to glucocorticoid treatment; there are responders and non-responders, or those that need higher dosages. Polymorphisms in the glucocorticoid receptor (GR) have been implicated in this variability. In this study, ninety-seven volunteers were surveyed for polymorphisms in the human GR-alpha (hGRα), the accepted biologically active reference isoform. One isoform identified in our survey, named hGR DL-2, had four single nucleotide polymorphisms (SNPs), one synonymous and three non-synonymous, and a four base pair deletion resulting in a frame shift and early termination to produce a 743 amino acid putative protein. hGR DL-2 had a decrease in transactivation potential of more than 90%. Upon further analysis of the individual SNPs and deletion, one SNP, A829G, which results in a lysine to glutamic acid amino acid change at position 277, was found to increase the transactivation potential of hGR more than eight times the full-length reference. Furthermore, the hGRα-A829G isoform had a differential hyperactive response to various exogenous steroids. Increasing our knowledge as to how various SNPs affect hGR activity may help in understanding the unpredictable patient response to steroid treatment, and is a step towards personalizing patient care. 相似文献
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H Br?uner-Osborne A A Jensen P O Sheppard P O'Hara P Krogsgaard-Larsen 《The Journal of biological chemistry》1999,274(26):18382-18386
The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19-25% sequence identity to the gamma-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu1a. The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6, and 3.9 mM for these cations, respectively) was very similar to that of the wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mM, respectively). For the mGlu1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved in CaR as Ser-147 and Ser-170, respectively. Each of these residues was mutated to alanines and tested pharmacologically using the endogenous agonist Ca2+. CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca2+ (4-fold increase in EC50) and maximal response (79% of wild-type response). CaR-S170A showed no significant response to Ca2+ even at 50 mM concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological profiles almost identical to that of the wild-type receptor. These data demonstrate that Ser-170 and to some extent Ser-147 are involved in the Ca2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors. 相似文献