PURIFICATION AND CHARACTERIZATION OF THE 180-kDa MEMBRANE GUANYLATE CYCLASE CONTAINING ATRIAL NATRIURETIC FACTOR RECEPTOR FROM RAT ADRENAL GLAND AND ITS REGULATION BY PROTEIN KINASE C
Rameshwar K. Sharma, Ravi B. Marala, and Teresa M. Duda Section of Regulatory Biology, Cleveland Clinic Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195-5068, USA
Corresponding author: Rameshwar K. Sharma Received October 11, 1989 Revised April 10, 1989
ABSTRACT
The original concept that cyclic GMP is one of the mediators of the hormone- dependent process of steroidogenesis has been strengthened by the characterization of a 180-kDa protein from rat adrenocortical carcinoma and rat and mouse testes. This protein appears to have an unusual characteristic of containing both the atrial natriuretic factor (ANF)-binding and guanylate cyclase activities, and appears to be intimately involved in the ANF-dependent steroidogenic signal transduction. In rat adrenal glands we now demonstrate: 1) the direct presence of a 180-kDa ANF-binding protein in GTP- affinity purified membrane fraction as evidenced by affinity cross-linking technique and by the Western blot analysis of the partially purified enzyme; 2) that the enzyme is biochemically and immunologically different from the soluble guanylate cyclase as there is no antigenic cross-reactivity of 180-kDa guanylate cyclase antibody with soluble guanylate cyclase; 3) in contrast to the soluble guanylate cyclase, the particulate enzyme is not stimulated by nitrite-generating compounds and hemin; and 4) protein kinase C inhibits both the basal and ANF-dependent guanylate cyclase activity and phosphorylates the 180-kDa guanylate cyclase. These results reveal the presence of a 180-kDa protein in rat adrenal glands and support the contention that: (a) this protein contains both the guanylate cyclase and ANF receptor; (b) the 180-kDa enzyme is coupled with the ANF- dependent cyclic GMP production; (c) the 180-kDa enzyme is biochemically distinct from the nonspecific soluble guanylate cyclase; and (d) there is a protein kinase C- dependent negative regulatory loop for the operation of ANF-dependent cyclic GMP signal pathway which acts via the phosphorylation of 180-kDa guanylate cyclase.
INTRODUCTION
Original studies with the model cell systems of isolated fasciculata of rat adrenal
cortex and rat adrenocortical carcinoma indicated that cyclic GMP is one of the physiological mediators of steroidogenic signal transduction. This work led to the proposal of a hypothetical working model in which membrane guanylate cyclase was the key enzyme in the transmembrane receptor-mediated cyclic GMP signal pathway [reviewed in (1)]. The concept however, was not readily accepted because at the time there was no conclusive biochemical evidence for the presence of any distinct cyclic GMP signal component in adrenal cortex. In addition, the paramount argument against the second messenger role of cyclic GMP in any receptor-mediated signal transduction process was the lack of a clear cut demonstration of a hormonal signal which resulted in the specific stimulation of particulate guanylate cyclase. The dogma was that there was only one guanylate cyclase, soluble enzyme, which was documented to be hormone- independent and nonspecifically activated by a variety of nitrite-generating compounds and agents that affect the oxidation-reduction potential of biological reactions (2,3). These reservations were overcome by the direct biochemical demonstration of cyclic GMP-dependent protein kinase and adrenocorticotropic hormone (ACTH)-dependent particulate guanylate cyclase in adrenal cortex, thus identifying the two individual key components of cyclic GMP signal system (1). The membrane guanylate cyclase was hormone specific and, unlike the soluble form of the enzyme, was not stimulated nonspecifically. Therefore, discovery of ACTH-dependent guanylate cyclase was a remarkable breakthrough, because this identified the key receptor-coupled enzyme which could be turned on by the hormone in catalyzing the formation of cyclic GMP from GTP, thus rationalizing the potential second messenger role of cyclic GMP in external surface receptor-mediated signal transductions.
