Cytoplasmic Protein mRNA Interaction Mediates cGMP-modulated Translational Control of the Asialoglycoprotein Receptor*

(Received for publication, December 19, 1996, and in revised form, January 29, 1997)

Richard J. Stockert Dagger and Qing Ren

From the Department of Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, New York 10461

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Expression of the asialoglycoprotein receptor by the human hepatocellular carcinoma cell line HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level. In a cell-free system, initiation of asialoglycoprotein receptor mRNA translation was dependent on the presence of the 7-methylguanylate cap site and was independent of 8-bromo-cGMP levels in which the cells were grown prior to RNA isolation. Stable transfection of COS-7 cells with deletion constructs of the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region (UTR). Addition of biotin (an activator of guanylate cyclase) induced the expression of beta -galactosidase present as a chimeric plasmid containing the H2b 187-nucleotide 5'-UTR. An RNA gel retardation assay identified a 37-nucleotide cognate sequence within this 187-nucleotide region. Titration of the 5'-UTR with a cytosolic fraction isolated from HuH-7 grown in the presence or absence of 8-bromo-cGMP or biotin provided direct evidence for an RNA-binding protein responsive to intracellular levels of cGMP. Based on these findings, it seems reasonable to propose that reduction of intracellular levels of cGMP by biotin deprivation results in a negative trans-acting factor associating with the 5'-UTR of asialoglycoprotein receptor mRNAs, thereby inhibiting translation.


INTRODUCTION

Regulated expression of cell-surface lectins has been implicated in such diverse processes as endocytosis, bacterial and viral infection, regulation of cell proliferation, homing of lymphocytes, and metastasis of cancer cells (1). The asialoglycoprotein receptor (ASGR)1 is the hepatocellular prototype of a cell-surface lectin responsive to the differentiated state of the liver cell (for review, see Ref. 2). In addition to being a model of receptor-mediated endocytosis (3), the presence of ASGR on hepatocytes provides a membrane-bound active site for cell-to-cell interactions (4, 5), has made possible the selective targeting of chemotherapeutic agents (6) and foreign genes (7), and has also been implicated as a site that mediates hepatitis B virus uptake (8).

A human hepatoma cell line (HepG2) has provided a convenient model to investigate ASGR biosynthesis. When HepG2 cells were grown to confluence in a minimal essential medium or in a chemically defined medium containing a variety of hormones and growth factors supplemented with dialyzed fetal bovine serum, expression of ASGR was reduced by 60-70% (9). The low molecular weight factor required for the restoration of ASGR expression was isolated, purified, and identified as biotin (10). Similar results were obtained with a second hepatocellular carcinoma cell line, HuH-7 (11), indicating that the effect was not cell line-specific. Though usually not considered as part of an induction pathway, the effects of biotin upon the steady state expression of ASGR could be mimicked by the addition of the second messenger 8-bromo-cGMP (8-Br-cGMP), and these additions were not additive (11, 12). This suggested that the effect of biotin may have been mediated through changes in the cGMP level via biotin activation of the membrane-associated guanylate cyclase (13).

Estimates of the steady state level of ASGR mRNA suggested that cGMP-regulated expression of ASGR was at the posttranscriptional level (11, 12). Polysome analysis of ASGR subunits H1 and H2 mRNAs indicated that the addition of 8-Br-cGMP caused a shift of ASGR mRNA from the ribonucleoprotein fraction into a translationally active membrane-associated polysomal pool. Although the biochemical mechanisms have not been determined, cGMP has been suggested to regulate the expression of other proteins at a translational level (13, 14) and has been shown to increase total protein synthesis in isolated hepatocytes (15).

In mammalian cells, translation of most mRNA species appears to occur by the association of the preinitiation complex at or near the 7-methylguanylate cap structure at the 5'-untranslated region (UTR) of mRNA and scanning downstream to the site of protein synthesis initiation (16). The 60 S ribosomal subunit is subsequently recruited to the complex, and translation begins. Recovery of the ASGR mRNA in the ribonucleoprotein fraction during biotin deprivation suggested that intracellular levels of cGMP may play a significant role in modulating the initiation phase of ASGR mRNA translation. The bimodal polysomal distribution of ASGR mRNA was characteristic of a class of mRNAs that were inefficiently translated (17). Current evidence suggests that mRNAs in these functionally distinct fractions differ structurally or through the proteins they interact with (18). Within this group of mRNAs, most interactions between RNA and cytosolic proteins were defined by motifs localized to the 5'-UTR (19).

