©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Variant of the Subunit of Soluble Guanylyl Cyclase Contains an Insert Homologous to a Region within Adenylyl Cyclases and Functions as a Dominant Negative Protein (*)

(Received for publication, June 5, 1995)

Sönke Behrends Christian Harteneck Günter Schultz Doris Koesling (§)

From the Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, D-14195 Berlin, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A variant of the alpha(2) subunit of soluble guanylyl cyclase (alpha) containing 31 additional amino acids was identified in a number of cell lines and tissues. The in-frame sequence of the insert was within the proposed catalytic domain of guanylyl cyclases and was homologous to a region within the putative catalytic domain of adenylyl cyclases. Messenger RNA for the new variant was detected in some but not all cell lines and tissues expressing the alpha(2) subunit. The novel form, as well as the alpha(2) subunit lacking the insert, were coexpressed with the beta(1) subunit in Sf9 and COS-7 cells; alpha(2)/beta(1) coexpression yielded a NO-sensitive recombinant protein, whereas the coexpressed alpha/beta(1) subunits exhibited no guanylyl or adenylyl cyclase activities. Because both subunits (alpha/beta(1)) copurified, the novel variant retains its ability to heterodimerize. In coexpression experiments, the alpha subunit competed with the alpha(2) subunit for dimerization with the beta(1) subunit, thereby reducing alpha(2)/beta(1)-catalyzed guanylyl cyclase activity. These data show that the novel variant functions as a dominant negative protein and that post-transcriptional mRNA processing represents a potential mechanism for regulation of NO-sensitive guanylyl cyclase acitivity.


INTRODUCTION

Guanylyl cyclases (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) exist in membrane-bound and cytoplasmic forms(1) . The known membrane-spanning forms are cell surface receptors for extracellular peptides and appear to exist as homodimers or other higher ordered structures. The cytoplasmic or soluble forms represent heme-containing heterodimers that are directly activated by nitric oxide. The cDNAs of two alpha and two beta subunits (alpha(1), alpha(2), beta(1), and beta(2)) have been identified(2, 3, 4, 5, 6, 7) . All subunits contain a putative catalytic domain homologous to the membrane-bound guanylyl cyclases and to the two putative catalytic domains of adenylyl cyclases (C and C)(2, 4, 5) . A heterodimer between an alpha and a beta subunit appears to be required for enzyme activity(8, 9) .

The mechanisms by which the soluble or membrane forms of guanylyl cyclase are regulated have been explored at both the protein and the gene level. In the case of the plasma membrane guanylyl cyclase A, an adenine nucleotide is required for hormonal stimulation of cyclase activity(10, 11, 12) . Guanylyl cyclase A, as well as other membrane forms of guanylyl cyclase, is also known to be regulated by phosphorylation/dephosphorylation(13, 14, 15) . A soluble form of guanylyl cyclase has been shown to be phosphorylated, but the functional significance remains unclear(16) . At the transcriptional level, regulation of expression or differential expression of the alpha and beta subunits of soluble guanylyl cyclase has been demonstrated(17, 18) .

Differential RNA splicing can serve to switch an active to an inactive protein, the most impressive example being the cascade of alternative splicing events that control somatic sexual differentiation in Drosophila(19) . A naturally occurring variant of FosB generated by alternative splicing (DeltaFosB) has been shown to inhibit Fos/Jun transcriptional activity(20) . This dominant negative effect is presumably due to competition with FosB at the step of heterodimer formation with Jun. Similarly, helix-loop-helix proteins have been described that negatively regulate other helix-loop-helix proteins through the formation of nonfunctional heterodimeric complexes(21) . Modulation of enzyme activity by alternative splicing within the catalytic region has been suggested for protein-tyrosine phosphatases (22, 23) .

Here, we present a variant form of the alpha(2) subunit of soluble guanylyl cyclase that contains an in-frame insert of 31 amino acids within the catalytic domain. The insert is homologous to a region within the apparent catalytic domain of adenylyl cyclases. Although the new variant heterodimerizes with the beta(1) subunit, an active enzyme is not formed. Thus, the alpha subunit effectively competes with alpha(2) lacking the insert, thereby functioning in a dominant negative manner. This suggests that guanylyl cyclase activity can be regulated at the level of RNA processing.


