The Chick Fibroblast Cell Surface after Transformation by Rous Sarcoma Virus

Robbins, Phillips W.; Wickus, Gary G.; Branton, Philip E.; Gaffney, Betty J.; Hirschberg, Carlos B.; Fuchs, Pinhas; Blumberg, Peter M.

doi:10.1101/sqb.1974.039.01.135

Cited by 72 publications

(37 citation statements)

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“…The present study suggests that several of these effects might be mediated by the oligosaccharide portions of AcChoR. It is possible that, as with the TM-treated cells, altered glycosylation and a consequent increase in proteolytic degradation underlie the decreased accumulation and accelerated degradation of AcChoR observed with muscle cell transformation (45,48,49). The stabilization of synaptic AcChoR by innervation might be analogous to the inhibition of AcChoR degradation elicited in the absence of neuronal influence by treatment of cultured muscle cells with Con A.…”

Section: Discussionmentioning

confidence: 90%

Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture.

Prives¹,

Olden²

1980

Proc. Natl. Acad. Sci. U.S.A.

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We have investigated the significance of protein glycosylation for metabolism of acetylcholine receptors (AcChoR) in primary cultures of embryonic chicken muscle cells. Tunicamycin, a specific inhibitor of the glycosylation of asparagine residues on glycoproteins, decreased AcChoR accumulation and accelerated its degradation. In contrast, there was no evidence that tunicamycin treatment affected AcChoR biosynthesis, intracellular transport, or incorporation into surface membranes. Leupeptin, an inhibitor of intracellular proteases, markedly increased accumulation of AcChoR on the external surface of muscle cells treated with tunicamycin. Our findings indicate that impairment of protein glycosylation prevents accumulation of AcChoR by increasing its susceptibility to degradation by cellular proteases.is strongly impaired by TM treatment. However, the proteolytic degradation of underglycosylated AcChoR is substantially increased under these conditions, resulting in marked diminution of AcChoR accumulation. The protease inhibitor leupeptin substantially reverses the decrease in AcChoR in TM-treated cultures, suggesting that glycosylation stabilizes AcChoR against proteolytic degradation. The possibility that the carbohydrate on AcChoR may function in regulating its stability is supported by our findings that: (i) impaired protein glycosylation results in accelerated degradation of AcChoR, and (ii) concanavalin A (Con A), a ligand of AcChoR (2,3), increases AcChoR stability in control cultures but not in TM-treated cells.The acetylcholine receptor (AcChoR) of skeletal muscle cells is an integral membrane glycoprotein that is uniquely well characterized both pharmacologically and biochemically (for recent reviews see refs. 1-3). Thus AcChoR may serve as a model for detailed study of the function of the carbohydrate components of glycoproteins in the regulation of well-defined plasma membrane properties. AcChoR is of particular interest because its synthesis, distribution on the muscle cell surface, and degradation are tightly regulated during differentiation and upon innervation (3). The striking effects induced under neuronal influence include the disappearance of AcChoR from extrasynaptic regions of the muscle cell surface and a marked decrease in the degradation rate of synaptic AcChoR (3). The regulation of AcChoR metabolism has recently been intensively studied in cell cultures of differentiating skeletal muscle (3, 4) using 25I-labeled a-bungarotoxin ('25I-a-Bgt), a specific ligand that binds to AcChoR with high specificity and low reversibility (5).The function of carbohydrate components of glycoproteins can be conveniently studied in cultured cells by the use of tunicamycin (TM), an antibiotic that specifically inhibits protein glycosylation (6-17). Treatment of cells with TM results in the synthesis of glycoproteins deficient in asparagine-linked oligosaccharides (6,18,19). TM has been utilized to study the contribution of the oligosaccharide components to the posttranslational processing (16, 17), i...

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Section: Discussionmentioning

confidence: 90%

Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture.

Prives¹,

Olden²

1980

Proc. Natl. Acad. Sci. U.S.A.