In the present study, the potential role of the 5'-UTR as the cis-acting element governing the cGMP-modulated expression of ASGR was established. In vitro transcription coupled with an RNA gel retardation assay defined the cis-acting element within a 37-nucleotide region. In addition, the effective concentration of a cytoplasmic protein trans-acting fraction was shown to be responsive to cGMP and biotin deprivation.


EXPERIMENTAL PROCEDURES

DNA Constructs

The 5' and 3'-UTR regions of the H2b cDNA of ASGR were deleted using polymerase chain reaction to introduce unique restriction sites (XbaI, 6 nucleotides upstream of ATG translation start site or BamHI, 9 nucleotides downstream of the translation stop site). The resulting constructs were subcloned into either pcDNA3 for selection of stable transfectants in COS-7 or pGEM-4Z for in vitro transcription. The 5'-UTR of the ASGR H2b was prepared by polymerase chain reaction with the addition of HindIII ends and cloned into a beta -galactosidase reporter vector (pSV-beta -galactosidase, Promega) 296 nucleotides upstream of the lacZ coding region start site. The nucleotide sequences of the polymerase chain reaction-generated 5' and 3' inserts were confirmed by the dideoxy chain termination method with Sequenase (DNA Sequence Facility, Albert Einstein College of Medicine).

Cell Culture, Transfection, and Analysis of Cell Extracts

COS-7 and HuH-7 cells were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) or dialyzed FBS with or without 8-Br-cGMP or biotin. Stable transfectants of COS-7 cells (20) resistant to 400 µg/ml G418 were subcloned, and those with the highest level of ASGR expression in MEM + 10% FBS as determined by Western blot (21) were utilized. Transient transfection of HuH-7 with the chimeric plasmid was mediated by LipofectAMINE (Life Technologies, Inc.) following the manufacturer's instructions. 5 hrs after transfection, cells were harvested by trypsinization and replated at a 1:3 ratio in MEM supplemented with 10% dFBS. 24 hrs later, the medium was changed to MEM + 10% dFBS with or without 10-7 M biotin. Three days after transfection, cells were harvested, and beta -galactosidase activity was measured following the manufacturer's instructions (Invitrogen). Near-confluent HuH-7 cultures (1 × 106 cells) were labeled with 200 µg/ml [35S]Met/Cys (Pro-mix, Amersham Corp.) for 1 h, followed by a 2-h chase. ASGR was immunoprecipitated, resolved on 10% SDS-PAGE, and the gel was processed for fluorography. Western blot and immunoprecipitation protocols as well as antibodies used in this study have been previously described (11).

Northern Blot Analysis

Total cytoplasmic RNA was isolated by extraction with guanidine thiocycanate (22) from approximately 5 × 107 cells (HuH-7 or transfected COS-7). For Northern blot analysis, RNA samples were resolved on 1% agarose-formaldehyde gels and transferred to Nytran membrane. The blots were hybridized at high stringency with random prime-labeled ([alpha -32P]dCTP) H1- and H2-specific probes (12, 23). Equal loading and transfer of RNA was verified by staining the membrane with methylene blue.

In Vitro Translation

Total RNA was isolated from HuH-7 cells grown in MEM supplemented with 10% FBS, 10% dFBS, or 10% dFBS plus 1.0 mM 8-Br-cGMP. The translation reaction was performed using a rabbit reticulocyte lysate as described by the manufacturer (Promega) in the presence or absence of 2 µg/assay of m7GpppG, an inhibitor of cap site recognition. Following a 90-min incubation at 30 °C, the [35S]-labeled (Pro-mix, Amersham Corp.) ASGR translation products were recovered by immunoprecipitation using a polyclonal antibody to affinity-purified human receptor (11). Samples were resolved on 10% SDS-PAGE, and gels were prepared for fluorography.

