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Altered Effects of Glucocorticoids on the Trafficking and Processing of Mouse Mammary Tumor Virus Glycoproteins Constitutively Expressed in Rat Hepatoma Cells in the Absence of Nonglycosylated Viral Components

Emily J. Platt^*, Laurie J. Goodman^*, Steven R. Kain, Karen Singer Zettl and Gary L. Firestone

Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley Berkeley, California 94720

Address requests for reprints to: Dr. Gary L. Firestone, Department of Molecular and Cell Biology, Box 591 LSA, University of California at Berkeley, Berkeley, California 94720.

Abstract

We have documented previously that glucocorticoid hormones modulate the posttranslational localization of cell surface mouse mammary tumor virus (MMTV) glycoproteins in the viral-infected M1.54 rat HTC hepatoma cell line. To determine whether glucocorticoids affect the trafficking of individually synthesized MMTV glycoproteins, HTC cells were transfected with a constitutively expressed MMTV glycoprotein gene lacking the viral phosphoprotein and polymerase genes. This construct also allows equivalent levels of MMTV glycoproteins to be compared in the presence or absence of glucocorticoids. Indirect immunofluorescence and immunoprecipitation of radiolabeled cells revealed that in transfected cells the transmembrane MMTV glycoproteins are efficiently expressed, transported to the cell surface, and proteolytically cleaved in the presence or in the absence of the synthetic glucocorticoid dexamethasone. Cell surface immunoprecipitation of [³⁵S]methionine-labeled cells showed that the level of plasma membrane gp78 appeared to be stimulated 2-fold after dexamethasone treatment, even though fluorescence-activated cell sorting revealed no discernible change in the total concentration of cell surface MMTV glycoproteins. Analysis of oligosaccharide side chain maturation through a pulsechase radiolabeling revealed that the rate of rough endoplasmic reticulum-Golgi transport was essentially identical in dexamethasone-treated and untreated transfected cells and was similar to that observed in dexamethasone-treated M1.54 cells. Thus, in contrast to viral-infected hepatoma cells, mostly constitutive cellular machinery mediates the trafficking and maturation of cell surface MMTV glycoproteins expressed outside of the proviral context. Taken together, our results suggest that the glucocorticoid-stimulated synthesis of nonglycosylated viral components may contribute to or be responsible for the regulated trafficking of MMTV glycoproteins observed in viral-infected rat hepatoma cells.

FOOTNOTES

This research was supported by NIH Grant DK-42799 and in part by funds awarded from the Lucille P. Markey Program in Biomolecular Structure and Design.

^* Predoctoral trainees supported by NIH National Research Service Grant CA-09041.

Supported by NCI Postdoctoral Research Fellowship F32-CA-09053.

Predoctoral trainee supported by NIH National Institute of General Medical Sciences Grant GM-07379 in Systems and Integrative Biology.

Received for publication June 24, 1991. Revision received September 4, 1991. Accepted for publication September 5, 1991.

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