©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Dominant Negative Mutations of the Guanylyl Cyclase-A Receptor
EXTRACELLULAR DOMAIN DELETION AND CATALYTIC DOMAIN POINT MUTATIONS (*)

(Received for publication, August 2, 1994; and in revised form, October 13, 1994)

Dana Kathryn Thompson (2) David L. Garbers (1)(§)

From the  (1)Howard Hughes Medical Institute and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050 and the (2)Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Guanylyl cyclase-A (GC-A), a receptor for A-type natriuretic peptide (ANP), contains an extracellular ligand-binding domain, a single transmembrane domain, and intracellular protein kinase-like and cyclase catalytic domains. Expression of the putative cyclase catalytic region (HCAT) resulted in the formation of an active enzyme that migrated as a homodimer on gel filtration columns; treatment with sodium trichloroacetate caused dissociation of the dimer and a loss of cyclase activity. Co-transfection of HCAT and full-length GC-A led to elevated basal intact cell cGMP concentrations and increased cell homogenate guanylyl cyclase activity. However, atrial natriuretic peptide-induced elevations of cGMP and cyclase activity were inhibited by the introduction of HCAT. Alanine scanning mutagenesis of highly conserved residues within HCAT identified one mutation (D893A) that destroyed enzyme activity but not the ability of the mutant subunit to form homodimers. The mutant subunit inhibited the cyclase activity of wild-type HCAT (approximately 70%) as well as that of full-length GC-A (approximately 85%) in co-expression studies where the amount of wild-type HCAT or full-length GC-A was not altered. Unlike co-transfection with wild-type HCAT, co-transfection of HCATD893A and GC-A did not result in elevated basal intact cell cGMP concentrations. For the first time we describe deletion and point mutations within the plasma membrane family of guanylyl cyclase receptors that result in the formation of effective dominant negative proteins.


INTRODUCTION

Effective inhibitors of specific guanylyl cyclase signaling pathways have not been available, but would prove valuable in determining the functions of cGMP and of the individual cyclase receptors in the many cells throughout the body where the role of this cyclic nucleotide is not understood.

Characteristic of the adenylyl and guanylyl cyclases is the existence of at least two cyclase catalytic consensus domains within an active enzyme. The various forms of vertebrate adenylyl cyclase contain two internal cyclase homology domains(1, 2, 3, 4, 5, 6) , where the separate expression of either region results in a loss of enzyme activity(7) . Cytoplasmic forms of guanylyl cyclase apparently require the co-expression of two subunits (alpha and beta) for activity(8, 9) , each of which contains a cyclase homology domain(10, 11, 12) , and plasma membrane forms of guanylyl cyclase have been suggested recently to exist as dimers or higher-ordered structures even in the absence of ligand(13, 14, 15) .

Since two consensus cyclase catalytic domains may be required for enzyme activity, the construction of cyclase mutants that retained the ability to oligomerize but not the ability to signal in response to ligand or the ability to form cGMP would act as dominant negative mutations. Such approaches have proved successful with various proteins, including the family of receptor tyrosine kinases(16, 17, 18, 19) . In these cases, truncation mutants have been constructed by deletion of the intracellular catalytic domain; the resulting extracellular domain fragment is a potent dominant negative subunit which can combine with the full-length receptor to form an inactive hybrid. The topological resemblance of membrane guanylyl cyclase and receptor tyrosine kinases led two groups to design a similar cytoplasmic truncation mutant of GC-A, (^1)and although the full-length and mutant receptors appeared to associate, the ANP-induced elevations of cyclic GMP were not effectively blocked at high concentrations of ANP (13, 14) . Dominant negative mutations have been generated of the soluble, heterodimeric form of guanylyl cyclase, but given their inherent differences in structure, these constructs are of no use in inhibiting membrane guanylyl cyclase.