EXPERIMENTAL PROCEDURES

Materials

2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA-NO) (^1)(24) was purchased from NCI Chemical Carcinogen Repository. 10-fold concentrated stock solutions were prepared in 10 mM NaOH. cDNA libraries were purchased from Stratagene (human fetal brain 936206, human T-cell 938200, human liver 937220, and human colon 937204) or Clontech (human endothelial cell HL1164b and human testis HL1010b). Vectors pcDNA3, PCR II, and pVL1393 were purchased from Invitrogen; pAcSGHisNTB was purchased from Dianova.

Isolation of RNA and First Strand cDNA Synthesis

Poly(A) RNA was isolated using the Oligotex Direct mRNA kit (QIAGEN). First strand cDNA synthesis was performed with SuperScript Plus RNase H reverse transcriptase (Life Technologies, Inc.) and 50-100 ng of poly (A) RNA using random oligonucleotides (100 ng/µl) at 45 °C.

Isolation of the alphaClone

For the polymerase chain reaction (PCR), a set of degenerate primers was designed from two highly conserved regions within the catalytic domain of guanylyl cyclases: A (TAYAARGTIGARACIRTIGGIGA) and B (CCRAAIARRCARTAICKIGGCAT). PCRs were performed for 35 cycles (94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min) using the wax-mediated hot start technique (Perkin-Elmer). Products were subcloned using the TA cloning system (Invitrogen). Subcloned PCR products were sequenced by the dideoxy chain termination method (25) using the Sequenase version 2.0 DNA sequencing kit (U. S. Biochemical). Both strands of the alpha PCR product were sequenced. Sequence data were analyzed using the Husar 3.0 program package at the Heidelberg Unix Sequence Analysis Resources.

Tissue Distribution of the alpha(2)/alphaSubunits

The alpha(2)-specific primers (C and D) were synthesized according to base pairs 1530-1553 (GCGACTGTCTACCCCGTTTGTGAT) and 1929-1906 (CTGTACTTGCTGCCCTTGCCATAA) of the alpha(2) cDNA sequence. alpha-specific primers (E and F) were chosen from within the in-frame insert (TTTTCTCCTTTCCTGTTTCCATCC) and from 275 base pairs to the 3`-end (ACGAGACCGCGGAATGAATG). Both primer pairs were used in the PCR under the conditions described above. PCR products were verified as alpha-specific by Southern gel-blot hybridization (26) using a nonisotopic nucleic acid detection kit (Gene Images, U. S. Biochemical). The biotinylated alpha(2)- or alpha-specific probes were synthesized by polymerase chain reaction. Hybridization and subsequent washing (0.5 SSC/0.1% (w/v) SDS) were performed at 65 °C.

Plasmid Construction

To construct the alpha(2) and alpha expression vectors, alpha(2) cDNA in pBR322 (7) was cut by EcoRV/BamHI and ligated into StuI/BglII-cut pAcSGHisNTB (Dianova). From this vector, the alpha(2) insert was cut by BstYI (position 1863) and BbsI (position 2220). The resulting 357-base pair fragment was cloned into the BamHI/BbsI-cut alpha PCR product in PCR II. From this intermediate, a 353-base pair SphI/BsmI fragment was cloned into the SphI/BsmI-cut alpha(2) cDNA in pAcSGHisNTB. Next, alpha(2) and alpha cDNAs in pAcSGHisNTB were cut by NaeI/BspMI and subcloned into SmaI/BglII-cut pVL1393 and into HindIII (Klenow fill-in of protruding overhang)/BamHI-cut pcDNA3. The beta(1) cDNA from pBluescript (8) was cloned into the HindIII/EcoRI-cut pcDNA3. Baculovirus transfer vectors for the alpha(1) and beta(1) subunits were constructed as described(27) . The baculovirus transfer vector for the beta(1) subunit containing an amino-terminal hexahistidine tag was cloned EagI/SmaI from pBluescript (8) into the NotI/SmaI sites of pAcSGHisNTB.

