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• W2169938286 abstract "Article15 December 1997free access Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for Ras in regulating the expression of muscarinic receptors and G proteins Albert P. Gadbut Albert P. Gadbut Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Leeying Wu Leeying Wu Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dongjiang Tang Dongjiang Tang Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Alexander Papageorge Alexander Papageorge Laboratory of Cellular Oncology, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author John A. Watson John A. Watson Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA Search for more papers by this author Jonas B. Galper Corresponding Author Jonas B. Galper Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Albert P. Gadbut Albert P. Gadbut Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Leeying Wu Leeying Wu Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Dongjiang Tang Dongjiang Tang Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Alexander Papageorge Alexander Papageorge Laboratory of Cellular Oncology, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author John A. Watson John A. Watson Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA Search for more papers by this author Jonas B. Galper Corresponding Author Jonas B. Galper Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Albert P. Gadbut1, Leeying Wu1, Dongjiang Tang1, Alexander Papageorge2, John A. Watson3 and Jonas B. Galper 1 1Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA 2Laboratory of Cellular Oncology, National Institutes of Health, Bethesda, MD, USA 3Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA The EMBO Journal (1997)16:7250-7260https://doi.org/10.1093/emboj/16.24.7250 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info We propose a novel mechanism for the regulation of the processing of Ras and demonstrate a new function for Ras in regulating the expression of cardiac autonomic receptors and their associated G proteins. We have demonstrated previously that induction of endogenous cholesterol synthesis in cultured cardiac myocytes resulted in a coordinated increase in expression of muscarinic receptors, the G protein α-subunit, G–αi2, and the inward rectifying K+ channel, GIRK1. These changes in gene expression were associated with a marked increase in the response of heart cells to parasympathetic stimulation. In this study, we demonstrate that the induction of the cholesterol metabolic pathway regulates Ras processing and that Ras regulates expression of G-αi2. We show that in primary cultured myocytes most of the RAS is localized to the cytoplasm in an unfarnesylated form. Induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of RAS. Studies of Ras mutants expressed in cultured heart cells demonstrate that activation of Ras by induction of the cholesterol metabolic pathway results in increased expression of G-αi2 mRNA. Hence farnesylation of Ras is a regulatable process that plays a novel role in the control of second messenger pathways. Introduction The relationship between the availability of low density lipoprotein (LDL) cholesterol and the control of cholesterol biosynthesis has been well established (Brown and Golststein, 1980). We have demonstrated previously that induction of endogenous cholesterol synthesis in embryonic chick heart cells by culture in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a 40% increase in total cell cholesterol (Haigh et al., 1988). This change in cholesterol was associated with profound effects on cardiac physiology and gene expression. Specifically, induction of the cholesterol metabolic pathway was associated with a 10-fold increase in the efficacy of the muscarinic cholinergic agonist carbamylcholine in decreasing the rate of myocyte contraction and a reciprocal decrease in the ability of the β-adrenergic agonist isoproterenol to increase the force of contraction (Haigh et al., 1988; Barnett et al., 1989). The increased responsiveness to carbamylcholine seen in heart cells following induction of cholesterol biosynthesis was associated with an increase in expression of components of the parasympathetic pathway, i.e. high affinity muscarinic receptors and the G protein α-subunit G-αi2 (Haigh et al., 1988), and an increase in