Here, we considered the possibility that an extracellular truncation mutant would continue to dimerize with the full-length receptor and in so doing act as a dominant negative protein. This turned out to be the case, but since the cytoplasmic fragment contained cyclase activity, intracellular cGMP was elevated in the basal state. We then tested a series of point mutants and found one (D893A) that inactivated the cyclase when combined with wild-type receptor. Thus, the introduction of the point mutant not only interrupts ligand signaling, but also blocks cGMP production by the receptor.


EXPERIMENTAL PROCEDURES

Construction of Plasmids (Fig. 1)

Full-length GC-A was cloned into the expression vectors pSVL and pCMV3 as described previously(20) . The deletion mutant, HCAT, was provided by Dr. Michael Chinkers (Vollum Institute) and contained the FLAG epitope (DYKDDDDK), the cyclase catalytic domain, a region defined as the ``hinge'' region (approximately 47 amino acids located between the catalytic and kinase-like domains), and a small portion (15 amino acids) of the carboxyl segment of the consensus protein kinase homology domain. The deletion mutant, CAT, which does not encode the regions defined as the hinge or protein kinase-like domains was constructed by cloning the HCAT sequence into the BamHI/PstI sites of M13mp19. The oligonucleotide AGAACACAAGCTGGTACCATGTATCTGGAGGAG was used to introduce a KpnI site and start codon immediately 5` of Y806; the CAT mutant was then removed by KpnI/PstI digestion and cloned into the expression vector pCMV5.


Figure 1: Schematic diagram of various GC-A constructs. Wild-type GC-A contains an extracellular ligand-binding domain, a transmembrane domain, and intracellularly, protein kinase homology (PKH), hinge, and catalytic homology domains. HCAT contains a small portion of the PKH domain and the entire hinge and catalytic domains. CAT encodes only the catalytic homology domain.



Expression and Size Exclusion Chromatography of HCAT

COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B (Life Technologies, Inc.). Cells were transiently transfected by the DEAE-dextran method (21) with 5 µg of either HCAT, CAT, or vector alone. After transfection (48 h), cells were collected by scraping into 10 ml of ice-cold phosphate-buffered saline (Life Technologies, Inc.), gently pelleted (1,000 times g, 5 min), resuspended in harvest buffer (100 mM NaCl, 25 mM HEPES, pH 7.4, 10 mM dithiothreitol, 2.5 µg/ml aprotonin, 10 µg/ml leupeptin, and 0.1 µM phenylmethylsulfonyl fluoride), and lysed by sonication. The lysate was centrifuged at 100,000 times g for 15 min (2 °C) in a Beckman TL 100 tabletop ultracentrifuge and the supernatant fluid collected.

In order to obtain a concentrated lysate, typically 20-40 100-mm plates of transfected cells were pooled and lysed into 2 ml of harvest buffer to yield a supernatant fraction of approximately 10 mg protein/ml. This solution (100 µl) was loaded onto a Bio-Rad Bio-Sil SEC-125 column. The elution buffer was identical to the harvest buffer. Fractions were collected at 0.2-min intervals (200 µl/fraction). Protein standards from Pharmacia Biotech Inc. and Bio-Rad were chromatographed to obtain a calibration curve.

Treatment with Chaotropic Salt

The supernatant fraction (100 µl) from pSVLHCAT- or pSVLCAT-transfected COS cells was loaded onto the size exclusion column which had been equilibrated with elution buffer containing 0.3 M sodium trichloroacetate. Fractions were collected (as above) and assayed for guanylyl cyclase activity and analyzed by SDS-PAGE (as described below). Protein standards also were run in elution buffer plus sodium trichloroacetate to generate a calibration curve. To assay the lysate for guanylyl cyclase activity in the presence of chaotrope, 10 µl of the supernatant fraction of the lysate was incubated for 15 min on ice in the absence or presence of 0.3 M sodium trichloroacetate or 0.3 M sodium acetate. Cyclase assays were continued and [P]cGMP production measured as described below.