Sf9 Cell Culture, Expression of Recombinant Guanylyl Cyclase, and Cytosol Preparation

Sf9 cells were propagated in TNM-FH medium (Sigma) supplemented with 10% (v/v) fetal calf serum. Spinner cultured cells were grown under the same conditions except that 0.5 lipid concentrate (Life Technologies, Inc.) was added to the medium. Recombinant viruses were generated by cotransfection of Sf9 cells with the expression vectors described above and with BaculoGOLD baculovirus DNA (Dianova) by the Lipofectin method(28) . Positive viral clones were isolated by plaque assay and were identified by their ability to direct the expression of the appropriate proteins as revealed by immunoblotting. The purified viruses were amplified. For expression of guanylyl cyclase, spinner cultures with densities of 1.5 10^6 cells/ml were infected with the respective viruses at the indicated multiplicities of infection (M.O.I.). Cells were collected by centrifugation 38-44 h after infection and washed in 1.1% (w/v) NaCl solution. The resulting pellets were resuspended in two volumes of 50 mM triethanolamine/HCl, pH 7.4, 2 mM dithiothreitol, 2 mM EDTA, and 0.2 µM benzamidine. The cells were lysed by ultrasonication monitored by light microscopy. The lysate was then centrifuged at 100,000 g for 45 min at 4 °C, and the resulting supernatant (cytosolic fraction) was used for determination of enzyme activity or purification. Expression of subunits in COS-7 cells and immunoblot analysis of the cytosolic proteins were performed as described(8) . Polyclonal antibodies raised against the carboxyl-terminal peptide of alpha(2) and alpha (KKVSYNIGTMFLRETSL) and against the carboxyl-terminal peptide of beta(1) (SRKNTGTEETEQDEN) were used for the detection of the respective subunits as described(8) .

Purification of the Recombinant alpha/betaHeterodimer

All purification steps were performed at 4 °C. Sf9 cells (6 10^8) were coinfected with 5 M.O.I. of the alpha(2) virus and 5 M.O.I. of the beta(1) virus containing the hexahistidine tag. Cytosolic protein (80 mg) was loaded onto a 10-ml column of DEAE-Sepharose Fast Flow (Pharmacia Biotech Inc.). The column was washed with six column volumes of 50 mM triethanolamine/HCl, pH 7.0. The elution was performed with a linear 0-400 mM NaCl gradient collected in 5-ml fractions. Both alpha and beta(1) subunits eluted at salt concentrations of approximately 200 mM NaCl, as monitored by immunoblotting. The respective fractions were pooled (DEAE pool) and applied to a 2-ml Ni-nitrilotriacetic acid-agarose column (QIAGEN). The column was washed three times with 10 ml of 50 mM triethanolamine/HCl, pH 7.0, containing increasing concentrations of imidazol (10, 25, and 40 mM). Protein was eluted with a linear gradient of 50- 200 mM imidazol (8 ml) collected in 1-ml fractions. The alpha/beta(1) subunits eluted as a heterodimer at imidazol concentrations of approximately 60 mM.

Determination of Protein Concentrations and Guanylyl/Adenylyl Cyclase Activities

Protein concentrations were determined by the method of Bradford (29) with bovine serum albumin as standard. Guanylyl cyclase activity of the obtained cytosolic fractions (80-200 µg/assay tube) was determined by incubation for 15 min at 37 °C in the presence of 50 mM triethanolamine/HCl buffer, pH 7.4, containing 3 mM dithiotreitol, 1 mM 3-isobutyl-1-methylxanthine, 1 mM cyclic GMP, 5 mM creatine phosphate, 4.6 units/tube creatine phosphokinase, 0.5 mM [alpha-P]GTP (about 1 µCi), and 3 mM MgCl(2) or 3 mM MnCl(2), with or without 10 µM DEA-NO in a total volume of 0.1 ml. Separation and purification of the enzyme-formed cGMP was as described previously(30) . Incubations for adenylyl cyclase activity were performed under identical conditions except that cyclic GMP and [alpha-P]GTP were replaced by cyclic AMP and [alpha-P]ATP. Separation and purification of the enzyme-formed cAMP was as described(31) .


RESULTS

HL-60 cell cDNA was amplified in the polymerase chain reaction using degenerate primers (A and B) corresponding to highly conserved regions of guanylyl cyclases. Among the subcloned PCR products, one clone (alpha) corresponded to the alpha(2) subunit with an additional in-frame insert of 93 base pairs. Independently, an identical clone was isolated using Y-79 cell cDNA. In both cell lines, cDNAs with or without insert were found. The nucleotide sequence did not reveal branch site nor donor or acceptor pre-mRNA consensus splicing signals, excluding the possibility of unprocessed pre-mRNA(32) . Identification of the remnants of the donor consensus that remain in the mRNA after splicing strongly suggests alternative inclusion of an additional exon (32) (Fig. 1A).