levels of mRNAs coding for the M2 muscarinic receptor, G-αi2 and the G protein-coupled inward rectifying K+ channel, GIRK1 (Gadbut et al., 1994). The decreased responsiveness to isoproterenol following induction of cholesterol biosynthesis was associated with a coordinated decrease in components of the sympathetic response system, i.e. a decrease in β-adrenergic receptors and the G protein α–subunit G-αs (Barnett et al. 1989). Mevinolin, a highly specific inhibitor of HMG CoA reductase (Figure 1), reversed all the effects of induction of endogenous cholesterol biosynthesis on cardiac autonomic physiology and receptor, G protein and K+ channel expression (Haigh et al., 1988; Barnett et al., 1989; Gadbut et al., 1994). These findings suggested the existence of a program of gene expression in which components of second messenger pathways are regulated by sterol metabolism. The experiments outlined in this study attempt to determine the mechanism by which regulation of the cholesterol metabolic pathway regulates expression of these genes. Figure 1.Cholesterol metabolic pathway. The schematic representation of the cholesterol biosynthetic pathway includes a number of cholesterol by-products including dolicholphosphate and ubiquinone. Note the sites of action of mevinolin, BZA and TMD. Download figure Download PowerPoint Farnesylpyrophosphate (FPP) is a cholesterol precursor as well as an intermediate in four other isoprenoid-dependent metabolic pathways. One of these FPP-dependent pathways is responsible for the post-translational modification and membrane localization of proteins such as Ras, Rac1 and nuclear lamin A and B (Figure 1; Hancock et al., 1989; for review, see Clarke, 1992). The requirement of FPP for the processing of these proteins has established a new link between cholesterol metabolism and the activity of these molecules. The farnesylation of Ras is catalyzed by farnesyltransferase (FPTase), a cytoplasmic heterodimer composed of a 48 kDa α-subunit and a 46 kDa β-subunit (Reiss et al., 1991). FPTase catalyzes the transfer of the 15 carbon farnesyl moiety of FPP to the carboxy-terminal cysteine of proteins expressing a CAAX motif, where C is cysteine, A is an aliphatic amino acid and X is either serine, glutamine or methionine. Proteins ending in CAAX, where X is leucine, are substrates for another enzyme, geranyl-geranyl transferase, which catalyzes the covalent linkage of a 20 carbon geranyl-geranyl moiety (Figure 1) to the carboxy-terminal cysteine (for review, see Zhang and Casey, 1996). Following farnesylation of Ras, the terminal tripeptide is cleaved and the resulting carboxy-terminal cysteine is carboxymethylated (Gutierrez et al., 1989). The farnesylated form of Ras serves as substrate for palmityl-thiotransferase, which catalyzes the binding of palmitic acid via a relatively labile thioester linkage to cysteines at positions 181 and 184 in H-Ras (Hancock et al., 1989). Although farnesylation is necessary for the association of Ras with the membrane, palmitoylation is required for efficient membrane binding (Casey et al., 1989; Guttierez et al., 1989; Dudler and Gelb, 1996). Palmitoylation, which takes place at the membrane, has been shown to be a rapidly reversible and regulatable process (Milligan et al., 1995; Wedegaertner et al., 1996). Until now, farnesylation of Ras, which involves the formation of a stable thioether linkage and takes place in the cytoplasm, has been presumed to rapidly follow protein synthesis and to be essentially complete and irreversible, followed by rapid association of Ras with the membrane. These observations supported the conclusion that farnesylaton is not a regulatable process (Kohl et al., 1993). However, most studies of Ras processing have been carried out in rapidly dividing transformed cells. Recently, Agarwal et al. (1993) demonstrated that in non-transformed murine epidermal cells, Ras may be found in the cytoplasm. If the processing of a cytoplasmic Ras is limiting in non-transformed cells, regulation of cholesterol metabolism might play a role in controlling the farnesylation of cytoplasmic Ras and its localization to the membrane. In this study, we will demonstrate that in a primary cultured cardiac myocyte, a large fraction of Ras is found unprocessed in the cytoplasm. The data further suggest that farnesylaton and membrane association of Ras are