SDS-PAGE Analysis and Guanylyl Cyclase Assays

For analysis of HCAT, each 50-µl fraction from the gel filtration column, with 10 µl of 6 times Laemmli sample buffer, was run on 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose (Vanguard) for immunostaining (as described below). From the same fractions used for SDS-PAGE, duplicate 50-µl aliquots were assayed for guanylyl cyclase activity. The assay at 37 °C was initiated by the addition of sample to 50 µl of assay mix to give final concentrations of: 10 µm unlabeled GTP (Boehringer Mannheim), 0.5 µCi of [alpha-P]-GTP (NEN, 3000 Ci/mmol, 10 mCi/ml), 5 mM MnCl(2), and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX). After incubation for 15 min, the reaction was stopped by the addition of 0.25% SDS (Boehringer Mannheim). cGMP was purified over alumina and Dowex columns as described previously(22) , and radioactivity was counted in 10 ml of Safety Solve (Research Products International) using a Beckman LS-6800 scintillation counter. Assays of co-transfection experiments were performed as above, except for unlabeled GTP (100 µM), Triton X-100 (1%), and equal added amounts of protein (typically 25-50 µg as determined by BCA protein estimations (Pierce) or Bradford assays(23) . For some experiments, MnCl(2), Triton X-100, and [P]GTP were omitted, 5 mM MgCl(2), 5 mM ATP, and various concentrations of ANP were included, and cGMP levels were determined by radioimmunoassay. Under all conditions, product formation was linear with respect to time (up to 20 min) and protein concentrations (up to 100 µg).

For electrophoresis of GC-A co-transfectants, 7.5% SDS-PAGE was used. For native gels, SDS was omitted from all buffers.

Mutagenesis of Selected Amino Acids

To target amino acids D877, K887, D893, G900, H909, R940, and H944 for conversion to alanine, the following oligonucleotides were synthesized: D877A, TACACCTGTTTTGCTGCTGTCATAGAC; K887A, TTTGATGTGTACGCGGTGGAGACCATT; D893A, GAGACCATTGGTGCTGCTTACATGGTG; G900A, ATGGTGGTGTCAGCGCTCCCAGTGCGG; H909A, AATGGACAACTCGCCGCCCGAGAGGTG; R940A, CAGCTGCGCTTGGCCATTGGCATCCAC; H944A, CGCATTGGCATCGCCACAGGTCCTGTG; and E974A, GCTAACAACCTGGCGGAACTGGTAGAG (the underlined nucleotides represent the site of conversion to alanine). Mutagenesis was performed using the Muta-Gene M13 Kit (Bio-Rad). The template used, M13mp19HCAT, was the same used to make the deletion mutant CAT. After dideoxy sequencing to confirm that only the intended mutations were introduced, the HCAT point mutants were cloned into the expression vector pCMV5. Point mutants were expressed in COS cells, assayed for guanylyl cyclase activity, and analyzed on a size exclusion column as described above for wild-type HCAT.

Antibody

The carboxyl-terminal peptide of GC-A (RTYWLLGERGCSTRG) was conjugated to keyhole limpet hemocyanin, and the conjugated peptide was then injected into rabbits at 2-week intervals. Rabbit anti-sera to GC-A were obtained after the second collection of blood.

Western Blot Analysis

After electrophoretic transfer, the nonspecific protein-binding sites of nitrocellulose membranes were blocked by incubating the membrane with 20 mM Tris-HCl (pH 7.5) containing 5% non-fat dry milk, 0.05% Tween-20, and 0.5 M NaCl for 1 h at 24 °C. After washing with a solution containing 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, and 0.05% Tween-20, the membrane was incubated with the antisera (1:200 dilution in Tris-buffered saline with 0.05% Tween-20 and 1% BSA) for 1 h at room temperature. The membrane was washed three times with Tris-buffered saline containing 0.05% Tween-20 and then incubated with horseradish peroxidase-conjugated anti-rabbit antibody (Bio-Rad) at a 1:10000 dilution for 45 min. After washing the membrane, the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Corp.) was used for detection.

