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: UNIPROT:Q07644 (polypeptide)
72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two point mutations were introduced by oligonucleotide-directed mutagenesis into the region of the Rous sarcoma virus envelope gene that encodes the hydrophobic transmembrane anchor of the receptor glycoprotein. Single-nucleotide substitutions ultimately converted a hydrophobic leucine, located centrally within the membrane-spanning domain, to either a similarly hydrophobic methionine or a positively charged arginine. The altered coding region was reinserted into an intact copy of the envelope gene, cloned into simian virus 40 late-replacement vector and expressed in primate cells. Analysis of envelope gene expression in CV-1 monkey cells revealed normal levels of synthesis of a membrane-spanning precursor for both the mutants; however, the arginine-containing mutant [mu 26(arg)] exhibited greatly reduced cell surface expression of mature protein, as determined by indirect immunofluorescence and 125I labeling of surface proteins. In experiments in which cells producing the mu 26(arg) polypeptide were pulsed with radioactive leucine and then chased for 5 h, no intracellular accumulation or extracellular secretion of mature products (gp85 and gp37) could be detected. Treatment of mu 26(arg)-infected cells with lysosomal enzyme inhibitors (chloroquine and leupeptin) resulted in the accumulation of gp85 and gp37, indicating that they were being degraded rapidly in lysosomes. The fact that terminally glycosylated and proteolytically cleaved env gene products were observed under these conditions showed that modifications associated with passage through the trans compartment of the Golgi apparatus occurred normally on the mutant polypeptide; thus insertion of a highly charged amino acid into the transmembrane hydrophobic region of gp37 results in the postGolgi transport to lysosomes. It is proposed that the insertion of this mutation into the transmembrane anchor of the envelope glycoprotein does not affect membrane association, orientation with respect to the membrane, or intracellular transport at early stages during maturation. At a step late in the transport pathway, however, the presence of the charged side chain alters the protein in such a manner that the molecules are transported to the lysosomes and degraded. It seems likely that transport of the protein from the trans-Golgi to the cell surface is either directly blocked, or that after expression on the cell surface the mature glycoprotein complex is unstable and rapidly endocytosed.
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PMID:A charged amino acid substitution within the transmembrane anchor of the Rous sarcoma virus envelope glycoprotein affects surface expression but not intracellular transport. 282 Oct 9

An alpha subunit cDNA of the mouse nicotinic acetylcholine receptor under transcriptional control of the Rous Sarcoma virus long terminal repeat was transfected into and expressed in a quail fibroblast cell line. The biosynthesis and post-translational modification of the alpha subunit protein made in this heterologous system have been studied using immunoprecipitation and ligand binding assays. The polypeptide is present at high steady-state levels and inserted in the correct transmembrane orientation. However, in the absence of assembly with other subunits the alpha subunit is confined to an intracellular membrane compartment and is not transported to the plasma membrane. Twenty percent of the newly synthesized alpha subunit acquired high affinity alpha bungarotoxin binding in a time-dependent process within 20 min of translation. Sucrose gradient fractionation demonstrated that both the polypeptide and toxin binding forms of the alpha subunit have a sedimentation coefficient of 5 s suggesting the absence of stable homo-oligomers. Quantitative binding assays demonstrated that the apparent affinity and rate of association of alpha bungarotoxin to the unassembled alpha subunit are greater than for native receptor. On the other hand, the affinities for the small ligands D-tubocurarine and gallamine are 10(3) lower than for native receptor; no detectable binding was observed for decamethonium, hexamethonium, or carbamylcholine. Thus, the acetylcholine receptor alpha subunit, independent of other subunits of the receptor, acquires a mature conformation and high affinity alpha bungarotoxin binding when expressed in a quail fibroblast cell line.
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PMID:Native folding of an acetylcholine receptor alpha subunit expressed in the absence of other receptor subunits. 282 54