regulatable under the control of the cholesterol biosynthetic pathway. We will demonstrate further that regulation of Ras processing represents a novel mechanism for the control of expression of genes coding for proteins involved in the modulation of the parasympathetic response of the heart. Results Determination of the mevalonate-dependent pathway responsible for increased expression of mRNAs coding for M2 and G-αi2 following induction of cholesterol synthesis To determine whether expression of mRNAs coding for M2 muscarinic receptors and G-αi2 was coordinately regulated by induction of the cholesterol metabolic pathway, total RNA from atrial myocytes cultured in medium supplemented with fetal calf serum (FCS) or LPDS was analyzed by RNase protection. Growth with LPDS resulted in a coordinated increase in levels of mRNAs coding for both M2 and G-αi2, compared with those in cells cultured in FCS (Figure 2a, lanes 2 and 3). Mevinolin, an inhibitor of HMG CoA reductase, reversed the increase in levels of mRNA coding for M2 and G-αi2 due to growth with LPDS to levels below those seen in cells grown in medium supplemented by FCS (Figure 2a, lane 4). These data are typical of five other experiments. Figure 2.(a) Effects of growth in medium supplemented with LPDS, LPDS + BZA-5B or LPDS + TMD on levels of mRNA coding for M2 and G-αi2. Atrial cells from hearts 14 days in ovo were cultured for 3 days in medium supplemented with FCS (lane 2), LPDS (lane 3), LPDS + 30 μM mevinolin (lane 4), LPDS + 50 μM TMD (lane 5) or LPDS + 50 μM BZA-5B (lane 6), added on the second day of culture. Antisense riboprobes were either undigested (lane 1) or hybridized to total RNA from cultures (lanes 2–6) or to tRNA (lane 7). In samples hybridized to M2 and G-αi2 riboprobes, 15 μg of total RNA were used. The GAPDH riboprobe was hybridized with 10 μg of total RNA. Radiographic exposure time for M2 and G-αi2 was 3 days and exposure time for GAPDH was 6 h. The intensity of the bands protected by the antisense riboprobe to GAPDH was identical for cells grown in FCS and LPDS, indicating equal loading of RNA. (b) Effects of TMD on levels of total cell cholesterol in cells grown in FCS and LPDS. Atrial cells were incubated in medium supplemented with FCS (bar 1), LPDS (bar 2). LPDS + 2 μg/ml TMD (bar 3) or LPDS + 500 ng/ml TMD (bar 4). TMD was added on the second day of culture. Levels of total cell cholesterol were determined by the method of Heider and Boyett (1978) on cells harvested on the third day of culture. Data are typical of two studies. Download figure Download PowerPoint The cholesterol metabolic pathway gives rise to FPP, which serves as a precursor to cholesterol and at least four other products (Clarke, 1992; Figure 1). Since mevinolin inhibits the synthesis of cholesterol at the level of mevalonate, it does not differentiate between any of the FPP–dependent pathways. To determine which of the FPP–dependent pathways was responsible for the increased expression of mRNAs coding for M2 and G-αi2, atrial myocytes were cultured in the presence of LPDS plus inhibitors of two of these pathways: TMD, an inhibitor of 2,3 oxido-squalene cyclase (Chang et al., 1979), which catalyzes the conversion of squalene to lanosterol; and BZA-5B, a specific inhibitor of FPTase (James et al., 1993), the enzyme which catalyzes the covalent binding of a farnesyl moiety to cellular proteins ending in the CAAX motif (Figure 1). TMD had no effect on levels of mRNAs coding for M2 and G-αi2 in cells cultured with LPDS (Figure 2a, column 5). This suggested that either the cholesterol branch of the pathway was not involved in regulating M2 and G-αi2 mRNA expression, or that TMD was incapable of inhibiting 2,3 oxido-squalene cyclase in these cells. However, while growth of cells in LPDS resulted in at least a 30% increase in total cell cholesterol compared with cells grown with FCS (Figure 2b, bars 1 and 2), TMD decreased cholesterol by 70% in these cells (Figure 2b, bars 3 and 4). Hence the increase in levels of mRNAs coding for M2 and G-αi2 following induction of the cholesterol metabolic pathway was not dependent on the level of cellular cholesterol. In cells grown in the presence of LPDS plus 50 μM BZA-5B, levels of mRNAs coding for M2 and G-αi2 decreased below those seen in control cells (Figure 2a, lane 6). These data suggested that a farnesylated