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:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Osteopontin (OPN) is an extracellular matrix protein that supports osteoclast adhesion to the bone by binding to integrin alpha v beta 3. We measured the binding between OPN and integrin alpha v beta 3 with recombinant human OPN and the urinary form of human OPN, uropontin. Recombinant OPN was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and cleaved from glutathione S-transferase with Factor Xa. The mass of this form of OPN (rOP27) is 27,046 Da. rOP27 is truncated at arginine residue 228, 69 amino acids short of the native carboxyl terminus. Uropontin and rOP27 support RGD-dependent cell adhesion and to bind purified integrin alpha v beta 3 with similar affinities. Further study showed that OPN is the only known naturally occurring RGD-containing protein with a much greater affinity for alpha v beta 3 than for the platelet integrin alpha IIb beta 3. Most importantly, we find that physiologic levels of Ca2+ block cell adhesion to OPN. Measurement of binding constants between rOPN and purified integrin alpha v beta 3 with surface plasmon resonance showed that the affinity between rOPN and alpha v beta 3 is 26-fold lower in Ca2+ (Kd = 1.1 x 10(-8) M) than in Mn2+ (Kd = 4.3 x 10(-10) M) and 9-fold lower than in Mg2+ (Kd = 1.3 x 10(-9) M). In bone, the resorbing osteoclast generates elevated levels of extracellular Ca2+, therefore the findings presented here suggest a previously unappreciated mechanism for the modulation of bone resorption by extracellular Ca2+.
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PMID:Ca2+ suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin alpha v beta 3. 753 71

Osteopontin (OPN) is an extracellular matrix protein that binds to integrin alpha v beta 3. Here we demonstrate that two other integrins, alpha v beta 1 and alpha v beta 5, are also receptors for OPN. Human embryonic kidney 293 cells adhere to human recombinant osteopontin (glutathione S-transferase-osteopontin; GST-OPN) using integrin alpha v beta 1. When the 293 cells are transfected with the beta 5 subunit, they can also adhere to GST-OPN using integrin alpha v beta 5. Divalent cations regulate the binding of GST-OPN to both alpha v beta 1 and alpha v beta 5. Mg2+ and Mn2+ support the binding of GST-OPN to these integrins but Ca2+ does not. The highest affinity is observed in Mn2+. In the presence of this ion, the affinity of GST-OPN for alpha v beta 1 is 18 nM and the affinity for alpha v beta 5 is 48 nM. The antibody 8A2, which is an agonist for beta 1, promotes the adhesion of 293 cells to GST-OPN even when Ca2+ is present. This observation suggests that cellular events could modulate the affinity of alpha v beta 1 for OPN. Collectively, these findings prove that integrins alpha v beta 1, alpha v beta 3, and alpha v beta 5 have similar affinity for OPN. Therefore, all three integrins must be considered when evaluating the biological affects of OPN.
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PMID:A biochemical characterization of the binding of osteopontin to integrins alpha v beta 1 and alpha v beta 5. 759 29

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.
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PMID:Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin. 891 Apr 76

Osteopontin (OPN) is a secreted glycoprotein implicated in cell adhesion. It contains the arginine-glycine-asparatic acid (RGD) cell adhesive domain and the thrombin cleavage sequence. Although thrombin cleavage of OPN has been shown to be of physiological importance, the function of C-terminal OPN fragment cleaved by thrombin remains unknown. To determine its role, we performed cell adhesion assays using glutathione S-transferase-OPN fusion protein fragments and full-length OPN fusion protein. The N-terminal fragment containing RGD motif promoted enhanced adhesion of mouse and human fibroblasts by 2.9 and 2.8 folds in comparison with full-length OPN, respectively. The enhanced adhesion of both cells mediated by N-terminal fragment was significantly suppressed by addition of C-terminal fragment lacking RGD motif that has less cell adhesive property than full-length OPN. These results suggest that the C-terminal domain may play a pivotal role in regulating OPN functions by suppressing the RGD-dependent cell adhesion.
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PMID:The carboxyl-terminal fragment of osteopontin suppresses arginine-glycine-asparatic acid-dependent cell adhesion. 989 40

The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely alphavbeta1, alphavbeta3, alphavbeta5, alpha9beta1, alpha8beta1, and alpha4beta1. We have investigated the interaction of alpha5beta1 integrin with OPN using K562 cells, which only express alpha5beta1. alpha5beta1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA. Treating K562 wild-type cells (K562-WT) with TPA stimulated an interaction between alpha5beta1 and OPN. No interaction was seen in the absence of TPA. alpha5beta1 selectively interacted with a GST fusion protein of the N-terminal fragment of OPN (aa17-168), which is generated in vivo by thrombin cleavage of OPN. Expression of the alpha4 integrin in K562 cells (K562-alpha4beta1) stimulated alpha5beta1-dependent binding to aa17-168 in the absence of TPA, suggesting that alpha4beta1 activates alpha5beta1 in K562 cells. Adhesion via alpha5beta1 is mediated by the Arg-Gly-Asp (RGD) motif of OPN, as mutating this sequence to Arg-Ala-Asp (RAD) blocked binding of both cell types. These data demonstrate that thrombin cleavage regulates the adhesive properties of OPN and that alpha5beta1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.
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PMID:A regulated interaction between alpha5beta1 integrin and osteopontin. 1067 66

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.
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PMID:Analysis of the alpha4beta1 integrin-osteopontin interaction. 1089 85