The above concept, cyclic GMP as one of the premium second messengers of receptor-mediated signal transductions, was consolidated by the observation that atrial natriuretic factor (ANF) selectively stimulates particulate guanylate cyclase activity (4,5), including that from the fasciculata cells of rat adrenal cortex and the rat adrenocortical
carcinoma (6). A protein subunit with an Mr of 180,000 apparently containing the extraordinary dual property of ANF receptor and guanylate cyclase was purified to homogeneity (7,8). The enzyme is biologically coupled to the ANF-dependent formation of cyclic GMP in rat testes (9). Because ANF in rat testes (10,11), as in rat adrenocortical fasciculata cells, stimulates the process of steroidogenesis, there is a good possibility that the 180-kDa guanylate cyclase is one of the steroidogenic signal transducers. Consistent with this idea, we now demonstrate the biochemical presence of this enzyme in rat adrenal glands, its biological coupling with ANF receptor-mediated cyclic GMP formation, its biochemical distinctness from soluble guanylate cyclase, and its negative regulation and phosphorylation by protein kinase C. Details of purification of 180-kDa guanylate cyclase and of the production and characterization of the polyclonal antibody against the 180-kDa enzyme are described in this paper.
MATERIALS AND METHODS
Materials
GTP, 3-[(3-cholaimidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), cyclic GMP, sodium nitroprusside, and bovine serum albumin were purchased from Sigma; GTP-agarose affinity resin was from P.L. Biochemicals; CNBr-activated Sepharose 4B was from Pharmacia; [12’I]Nal was from New England Nuclear and [7-32P]ATP was purchased from Amersham. Synthetic ANF consisting of the 26-amino- acid peptide H-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln- Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH used in these studies was a kind gift of Dr. R. Nutt. All other reagents were of analytical grade.
Methods
Guanylate Cyclase Assay. For crude and partially purified samples, guanylate cyclase was assayed as described previously (12). Briefly, the reaction mixture (100 L) contained 50 mM Tris-HCI, pH 7.5, 10 mM theophylline, 1 mM CaCl2, 15 mM creatine phosphate, 20 µg (140 units/mg) of creatine phosphokinase, and 20 uL of sample. The assay mixtures for the purified samples did not contain theophylline and the GTP- generating system (creatine phosphate and phosphokinase). The reaction was initiated by the addition of 1 mM GTP and 4 mM MnCl2, incubated at 37 C, and terminated by the addition of 900 uL of ice-cold 50 mM sodium acetate buffer, pH 6.2, followed by heating at 100 C for 3 min. The samples were centrifuged and the supernatant was assayed for cyclic GMP by radioimmunoassay.
Phosphorylation of the 180-kDa Guanylate Cyclase. The GTP-affinity purified fractions of guanylate cyclase were incubated with protein kinase C in the presence or
absence of 180-kDa guanylate cyclase antibody as described above, and the phosphorylated samples were analyzed on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by radioautography. 20 uL of partially purified protein kinase C (nearly equal to 6.5% purity) purified up to phenyl-Sepharose chromatography step as in reference 13, was used with 1 mM ATP, phosphatidylserine (50 µg/sample), diolein (8 µg/sample), and [y-32PJATP (0.5 uCi/sample).
SDS-PAGE. The samples were dissolved in buffer containing 62.5 mM Tris-HCI (pH 6.8), 3% sodium dodecyl sulfate, 10% glycerol, and 100 µg/mL of bromophenol blue, applied to 7.5% acrylamide containing 0.1% SDS and 0.3% bisacrylamide (14). Electrophoresis was conducted in the presence of Tris-glycine buffer (pH 8.1). The gel was stained with Coomassie Brilliant Blue.
Rabbit Anti-Particulate Guanylate Cyclase Antibody. The SDS-PAGE slices containing the isolated 180-kDa membrane guanylate cyclase were emulsified in Freunds complete adjuvant and distributed subcutaneously into five sites in the nuchal region, and 1.0 mL was injected intramuscularly into each hind leg muscle following a previously described protocol (7). The intramuscular injections were repeated at 3-week intervals using Freunds incomplete adjuvant. Rabbits were bled from the ear veins about 10 days after each injection and the serum separated. Gamma globulins were separated by repeated ammonium sulfate precipitation and extensive dialysis. These IgG fractions from immune and normal serum pools were used for all experiments. The antibody titer was determined by solid phase RIA using 125I-labeled goat anti-rabbit IgG.