ANP Binding and ANP Stimulation

Binding to cell homogenates was determined with I-ANP as described(24) , in the presence or absence of 2.5 µM unlabeled ANP. For binding to intact cells, cells were split to 24-well plates 24 h after transfection. 48 h after transfection, the plates were transferred to a 37 °C slide warmer for the duration of the experiment, and cells were washed twice with 250 µl of DMEM containing 0.1% BSA. Cells were then incubated in DMEM, 0.1% BSA plus various concentrations of I-ANP, and the presence or absence of 2.5 µM non-radioactive ANP (to determine nonspecific binding), for 1 h. The medium was then gently aspirated and the cells washed four times with 250 µl phosphate-buffered saline, 0.1% BSA. Finally, 0.5 ml of Trypsin-EDTA was added for 15 min, and the cells were collected and radioactivity was counted. Data were analyzed using the LIGAND program(25) .

For whole-cell stimulations, transfected cells were split to 6-well (35-mm) plates, washed with serum-free medium, incubated at 37 °C in medium + 0.1 M IBMX for 10 min, and then incubated for 10 min in 1 ml of fresh medium, 0.1 mM IBMX plus various concentrations ANP. Stimulations were stopped with 1 ml 1 N perchloric acid, and cGMP was measured by radioimmunoassay, following cyclic nucleotide purification, as described previously(26) .


RESULTS AND DISCUSSION

Molecular Size of the Active Catalytic Domain

In order to design dominant negative mutants of GC-A, several preliminary questions needed to be addressed, such as whether or not the cyclase consensus domain contained enzyme activity when expressed without the upstream domains, and if so, whether activity would be found in a monomeric or oligomeric species. Recent studies have suggested that full-length GC-A exists as an oligomer in the presence or absence of ligand(13, 14) , but questions remained as to whether this oligomerization results in a physical association of the catalytic domains. The carboxyl-terminal 282 amino acids of GC-A (HCAT), after expression in COS-7 cells, retained enzyme activity, and essentially all activity migrated on gel filtration columns in the presence of 10 mM dithiothreitol at the position of a homodimer (Fig. 2A). Immunoreactive HCAT was also seen principally at the position of catalytic activity, although some immunoreactive material was present at the column void volume, presumably as a high molecular mass aggregate of denatured protein. To ascertain whether the homodimer could be dissociated, and the effects of such disruption on cyclase activity, HCAT was chromatographed in elution buffer containing 0.3 M sodium trichloroacetate, a chaotropic salt (Fig. 2B; the protein standards also were analyzed in the presence of sodium trichloroacetate). The chaotrope caused the protein standards and the general profile of COS cell proteins to elute earlier than in the absence of the chaotrope. In contrast, HCAT immunoreactivity migrated principally at the expected position of a monomer; there was not detectable enzyme activity in these fractions. A small amount of immunoreactive material eluted at the position expected of the homodimer, and a low amount of cyclase activity continued to be detected in these fractions. Although results from the use of sizing columns must be interpreted cautiously because 1) HCAT eluted precisely at the fraction predicted for a dimer and not ambiguously between the dimer and monomer fractions, and 2) treatment with chaotrope caused the protein standards to elute much earlier but HCAT to run counter to this trend and elute much later in the predicted monomer fraction, the conclusion is strongly supported that HCAT forms a homodimer. The failure to detect monomers of HCAT in the absence of the chaotropic salt suggests that dimerization is strongly promoted by this region of GC-A, and barring a repulsive effect of the protein kinase-like domain, that dimers or higher-ordered structures occur normally in wild-type GC-A as a consequence, at least in part, of dimerization domain(s) within this portion of the receptor. The previously observed oligomerization of full-length GC-A now is explained, at least in part, by these findings as a requirement for catalysis.