Smad2 and Smad3 are downstream transforming growth factor-beta (TGF-beta) signaling molecules. Upon phosphorylation by its type I receptor, Smad2 or Smad3 forms a complex with Smad4 and translocates to the nucleus where the complex activates target gene transcription. In the present study, we report that Smad3 binds directly to the osteopontin (OPN) promoter and that Smad4 interacts with the Hox protein and displaces it from its cognate DNA binding site in response to TGF-beta stimulation. In gel shift assays, the glutathione S-transferase-Smad3 fusion protein was found to bind to a 50-base pair DNA element (-179 to -229) from the OPN promoter. Also, we found that both Hoxc-8 and Hoxa-9 bound to a Hox binding site adjacent to Smad3 binding sequence. Interestingly, Smad4, the common partner for both bone morphogenic protein and TGF-beta signaling pathways, inhibited the binding of Hox protein to DNA. FLAG-tagged Smad4 coimmunoprecipitated with HA-tagged Hoxa-9 from cotransfected COS-1 cells, demonstrating an interaction between Smad4 and Hoxa-9. Transfection studies showed that Hoxa-9 is a strong transcriptional repressor; it suppresses the transcription of the luciferase reporter gene driven by a 124-base pair OPN promoter fragment containing both Smad3 and Hox binding sites. Taken together, these data demonstrate a unique TGF-beta-induced transcription mechanism. Smad3 and Smad4 exhibit different functions in activation of OPN transcription. Smad3 binds directly to the OPN promoter as a sequence-specific activator, and Smad4 displaces the transcription repressor, Hoxa-9, by formation of Smad4/Hox complex as part of the transcription mechanism in response to TGF-beta stimulation.
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PMID:Hoxa-9 represses transforming growth factor-beta-induced osteopontin gene transcription. 1104 72

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130(Cas), focal adhesion kinase, integrin alpha(v)beta(3), vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of alpha(v)beta(3)-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.
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PMID:Phosphatidylinositol 3,4,5-trisphosphate directs association of Src homology 2-containing signaling proteins with gelsolin. 1157 4

The secreted phosphoprotein osteopontin (OPN), when immobilized on a surface, supports cell adhesion, prevents apoptosis of endothelial cells, and is a ligand for the alpha(v)beta(3) integrin, which is important in endothelial cell biology and neovascularization. OPN synthesized by tumor cells stimulates tumor growth, but the mechanism by which the protein acts remains unclear. One possibility, therefore, is that OPN may exert its effects on tumor growth by enhancing angiogenesis. While OPN is found at high levels in bone, where it is a component of the mineralized matrix, we have asked here whether OPN present in tumors is similarly extracellular matrix associated. We have shown that OPN is detectable in tumor extracts and in serum of tumor-bearing mice, and that the protein in tumors and in serum can be synthesized by both tumor and the host cells. Biochemical fractionation of tumor tissue confirmed that there is little if any association of OPN with the insoluble fraction. Immunochemical analysis of murine mammary tumors shows no co-localization of OPN with the extracellular matrix, identified by laminin staining. Ras-transformed cells in culture produce abundant OPN, however, the protein was found to be associated with the cell fraction but not with the matrix fraction. An enzyme-linked immunosorbent assay was used to demonstrate that OPN in conditioned medium from these cells fails to associate with extracellular matrix components, including laminin and fibronectin, in vitro. Recombinant OPN (GST-OPN) when coated onto a plastic surface can support human umbilical vein endothelial cell adhesion, suppressing apoptosis and allowing cell cycle progression, at concentrations from 1 to 50 microg/ml. Soluble GST-OPN in the same concentration range has no effect on HUVECs held in suspension. Thus, we conclude that OPN associated with tumors is primarily soluble, and that soluble OPN can neither support endothelial cell proliferation nor prevent apoptosis of these cells in the absence of adhesion.
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PMID:Tumor-derived osteopontin is soluble, not matrix associated. 1174 94

Isoforms of the thyroid hormone receptor (TR)alpha and TRbeta genes mediate thyroid hormone action. How TR isoforms modulate tissue-specific thyroid hormone (TH) action remains largely unknown. The steroid receptor coactivator-1 (SRC-1) is among a group of transcriptional coactivator proteins that bind to TRs, along with other members of the nuclear receptor superfamily, and modulate the activity of genes regulated by TH. Mice deficient in SRC-1 possess decreased tissue responsiveness to TH and many steroid hormones; however, it is not known whether or not SRC-1-mediated activation of TH-regulated gene transcription in peripheral tissues, such as heart and liver, is TR isoform specific. We have generated mice deficient in TRalpha and SRC-1, as well as in TRbeta and SRC-1, and investigated thyroid function tests and effects of TH deprivation and TH treatment compared with wild-type (WT) mice or those deficient in either TR or SRC-1 alone. The data show that 1) in the absence of TRalpha or TRbeta, SRC-1 is important for normal growth; 2) SRC-1 modulates TRalpha and TRbeta effects on heart rate; 3) two new TRbeta-dependent markers of TH action in the liver have been identified, osteopontin (upregulated) and glutathione S-transferase (downregulated); and 4) SRC-1 may mediate the hypersensitivity to TH seen in liver of TRalpha-deficient mice.
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PMID:Specificity of thyroid hormone receptor subtype and steroid receptor coactivator-1 on thyroid hormone action. 1238 68


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