A distinct glucocorticoid hormone response regulates phosphoprotein maturation in rat hepatoma cells.

Abstract

Glucocorticoid hormone-dependent maturation of the mouse mammary tumor virus (MMTV) Glucocorticoid hormones elicit many types of responses in a variety of target tissues and in vitro-cultured cell lines (1, 4, 24; K. R. Yamamoto, Annu. Rev. Genet., in press). These hormonal effects appear to be mediated by specific intracellular receptor proteins; the steroid-receptor interaction potentiates a functional change in the receptor that results in its increased DNA-binding activity and selective recognition of enhancer-like sites at or around genes under glucocorticoid control (1,7,10,18,26,(31)(32)(33)(34)(35)(36)(37)44,52; Yamamoto, in press). As a result of this apparent sequence specificity for glucocorticoid-receptor complexes, the rate of transcription of glucocorticoid-regulated genes is selectively modulated and accounts for gene expression changes of certain steroid responses (1; Yamamoto, in press). However, the mechanisms of most glucocorticoid responses are unknown, and many responses may result from secondary or regulatory steroid effects in which functional gene products under direct transcriptional control affect the expression of other biologically active genes at posttranscriptional or posttranslational levels. For example, the accumulation of alpha,-acid glycoprotein mRNA appears to result from selective message stabilization after glucocorticoid treatment (50), while steroid-mediated changes in the levels or activities of specific processing enzymes have also been reported (12, 27, 45); mechanisms of these regulated processes are largely unknown. We have been using the expression and subsequent maturation of mouse mammary tumor virus (MMTV) precursor polyproteins in virally infected cell lines as versatile and sensitive probes to explore posttranslational regulatory circuits under steroid control (16,17 (17).To begin to assess the molecular details and cellular requirements of the steroid-regulated posttranslational path-574 on May 10, 2018 by guest

Glucocorticoid-regulated compartmentalization of cell surface-associated glycoproteins in rat hepatoma cells: evidence for an independent response that requires receptor function and de novo RNA synthesis.

“…One mechanism consistent with the observed glucocorticoid-dependent production of cell surface viral glycoproteins is that the hormone-mediated increase in absolute glycoprotein concentrations, via induction of MMTV RNA synthesis (21,36,42,44), may bring the concentration of the precursor above a critical Km range of a pre-existing sorting or maturation factor. To test this notion genetically, we examined hormone-regulated expression of viral glycoproteins in glucocorticoid-responsive transcriptional variants derived by complement cytolysis from wild-type M1.54.…”

“…5, graph). Half-maximal expression of cell surface-associated MMTV species occurred at approximately 50 nM dexamethasone, whereas total MMTV proteins were produced with half-maximal induction of approximately 20 nM, which closely correlated with half-maximal receptor occupancy (21). Half D, I, d, and i).…”

“…The dependency on glucocorticoid receptors for regulation of MMTV glycoprotein compartmentalization was in- [2,21]) and production of cell surface viral glycoproteins (half-maximal response, 40 to 50 nM), although half-maximal production of cell surface viral glycoproteins was shifted to slightly higher concentrations of dexamethasone. Furthermore, steroid-regulated expression of cell surface MMTV glycoproteins correlated with the biological properties of known glucocorticoid agonists and antagonists.…”

“…In both induced and uninduced cells, the glycosylated MMTV polyprotein REGULATED TRAFFICKING OF GLYCOPROTEINS (encoded by the viral env gene) is translated from a subgenomic 3.8-kilobase MMTV RNA transcript while a phosphorylated precursor species is synthesized from the genomic 7.8-kilobase transcript. In addition to increasing the absolute level of each precursor polyprotein, dexamethasone is also required for their full posttranslational processing and maturation (13,14,21). Thus, in the presence of hormone, three new cell surface glycoproteins (gp78, gp7O, and gp32) and an extracellular species (gp7O) are derived from a common glycosylated polyprotein (Pren,V).…”

“…Moreover, two new intracellular phosphoproteins (p35 and p24), which are proteolytically derived from the phosphorylated precursor, can also be detected. Selection and characterization of M1.54 variants that fail to produce the cell surface MMTV glycoproteins have demonstrated that glucocorticoid-regulated production of MMTV cell surface and secreted glycoproteins can be genetically uncoupled from the hormonal effects on MMTV transcription and phosphoprotein maturation (13)(14)(15)21 Immunoadsorption of detergent-solubilized cells. We solubilized radiolabeled cells by sonicating cell pellets (107 cells) in 1 ml of 1% Triton X-100-0.5% deoxycholate-5 mM EDTA-250 mM NaCl-25 mM Tris hydrochloride (pH 7.5) (solubilization buffer); cell homogenates were centrifuged at 20,000 x g for 10 min, and postnuclear supernatants were isolated as solubilized cellular fractions.…”