Characterization of hepatitis C virus (HCV) quasispecies dynamics upon short-term dual therapy with the HCV NS5B nucleoside polymerase inhibitor mericitabine and the NS3/4 protease inhibitor danoprevir.

In the INFORM-1 study, 73 patients with chronic hepatitis C virus infection received mericitabine plus danoprevir for up to 13 days. Seventy-two patients experienced a continuous decline in HCV RNA levels during treatment, and of these patients, 14 had viral loads that remained >1,000 IU/ml by day 13 and 1 met the definition for viral breakthrough. In-depth NS5B and NS3/4A population and clonal sequencing studies and mericitabine and danoprevir drug susceptibility testing were performed to assess the variability and quasispecies dynamics before and upon monotherapy or dual therapy. Sequence analysis of the viral quasispecies indicated that the mericitabine resistance mutation S282T was not present at baseline, nor was it selected (even at a low level) during treatment. Protease inhibitor resistance mutations, either as predominant or as minority species, were detected in 18 patients at baseline. No enrichment of minority protease inhibitor-resistant variants present at baseline was observed during treatment; viral population samples were fully susceptible to mericitabine and/or danoprevir, despite the presence within their quasispecies of minority variants confirmed to have reduced susceptibility to danoprevir or other protease inhibitors. It was also observed that certain NS3 amino acid substitutions affected protease inhibitor drug susceptibility in a compound-specific manner and varied with the genetic context. In summary, the slower kinetics of viral load decline observed in some patients was not due to the selection of danoprevir or mericitabine resistance during treatment. Over 2 weeks' therapy, mericitabine suppressed the selection of danoprevir resistance, results that could differ upon longer treatment periods.

Developmental and metabolic regulation of the Drosophila melanogaster 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in Drosophila melanogaster synthesizes mevalonate for the production of nonsterol isoprenoids, which are essential for growth and differentiation. To understand the regulation and developmental role of HMG CoA reductase, we cloned the D. melanogaster HMG CoA reductase gene. The nucleotide sequence of the Drosophila HMG CoA reductase was determined from genomic and cDNA clones. A 2,748-base-pair open reading frame encoded a polypeptide of 916 amino acids (Mr, 98,165) that was similar to the hamster HMG CoA reductase. The C-terminal region had 56% identical residues and the N-terminal region had 7 potential transmembrane domains with 32 to 60% identical residues. In hamster HMG CoA reductase, the membrane regions were essential for posttranslational regulation. Since the Drosophila enzyme is not regulated by sterols, the strong N-terminal similarity was surprising. Two HMG CoA reductase mRNA transcripts, approximately 3.2 and 4 kilobases, were differentially expressed throughout Drosophila development. Mevalonate-fed Schneider cells showed a parallel reduction of both enzyme activity and abundance of the 4-kilobase mRNA transcript.

Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

Characterization of the nuclear export signal of human T-cell lymphotropic virus type 1 Rex reveals that nuclear export is mediated by position-variable hydrophobic interactions.

We previously determined that amino acids 64 to 120 of human T-cell lymphotropic virus type 1 (HTLV-1) Rex can restore the function of an effector domain mutant of human immunodeficiency virus type 1 (HIV-1) Rev (T. J. Hope, B. L. Bond, D. McDonald, N. P. Klein, and T. G. Parslow, J. Virol. 65:6001-6007, 1991). In this report, we (i) identify and characterize a position-independent 17-amino-acid region of HTLV-1 Rex that fully complements HIV-1 Rev effector domain mutants and (ii) show that this 17-amino-acid region and specific hydrophobic substitutions can serve as nuclear export signals. Mutagenesis studies revealed that four leucines within the minimal region were essential for function. Alignment of the minimal Rex region with the HIV-1 Rev effector domain suggested that the position of some of the conserved leucines is flexible. We found two of the leucines could each occupy one of two positions within the context of the full-length HTLV-1 Rex protein and maintain function. The idea of flexibility within the Rex effector domain was confirmed and extended by identifying functional substitutions by screening a library of effector domain mutants in which the two regions of flexibility were randomized. Secondly, the functional roles of the minimal Rex effector domain and hydrophobic substitutions were independently confirmed by demonstrating that these effector domains could serve as nuclear export signals when conjugated with bovine serum albumin. Nuclear export of the wild-type Rex conjugates was temperature dependent and sensitive to wheat germ agglutinin and was blocked by a 20-fold excess of unlabeled conjugates. Together, these studies reveal that position-variable hydrophobic interactions within the HTLV-1 Rex effector domain mediate nuclear export function.