Immunoblot Analysis. Utilizing rabbit anti-particulate guanylate cyclase, authenticity of the guanylate cyclase in GTP-affinity purified fraction was checked by Western blot analysis (15). The crude enzyme was run on SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (15). The excess of protein binding sites was blocked with 2% bovine serum albumin in 50 mM sodium phosphate buffer (pH 7.0) containing 0.85% NaCl. The nitrocellulose membrane was treated with antisera (1:200-dilution) in phosphate-buffered saline and the membrane was extensively washed with the same buffer and treated with 1 in 1,000 dilution of goat anti-rabbit IgG conjugated to peroxidase. The nitrocellulose membrane was treated with 4-chloro- 1-naphthol and hydrogen peroxide to reveal the antibody cross-reactivity protein band.
Affinity Cross-linking Technique. The appropriate enzyme fraction (100 - 200 ug protein) was incubated at 4 C for 20 h in ANF binding buffer (125 mM NaCl, 25 mM 4-[2-hydroxyethyl]-1-piperazine-ethanesulfonic acid (Hepes), 1 mM bacitracin, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 g/dL bovine serum albumin (BSA)) with 0.2 uCi 125I-ANF in the absence or presence of 1 uM unlabeled ANF in a total volume of 100 uL. To the mixture was added 11 µL of 10 mM disuccinimidyl suberate in dimethyl sulfoxide (DMSO), and incubated on ice for 15 min. The reaction was quenched with 56 uL 1 M Tris, 0.2 M EDTA, pH 6.8. To the sample was added 35 uL sample buffer for SDS-polyacrylamide gel electrophoresis containing 2% ß- mercaptoethanol as the reducing agent, boiled for 3 min, resolved on a 7.5% SDS-PAGE (14), and autoradiographed.
Protein Determination. The protein was determined by the method of Smith et al (16) marketed by Pierce utilizing bovine serum albumin as a standard.
Purification of the 180-kDa Guanylate Cyclase. Unless otherwise stated, procedures were performed at 0-4 C using deionized water.
Step 1: Solubilization. A zwitterionic detergent (CHAPS) was used for the solubilization of particulate guanylate cyclase. In a typical preparation 3 g of rat adrenal glands (or rat adrenocortical carcinoma) were solubilized as in (17). To the membrane suspension (2.7 mg/mL) was added 50 µg/mL lima bean trypsin inhibitor, 0.01% dimethyl sulfoxide, and 10 mM CHAPS (final concentration). This was stirred vigorously on ice for 1 h and then centrifuged at 105,000 x g for 60 min.
Step 2: GTP-Agarose Affinity Chromatography. GTP-agarose was washed successively with deionized water, 10 volumes of 20% ethyl alcohol, and several volumes of deionized water to remove preservatives and contaminants. The resin was equilibrated with 25 mM triethanolamine hydrochloride (pH 7,6), 5 mM MnCl,, and I mM CHAPS (Buffer A). A 80 mM MnCl2 solution was added to the solubilized enzyme to a final concentration of 5 mM. The solubilized enzyme was treated with the GTP- agarose affinity resin by inverting overnight in a mechanical shaker. The resin was loaded onto a small (1.6 x 8 cm) column, which was washed extensively with Buffer A until there was no detectable protein (absorbance at 280 nm). The guanylate cyclase was eluted at room temperature with 25 mM triethanolamine, pH 7.6, 1 mM CHAPS, and 2 mM EDTA. The fractions were collected at 30 min intervals, assayed for guanylate cyclase activity, and the active ones pooled.
Step 3: Cyclic GMP Affinity Chromatography. This purification step technique, originally utilized for the complete purification of 180-kDa rat adrenocortical carcinoma membrane guanylate cyclase, has earlier been described briefly (7), and is detailed below: Cyclic GMP-Sepharose resin was extensively washed with deionized water and equilibrated with Buffer A. The GTP-agarose enzyme was adjusted to 5 mM Mn2+ with 80 mM MnCl2 The enzyme was adsorbed to the cyclic GMP-Sepharose by mixing overnight. The resin was loaded onto a small column (1.6 x 8 cm), flow-through was once cycled back on the column, and the column was washed extensively with Buffer A. Elution of guanylate cyclase from cyclic GMP-Sepharose was done using 25 mM triethanolamine (pH 7.6), 1 mM CHAPS, and 2 mM EDTA at room temperature. The fractions were assayed for guanylate cyclase activity and active fractions were pooled.