protein was critical for the increased expression of G-αi2 and M2 mRNAs in response to induction of the cholesterol metabolic pathway. Effect of induction of the cholesterol metabolic pathway on post-translational modification and cellular distribution of Ras Of those proteins which require farnesylation for their association with the membrane, we reasoned that Ras was the protein most likely to play a role in the regulation of second messenger pathways. Since farnesylation of Ras has been shown to be necessary for both translocation of Ras to the cell membrane and for Ras activity (Hancock et al., 1989; Lowy and Willumsen, 1993), we further reasoned that if BZA-5B decreased the expression of M2 and G-αi2 mRNAs via inhibition of FPTase, then induction of the cholesterol metabolic pathway might increase the expression of M2 and G-αi2 mRNA via an increase in the farnesylation of Ras. To test this hypothesis, we first determined the effect of stimulation of the cholesterol pathway on the processing and localization of Ras. Analysis of the distribution of Ras between cytoplasmic and membrane fractions demonstrated that most of the Ras from cells cultured in FCS was found in the cytoplasm and migrated as a doublet on polyacrylamide gel electrophoresis (PAGE) (Figure 3a, lane A). The small amount of Ras found in the membrane fraction (Figure 3a, lane B) migrated faster than that in the cytoplasm and also often migrated as a doublet. Since Ras antibody Y-238 used in these studies cross-reacts with both H- and K-Ras (Shen et al., 1987), the cytoplasmic doublet in Figure 3a could represent unfarnesylated H- and K-Ras. Several alternative explanations are possible, however, Western blot analysis of cytoplasmic extracts with commercially available K-Ras (B) antibody did not give interpretable results. It has been demonstrated that on PAGE, farnesylated Ras migrated ahead of unprocessed Ras. Hence, the doublet could represent processed and unprocessed Ras. Furthermore, partial palmitoylation of cytoplasmic Ras might also lead to a doublet on PAGE. Finally, several studies have suggested that treatment of cells with phorbol esters might lead to the generation of a phosphorylated Ras, which migrated behind unphosphorylated Ras (Ballester et al., 1987; Jeng et al., 1987). Figure 3.(a) Effects of LPDS on the farnesylation and localization of Ras. Atrial cells were cultured for 2 days in medium supplemented with either FCS (lanes A and B), LPDS (lanes C and D) or LPDS + 50 μM BZA-5B (lanes E and F). On the second day of culture, cells were metabolically labeled with [35S]methionine for 16 h. On the third day, cells were homogenized, and membrane and cytoplasmic fractions prepared as described in Materials and methods. Extracts were immunoprecipitated with monoclonal antibody Y-238 specific for H–Ras, but which also cross-reacted with K-Ras. As a control, homogenates were incubated with an equal volume of DMEM supplemented with 6% FCS (data not shown). Samples were analyzed by PAGE followed by autoradiography. Between 21 and 35 kDa, only two bands were precipitated specifically by Y-238. This gel is representative of five separate experiments. Lanes A, C and E are from the cytoplasmic fraction; lanes B, D and F are from the membrane fraction. (b) In vivo labeling of Ras by [3H]mevalonic acid. Embryonic chick atrial cells 14 days in ovo were cultured for 2 days in medium supplemented with FCS. On the second day of culture, medium was made 0.9 mCi/ml in [3H]mevalonate. On the third day, cells were washed, and cytoplasm and membrane were prepared followed by immunoprecipitation, PAGE and autoradiography as described in Materials and methods. Lane A, cytoplasm; lane B, membrane. Data are typical of three experiments. Download figure Download PowerPoint In cells cultured in LPDS, membrane-associated Ras was markedly increased compared with that in membranes from cells cultured in FCS (Figure 3a, compare lanes B and D). One interpretation of these findings is that in cultured chick atrial cells, unprocessed Ras accumulates in the cytoplasm and the induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of cytoplasmic Ras. If such a mechanism for the regulation of Ras localization did exist, then cytoplasmic Ras must be unprocessed and farnesylatable, and membrane-associated Ras must be farnesylated. Processing of cytoplasmic and