An artificial membrane system was developed to study the molecular basis for interaction of pp60v-src, the Rous sarcoma virus transforming protein, with lipid bilayers. pp60v-src was extracted from cell membranes by detergent solubilization and reincorporated into phospholipid vesicles. Reconstituted pp60v-src retained tyrosine kinase activity and was integrally associated with the liposome through a 10-kilodalton (kDa) amino-terminal domain. The same 10-kDa domain was shown to anchor pp60v-src to the plasma membrane of transformed cells. Reconstitution experiments performed with nonmyristylated pp60v-src proteins revealed that these polypeptides did not interact with phospholipid vesicles. In contrast, myristylated, soluble pp60v-src molecules (including a highly purified pp60v-src preparation) could be reconstituted into liposomes, but their interaction with the liposomal bilayer was not mediated by the 10-kDa amino-terminal domain. When membrane proteins were included during reconstitution of purified pp60v-src, binding through the 10-kDa anchor was restored. A model is presented to accommodate the different types of interactions of pp60v-src with liposomes; the model postulates the existence of an additional membrane component that anchors the pp60v-src polypeptide to the phospholipid bilayer.
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PMID:Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles. 283 37

Protein I from intestinal epithelium is biochemically and immunologically related to the fibroblast 36-kDa substrate of the Rous sarcoma virus-encoded tyrosine protein kinase (Gerke and Weber (1984) EMBO J. 3, 227-233). Protein I is a Ca2+-binding protein containing two copies each of a 36- and 10-kDa subunit. Denaturation/renaturation experiments show that the 36-kDa subunit is a monomer, whereas the 10-kDa subunit forms a dimer. Mixing of the subunits leads to reconstituted protein I. Physicochemical properties of protein I and its isolated subunits reveal a Ca2+-dependent conformational change in the 36-kDa subunit which involves the exposure of 1 or more tyrosine residues to a more aqueous environment. This change points to a Ca2+ binding constant of about 10(4) M-1 in the presence of 2 mM Mg2+ and induces the ability of protein I and the 36-kDa subunit to bind in vitro to F-actin and nonerythroid spectrin. The same high Ca2+ requirement has been reported for the in vitro tyrosine phosphorylation of a 35-kDa protein from A-431 carcinoma cells by the epidermal growth factor receptor kinase (Fava and Cohen (1984) J. Biol. Chem. 259, 2636-2645). Here we show that this 35-kDa substrate is biochemically and immunologically related to the 36-kDa subunit of protein I, which in turn corresponds to the substrate of the Rous sarcoma virus kinase. The protein of A-431 cells exists not only as a monomer but also as a dimer. The latter fraction contains a 10-kDa polypeptide immunologically related to the corresponding subunit of protein I. Given past results on the A-431 system, we speculate that the monomer rather than the dimer is the preferred in vitro substrate for the epidermal growth factor receptor kinase. Thus, the 10-kDa subunit, which induces dimerization of the phosphorylatable large subunit, may act as an inhibitor.
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PMID:Calcium-dependent conformational changes in the 36-kDa subunit of intestinal protein I related to the cellular 36-kDa target of Rous sarcoma virus tyrosine kinase. 298 69

A simple and effective purification method for the src kinase, the transforming gene product of Rous sarcoma virus, has been developed by using affinity chromatography on casein-agarose and tyrosine-agarose columns. NaDodSO4/polyacrylamide gel electrophoresis and silver staining analysis showed that the purified kinase preparation was composed of a predominant polypeptide of 60,000-Da. In most of the preparations, however, three minor proteins (54,000, 52,000, and 15,000 Da) were also detected, and they were partially characterized. As one of the exogenous substrates, calmodulin was found to be phosphorylated on tyrosine by the purified src kinase.
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PMID:Purification of the Rous sarcoma virus src kinase by casein-agarose and tyrosine-agarose affinity chromatography. 298 51

Rous sarcoma virus expresses a transcriptional activator that affects the LTR as well as other promoters. We discern this activity as a stimulation of the transient expression of an LTR-promoted hybrid transcriptional unit and also of the rat preproinsulin II gene in transfected NIH 3T3 cells. We map the activity to an alternate reading frame in the p19-p10 region of the gag gene and identify a mRNA whose spliced structure would direct translation of this reading frame from the Pr76gag initiation codon. This mRNA probably differs from genomic RNA only by the 282 nucleotide splice. The predicted translation product is a 124 residue polypeptide; the first six amino acids arise from gag. The target for the action of this transcriptional modulator at the LTR lies between 111 and 620 nucleotides upstream of the cap site.
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PMID:Rous sarcoma virus encodes a transcriptional activator. 298 97