The rationale for adoption of this purification scheme was based on a novel concept of cross-affinity chromatography (18). Theoretically, guanylate cyclase catalysed transformation of GTP into cyclic GMP accompanied by the formation of pyrophosphate — a high energy bond — is a reversible reaction. This suggests that guanylate cyclase should have binding sites for the substrate GTP and the product cyclic GMP. (Our unpublished results indicate that this indeed is the case.) Therefore, if the two immobilized ligands, GTP and cyclic GMP, are used in sequence, selective purification of guanylate cyclase should be possible. We call this method “crossed- affinity chromatography” because the protein eluted from the second column represents the cross-product of those proteins binding both substrate and product. By this technique, in excess of 273,000-fold purification to apparent homogeneity of rat adrenocortical carcinoma was achieved (7,18).
Immunoaffinity Purification of the 180-kDa Guanylate Cyclase. The rat adrenocortical carcinoma 180-kDa guanylate cyclase immunoglobulins raised in the rabbit as described above were covalently linked to CNBr-activated Sepharose 4B according to the specified directions (19). This immunoaffinity column was used to further purify the GTP-affinity step purified membrane guanylate cyclase. A 2-mL (1.2 mg protein) aliquot of GTP-affinity purified fraction was mixed with 1 mL of immunoaffinity gel overnight at 4 C over a mixer, poured into a small column (1.6 x 8 cm), which was then washed extensively with buffer containing 0.25 M triethanolamine, 1 mM CHAPS, and 4 mM MnCl,. The enzyme was eluted with 0.1 M acetate buffer, pH 3.5, containing 1 mM CHAPS and 2 mM EDTA. The eluted guanylate cyclase
fractions were immediately neutralized by the addition of 0.10 M Tris, pH 8.5. The activity of immunoaffinity purified fraction was calculated by immunoslot blot analyses. Briefly, serial dilutions (0.05 to 3.5 µg protein) of the GTP-affinity purified fraction with known activity were applied to nitrocellulose membrane as slot blots and were processed for Western blot analysis (as described earlier) with some modification: anti- rabbit IgG conjugated to alkaline phosphatase (1:5,000 dilution) was used instead of anti-rabbit IgG conjugated to peroxidase. The color was developed using 5-bromo-4- chloro-3-indolyl phosphate (BCIP) and p-nitro blue tetrazolium chloride (NBT) as substrates. 0.001 ug of immunoaffinity purified enzyme was applied to nitrocellulose membrane and processed as above. The intensities of the colored slot blots were analyzed in a densitometer. 0.001 µg of immuno-affinity purified enzyme gave same color intensity on slot blot as 0.429 ug of GTP-affinity purified enzyme. The activity of immunoaffinity purified enzyme was calculated by multiplying the activity of GTP- affinity purified enzyme by a factor of 429.5.
RESULTS
Titer of Rat Adrenocortical Carcinoma 180-kDa Guanylate Cyclase Antibodies Determined by Solid Phase Radioimmunoassay - The titer of 180-kDa guanylate cyclase antibodies was determined to be 1:25,600 (Fig. 1).
Purity and Characterization of a 180-kDa Guanylate Cyclase from Rat Adrenal Glands - We have previously reported on the purification and characterization of a 180- kDa guanylate cyclase from the rat adrenocortical carcinoma that apparently contains ANF receptor. In order to scrutinize the presence of this enzyme in rat adrenal glands, we utilized the tumor 180-kDa guanylate cyclase immunoaffinity column to purify the GTP-affinity step rat adrenal protein. For biochemical comparison, the tumor enzyme was also prepared under identical conditions. SDS-PAGE revealed a single Coomassie Blue- and silver-stained band with a molecular mass of 180-kDa in both the normal and malignant rat adrenal cortex, which was indistinguishable in its migration pattern from that of the originally purified (7,8) crossed-affinity purified tumor enzyme (Fig. 2A). This interpretation is further supported by the results of the Western blot analysis: GTP- affinity purified enzyme fractions of the rat adrenocortical carcinoma and rat adrenal gland revealed a single 180-kDa immunoreactive band, although the SDS-PAGE of these fractions in each case showed multiple Coomassie-stained bands (Fig. 2B). These results
5000
4000
3000
cpm
2000
1000
0
10
1
102
10
3
10
4
10
5
Antibody Dilution
demonstrated apparent homogeneity of the rat adrenal 180-kDa protein which was biochemically and immunologically indistinguishable from the previously characterized 180-kDa rat adrenocortical carcinoma membrane guanylate cyclase (7).