Gel Retardation Assay

A nested set of RNA probes were prepared by linearization of the full-length H2b cDNA using restriction sites localized in the 5'-UTR as templates for in vitro transcription from the Sp6 promoter of pGEM-4Z. The full-length (187-nucleotide) fragment was labeled by the incorporation of [alpha -32P]UTP during transcription. The gel retardation assay was adapted from that described by Leibold and Munro (24). HuH-7 cells were homogenized, the cytosol (S-100) was prepared by centrifugation at 100,000 × g for 1 h, and the aliquots were stored at -135 °C. S-100 was preincubated at 0 °C for 10 min in assay buffer with a 100-fold molar excess or without unlabeled transcripts prior to the addition of the labeled 187-nucleotide transcript, and incubation continued for an additional 10 min. Inclusion of proteinase K (100 µg/ml) in the assay mixture completely abolished this protein-dependent assay. The mixture was resolved on a 4% low cross-linked PAGE and prepared for autoradiography.


RESULTS

Short Term cGMP-regulated Expression of ASGR

Based on our previous findings that biotin was required for expression of ASGR by HepG2 and HuH-7 cell lines, we proposed that the mechanism of biotin regulation was mediated by maintaining the intracellular level of cGMP via the activation of guanylate cyclase (11). Our original experimental protocol was modified from the steady state determination of receptor concentrations via Western blot (11) to the measurement of biosynthesis rate by pulse labeling with [35S]Met/Cys. This change in protocol allowed the reduction of 8-Br-cGMP and atrial natriuretic factor (ANF) concentrations used in the present experiments to the level previously employed by others in short term protocols in cultured or isolated hepatocytes (25-29). HuH-7 cells were grown to near-confluence in MEM supplemented with either 10% FBS or 10% dFBS to which 10 to 1000 µM 8-Br-cGMP, 10 nM ANF, or 100 µM sodium nitroprusside (SNP) (shown to produce nitric oxide (25), an activator of soluble guanylate cyclase (29)) were added (Fig. 1A). Within 1 h of the addition of 500 µM 8-Br-cGMP and activators of both the particulate (ANF) and soluble (SNP) guanylate cyclases (25-29), the biosynthetic rate of ASGR was increased by 6.7-, 8.3-, and 4.2-fold, respectively, when compared with untreated cells in dFBS alone. No difference in the abundance of specific mRNAs was detected by Northern blot analysis, regardless of whether cells were maintained in MEM supplemented with FBS or dFBS with or without 8-Br-cGMP or its inducers (Fig. 1B). These results strongly support our hypothesis that intracellular levels of cGMP regulate the expression of the ASGR at a posttranscriptional level. When taken together with our earlier studies (11, 12), these results clearly demonstrated that the molecular level of control was translational.


Fig. 1. A, induction of ASGR synthesis. HuH-7 cells were grown to near-confluence in MEM supplemented with 10% FBS (control) or dFBS. Culture medium was replaced with MEM/dFBS containing increasing concentrations of 8-Br-cGMP (10-1,000 µM), 10 nM ANF, or 100 µM SNP for 1 h before pulse labeling for 1 h with Pro-mix ([35S]Met/Cys), 200 µCi/ml. Cells were harvested following a 2-h chase in medium supplemented with 0.1 mM methionine/cysteine. ASGR immunoprecipitated from aliquots of cell lysate containing equal amounts of radiolabeled protein was resolved on a 10% SDS-PAGE, and the resulting fluorograms were quantitated by densitometric scanning. The mean and standard deviation from three independent experiments as a percent of control are shown. B, effect of 8-Br-cGMP, ANF, and SNP on the steady state concentration of the H1 and H2 subunit-related mRNA. Total RNA was isolated from cells grown in (1) FBS, (2) dFBS control cells, or cells treated with (3) 500 µM 8-Br-cGMP, (4) ANF, or (5) SNP as above. Northern blot analysis was performed using 10 µg of total RNA/lane, and the transferred RNA was sequentially hybridized with probes for H1 and H2 ASGR subunits. The blot was stained with methylene blue to confirm that equal amounts of RNA had been transferred.
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In Vitro Translation of ASGR