Phosphorylation of the src gene product pp60v-src was studied in plasma membrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [gamma-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60v-src. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 microM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp60v-src. However, at higher ATP concentrations (100 microM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp60v-src was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp60v-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.
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PMID:Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes. 298 81

Co-infection of Ehrlich ascites carcinoma (EAC)-bearing mice with Sendai virus and Rous sarcoma virus (RSV) did not result in the formation of complete RSV. Sendai virus could be, however, propagated in this system over 8 serial passages. As demonstrated by immunofluorescence and complement fixation reactions, antigens specific to each virus were synthesized in EAC cells following either single or mixed virus infection. The virus progens also contained antigenic fractions incorporated from the host cell. The incomplete progens synthesized when RSV inoculation preceded that of Sendai virus possessed three polypeptide fractions characteristic of Sendai virus and one RSV-specific fraction.
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PMID:Virus - tumor cell relationships. In vivo cocultivation of para-influenza type 1 (Sendai) virus and of Rous sarcoma virus (Schmidt-Ruppin strain) in mouse Ehrlich ascites carcinoma. 298 91

The enzymatic domains of the avian retrovirus polymerase (pol) gene have been mapped by the use of peptide antibodies and COOH-terminal amino acid analysis. The processed pol beta polypeptide is cleaved in vivo to yield alpha and pp32. Rabbit antibodies were directed against synthetic peptides whose sequence was deduced from the known pol sequence of Rous sarcoma virus, Prague C (Schwartz, D.E., Tizard, R., and Gilbert, W. (1983) Cell 32, 853-869). The RNase H active site of pol was located in the NH2-terminal region of the alpha DNA polymerase subunit. The COOH terminus of the alpha subunit was found to be immediately adjacent to the NH2 terminus of the pp32 pol protein. COOH-terminal amino acid analysis of pp32 revealed that this protein is also processed. From the deduced amino acid sequence of pol, it appears likely that pol encodes an additional 4100-dalton polypeptide located at its extreme COOH terminus. The enzymatic domains on beta appear to map in the following order: RNase H-DNA polymerase-DNA endonuclease. Hydrophilicity analysis and secondary structure predictions of wild type Rous sarcoma virus pol products and mutated pp32 possessing single amino acid changes permit further structural evaluation of the multifunctional pol protein.
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PMID:Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains. 298 84

The Rous sarcoma virus (RSV)-transforming protein, pp60src, is a plasma membrane-associated tyrosine-specific protein kinase. A 36,000-Da cellular polypeptide (p36) which is phosphorylated at tyrosine in RSV-transformed chicken embryo fibroblasts (RSV-CEF) is also plasma membrane associated. To determine if p36 is directly phosphorylation and kinase activity in situ in the plasma membrane, src-dependent protein phosphorylation in membranes isolated from RSV-CEF has been characterized. These membrane preparations contained high ATPase and phosphoprotein phosphatase activities; but when sufficient concentrations of [gamma-32P]ATP were used, the phosphorylation of pp60src and the phosphorylation of p36 were linear for 1 min or more, and the initial rates of phosphorylation could therefore be determined. In membranes from RSV-CEF pp60src and p36 became phosphorylated predominantly at tyrosine, while in membranes from uninfected cells p36 was phosphorylated at low levels at serine. When membranes from RSV-CEF were preincubated with tumor-bearing rabbit (TBR) serum, the IgG became phosphorylated while the phosphorylation of p36 was inhibited, suggesting that p36 is directly phosphorylated by pp60src. Phosphorylation of pp60src, p36, and TBR-IgG was dependent on growth temperature in membranes from cells infected by a temperature-sensitive mutant, tsNY68, although some dependence on growth temperature was observed even with membranes from wild-type RSV-infected cells. However, at the nonpermissive temperature, tsNY68 pp60src retained 20-40% of its kinase activity, providing supporting for the proposal (B. M. Sefton, T. Hunter, and K. Beemon (1980, J. Virol, 33, 220-229) that transformation may result from a small quantitative change in pp60src activity. The phosphorylation of pp60src and its kinase activity were not coordinately affected by growth temperature or mutations within src, indicating that different factors affect the phosphoacceptor capacity and kinase activity of the protein.
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PMID:pp60src-dependent protein phosphorylation in membranes from Rous sarcoma virus-transformed chicken embryo fibroblasts. 299 19


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