membrane-associated Ras If membrane-associated Ras in chick atrial cells is farnesylated, then inhibition of FPTase by BZA-5B should result in a decrease in the Ras localized to the membrane. Incubation of cells in LPDS plus 50 μM BZA-5B resulted in the almost complete disappearance of membrane-associated Ras and a marked increase in cytoplasmic Ras (Figure 3a, lanes E and F). The Ras in the cytoplasm of BZA-5B-treated cells also migrated as a doublet. However, these bands appeared to migrate more slowly than those in control cells and in cells incubated with LPDS (Figure 3a, compare lane E with lanes A and C). Cytoplasmic Ras in cultured atrial cells is unprocessed and farnesylatable The finding that inhibition of FPTase with BZA-5B did not decrease the level of cytoplasmic Ras, but rather caused it to increase (Figure 3a, lanes E and F), supports the conclusion that cytoplasmic Ras is unfarnesylated. To test directly the hypothesis that cytoplasmic Ras is unfarnesylated, cells were incubated for 16 h in FCS plus 0.9 mCi/ml of 5′ [3H]mevalonate. Fractionation of cells into membrane and cytoplasm followed by immunoprecipitation with antibody Y-238 demonstrated that >95% of the [3H]mevalonate incorporated into Ras was found in the membrane fraction (Figure 3b). Furthermore, in cells labeled with [3H]mevalonate and incubated in LPDS plus 50 μM BZA-5B, no [3H]mevalonate was incorporated into cytoplasmic Ras (data not shown). To determine further whether Ras accumulated in the cytoplasm of cultured atrial myocytes in an unprocessed and farnesylatable form, we studied the ability of cytoplasmic Ras to serve as a substrate for FPTase. Equal amounts of cytoplasmic protein from cultured atrial cells or NIH-3T3 cells were incubated with purified FPTase and [3H]FPP followed by PAGE and autoradiography (Figure 4a). Atrial cell cytoplasmic extracts demonstrated a radiolabeled band which migrated in parallel with purified Ras radiolabeled with [3H]FPP (Figure 4a, lanes A and B). No Ras band was detected in NIH-3T3 cell cytoplasm (Figure 4a, lane C), a cell in which Ras is in high abundance and almost fully farnesylated (Kohl et al., 1993). Since FPTase might catalyze the farnesylation of other low molecular weight proteins, the Ras from [3H]FPP-labeled cytoplasm was immunoprecipitated with antibody Y-238 followed by PAGE and autoradiography. Radiolabeled bands migrated as expected for Ras (data not shown). A farnesylatable Ras could also be detected in the cytoplasm of atrial cells cultured in medium supplemented with LPDS (data not shown). Although it was not possible to quantitate the fraction of total atrial cytoplasmic Ras which was farnesylatable, these data demonstrate that a significant fraction of cytoplasmic Ras in cultured atrial cells was in the native unfarnesylated form. Figure 4.(a) In vitro farnesylation of atrial cell cytoplasmic Ras. Cytoplasm was prepared from atrial cells 14 days in ovo cultured in medium supplemented with FCS. Cytoplasm was dialyzed and concentrated on a Centriprep-10 microconcentrator (Amicon, Beverly, MA) and 88 μg of cytoplasmic protein from heart cell or NIH-3T3 cell cytoplasm were incubated with [3H]FPP (22 Ci/mM, Dupont NEN) and purified FPTase for 30 min at 37°C, followed by PAGE and autoradiography. Lane A, 25 ng of purified Ras; lane B, chick atrial cytoplasm; lane C, 3T3 cell cytoplasm. (b) Distribution of [3H]palmitate-labeled Ras between cytoplasm and membrane. Embryonic chick atrial cells were incubated in medium supplemented with FCS. On the second day of culture, medium was made 0.5 mCi/ml in [3H]palmitate. On the third day, cells were harvested, membrane and cytoplasm prepared and Ras immunoprecipitated followed by PAGE and autoradiography. Lane A, cytoplasm; lane B, membrane fraction. Data are typical of three similar experiments. Download figure Download PowerPoint Cytoplasmic Ras is neither palmitoylated nor phosphorylated The absence of [3H]mevalonate incorporation into cytoplasmic Ras strongly supports the conclusion that the cytoplasmic Ras doublet demonstrated in Figure 3a does not represent a mixture of farnesylated and unfarnesylated Ras or processed Ras which had become depalmitoylated and cycled back to the cytoplasm. To rule out further the possibility that cytoplasmic Ras represents a partially processed form of Ras, a further series of experiments was