In mammalian cells, translation of most mRNAs appears to occur by association of a preinitiation complex at a 7-methylguanylate cap site and subsequent scanning to the translation initiation site (16). To establish the cap site status of ASGR mRNA in cells grown in FBS as compared with dFBS and dFBS supplemented with 500 µM cGMP, the extent of cap-dependent in vitro translation was determined. Total mRNA was isolated from HuH-7 cells (5 × 107) and translated in a rabbit reticulocyte lysate in the presence or absence of m7GpppG, an inhibitor of cap-dependent initiation (30). The labeled ASGR translation product was recovered by immunoprecipitation using a polyclonal antibody to affinity-purified human receptor. Resolution on 10% SDS-PAGE and subsequent fluorography indicated that inclusion of m7GpppG reduced translation of ASGR mRNA isolated from both control and biotin-deprived cells with or without 8-Br-cGMP to an equal extent (>90%) (Fig. 2). These results indicated that initiation of ASGR mRNA translation was cap-dependent and that addition of a 7-methylguanylated cap to ASGR mRNA was independent of biotin deprivation.


Fig. 2. Cap-dependent translatability of ASGR H2 subunit mRNA isolated from biotin-deprived HepG2. Cells were maintained in MEM supplemented with 10% FBS or 10% dFBS with or without 500 µM 8-Br-cGMP for 24 h before the isolation of mRNA. Equal amounts of mRNA (2 µg) were added to the translation mixture plus or minus m7GpppG. ASGR H2 translation product was recovered by immunoprecipitation using a subunit-specific antibody, as described previously (49). The resulting fluorogram was quantified by densitometry.
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Localization of the Cis-acting H2 mRNA Cognate Sequence

To localize the cis-acting element, mutated H2 cDNAs from which the entire 5' or 3'-UTR was deleted were constructed by polymerase chain reaction amplification. These constructs, along with the full-length H2 cDNA, were cloned into the eukaryotic expression vector pcDNA3 carrying a neomycin resistance gene. Stable transfectants of COS-7 cells were selected with 400 µg/ml G418. As shown in Fig. 3, deletion of the 5'-UTR resulted in loss of the cGMP requirement for H2 expression. In contrast, deletion of the 3'-UTR was without effect. These results indicated that the cGMP-responsive element was located in the 5'-UTR of the H2 mRNA. Northern blot analysis indicated that there was no significant difference in mRNA levels to account for this differential response to biotin deprivation or supplementation with 8-Br-cGMP (Fig. 3), supporting translational regulation in the transfected COS-7 cell lines.


Fig. 3. Expression of the H2 subunit of ASGR in stably transfected COS-7 cells. The full-length (H2FL) and the 5' (H25D) or 3' (H23D) deleted UTR sequences of the H2b of ASGR cDNAs were subcloned into pcDNA3 vector and stable transfectants selected with 400 µg/ml of G418. The cell lines were grown to near-confluence in MEM supplemented with 10% FBS or dFBS with or without 1.0 mM cGMP. Cells were metabolically labeled with [35S]Met/Cys, and the extent of ASGR polypeptide synthesis was estimated as described in Fig. 1. The failure of growing cells in dFBS to inhibit ASGR expression by the H25D line strongly indicates that the cGMP cognate sequence was located in the 5'-UTR of H2b mRNA. The film was exposed for 72 h for H23D and H2FL and for 18 h for the H25D cell line. Northern blot analysis was performed using 10 µg total RNA/lane, and the transfer was hybridized with an H2-specific probe. The blot was stained with methylene blue to confirm that equal amounts of RNA had been transferred.
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Transient transfection of HuH-7 with the chimeric plasmid confirmed that the putative cis-acting element was localized within the 5'-UTR (Table I). Addition of biotin to the culture medium resulted in a two-fold increase in beta -galactosidase activity. Interestingly, the presence of the 5'-UTR in the plasmid reduced beta -galactosidase expression when compared with the original or a chimeric plasmid in which the 5'-UTR was inserted in the antisense orientation by almost 35%, even when biotin was added. This finding was consistent with the reduced level of H2b translation when compared with the H1 ASGR subunit under normal physiologic conditions (2).