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“…Neoplastic transformation of fibroblastic cells results in decreased amounts of a major cell surface protein (CSP), also called large external transformation-sensitive protein (LETS protein) (1)(2)(3)(4)(5)(6)(7)(8), and in decreased amounts of a major extracellular protein, collagen (9)(10)(11). The altered shape and adhesive properties of transformed chick embryo fibroblasts (CEF) and some other cells have been attributed to their diminished CSP content (7,12,13).…”

mentioning

confidence: 99%

Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts.

Adams¹,

Sobel²,

Howard³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

281

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Transformation of chick embryo fibroblasts by Rous sarcoma virus results in decreased amounts of a major cell surface protein and of collagen. To determine the mechanism accounting for the decreased production of these proteins, we have measured the relative amounts of functional mRNAs for these and other transformation-sensitive proteins. Total cellular RNAs extracted from normal cells and from cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus were translated in a cell-free system derived from wheat germ. Analysis of the in vitro translation products of RNAs from normal and transformed chick embryo fibroblasts shows a 5-fold reduction in the translatable mRNA for cell surface protein and a 10-fold reduction in translatable mRNA for two collagen precursors. In addition, increases in functional mRNA are observed for myosin and for two membrane polypeptides with molecular weights of 95,000 and 78,000; the later two proteins increase on transformation, but the increases are in large part secondary to the depletion of glucose from the medium of transformed cells. Our data suggest that some of the major cellular changes induced by oncogenic viruses are due to changes in the activity of specific cellular genes.Neoplastic transformation of fibroblastic cells results in decreased amounts of a major cell surface protein (CSP), also called large external transformation-sensitive protein (LETS protein) (1-8), and in decreased amounts of a major extracellular protein, collagen (9-11). The altered shape and adhesive properties of transformed chick embryo fibroblasts (CEF) and some other cells have been attributed to their diminished CSP content (7,12,13). Furthermore, loss of CSP has been correlated with the ability of many transformed cell lines to form tumors (14). Several mechanisms have been proposed to explain why transformed cells have less CSP. These include decreased biosynthesis (4), increased degradation due to the increased proteolytic activity of transformed cells (3, 15), and increased release from the cell surface into the medium (16). Recent experiments by Olden and Yamada (17) using metabolic labeling techniques indicate that a decrease in the synthetic rate of CSP is the major mechanism for the reduction of CSP in transformed CEF. Peterkofsky (10) and Levinson et al. (11) have shown that the decreased collagen content of transformed cells may also be attributed to decreased synthesis. Other proteins have been found to increase after transformation of CEF; among these are two glucose-regulated membrane proteins with molecular weights of 95,000 (GRP-95) and 78,000 (GRP-78) (4,(18)(19)(20).In this study, we have measured the relative amounts of translatable mRNA coding for these transformation-sensitive proteins by extracting RNA from normal and transformed CEF and translating the mRNA in a cell-free system prepared from wheat germ. The translation products were analyzed by immunoprecipitation, collagenase digestion, and sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electropho...

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“…Recently, a cell surface iodinated protein with nominal molecular weight of about 250,000 was shown to be either undetectable or reduced in various viral-transformed fibroblasts (14)(15)(16)(17)(18)(19)(20). This protein has been designated as LETS (large external protein that is transformation sensitive) protein (21) or Z protein (16) or CSP (cell surface protein) (19).…”

mentioning

confidence: 99%

“…This protein has been designated as LETS (large external protein that is transformation sensitive) protein (21) or Z protein (16) or CSP (cell surface protein) (19). More recently it has been shown (22) that LETS protein is closely related to a previously identified plasma protein, cold insoluble globulin (CIG) (23,24).…”

mentioning

confidence: 99%

Correlation between tumor induction and the large external transformation sensitive protein on the cell surface.

Chen¹,

Gallimore²,

McDougall³

1976

Proc. Natl. Acad. Sci. U.S.A.

181

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The Chick Fibroblast Cell Surface after Transformation by Rous Sarcoma Virus

Cited by 72 publications

References 0 publications

Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture.

Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture.

Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts.

Correlation between tumor induction and the large external transformation sensitive protein on the cell surface.

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