Increased oligonucleotide permeability in keratinocytes of artificial skin correlates with differentiation and altered membrane function.

We tested the permeability of fluorescent oligonucleotides in cultured human epidermal keratinocyte monolayers and keratinocytes grown in a 3-dimensional skin model. Oligonucleotide permeability in living cells was determined by confocal microscopy after either simple addition to the culture medium or topical application via oligonucleotide-saturated filters placed atop the artificial skin. In cultured monolayers, few keratinocytes (9%) were found to acquire intracellular oligonucleotides that were primarily localized to the nucleus. In contrast, keratinocytes grown in an artificial, 3-dimensional skin matrix acquired extensive oligonucleotide permeability as differentiation progressed. About 95% of the granular cells showed nuclear accumulation of oligonucleotides. About 70% of the oligonucleotide-permeable granular cells were viable as verified by a mitochondria-specific, potential-sensitive dye, tetramethyl rhodamine ethyl ester. A marker used to study apoptotic cells with altered membrane potential, merocyanine 540, was found elevated in the cytoplasm of granular cells. In contrast, cultured keratinocyte monolayers or basal keratinocytes of skin showed a membrane staining pattern typical of undifferentiated cells. Few cells (<3%) of the basal layer had nuclear oligonucleotides, but none of the labeled cells were viable. These results suggest that the development of oligonucleotide and merocyanine 540 permeability in differentiated granular cells parallels the changes in membrane permeability found in other apoptotic systems.

Increased oligonucleotide permeability in keratinocytes of artificial skin correlates with differentiation and altered membrane function.

We tested the permeability of fluorescent oligonucleotides in cultured human epidermal keratinocyte monolayers and keratinocytes grown in a 3-dimensional skin model. Oligonucleotide permeability in living cells was determined by confocal microscopy after either simple addition to the culture medium or topical application via oligonucleotide-saturated filters placed atop the artificial skin. In cultured monolayers, few keratinocytes (9%) were found to acquire intracellular oligonucleotides that were primarily localized to the nucleus. In contrast, keratinocytes grown in an artificial, 3-dimensional skin matrix acquired extensive oligonucleotide permeability as differentiation progressed. About 95% of the granular cells showed nuclear accumulation of oligonucleotides. About 70% of the oligonucleotide-permeable granular cells were viable as verified by a mitochondria-specific, potential-sensitive dye, tetramethyl rhodamine ethyl ester. A marker used to study apoptotic cells with altered membrane potential, merocyanine 540, was found elevated in the cytoplasm of granular cells. In contrast, cultured keratinocyte monolayers or basal keratinocytes of skin showed a membrane staining pattern typical of undifferentiated cells. Few cells (<3%) of the basal layer had nuclear oligonucleotides, but none of the labeled cells were viable. These results suggest that the development of oligonucleotide and merocyanine 540 permeability in differentiated granular cells parallels the changes in membrane permeability found in other apoptotic systems.

Inhibition of human immunodeficiency virus type 1 Rev-Rev-response element complex formation by complementary oligonucleotides.

Complementary 18-mer oligodeoxynucleotides (oligonucleotides) specifically inhibited the formation of human immunodeficiency virus Rev-Rev-response element (RRE) complexes. Inhibition of Rev-RRE binding required blockage of G-7819 to G-7820 in band shift assays. Structural studies revealed both local and distal effects. RRE structure was also disrupted by oligonucleotides targeted to other minor stems, by altering RNA renaturation conditions, or by reducing Rev concentrations--indicating a dynamic RRE structure and involvement of a minor RRE stem in the maturation of initial Rev-RRE complexes. Thus, complementary oligonucleotides alter RRE structure and may prove useful for the design of therapeutic anti-RRE oligonucleotides.

Human immunodeficiency virus type 1 Tat does not transactivate mature trans-acting responsive region RNA species in the nucleus or cytoplasm of primate cells.