Figure 1: Nucleotide sequence of the additional exon in the alpha subunit and amino acid alignment. A, given is the nucleotide sequence and the deduced amino acid sequence of the additonal exon in the alpha subunit (underlined). The letters printed in bold correspond to the remnants of the donor consensus sequences that remain in the mRNA after splicing ((C/A)AG). B, shown is a comparison of a sequence within the novel variant exon of the alpha subunit (sGC alpha) to the analogous region within the apparent catalytic domain of adenylyl cyclases (AC I-VIII, C-domain). Identical residues are shaded. The accession numbers with the position of the aligned amino acids are as follows: sGCalpha, 620-639; type I, Swissprot Q08828, 674-693; type II, Swissprot Q08462, 314-333; type III, Swissprot P21932, 1019-1038; type IV, Swissprot P26770, 951-970; type V, Swissprot Q04400, 991-1010; type VI, PIR A46187, 1059-1078; and type VIII, PIR PQ0227, 490-509.



The 31 additional deduced amino acids are inserted after amino acid 612 of the alpha(2) subunit within the apparent catalytic region. Amino acid alignments of the catalytic regions of guanylyl and adenylyl cyclases show a gap in the same position(33) . This prompted us to look for further sequence homologies. Within the same region of the catalytic consensus sequence of adenylyl cyclases (C domain), a sequence homologous to the insert was apparent (Fig. 1B). The homology was further evaluated by the method of Needleman and Wunsch(34) , as implemented by Dayhoff (35) and Doolittle et al.(36) using the computer program PCOMPARE (PCGENE 6.60, IntelliGenetics Inc.). The comparisons of the insert in the alpha subunit with the analogous sequences in human type 1 and type 2 adenylyl cyclases yielded statistically significant alignment scores (4.3 and 3.9, respectively, using the genetic code matrix and a number of random runs of 100).

Preliminary reverse transcription PCR studies showed an almost ubiquitous expression of the alpha(2) subunit along with the alpha(1) and beta(1) subunits (data not shown). To test whether alternative splicing of alpha(2) pre-mRNA occurs in all or only in some cell lines or tissues, cDNAs containing alpha(2) mRNA were chosen (Fig. 2, upper panel) and analyzed with respect to the presence of alpha mRNA. Messenger RNA coding for the alpha subunit could be demonstrated in human cDNA libraries from liver, colon, and endothelium (Fig. 2, lower panel). The alternatively spliced transcript alpha was also present in the cDNAs of the human promyelocytic leukemia cell line HL-60, in the human retinoblastoma cell lines Y-79 and WERI, and in the human megakaryocytic leukemia cell line MEG01. In contrast, mRNA coding for the alpha subunit was neither detectable in human cDNA libraries from fetal brain, testis, and the Jurkat T-cell line nor in the human erythroleukemia cell lines HEL and K562.


Figure 2: Comparison of alpha(2) and alpha expression in various human tissues and cell lines. PCR was performed using alpha(2)-specific primers C and D (upper panel) and alpha-specific primers E and F (lower panel). Reverse transcribed mRNA (5 ng) of the respective cell lines or cDNA library solution (10^6-10^7 plaque-forming units) of the respective tissues were used as template. PCR reactions were carried out as described under ``Experimental Procedures.'' Products were separated on a 1% (w/v) agarose gel, blotted, and detected with an alpha(2)- (upper panel) or an alpha-specific probe (lower panel). bp, base pairs.



Expression of the alpha subunit in COS-7 cells yielded a protein with an apparent molecular mass of 81 kDa that was recognized by polyclonal antibodies against the carboxyl terminus of the alpha(2) subunit (data not shown). However, coexpression of the novel variant with the beta(1) subunit did not result in detectable basal or nitric oxide-stimulated guanylyl cyclase activity in the COS expression system (data not shown). Because higher expression levels can be achieved in the Sf9-baculovirus expression system, coexpression experiments of the alpha(1)/beta(1), alpha(2)/beta(1), and alpha/beta(1) subunits were performed. As shown in Table 1, the highest guanylyl cyclase activity was demonstrated in the cytosol from Sf9 cells coinfected with the viruses coding for the alpha(1) and beta(1) subunits. In comparison, the alpha(2)/beta(1) heterodimer exhibited 3-9-fold lower activity, whereas no guanylyl cyclase activity could be detected in Sf9 cells infected with the alpha and beta(1) viruses. Because formation of cAMP has been reported for guanylyl cyclase prepared from rat liver (37) , adenylyl cyclase activity was also assessed (see Table 1). Adenylyl cyclase activity was at least 1 order of magnitude lower than guanylyl cyclase activity. It was highest in the presence of Mn and NO. Again, the alpha(1)/beta(1) enzyme displayed higher activity than the alpha(2)/beta(1) form, whereas no adenylyl cyclase activity could be detected for the coexpressed alpha and beta(1) subunits.