The specific activity of the rat adrenal enzyme is ~ 21,211 nmol/min/mg of protein, which is about 10-fold higher than that of the rat adrenocortical carcinoma and compares favorably with those reported for the bovine adrenal cortex (20,21) and the lung enzyme (22). That the enzyme is an authentic guanylate cyclase is shown by the immunological studies, which show that the antibody to the 180-kDa protein blocks up to 42% of the partially purified guanylate cyclase activity. It is noteworthy that as is the case in the adrenocortical carcinoma and rat and mouse testes (9), the antibody does not significantly inhibit the crude membrane guanylate cyclase activity (Fig. 3). As suggested earlier (9), one possible explanation for this is that the basal activity of the crude membrane enzyme comprises the accumulative activity of as-yet-unknown
Mr x 10
- 3
TOP
Mr x 10
- 3
TOP
200
180
180
116
94
68
43
DYE
DYE
1
2
3
1
2
3
A
B
0.5
Guanylate cyclase activity nmol / mg protein / min
0.4
0.3
0.2
☐ Enz. only
0.1
Enz. + Ab
Enz. + Ps
0.0
Enz. + ANF
Membrane
Solubilized
Enz. + ANF + Ps
Enz. + ANF + Ab
70
Guanylate cyclase activity nmol / mg protein / min
60
50
Abbreviations:
40
Enz. = enzyme Ab = antibody Ps = preimmune serum
30
20
ANF = atrial natriuretic factor
10
0
GTP-Affinity
multiple isozyme forms, among which the 180-kDa enzyme represents a minor component. This interpretation is supported by the fact that antibody to the 180-kDa protein blocks almost all of the guanylate cyclase activity of the purified enzyme (8,18).
ANF Binding to a 180-kDa Protein - Affinity crosslinking studies were done to determine the apparent molecular mass of ANF binding protein in rat adrenocortical
carcinoma membranes. Assessment by SDS-PAGE revealed that a 180-kDa protein bound ANF specifically in malignant rat adrenal GTP-affinity purified fraction. In addition an 85-kDa protein was specifically labeled with 125I-ANF in the adrenocortical carcinoma enzyme fraction (Fig. 4). We do not know the nature of this 85-kDa ANF binding site.
Blockage of ANF-dependent Guanylate Cyclase Activity by the Antibody to the 180-kDa Rat Adrenocortical-Carcinoma Membrane Guanylate Cyclase - ANF-stimulated crude membrane guanylate cyclase from rat adrenal glands, and the hormonal stimulation was blocked by the antibody to the 180-kDa rat adrenocortical-carcinoma membrane guanylate cyclase (Fig. 3), indicating the hormonal dependence of the 180- kDa enzyme.
Loss of ANF-Dependent Guanylate Cyclase Activity on Solubilization and Further Purification of the 180-kDa Guanylate Cyclase - A common feature of the rat adrenocortical carcinoma 180-kDa guanylate cyclase (7,18), 120-kDa lung guanylate cyclase (22), and the 130-kDa bovine adrenocortical enzyme (20) is that all these proteins upon solubilization and further purification lose ANF-dependence in catalyzing the formation of cyclic GMP. Similarly, the hormonal dependence is lost upon solubilization and further purification of the 180-kDa guanylate cyclase isolated from rat adrenal glands (Fig. 3).
Lack of Immunocrossreactivity of the 180-kDa Membrane Guanylate Cyclase Antibody with the Soluble Guanylate Cyclase - In contrast to the complete blockage of both the hormone-dependent activity and the total guanylate cyclase activity of the purified membrane enzyme (Fig. 3), the antibody to the 180-kDa membrane guanylate cyclase neither blocked the soluble guanylate cyclase activity (Fig. 5A) nor showed any cross-reactivity by ELISA or by Western blot analysis. This represents the immunological distinctness of the 180-kDa membrane guanylate cyclase from the soluble
- 3
Mr x 10
TOP
180
85
DYE
1
2
guanylate cyclase. Because the antibody also does not block adenylate cyclase activity (Fig. 5B), this indicates that the 180-kDa guanylate cyclase is also immunologically Lack of Stimulation of the 180-kDa Guanylate Cyclase Activity by the Nitrite
Generating and Other Agents Which Stimulate Soluble Guanylate Cyclase - Nitric oxide generating compounds stimulate soluble and most of the particulate guanylate cyclases (2, 3). Sodium nitroprusside and sodium azide did not activate the adrenocortical carcinoma membrane guanylate cyclase; on the other hand, soluble guanylate cyclase was stimulated by these agents in a concentration-dependent fashion (Figs. 6A & 6B).