carried out. Cultured atrial cells were incubated overnight with [3H]palmitate, and incorporation into cytoplasmic and membrane-associated Ras was determined. Data summarized in Figure 4b demonstrate that all of the palmitate incorporated into Ras was found in the membrane fraction. To rule out the possibility that the cytoplasmic doublet represents phosphorylated and unphosphorylated Ras, cells were metabolically labeled with either [35S]methionine or 32PO4, followed by immunoprecipation with antibody Y–238. Although membrane and cytoplasmic Ras were labeled with [35S]methionine, no phosphorylated Ras could be detected in either the cytoplasmic or membrane fraction (data not shown). The finding that cytoplasmic Ras in BZA-5B-treated cells appeared to migrate more slowly than Ras from the cytoplasm of control cells or cells incubated with LPDS (Figure 3a, lane E) suggested that BZA-5B treatment might result in the accumulation of a processed form of Ras in the cytoplasm. As described above, no [3H]mevalonate-labeled Ras could be detected in the cytoplasm of cells incubated in LPDS plus BZA-5B. Furthermore, incubation of cells with BZA-5B and [3H]palmitate or 32PO4 did not result in the appearance of either farnesylated, palmitoylated or phosphorylated Ras in the cytoplasm (data not shown). Thus, the shift in electrophoretic mobility in Ras from the cytoplasm of BZA-5B-treated cells is not due to any known modification of Ras. These data support the conclusion that most of the Ras in atrial cytoplasm represents two species which are neither farnesylated, palmitoylated nor phosphorylated. At least a portion of this cytoplasmic Ras may serve as a substrate for FPTase. The finding that no farnesylateable Ras could be found in the cytoplasm of rapidly dividing NIH-3T3 cells suggested that unfarnesylated Ras might not be found in rapidly dividing cells. The finding that Ras accumulated in an unfarnesylated form in the cytoplasm of primary cultured heart cells and that induction of the cholesterol metabolic pathway resulted in an increase in the level of farnesylated membrane-associated Ras supported the conclusion that the cholesterol metabolic pathway may control Ras activity by regulating the processing of cytoplasmic Ras. Regulation of G-αi2 promoter activity by induction of the cholesterol metabolic pathway Data presented in Figures 2a and 3a demonstrated a striking parallel between changes in expression of mRNAs coding for M2 and G-αi2 and changes in the membrane association of Ras in response to both induction of the cholesterol metabolic pathway and to inhibition of FPTase activity by BZA-5B. To determine whether an increase in membrane-associated Ras actually played a role in the increased expression of M2 and G-αi2, the effect of LPDS and Ras mutants on G-αi2 promoter activity in atrial myocytes was studied. Cultured chick atrial cells 14 days in ovo were transfected with a construct containing 1.8 kb of 5′ flanking sequence of chick G-αi2 genomic DNA ligated to a luciferase reporter (αi2-Luc). Growth of these cells in medium supplemented with LPDS resulted in a 2.5 ± 0.5- (± SEM, n = 7) fold increase in luciferase activity compared with cells grown in the presence of FCS (Figure 5, bars 1 and 2). Luciferase activity in cells transfected with a promoterless luciferase was no different from baseline in either FCS or LPDS. Hence, induction of the cholesterol metabolic pathway increased G-αi2 expression at least in part by an effect on the G-αi2 promoter. Figure 5.Effect of LPDS and Ras mutants on G-αi2 promoter activity. Embryonic chick atrial cells were co-transfected with αi2-Luc and a human placental alkaline phosphatase (PAP) with or without Val12 Ras or Asn17 Ras. On the third day of culture, cells were harvested and luciferase activity was determined and normalized to human PAP activity. Data are reported as the ratio of luciferase activity to PAP with cells cultured in FCS taken as 1. Bars: 1, cells cultured in FCS; 2, cells cultured in LPDS; 3, cells cultured in FCS and transfected with Val12 Raas and inhibited with 50 μM BZA-5B added 16 h before harvesting; 4, cells cultured in FCS and transfected with Val12 Ras; 5, cells cultured with LPDS and transfected with Asn17 Ras. Download figure Download PowerPoint Effect of Ras mutants on Gαi2 promoter activity In order to determine whether the stimulation