To test whether the alpha subunit retains its ability to form a heterodimer, the alpha subunit was coexpressed with a modified beta(1) subunit containing an amino-terminal hexahistidine tag. Subsequently, a two-step purification over DEAE anion exchange and Ni-nitrilotriacetic acid-agarose columns was performed. The purified protein was analyzed on a 8% (w/v) polyacrylamide gel and revealed two major bands (Fig. 3, lane d). The apparent molecular masses corresponded well with those of the alpha and the beta(1) subunits. The identity of the purified proteins as alpha and beta(1) was additionally verified by immunoblotting (data not shown).


Figure 3: SDS-polyacrylamide gel of the purified alpha/beta(1) heterodimer. Shown is a Coomassie Blue-stained 8% (w/v) polyacrylamide gel. Lane a, molecular mass markers; lane b, 10 µg of protein of a 100,000 g supernatant of recombinant baculovirus-infected Sf9 cells; lane c, 10 µg of protein of pooled fractions after DEAE chromatography; lane d, 2 µg of protein of purified alpha/beta(1) heterodimer.



The alpha subunit can apparently form heterodimers with the beta(1) subunit. Therefore, we investigated whether the novel variant could compete with the alpha(2) subunit for dimerization with the beta(1) subunit, thereby exerting a dominant negative effect. In fact, coexpression of increasing amounts of the alpha subunit with constant amounts of the alpha(2) and beta(1) subunits in Sf9 cells resulted in a decrease of both basal and nitric oxide-stimulated enzyme activity (Fig. 4A). To rule out that this inhibition was merely due to a decrease in the expression level of the alpha(2) or beta(1) subunits, the expression levels were monitored by immunoblotting. Fig. 4B shows the constant expression of the alpha(2) (79 kDa, upper panel) and beta(1) subunits (61 kDa, lower panel), which is not affected by the increasing expression of the alpha subunit. Due to its slightly higher molecular mass (81 kDa), the band representing the alpha subunit can be identified closely above the alpha(2) subunit (see Fig. 4B, lane 2-7, upper panel). The lower apparent molecular mass of the beta(1) subunit (61 kDa versus 70 kDa in Fig. 3) is caused by the addition of 4 M urea to the 8% (w/v) polyacrylamide gel that had to be used to separate the alpha and alpha(2) subunits.


Figure 4: Dominant negative effect of the alpha subunit on basal and NO-stimulated guanylyl cyclase activity. Sf9 cells (15 10^6) were coinfected with recombinant baculoviruses coding for the alpha(2) and beta(1) subunits (1 M.O.I. each) and increasing amounts of alpha-virus (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 M.O.I.). A, enzyme activity in the cytosol of the coinfected cells was determined as described under ``Experimental Procedures.'' Shown are data of one representative experiment out of three. Determinations were performed in duplicate with cytosol from independent Sf9 cell infections. Basal activity was determined in the presence of 3 mM Mn (open circles, right ordinate) or nitric oxide-stimulated activity in the presence of 3 mM Mg and 10 µM DEA-NO (filled circles, left ordinate). Endogenous enzyme activity was determined in cytosol from Sf9 cells infected with 5 M.O.I. of beta(1) (see also lane c in B). It was below 10 pmol min mg and was subtracted as nonspecific activity. B, shown are immunoblots of the same cytosolic protein preparations that were used for the determination of enzyme activity. Cytosolic protein (25 µg/lane) was separated on a 8% (w/v) polyacrylamide gel containing additional 4 M urea. After transfer to nitrocellulose membranes, guanylyl cyclase subunits were detected using an antibody against the carboxyl-terminal peptide of the alpha(2) and alpha subunit (upper panel) and an antibody against the carboxyl-terminal peptide of the beta(1) subunit (lower panel).