Lack of Inhibition of the 180 kDa Guanylate Cyclase Activity by the Thiol Reagents Which Inhibit Soluble Guanylate Cyclase Activity - Compounds like N- ethylmaleimide, methylene blue, and cadmium, known to react with -SH groups and inhibit guanylate cyclase (23), did not alter the 180-kDa membrane guanylate cyclase activity (Fig. 7). Similarly, the presence or absence of 2-mercaptoethanol, a sulfydril agent, in the SDS-PAGE buffer did not alter the migration pattern of the 180-kDa guanylate cyclase (18).
Lack of Stimulation of the 180-kDa Guanylate Cyclase by Heme Compounds - Heme compounds are known activators of the soluble form as well as of certain particulate forms of the guanylate cyclase, apparently including the ANF receptor containing lung enzyme (24). Hemin and hemoglobin, however, had no effect on the pure 180-kDa guanylate cyclase activity (Fig. 8).
Phosphorylation of the 180-kDa Membrane Guanylate Cyclase by Protein Kinase C - To determine the protein kinase C-dependent phosphorylation of the 180-kDa guanylate cyclase, the GTP-affinity purified 180-kDa guanylate cyclase fraction was incubated with protein kinase C (~ 6.5% pure) in the presence of diolein and phosphatidylserine. Activated protein kinase C phosphorylated the 180-kDa guanylate cyclase and the antibody blocked the phosphorylation (Fig. 9). Another protein of Mr 102 to 110-kDa was also phosphorylated by protein kinase C and the phosphorylation inhibited by the antibody, suggesting that this could be a degraded 180-kDa membrane guanylate cyclase.
Soluble Guanylate Cyclase
120
A
cyclic GMP ( % of basal )
100
80
60
40
20
0
Basal
Normal serum
Antiserum
Adenylate cyclase
120
B
cyclic AMP ( % of basal )
100
80
60
40
20
0
Basal
Normal serum
Antiserum
300
A
cyclic GMP ( % change of control )
200
100
0
-100
0
10
-5
10
-4
10
-3
Sodium Nitroprusside ( M )
Soluble GC
Membrane GC
200
B
cyclic GMP ( % change of control )
100
0
-100
0
10
-5
10
-4
10
-3
Sodium Azide ( M )
120
Guanylate cyclase activity Percent of basal
100
80
Cadmium
60
NEM
Methylene blue
40
20
0
Basal
1
10
100
1000
Micromolar
120
Guanylate cyclase activity Percent of basal
100
80
60
40
20
0
Basal
Hemin
Catalase
Hemoglobin
Mr x 10
- 3
TOP
200
180
116
94
68
43
DYE
1
2
3
DISCUSSION
In our previous publication we described the purification and characterization of a 180-kDa protein from rat adrenocortical carcinoma whose apparent intriguing characteristic is that it has dual functions: it is both the ANF receptor and guanylate
cyclase (7). Utilizing the tumor 180-kDa membrane guanylate cyclase antibody-affinity column, we have now purified from the GTP-affinity fraction of solubilized membranes of rat adrenal glands a 180-kDa protein that is biochemically and immunologically indistinguishable from the previously characterized tumor 180-kDa ANF receptor containing guanylate cyclase (7). The specific activity of the rat adrenal guanylate cyclase is ~10-fold higher than the tumor guanylate cyclase (7, 18) (21,211 nmol/min/mg as compared to the tumor 1,800 nmol/min/mg of protein). Blockage of the ANF- dependent guanylate cyclase activity by the 180-kDa guanylate cyclase antibody indicates that the enzyme is biologically coupled to the ANF-dependent formation of cyclic GMP. Based on the tumor 180-kDa guanylate cyclase characteristic that it binds ANF stoichiometrically, it was concluded that the 180-kDa protein is both a guanylate cyclase and ANF receptor (7). This conclusion is further supported by our present affinity cross-linking studies; a 180-kDa ANF receptor is labeled in the tumor and this protein shows cross-reactivity with the 180-kDa guanylate cyclase antibody. With the identity of this protein in rat adrenal glands, its presence has now been authenticated in two major steroidogenic systems, testes (rat and mouse) and rat adrenal glands.