Figure 2: Migration of HCAT on gel permeation columns. A, migration of HCAT in the presence of 100 mM NaCl, 25 mM HEPES, pH 7.4, and 10 mM dithiothreitol. The soluble fraction (100 µl) of pSVLCAT-transfected COS cells was loaded onto a Bio-Sil SEC-125 column, and fractions (200 µl) were collected at 0.2-min intervals. Main panel, enzyme activity of the various fractions collected. Aliquots (50 µl) were assayed as described under ``Experimental Procedures.'' Elution positions of molecular mass standards are indicated. Inset, immunoreactivity of the same fractions. Aliquots (50 µl) of each fraction were run on 10% SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal antibody R1215, which recognizes the carboxyl terminus of HCAT. B, migration of HCAT in the presence of the sodium trichloroacetate. Gel filtration was performed as in part A, except the column was equilibrated with elution buffer containing 0.3 M sodium trichloroacetate. Samples were run on 10% SDS-PAGE and transferred to nitrocellulose for Western blot analysis. Molecular mass standards, also run in the presence of sodium trichloroacetate, are indicated.



CAT, the construct containing 58 fewer amino acids at the amino terminus was devoid of catalytic activity, and all immunoreactive material migrated at the void volume of the gel filtration column, suggesting that it was denatured (data not shown). Thus, amino acids contained within the hinge domain (or possibly at the very carboxyl terminus of the defined protein kinase homology domain) are integral to forming a properly folded active catalytic site, at least in COS cells. The hinge domain is highly conserved among membrane forms of guanylyl cyclase and resembles the coiled coil sequences which mediate association in some oligomeric proteins(27) ; thus it is possible that this region mediates dimerization of two catalytic domains to form an active site.

Dominant Negative Effect of HCAT

That HCAT formed homodimers raised the question of whether HCAT could form a dimer with full-length GC-A. However, owing to the deletion of both the extracellular ligand-binding domain as well as the kinase homology domain, which has been shown to be required for signaling, HCAT could potentially interrupt ANP signaling in GC-A/HCAT heterodimers. COS-7 cells, co-transfected with GC-A, and vector displayed low basal cGMP levels and responded to ANP with typical elevations of cGMP (approximately 3200-fold at 10M ANP). In contrast, basal cGMP levels of cells co-transfected with GC-A and HCAT were elevated 45-fold in the absence of ANP (Fig. 3); this is likely due to increased cyclic GMP formation due to the presence of HCAT/HCAT, HCAT/GC-A, and GC-A/GC-A dimers. HCAT/HCAT and HCAT/GC-A would not be expected to respond to ANP and therefore stimulation by ANP should be severely blunted, as in fact was seen (Fig. 3A). These results were essentially mirrored when cyclase activity of cell homogenates was measured in the presence of 5 mM MgCl(2), 1 mM ATP, and in the absence or presence of various concentrations of ANP (Fig. 3B). Basal cyclase activity of GC-A/HCAT transfectants was again increased compared to GC-A/vector homogenates (1.6 versus 0.3 pmol of cGMP formed min per mg protein, respectively), again, probably due to formation of HCAT/HCAT, HCAT/GC-A, and GC-A/GC-A active dimers. However, neither HCAT/HCAT nor HCAT/GC-A dimers would be expected to respond to ANP, and as shown in Fig. 3B, responsiveness to ANP was severely blunted by the introduction of HCAT.


Figure 3: The effects of co-transfection of HCAT and of GC-A on cGMP concentrations and guanylyl cyclase activity in COS cells. Cells were transfected with pCMV-GC-A and either pCMV5 or pCMV5-HCAT. A, intact cells were treated with various concentrations of ANP for 10 min, and cGMP concentrations were measured by radioimmunoassay. B, cells were homogenized and assayed for cyclase activity in the presence of 100 µM GTP, 5 mM MgCl(2), 1 mM ATP, and various concentrations of ANP, and cGMP levels were measured by radioimmunoassay. For both experiments, data for experimental co-transfectants (GC-A + HCAT) are presented as percent of control co-transfectants (GC-A + vector), and points represent duplicate or triplicate determinations within a single representative experiment.