DISCUSSION

A novel variant cDNA coding for a guanylyl cyclase subunit (alpha) was obtained from different human cell lines using PCR with degenerate primers based on conserved sequences of the catalytic domain of mammalian guanylyl cyclases. The newly identified sequence represents an alternatively spliced transcript of the alpha(2) subunit of soluble guanylyl cyclase, which is present in some but not all tissues and cell lines containing alpha(2) mRNA. On comparison with mammalian adenylyl cyclases, the variant exon shows homology with the catalytic C domain. Coexpression experiments in Sf9 cells suggest that the alpha subunit acts as a negative regulator of NO-sensitive guanylyl cyclase activity. This dominant negative effect of the alpha subunit is due to competition with alpha(2) lacking the insert and the formation of nonfunctional heterodimers. Our data support the hypothesis that NO-sensitive guanylyl cyclase activity can be regulated by post-transcriptional modification of the alpha(2) pre-mRNA.

In a previous study, we showed that the beta(1) subunit can form heterodimers with alternative partners (alpha(1)/alpha(2))(7) . Highest enzyme activity was found in COS-7 cells expressing the alpha(1)/beta(1) heterodimer. In contrast, the alpha(2)/beta(1) heterodimer was shown to be 3-6 times less active. Here, the lower enzyme activity of the alpha(2)/beta(1) form is confirmed in the Sf9-baculovirus expression system. In addition, tissue screening by reverse transcription PCR indicates virtually ubiquitous distribution of the alpha(1), alpha(2), and beta(1) subunits, arguing against exclusive expression of either heterodimer. Thus, it is conceivable that a given cell modulates its degree of NO responsiveness by the expression of differing amounts of alternative alpha subunits. In the present study, we show that alternative splicing of the alpha(2) pre-mRNA converts the translational product into a dominant negative protein. This dramatically increases the regulatory potential of alternative heterodimer formation. Differential splicing leading to a dominant negative protein in heterodimeric complexes is not without precedence: DeltaFosB, a member of the AP-1 family of transcription factors, is a truncated form of FosB that arises by alternative splicing of the FosB transcript(20) . DeltaFosB retains its ability to form heterodimers with each of the Jun proteins but does not activate a target gene, thus acting as a negative regulator.

The cyclase catalytic consensus sequence is conserved across the guanylyl and adenylyl cyclase families(38) . In particular, there is analogy between soluble guanylyl cyclases and adenylyl cyclases; in both cases, there is evidence that catalytic activity is dependent on the interaction of two homologous but not identical catalytic consensus sequences (located on alpha/beta and C/C, respectively)(8, 9, 39) . Beyond this structural resemblance, soluble guanylyl cyclase preparations exhibit adenylyl cyclase activity(37) . Because a sequence within the additional exon of the alpha subunit shows homology with the C domain of adenylyl cyclases, our initial hypothesis was that alternative inclusion of the additional exon could convey higher adenylyl cyclase activity upon the soluble enzyme. Measurement of adenylyl cyclase in alpha/beta(1)-coinfected Sf9 cells shows that on the contrary, adenylyl cyclase activity is abolished (see Table 1). Therefore, whereas the novel sequence strengthens the similarity between the two enzyme families, inclusion of the optional exon does not lead to a transition from guanylyl to adenylyl cyclase activity.

In a recent study, Ujiie et al. showed desensitization of soluble guanylyl cyclase in cultured rat medullary interstitial cells (18) . Pretreatment of these cells with the NO-releasing compound sodium nitroprusside resulted in a decrease in guanylyl cyclase activity and a concomitant slight decrease in the mRNAs coding for the alpha(1) and beta(1) subunits as detected by Northern blotting. In view of our results, it is also important to consider desensitization caused by a post-transcriptional mechanism. Thus, alternative processing of the alpha(2) premRNA in human tissues could contribute to the development of tolerance.

In conclusion, we propose a model where differential expression of alpha isoforms (alpha(1), alpha(2), and alpha) modulates NO-sensitive guanylyl cyclase activity. Expression of the alpha(1)/beta(1) heterodimer yields high guanylyl cyclase activity. Increasing transcription of the alpha(2) mRNA at the expense of the alpha(1) mRNA gradually decreases enzyme activity. On the post-transcriptional level, alternative splicing of the alpha(2) pre-mRNA offers an additional potent mechanism to down-regulate NO-sensitive guanylyl cyclase activity.


FOOTNOTES

*
This study was supported by funds from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-30-838-6259; Fax: 49-30-831-5954.

(^1)
The abbreviations used are: DEA-NO, 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt; M.O.I., multiplicity of infection; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Jürgen Malkewitz for cell culture and valuable technical assistance.


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