Table I.

5'-Untranslated region mediates biotin-dependent expression of beta -galactosidase activity in chimeric plasmid

HuH-7 cells were transfected with pSV-beta -gal plasmid without (control) or with the H2b 5'-UTR insert and growth for 24 h in MEM supplemented with 10% dialyzed FBS. Cells were harvested and replated at a 1:3 ratio in the same medium with or without 10-8 M biotin supplementation. When the cells reached near-confluence (72 h postplating), the levels of beta -galactosidase activity in cell lysates were determined.


Plasmid Biotin
+  -

%
pSV-beta -gal 100 92  ± 12
  + 5'-UTR sense orientation 68  ± 11 32  ± 4
  + 5'-UTR antisense orientation 83  ± 14 102  ± 17

a Values shown are means ± S.D. of three independent transfections normalized to lysate protein and are expressed as a percent of the beta -galactosidase activity present in HuH-7 cells transfected with the pSV-beta -gal plasmid and grown in the presence of biotin.

RNA Gel Retardation Assay

The 5'-UTR (187-base pair) cDNA fragment of the H2b of ASGR was directionally cloned into pGEM-4Z vector for the generation of a nested set of 5'-UTR mRNA fragments by in vitro transcription for an RNA-protein binding assay (Fig. 4). RNA fragments of the 5'-UTR were added in 100-fold molar excess prior to the addition of the full-length 187-nucleotide-labeled RNA probe and resolution on 4% PAGE. As illustrated in Fig. 5, the failure of the Sp6-FokI transcript to inhibit the band shift assay indicated that a cognate sequence lies between 70 and 110 nucleotides relative to the Sp6 promoter. Since translational regulation due to protein-protein interactions between two regions of a transcript has been reported (33), a potential and equally critical role for the upstream 1-70-nucleotide (Sp6-FokI) region cannot be eliminated by the present study. Failure of the 208-nucleotide 3'-UTR fragment of the H2 mRNA and the glyceraldehyde-3-phosphate dehydrogenase open reading frame mRNA to inhibit the gel retardation assay further supported the specificity of this assay (Fig. 5B).


Fig. 4. Restriction map of 5'-UTR of H2. A nested set of RNA fragments were prepared by in vitro transcription and used to define the cognate sequence by an inhibition gel retardation assay as illustrated in Fig. 5.
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Fig. 5. Localization of the putative cognate sequence of the RNA transcript by an inhibition gel shift assay. A, unlabeled RNA fragments as indicated by restriction cut sites (Fig. 4) were added in 100-fold molar excess to the S-100 incubation mixture 10 min prior to the addition of the Sp6-SmaI 32P-labeled probe (0.5 ng). The failure of the Sp6-FokI fragment to inhibit the probe retardation indicates that a cognate sequence lies between the FokI and HinfI fragment. B, the addition of a 100-fold molar excess of full-length 5'-UTR was compared with a 100-fold molar excess of the 208-nucleotide 3'-UTR and glyceraldehyde-3-phosphate dehydrogenase open reading frame mRNAs. The failure to inhibit the gel retardation by the 3'-UTR and GAPD mRNA fragments confirmed the specificity of the assay.
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As shown in Fig. 6, incubation of the 187-nucleotide 5'-UTR probe with increasing amounts of the S-100 fraction isolated from HuH-7 cells grown to confluence in MEM supplemented with FBS, dFBS + 1.0 mM 8-Br-cGMP (conditions required for normal ASGR expression), or dFBS alone showed a concentration-dependent gel retardation. As the concentration of S-100 protein was increased to 1 µg/assay, it became evident that the S-100 fraction isolated from HuH-7 cells grown in dFBS had a higher effective concentration of RNA-binding protein than cells grown in FBS and that inclusion of 8-Br-cGMP to the cell culture reduced the concentration to the control level. This quantitative (as opposed to a qualitative difference) was consistent with the bimodal distribution of ASGR mRNA between the translationally active polysomes isolated from cells grown in FBS and the shift to the repressed state when cells were grown in dFBS (11, 12).