Such inhibition by HCAT is most likely due to the formation of GC-A/HCAT heterodimers which are reduced or deficient in the ability to respond to ANP. However, to rule out the possibility that inhibition of HCAT was due to a reduction in expression of GC-A, immunoblots of cell homogenates were examined. Cells co-transfected with GC-A and either vector or HCAT each expressed the same levels of GC-A (Fig. 4). Routing of GC-A to the cell surface apparently also was not altered, since immunoreactive GC-A in purified plasma membranes remained constant as well (not shown). Binding of I-ANP to whole cells was equivalent between GC-A/HCAT co-transfectants and cells expressing GC-A alone. For GC-A/HCAT versus GC-A cells, K(d) values were 4.2 times 10 and 3.1 times 10M, respectively, and B(max) values were 2.4 times 10 and 2.0 times 10M, respectively. Therefore, since the amount of GC-A (estimated from immunoblots) is not reduced by co-expression of HCAT, the equivalent binding values suggest that ANP binds equally as well to monomeric as to oligomeric GC-A. HCAT, therefore, did not inhibit cyclic GMP elevations or cyclase activation by ANP as a result of an inhibition of ligand binding.


Figure 4: Western blot of co-transfectants to monitor protein expression. COS cells were transfected with pCMVGC-A and either vector (A), pCMV5HCAT (B), or PCMV5HCATD893A (C). Cells were harvested, as described under ``Experimental Procedures,'' and equal amounts of protein were run on 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal antibody R1215, which recognizes the carboxyl termini of both the fragments and full-length GC-A. Molecular weight markers are indicated, and GC-A and the fragments are designated by bullets.



Alanine Scanning Mutagenesis of Conserved Amino Acids within the Catalytic Domain

The dominant negative effect of the extracellular domain deletion mutant was of potential utility, but basal cGMP concentrations and cyclase activity were elevated in co-transfection experiments. Therefore, a series of experiments was designed to determine whether or not a point mutation could be produced in which enzyme activity of the heteromer was destroyed.

Several invariant or highly conserved residues throughout the catalytic domain were targeted for alanine scanning mutagenesis; this included Gly, an amino acid in the same relative position as the glycine of rutabaga, the Drosophila adenylyl cyclase mutant without activity, where glycine is mutated to arginine (28) . Gly, along with charged residues Asp, Lys, Asp, His, Arg, His, and Glu were converted to alanine in HCAT. The HCAT point mutants were then transfected transiently into COS cells and examined by immunodetection to confirm protein size (data not shown) and assayed for guanylyl cyclase activity (Fig. 5). One mutant protein, E974A, displayed substantially higher activity (approximately 30-40-fold higher activities at 100 µm of MnGTP and equivalent amounts of enzyme protein based on immunoblots) as compared to wild-type HCAT. The remaining mutations, including G900A, resulted in no detectable cyclase activity.


Figure 5: Specific activity of various point mutants of HCAT. Point mutants were introduced into HCAT as described under ``Experimental Procedures,'' transiently transfected in COS-7 cells, and assayed for guanylyl cyclase activity in the presence of MnCl(2). Bars represent the mean of triplicate determinations in a single representative experiment. The specific activity of vector-transfected cells was less than 1 pmol min/mg protein.



Each of the mutant proteins was analyzed by gel filtration to determine apparent size. E974A, the superactive mutant, and one other mutant, D893A, eluted as homodimers (data summarized in Fig. 5). The remaining point mutants D877A, K887A, H909A, R940A, and H944A, as well as the mutant corresponding to rutabaga G900A, eluted in the void volume as high molecular weight aggregates, presumably due to protein denaturation. These conserved residues, as well as the rutabaga glycine, therefore, could play critical roles in protein folding.