of the G–αi2 promoter by LPDS was dependent on Ras, cells co-transfected with αi2-Luc and a dominant-negative Ras mutant (Asn17 Ras, Feig and Cooper, 1988) were cultured in LPDS. Co-expression of αi2-Luc plus Asn17 Ras resulted in a decrease in luciferase activity to the levels seen in cells grown in FCS. Hence expression of Asn17 Ras reversed the effect of induction of the cholesterol metabolic pathway on stimulation of G-αi2 promoter activity (Figure 5, bars 1, 2 and 5). Co-expression of Asn17 Ras and αi2-Luc in cells cultured in medium supplement with FCS resulted in a 20% decrease in luciferase activity compared with cells transfected with αi2-Luc alone (data not shown). These data suggest that only a fraction of basal Gαi2 promoter activity is Ras dependent. In order to determine whether Ras mimicked the effect of growth in LPDS on G-αi2 promoter-driven luciferase activity, cells were cultured in FCS and co-transfected with αi2-Luc plus a dominant active Ras (Val12 Ras; Lowy and Willumsen, 1993). Expression of Val12 Ras increased luciferase activity 2.4 ± 0.3- (± SEM, n = 5) fold compared with cells transfected with αi2-Luc alone (Figure 5, bar 4). Hence, Val12 Ras mimicked the effect of growth in LPDS on regulation of the G-αi2 promoter. Co-transfection of atrial cells cultured in medium supplemented with FCS with αi2-Luc plus Val12 Ras followed by an overnight incubation with 50 μM BZA-5B resulted in a 70% decrease in Val12 Ras-stimulated luciferase activity (Figure 5, bar 3). Although it is not possible quantitatively to compare steady-state levels of αi2 mRNA and transcription rates as measured by αi2 promoter-driven Luciferase activity, these data support the conclusion that like LPDS-stimulated αi2 production, the stimulation of αi2-Luc in response to Val12 Ras was also BZA-5B inhibitable and hence dependent on FPTase activity and hence must require membrane association of the mutant Ras. Co-transfection of cells cultured in LPDS with both Val12 Ras and αi2-Luc did not increase luciferase activity significantly above that in cells cultured with LPDS and tr" @default.
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• W2169938286 title "Induction of the cholesterol metabolic pathway regulates the farnesylation of RAS in embryonic chick heart cells: a new role for Ras in regulating the expression of muscarinic receptors and G proteins" @default.
• W2169938286 cites W1488144593 @default.
• W2169938286 cites W1490645937 @default.
• W2169938286 cites W1520481055 @default.
• W2169938286 cites W1522080250 @default.
• W2169938286 cites W1538938046 @default.
• W2169938286 cites W1546578920 @default.
• W2169938286 cites W1549467326 @default.
• W2169938286 cites W1558848958 @default.
• W2169938286 cites W1584939531 @default.
• W2169938286 cites W1607745865 @default.
• W2169938286 cites W1613577755 @default.
• W2169938286 cites W1679229316 @default.
• W2169938286 cites W1775749144 @default.
• W2169938286 cites W1819297632 @default.
• W2169938286 cites W1860242155 @default.
• W2169938286 cites W1935382092 @default.
• W2169938286 cites W1980290321 @default.
• W2169938286 cites W1993817333 @default.
• W2169938286 cites W2002701625 @default.
• W2169938286 cites W2003267467 @default.
• W2169938286 cites W2020341627 @default.
• W2169938286 cites W2036082581 @default.
• W2169938286 cites W2037480365 @default.
• W2169938286 cites W2041188540 @default.
• W2169938286 cites W2050460102 @default.
• W2169938286 cites W2053854358 @default.
• W2169938286 cites W2054017818 @default.
• W2169938286 cites W2061398096 @default.
• W2169938286 cites W2073040909 @default.
• W2169938286 cites W207418012 @default.
• W2169938286 cites W2089906643 @default.
• W2169938286 cites W2091948299 @default.
• W2169938286 cites W2092626728 @default.
• W2169938286 cites W2133362776 @default.
• W2169938286 cites W2139802777 @default.
• W2169938286 cites W2141273003 @default.
• W2169938286 cites W2144718273 @default.
• W2169938286 cites W2158608327 @default.
• W2169938286 cites W2166483827 @default.
• W2169938286 cites W2173344695 @default.
• W2169938286 cites W2176744910 @default.
• W2169938286 cites W2178557511 @default.
• W2169938286 cites W2204574216 @default.
• W2169938286 cites W2403390356 @default.
• W2169938286 cites W2407292624 @default.
• W2169938286 cites W759408661 @default.
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