Fig. 6. Titration of 5'-UTR-binding protein by band-shift assay. Increasing amounts of the S-100 cytosolic protein were incubated with the 187-nucleotide in vitro transcription product (10,000 cpm) before resolution on a 4% native gel. Among various preparations of S-100 isolated from cells grown in MEM supplemented with FBS or dFBS + cGMP when compared with dFBS alone, the two-fold difference in protein concentration required for a positive band shift was the minimum observed. At intermediate concentrations, both shifted and free probe were presented in the lane (data not shown). The gel was dried, and the bands were localized by autoradiography.
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DISCUSSION

In mammalian cells, translation of most mRNA species appears to occur by the association of a preinitiation complex at a 7-methylguanylate cap site and subsequent scanning downstream to the site of protein synthesis initiation (16, 31). Our studies showed that cap site addition to ASGR mRNA was independent of biotin deprivation (Fig. 2). However, the recovery of the ASGR message in the ribonucleoprotein fraction during biotin deprivation (11, 12) suggested that intracellular levels of cGMP play a significant role in modulating the initiation phase of translation (31).

Perhaps the best-defined example of translational regulation is that of ferritin synthesis in iron-deficient cells (16, 30). Analysis of the cis-acting mRNA sequences led to the definition of the iron-responsive element with a putative stem-loop structure in the 5'-UTR (30). Modeling of the ASGR H2b 5'-UTR indicated the presence of two potential regions of secondary structure. The free energy levels (-7.4 and -8.5 kcal/mol) of these two stem-looped regions was far below that usually considered necessary to prevent recognition of a cap site (-50 kcal/mol). However, they might provide the loop structure necessary for specific recognition by exposing the RNA backbone and bases to interaction with protein groups (32, 33).

In the absence of a highly ordered 5'-UTR stem-loop structure, translation may be regulated by a short linear sequence (16). A highly conserved CCAUCNN sequence localized within the 5'-UTR of both ASGR subunit mRNAs isolated from either human or rat has been identified as a conserved RNA-binding protein cognate sequence within the 5'-UTR of ornithine decarboxylase (34). The presence of this conserved sequence within the putative cis-acting element as indicated by gel retardation assay (Fig. 5) supports the possibility that it may serve as a recognition motif for the cGMP responsive trans-acting factor.

One plausible explanation for cGMP-regulated expression of ASGR would be modulation of a trans-acting factor phosphorylation status. Although the modulation of ASGR by cGMP was not liver-specific (Fig. 6), it should be viewed in the context of the original finding in HepG2 cells (11, 12). Since there was little, if any, cGMP-dependent protein kinase detected in hepatocytes (35, 36), the classic cGMP signal transduction pathways mediated by cGMP-dependent protein kinase was presumed to be absent in liver cells (36). Therefore, if a phosphorylation/dephosphorylation signal transduction pathway was involved in translational regulation of ASGR expression, one of the cGMP-binding phosphodiesterases (PDEs) would be the most likely effector target. As opposed to cGMP-dependent protein kinase, the various cGMP PDEs are regulated allosterically by the binding of cGMP to noncatalytic binding sites (37). Modulation of cGMP levels can either inhibit or stimulate PDE hydrolytic activity, increasing or decreasing intracellular cGMP itself or cAMP (38). Indeed, a number of recent studies have suggested that cGMP-stimulated PDE may play a central role in regulating the intracellular concentrations of cAMP (39, 40). Based on our previous findings that increased levels of cAMP resulted in the down-regulation of ASGR (41), induction of a cGMP-stimulated PDE resulting in a protein dephosphorylation via reduction of cAMP is a reasonable mechanism for cGMP-regulated expression of ASGR.