Human immunodeficiency virus (HIV)-encoded transactivator Tat is essential for viral gene expression and replication. By interacting with a nascent RNA stem-loop called the trans-acting responsive region (TAR). Tat increases rates of initiation and/or elongation of HIV transcription. Several reports have also suggested that Tat has additional effects on mature HIV RNA species including modification of primary transcripts in the nucleus and their increased translation in the cytoplasm. These posttranscriptional effects are most pronounced in the Xenopus oocyte. To investigate directly whether Tat has similar effects on viral transcripts in cells that are permissive for HIV replication, we cotransfected and microinjected human and monkey cells with Tat and TAR in the form of DNA or RNA. Whereas Tat transactivated TAR DNA targets, it did not transactivate TAR RNA targets in the nucleus of microinjected cells or in the cytoplasm of transfected cells. We conclude that in cells permissive for viral replication, Tat exerts its effect primarily at the level of HIV transcription.

Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) is a single polypeptide chain with two contiguous domains: a soluble domain (548 amino acids) that catalyzes the rate-controlling step in cholesterol synthesis and a membrane-bound domain (339 amino acids) that anchors the protein to the endoplasmic reticulum (ER). HMG CoA reductase is degraded at least 10-fold more rapidly than other ER proteins; degradation is accelerated in the presence of cholesterol. To understand this controlled degradation, we transfected reductase-deficient Chinese hamster ovary (CHO) cells with a plasmid expression vector containing a reductase cDNA that lacks the segment encoding the membrane domain. The plasmid produced a truncated reductase (37 kd smaller than normal) that was enzymatically active with normal kinetics; most of the truncated enzyme was found in the cytosol. The truncated enzyme was degraded one-fifth as fast as the holoenzyme; degradation was no longer accelerated by sterols. We conclude that the membrane-bound domain of reductase plays a crucial role in the rapid and regulated degradation of this ER protein.

Inhibition of human immunodeficiency virus type-1 env expression by C-5 propyne oligonucleotides specific for Rev-response element stem-loop V.

The binding of Rev to the Rev-response element (RRE) of the human immunodeficiency virus (HIV) is essential for RNA transport and expression of structural proteins such as gp160 encoded by env. To determine if env expression could be disrupted by complementary oligodeoxynucleotides (ODNs), band-shift studies were used to identify RRE sites that are essential for the formation of Rev-RRE complexes [Chin, D. J. (1992) J. Virol. 66, 600-607] or the stability of preformed complexes. In this report, we describe complete disruption of preformed Rev-RRE complexes by a subset of 15 ODNs complementary to stem-loop V. The most potent ODN complementary to bases CUGGGGCAUCAAGC disrupted 50% of preformed complexes at 1.2 microM, a 400-fold molar excess over the RNA. Expression of env in COS7 cells was blocked by nuclear microinjection of ODNs with C-5 propyne-modified pyrimidines and phosphorothioate linkages. Inhibition was highly dependent upon RNA target position, internucleotide chemistry, ODN sequence, and concentration. Unmodified phosphodiester or phosphorothioate ODNs were inactive. For the most potent ODN, 50% of the injected cells' env expression (I50) was blocked with 0.1 microM. A translational block is unlikely since these ODNs blocked expression of a luciferase vector in which the RRE was placed downstream of the termination codon. Consistent with their in vitro effects upon Rev-RRE complexes, stem-loop V ODNs were 9-fold more active than stem-loop II ODNs in blocking env expression while having a reduced (I50 = 0.27 microM) but equivalent potency against luciferase-RRE. These results suggest that disruption of Rev-RRE complexes may assist in blocking env expression.

100-kDa polypeptides in peripheral clathrin-coated vesicles are required for receptor-mediated endocytosis.

The role of the 100-kDa polypeptide components of clathrin-coated vesicles in endocytosis was investigated by microinjection of specific monoclonal antibodies. Receptor-mediated uptake of transferrin and liposomes was quantitatively inhibited. These results show that the 100-kDa polypeptides are directly involved in localized clathrin assembly at the cell periphery and are markers for the endocytic pathway. This demonstrates an in situ function of these polypeptides and the protein complexes in which they are found.

Rapid nuclear accumulation of injected oligodeoxyribonucleotides.