Dominant Negative Effect of D893A on Cyclase Activity

That D893A formed a homodimer without activity raised the question of whether or not it could combine with, and disrupt the activity of, wild-type enzyme. Initially, this was tested by the co-transfection of HCATD893A and HCAT into COS-7 cells. Cells were simultaneously transfected with 1 µg of pSVLHCAT and various amounts (0.1, 0.5, 1.0, or 2.0 µg) of either pCMV5 or pCMV5 HCATD893A (Fig. 6). Increasing amounts of vector did not diminish cyclase activity (Fig. 6) or protein expression as detected by immunoblots (data not shown); however, increasing amounts of the point mutant HCATD893A dramatically reduced HCAT cyclase activity by as much as 73%. Since HCAT and HCATD893A migrated identically on SDS-PAGE, it was not possible to distinguish between the two on immunoblots in order to monitor the expression of wild-type HCAT in the HCATD893A co-transfected cells. Therefore, co-transfection experiments were carried out using full-length GC-A (in pCMV5, 0.2 µg) and 0.1 or 0.5 µg of either pCMV5 or pCMV5 HCATD893A. HCATD893A caused a substantial decrease in guanylyl cyclase activity, as great as 85% when co-transfected with full-length GC-A (Fig. 7). The inhibition of cyclase activity was observed when cell lysates were assayed either in the presence of Mn and Triton X-100 (which represents the greatest stimulation of activity) or in the presence of Mg and ATP without detergent (Table 1). The loss of activity under these conditions can be attributed directly to the point mutation, since cells co-expressing GC-A and wild-type HCAT and assayed in the presence of Mn and Triton demonstrate an elevation, not a reduction, of cyclase activity (not shown). Importantly, the expression of wild-type GC-A, as detected on immunoblots, remained unchanged in the presence of either vector alone or the point mutant (Fig. 4). Furthermore, specific I-ANP binding to whole cells and to cell homogenates (Table 1) and distribution among membrane fractions of immunoreactive GC-A (data not shown) was not different between control and experimental co-transfectants, providing additional evidence that expression of wild-type GC-A remained constant.


Figure 6: Co-transfection of HCAT and the point mutant HCATD893A. COS-7 cells were transiently transfected with 1 µg of HCAT and either no additional DNA, increasing amounts of HCATD893A, or increasing amounts of vector. Cells were harvested and assayed for cyclase activity in the presence of Mn/Triton X-100 as described under ``Experimental Procedures.'' Bars represent the means ± standard error of triplicate determinations of a single representative experiment.




Figure 7: Co-transfection of GC-A and HCATD893A. COS-7 cells were transiently transfected with 0.1 µg of GC-A, and 0.1 or 0.5 µg of either HCATD893A or vector. Cells were harvested and assayed for cyclase activity in the presence of MnCl(2)/Triton X-100 as described under ``Experimental Procedures.'' Bars represent the means ± standard error of triplicate determinations of a single representative experiment.





Dominant Negative Effect of D893A on ANP-induced Elevations of Cyclic GMP in Intact Cells

The dominant negative effects of the point mutant, D893A, were evident when cyclic GMP responses of intact cells were determined (Fig. 8A). Cells co-transfected with wild-type GC-A and vector displayed a typical dose-response to ANP, with half-maximal stimulation at approximately 1 µM ANP and maximal stimulation at 100 µM ANP. Cells co-transfected with GC-A and HCATD893A showed a reduction in cGMP accumulation at all concentrations of ANP. A similar inhibition by HCATD893A on GC-A was seen in co-transfectants homogenized and assayed for ANP-stimulated cyclase activity (Fig. 8B).