Presently, it may be premature to speculate that changes in the phosphorylation state of any protein via PDE mediates the effects of cGMP on translation of ASGR, especially in the light of the recent discovery of new types of cyclic nucleotide receptors that include other non-catalytic sites (37). Our understanding of the potential cGMP cascade in liver is still in its infancy, and there may yet be other schemes to account for cGMP action, such as Ca+2 ion flux or induction of inositol triphosphate (42, 43). Whatever the biochemical mechanism of the cGMP action may be, it is reasonable to speculate that in the absence of cGMP, a negative trans-acting factor associates with the 5'-UTR of the ASGR mRNA, thereby inhibiting translation. Purification of the ASGR mRNA-binding protein should provide new insight into the physiologic affect and molecular target of cGMP in the hepatocyte.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants DK32972 and DK41296.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Ullmann 517, New York, NY 10461. Tel: 718-430-3644; Fax: 718-430-8975.
1   The abbreviations used are: ASGR, asialoglycoprotein receptor; UTR, untranslated region; dFBS, dialyzed fetal bovine serum; ANF, atrial natriuretic factor; SNP, sodium nitroprusside; H2b, human hepatic lectin subunit; PDE, phosphodiesterase; 8-Br-cGMP, 8-bromo-cGMP; MEM, Eagle's minimal essential medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis.