The intracellular transport and fate of nucleic acids is poorly understood. To study this process, we injected fluorescent oligodeoxyribonucleotides (oligos) into the cytoplasm of CV-1 epithelial cells and primary human fibroblasts. Rapid nuclear accumulation was found with the phosphodiester (PD), phosphorothioate (PT), and methylphosphonate (MP) forms of a 28-mer oligo complimentary to the rev mRNA of the human immunodeficiency virus type 1. Migration of the oligos in the cytoplasm was slower than diffusion of a coinjected dextran, but the oligos freely diffused into the nucleus. Nuclear incorporation was temperature but not energy dependent. The intranuclear distribution of the oligos was influenced by the chemistry of internucleoside linkages. The PD oligos and, to a lesser extent, the PT oligos colocalized with small nuclear ribonucleoproteins (snRNPs), whereas the MP oligos colocalized with concentrated regions of genomic DNA. These data have important implications for our understanding of the transport and accumulation of exogenous nucleic acids in mammalian nuclei, and the assay described could potentially be used for testing the efficacy of oligos designed as therapeutic agents.

Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET).

Fluorescence resonance energy transfer (FRET) was used to study hybrid formation and dissociation after microinjection of oligonucleotides (ODNs) into living cells. A 28-mer phosphodiester ODN (+PD) was synthesized and labeled with a 3' rhodamine (+PD-R). The complementary, antisense 5'-fluorescein labeled phosphorothioate ODN (-PT-F) was specifically quenched by addition of the +PD-R. In solution, the -PT-F/+PD-R hybrid had a denaturation temperature of 65 +/- 3 degrees C detected by both absorbance and FRET. Hybridization between the ODNs occurred within 1 minute at 17 microM and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T1/2 approximately 3 h) in the presence of a 300-fold excess of the unlabeled complementary ODN and could be degraded by DNAse I. Upon microinjection into the cytoplasm of cells, pre-formed fluorescent hybrids dissociated with a half-time of 15 minutes, which is attributed to the degradation of the phosphodiester. Formation of the hybrid from sequentially injected ODNs was detected by FRET transiently in the cytoplasm and later in the cell nucleus, where nearly all injected ODNs accumulate. This suggests that antisense ODNs can hybridize to an intracellular target, of exogenous origin in these studies, in both the cytoplasm and the nucleus.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and its mRNA in rat liver as studied with a monoclonal antibody and a cDNA probe.

A monoclonal antibody directed against 3-hydroxy-3-methylglutaryl Coenzyme A reductase and a cDNA to reductase mRNA were used to study the subunit structure of the enzyme and the regulation of its mRNA in rat liver. Although the monoclonal antibody and the cDNA were made with materials from cultured hamster cells, the two reagents cross-reacted with reductase protein and mRNA from rat liver. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibody, the subunit molecular weight of rat liver reductase was 90,000. When the enzyme was solubilized from microsomes by freeze-thawing, the subunit molecular weight was reduced to 52,000-58,000, owing to proteolysis. This proteolysis was inhibited by EGTA and leupeptin. The cDNA probe for reductase, radiolabeled with 32P, hybridized to restriction fragments of genomic DNA from rat liver, as visualized by Southern blot analysis. In the livers of control rats, no reductase mRNA was detected when the 32P-cDNA was blot-hybridized to poly(A+) RNA. Hepatic reductase activity was increased 45-fold when rats were fed cholestyramine and mevinolin. Under these conditions, the amount of immunodetectable reductase protein rose by 33-fold, and the reductase mRNA became visible by blot hybridization as a band of approximately 4 kilobases in length. When the mevinolin/cholestyramine-treated rats were fed cholesterol, reductase activity and immunodetectable protein declined markedly and the reductase mRNA was reduced to barely detectable levels. We conclude that treatment with cholestyramine and mevinolin increases the amount of reductase protein in rat liver by elevating the amount of its mRNA and that cholesterol feeding to such induced rats lowers the amount of hepatic reductase protein by decreasing the level of its mRNA.

Regulation of synthesis and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by low density lipoprotein and 25-hydroxycholesterol in UT-1 cells.