Figure 8: Effect of GC-A/HCATD893A co-transfection on cGMP concentrations of intact cells and on guanylyl cyclase activity. Cells were transfected with pCMV-GC-A and either vector or pCMVHCATD893A. A, intact cells were treated with various concentrations of ANP for 10 min, and cGMP concentrations were measured by radioimmunoassay. B, cells were homogenized and assayed for cyclase activity in the presence of 100 µM GTP, 5 mM MgCl(2), 1 mM ATP, and various concentrations of ANP, and cGMP levels were measured by radioimmunoassay. For both experiments, data for experimental co-transfectants (GC-A + HCATD893A) are presented as percent of control co-transfectants (GC-A + vector), and points represent duplicate or triplicate determinations in a single representative experiment.



Thus, like HCAT, HCATD893A is a dominant negative inhibitor of signal transduction by GC-A. In addition, and unlike HCAT, HCATD893A is also a dominant negative inhibitor of cyclase catalysis. Presumably, aspartic acid 893 is critical to enzyme activity, perhaps by contributing to the structure of the active site, or participating, by benefit of its charge, to catalysis itself. The importance of this residue is borne out not only by its high conservation among all cyclases, but more recently by the finding that alanine substitution made at the analogous aspartic acid in the alpha subunit of soluble guanylyl cyclase serves as a potent dominant negative inhibitor when recombined with wild-type beta subunit(29) . Elsewhere, it has been demonstrated that catalysis by guanylyl and adenylyl cyclases proceed through the same stereochemical course(30) , thus it will be interesting to see if mutation of the analogous residue in adenylyl cyclase confers the same inhibition.

Mutation by truncation is commonly used to engineer dominant negative mutants and has been attempted previously for membrane guanylyl cyclase. Chinkers and Wilson (13) and Lowe (14) each expressed the extracellular domain of GC-A, and when co-transfected with full-length GC-A it caused a shift to the right of the ANP dose-response curve. However, maximal cGMP elevations were not suppressed by the mutation. Given that GC-A may form tetramers, it remains possible that dimers of full-length/intracellular domain forms are capable of forming fully active tetramers.

Here, an intrinsically active intracellular fragment functions as a dominant negative inhibitor of signal transduction by full-length GC-A, even at high levels of ANP. Further mutation, alanine substitution at a highly conserved aspartic acid, also serves as a dominant negative inhibitor, in this case, of catalysis. Given the widespread expression of GC-A and its involvement in many endocrine and possibly paracrine functions, it would be desirable to examine this receptor in intact animals. These mutants may serve as useful reagents for examining GC-A function in vivo, much like dominant negatives have been used in analogous studies to examine the role of fibroblast growth factor acting through the fibroblast growth factor receptor (18) and RAF-1 kinase (31) to induce mesoderm development. In the regulation of blood pressure and volume via natriuresis and diuresis in the kidney, smooth muscle relaxation in the cardiovasculature, and aldosterone secretion by the adrenals, the importance of membrane guanylyl cyclase has been acknowledged, although the pathways have not been precisely detailed. The selection of tissue-specific promoters could lead to a dominant negative effect in limited areas in transgenic animals, allowing one to dissect the role of guanylyl cyclase receptors in multiple selected tissues. Finally, construction of distinct dominant negative mutants specific for either membrane or soluble forms of guanylyl cyclase should permit the two pathways to be studied independently, even if they are present in the same cell.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HD10254 (to D. L. G.) 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.: 214-648-5090; Fax: 214-648-5087.

(^1)
The abbreviations used are: GC-A, guanylyl cyclase-A: ANP, atrial natriuretic peptide; DMEM, Dulbecco's modified Eagle's medium; IBMX, 3-isobutyl-1-methylxanthine; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.


ACKNOWLEDGEMENTS

We thank Deborah Miller and Cecelia Green for assistance with DNA sequencing and radioimmunoassays and Juanita Coley for assistance with the manuscript.


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