REFERENCES

  1. Drickamer, K., and Carver, J. (1992) Curr. Opin. Struct. Biol. 2, 653-654
  2. Stockert, R. J. (1995) Physiol. Rev. 75, 591-609 [Abstract/Free Full Text]
  3. Ashwell, G., and Harford, J. (1982) Annu. Rev. Biochem. 51, 531-554 [CrossRef][Medline] [Order article via Infotrieve]
  4. Paietta, E., Stockert, R. J., McManus, M., Thompson, D., Schmidt, S., and Wiernik, P. H. (1989) J. Immunol. 143, 2850-2857 [Abstract/Free Full Text]
  5. Weisz, O. A., and Schnaar, R. L. (1991) J. Cell Biol. 115, 495-504 [Abstract]
  6. Wu, G. Y., Rubin, M. I., Wu, C. H., and Stockert, R. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3078-3080 [Abstract]
  7. Wu, G. Y., and Wu, C. H. (1991) Biotherapy 3, 87-95 [Medline] [Order article via Infotrieve]
  8. Treichel, U., Meyer Zum Buschenfeld, K-H., Stockert, R. J., Poralla, T., and Gerken, G. (1994) J. Gen. Virol. 75, 3021-3029 [Abstract]
  9. Collins, J. C., Wolkoff, A. W., Stockert, R. J., and Morell, A. G. (1988) Hepatology 8, 108-115 [Medline] [Order article via Infotrieve]
  10. Collins, J. C., Paietta, E., Green, R., Morell, A. G., and Stockert, R. J. (1988) J. Biol. Chem. 263, 11280-11283 [Abstract/Free Full Text]
  11. Stockert, R. J., and Morell, A. G. (1990) J. Biol. Chem. 265, 1841-1846 [Abstract/Free Full Text]
  12. Stockert, R. J., Paietta, E., Racevskis, J., and Morell, A. G. (1992) J. Biol. Chem. 267, 56-59 [Abstract/Free Full Text]
  13. Spence, J. T., Merrill, M. J., and Pitot, H. C. (1981) J. Biol. Chem. 256, 1598-1603 [Abstract/Free Full Text]
  14. Spence, J. T., and Koudelka, A. P. (1984) J. Biol. Chem. 259, 6393-6396 [Abstract/Free Full Text]
  15. Rashed, H. M., Nair, B. G., and Patel, T. B. (1992) Arch. Biochem. Biophys. 298, 640-645 [Medline] [Order article via Infotrieve]
  16. Hershey, J. W. B. (1991) Annu. Rev. Biochem. 60, 717-755 [CrossRef][Medline] [Order article via Infotrieve]
  17. Walden, W. E., and Thach, R. E. (1986) Biochemistry 25, 2033-2041 [Medline] [Order article via Infotrieve]
  18. Meyuhas, O., Thompson, E. A., and Perry, R. P. (1987) Mol. Cell. Biol. 7, 2691-2699 [Medline] [Order article via Infotrieve]
  19. Berger, L. C., Bag, J., and Sells, B. H. (1992) Biochem. Cell Biol. 70, 770-778 [Medline] [Order article via Infotrieve]
  20. Sussman, D. J., and Milman, G. (1984) Mol. Cell. Biol. 4, 1641-1643 [Medline] [Order article via Infotrieve]
  21. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  22. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  23. Paietta, E., Gallagher, R., Wiernik, P. H., and Stockert, R. (1988) Cancer Res. 48, 280-287 [Abstract]
  24. Leibold, E. A., and Munro, H. N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2171-2175 [Abstract]
  25. Curran, R. D., Ferrari, F. K., Kispert, P. H., Stadler, J., Stuehr, D. J., Simmons, R. J., and Billar, T. R. (1991) FASEB J 5, 2085-2092 [Abstract/Free Full Text]
  26. Pittner, R. A., Fears, R., and Brindley, D. M. (1986) Biochem. J. 240, 253-257 [Medline] [Order article via Infotrieve]
  27. Gudoshnikov, V. I., Baranova, I. N., and Fedotov, V. P. (1991) Biull. Eksp. Biol. Med. 111, 478-480 [Medline] [Order article via Infotrieve]
  28. Brass, E. P., and Vetter, H. W. (1993) Pharmacol. & Toxicol. 72, 369-372 [Medline] [Order article via Infotrieve]
  29. Curran, R. D., Ferrari, F. K., Kispert, P. H., et al. (1991) FASEB J. 5, 2085-2092 [Abstract/Free Full Text]
  30. Hentze, M. W., Caugman, S. W., Rouault, T. A., Barriocanal, J. G., Dancis, A., Harford, J. B., and Klausner, R. D. (1987) Science 238, 1570-1573 [Medline] [Order article via Infotrieve]
  31. Jansen, M., DeMoor, C. H., Sussenbach, J. S., and Van Den Brande, J. L. (1995) Pediatr. Res. 37, 681-685 [Abstract]
  32. Rogers, J., and Munro, H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2277-2281 [Abstract]
  33. McCarthy, J. E. G., and Koolmus, H. (1995) Trends Biochem. Sci. 233, 191-197 [CrossRef]
  34. Kean, D. J., Query, C. C., and Keene, J. (1991) Trends Biochem. Sci. 16, 214-220 [CrossRef][Medline] [Order article via Infotrieve]
  35. Manzella, J. M., and Blackshear, P. J. (1992) J. Biol. Chem. 267, 7077-7082 [Abstract/Free Full Text]
  36. Lincoln, T. M., and Cornwell, T. L. (1993) FASEB J. 7, 328-338 [Abstract/Free Full Text]
  37. Lincoln, T. M., and Corbin, J. D. (1983) Adv. Cyclic Nucleotide Res. 15, 139-192
  38. Thomas, M. K., Francis, S. H., Beebe, S. J., Gettys, T. W., and Corbin, J. D. (1992) Adv. Second Messenger Phosphoprotein Res. 25, 45-53 [Medline] [Order article via Infotrieve]
  39. Stroop, S. D., and Beavo, J. A. (1992) Adv. Second Messenger Phosphoprotein Res. 25, 55-71 [Medline] [Order article via Infotrieve]
  40. Whalin, M. E., Scammell, J. G., Strada, S. J., and Thompson, W. J. (1991) Mol. Pharmacol. 39, 711-717 [Abstract]
  41. Stockert, R. J. (1993) J. Biol. Chem. 268, 19540-19544 [Abstract/Free Full Text]
  42. Thomas, M. K., Francis, S. H., Beebe, S. J., Gettys, T. W., and Corbin, J. D. (1992) Adv. Second Messenger Phosphoprotein Res. 25, 45-53 [Medline] [Order article via Infotrieve]
  43. Shabb, J. B., and Corbin, J. D. (1992) J. Biol. Chem. 267, 5723-5726 [Free Full Text]

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