UT-1 cells are a clone of Chinese hamster ovary cells that were selected to grow in the presence of compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. These cells have 100- to 1,000-fold more immunoprecipitable reductase than normal. The enzyme activity is rapidly decreased when low density lipoprotein (LDL) or 25-hydroxycholesterol is added to the culture medium. In this current study, a quantitative immunoprecipitation assay was used to determine whether LDL and 25-hydroxycholesterol inhibit the synthesis or stimulate the degradation of reductase in UT-1 cells. Each of these agents inhibited the incorporation of [35S]methionine into immunoprecipitable reductase by more than 98%. Pulse-chase experiments showed that reductase was degraded with a half-life of 10-13 hr in UT-1 cells and that the rate of degradation of preformed enzyme was increased 3-fold by the addition of either LDL or 25-hydroxycholesterol. We conclude that the predominant mechanism by which LDL and 25-hydroxycholesterol decrease reductase activity in UT-1 cells is a profound suppression of synthesis of the enzyme.

Identification of a cholesterol-regulated 53,000-dalton cytosolic protein in UT-1 cells and cloning of its cDNA.

UT-1 cells, a clone of Chinese hamster ovary (CHO) cells, have a 100- to 1,000-fold elevation in the amount of 3-hydroxy-3-methylglutaryl CoA reductase and therefore grow in the presence of compactin, an inhibitor of reductase. In this paper, we report that UT-1 cells also have a markedly increased amount of another protein with a Mr of 53,000 and an isoelectric point of approximately equal to 6. Whereas the reductase is an enzyme of the endoplasmic reticulum, the 53,000-dalton protein (termed the "53k" protein) is in the cytosol. It is not precipitated by an antireductase antibody. Synthesis of the 53k protein, like that of the reductase, is suppressed when UT-1 cells are incubated with plasma low density lipoprotein (LDL). We prepared a library of recombinant plasmids containing double-stranded cDNAs from UT-1 cells. Using differential colony hybridization, we identified recombinant plasmids containing double-stranded cDNA inserts encoding mRNAs expressed at high levels in UT-1 cells as compared with CHO cells. One of the plasmids, designated p53k-3, contained a 0.97-kilobase double-stranded cDNA that hybridized to a 3.8-kilobase mRNA. When translated in vitro, this 3.8-kilobase mRNA directed the synthesis of a protein identical to the cellular 53k protein as determined by two-dimensional gel electrophoresis. Hybridization studies showed that the mRNA for the 53k protein was present in much larger amounts in UT-1 cells than in parental CHO cells. In both cell types, the content of this mRNA decreased markedly when the cells were incubated with LDL. Although the function of the 53k protein is not known, circumstantial evidence suggests that it may represent cytosolic 3-hydroxy-3-methylglutaryl CoA synthase, the enzyme preceeding the reductase in the cholesterol biosynthetic pathway. The current data indicate that the synthesis of at least two proteins, the reductase and the 53k protein, are induced to high levels in compactin-resistant UT-1 cells and that the synthesis of both is suppressed coordinately by LDL.

Appearance of crystalloid endoplasmic reticulum in compactin-resistant Chinese hamster cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We have developed a line of Chinese hamster ovary cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the membrane-bound enzyme that controls cholesterol synthesis. This line, designated (UT-1, was obtained by stepwise adaptation of cells to growth in increasing concentrations of compactin, a competitive inhibitor of reductase. Reductase accounts for approximately 2% of total cell protein in UT-1 cells, as calculated from enzyme specific activity and by immunoprecipitation of reductase after growth of cells in [35S]methionine. After solubilization in the presence of the protease inhibitor leupeptin and electrophoresis in NaDodSO4/polyacrylamide gels, reductase subunits from UT-1 cells were visualized by immunoblotting as a single band (Mr = 62,000). To accommodate the increased amounts of reductase, UT-1 cells developed marked proliferation of tubular smooth endoplasmic reticulum (ER) membranes, as revealed by immunofluorescence and electron microscopy. The ER tubules were packed in crystalloid hexagonal arrays. When UT-1 cells were incubated with low density lipoprotein, reductase activity was suppressed by 90% in 12 hr and the crystalloid ER disappeared. UT-1 cells should be useful for studies of the regulation of reductase and also for studies of the synthesis and degradation of smooth ER.

Molecular cloning of 3-hydroxy-3-methylglutaryl coenzyme a reductase and evidence for regulation of its mRNA.

A recombinant plasmid containing a 1.2-kilobase cDNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated from a cDNA library prepared from UT-1 cells, a clone of Chinese hamster ovary cells that has markedly elevated reductase activity. This plasmid, designated pRed-10, was identified by differential colony hybridization and hybrid-selected mRNA translation. The mRNA that hybridized to pRed-10 directed the synthesis in vitro of a 90,000-dalton protein that was immunoprecipitated by an antireductase antibody. The same 90,000-dalton protein was immunoprecipitated when UT-1 cells were pulse labeled with [35S]methionine in vivo and rapidly solubilized with boiling NaDodSO4. By blot hybridization, pRed-10 hybridized to mRNAs of 4.2 and 4.7 kilobases in UT-1 cells. Both mRNAs were reduced to undetectable levels when low density lipoprotein, a suppressor of the reductase, was present in the culture medium. These data indicate that the primary translation product of reductase mRNA is a 90,000-dalton protein and that LDL suppresses the reductase in UT-1 cells by drastically reducing the level of its mRNA.

Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells.

32P-labeled cDNA probes were used to study levels of genomic DNA and regulation of mRNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT-1 cells, a clone of compactin-resistant Chinese hamster ovary cells that have a 100-1000-fold increase in the amount of reductase protein. Similar measurements were made for the 53-kDa protein, a cytosolic protein of unknown function that is also expressed at high levels in UT-1 cells. The number of copies of the gene for reductase was increased by 15-fold in UT-1 cells as compared to the parental Chinese hamster ovary cells, as judged from Southern gel analysis of restriction endonuclease-digested genomic DNA. In contrast, there was no detectable increase in the number of gene copies for the 53-kDa protein. The amount of cytoplasmic mRNA for both proteins was markedly elevated in UT-1 cells, as determined by filter hybridization studies using 32P-labeled cDNA probes. The amount of mRNA for both reductase and the 53-kDa protein declined in parallel after addition of low density lipoprotein, 25-hydroxycholesterol, or mevalonate to the culture medium. The decline in reductase mRNA was associated with a marked decrease in the rate of [3H]uridine incorporation into hybridizable cytoplasmic mRNA. When UT-1 cells were grown for 3-4 months in the absence of compactin, the level of reductase mRNA and enzymatic activity decreased markedly, but the number of copies of the reductase gene did not decline. When the compactin-withdrawn cells were rechallenged with compactin, high levels of reductase mRNA and enzymatic activity promptly returned. We conclude that the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, has been stably amplified in UT-1 cells. Despite this differential gene amplification, the levels of cytoplasmic mRNA for both gene products are markedly elevated, and both are reduced in parallel by either sterols (low density lipoprotein-cholesterol or 25-hydroxycholesterol) or mevalonate, the product of the reductase-catalyzed reaction.

Association of IRS-1 with the insulin receptor and the phosphatidylinositol 3'-kinase. Formation of binary and ternary signaling complexes in intact cells.

Insulin stimulates the formation of binary and ternary signaling complexes between the phosphatidylinositol (PtdIns) 3'-kinase, IRS-1, and the insulin receptor in vivo. Binary complex formation between IRS-1 and the PtdIns 3'-kinase occurs in intact cells and requires the tyrosyl phosphorylation IRS-1, as mutant insulin receptors which weakly phosphorylate IRS-1 in vivo do not mediate formation of IRS-1/PtdIns 3'-kinase complexes in transfected CHO cells. Association with IRS-1 involves as much as 70% of total cellular PtdIns 3'-kinase activity. Insulin also stimulates the formation of ternary signaling complexes, as both IRS-1 and the PtdIns 3'-kinase are present in anti-insulin receptor immunoprecipitates from insulin-stimulated cells. Overexpression of IRS-1 in CHO cells increases the amount of PtdIns 3'-kinase activity in alpha IR immunoprecipitates, and IRS-1 markedly increases the in vitro binding of p85 alpha and PtdIns 3-kinase activity to anti-receptor immunoprecipitates. The mechanism for this association is unknown, but appears to involve the binding of IRS-1/PtdIns 3'-kinase complexes to the insulin receptor. The formation of binary and ternary complexes between the insulin receptor, IRS-1 and the PtdIns 3'-kinase may play a critical role in transmission of the insulin signal.