Similar to the situation in rat adrenocortical carcinoma (25,26), protein kinase C inhibits the ANF-dependent guanylate cyclase activity with the concomitant phosphorylation of a 180-kDa protein in the particulate fraction of rat adrenal glands. Blockage of this phosphorylation and of the protein kinase C-dependent phosphorylation of the homogeneous 180-kDa membrane guanylate cyclase provides compelling evidence that the 180-kDa guanylate cyclase is dually regulated in opposing fashions by ANF and protein kinase C signals; ANF stimulates the enzyme and its phosphorylation by protein kinase C uncouples the hormonal stimulation. Such an interpretation is in accord with our recently proposed model depicting the protein kinase C regulation of atrial natriuretic factor-dependent 180-kDa membrane guanylate cyclase (27).
Antibody to the 180-kDa membrane guanylate cyclase has been invaluable for
| Fractions | Total Protein (mg) | Specific Activity (nmol/mg/min) | Total Activity | Recovery (pmol/min) | Fold Purification (%) |
|---|---|---|---|---|---|
| Membrane | 32.4 | 0.251ª | 8,132 | 100 | 1 |
| Solubilized | 10.8 | 0.431ª | 4,655 | 57.2 | 1.71 |
| GTP-Agarose | 0.061 | 49.38ª | 3,012 | 37.0 | 197 |
| Affinity | |||||
| Immuno- Affinity | 0.0002 | 21,211b | 424 | 5.2 | 84,507 |
Specific activity was determined by guanylate cyclase assay as described in Materials and Methods.
b Specific activity was determined by the immunoslot blot analysis as described in Materials and Methods.
| Conditions | Particulate | Soluble | Ref. |
|---|---|---|---|
| ACTH | Stimulation | No effect | (23) |
| ANF | Stimulation | No effect | |
| ANF binding | Stoichiometric | None | |
| Sodium nitro- prusside | No effect | Stimulation | |
| Sodium azide | No effect | Stimulation | |
| Tufstin | No effect | Stimulation | (23) |
| Dithiothreitol | No effect | Stimulation | (23) |
| Heme | No effect | Stimulation | (30) |
| Arachidonic acid | No effect | Stimulation | (18) |
| Cd2+ | No effect (low | Inhibition | (23) |
| concentration) | (low concen- tration) | ||
| Molecular weight (SDS-PAGE) | 180,000 | 72,000 | (28-31) |
| Isoelectric point | 4.7 | 6 | (24) |
| Anti-particulate guanylate cyclase antibody cross- | |||
| reactivity | + |
characterizing and establishing the ANF and protein kinase C dependence of the 180- kDa guanylate cyclase. It is therefore essential that this antibody is highly specific towards the immunological determinants of the 180-kDa protein. That this indeed is the case is evidenced by the following criteria: a) Antibody titer curve shows that a dilution of antibody as high as 1:25,600 shows cross-reactivity with the 180,000 protein antigen (Fig. 1); b) Western blot analysis of the partially purified 180-kDa preparation (GTP- affinity fraction) from the mouse and rat testes (9) and rat adrenal membranes reveals a single 180-kDa immunogenic band, although the SDS-PAGE of the GTP-affinity proteins shows multiple proteins (Fig. 9); c) the antibody blocks up to 90% of the guanylate cyclase activity of the pure 180-kDa enzyme (7,8,18), without blocking the guanylate cyclase activity of the soluble form of the enzyme. Similarly the antibody does not interfere with the activity of the adenylate cyclase (Fig. 5B); d) the antibody blocks the ANF-dependent guanylate cyclase activity. It is noteworthy that the antigen (the 180-kDa adrenocortical carcinoma protein) used to prepare the antibody in rabbits was a single protein, as evidenced by SDS-PAGE and isoelectric profile (7), indicating homogeneity both by the criteria of size and charge. These results collectively demonstrate the immunogenic specificity of the antibody towards the 180-kDa protein in general, and they also attest to its specificity against the catalytic domain of guanylate cyclase. It is remarkable that the antibody has no cross-reactivity against the catalytic domain of soluble guanylate cyclase. It can thus be used effectively as a probe to distinguish between the structural domains of the membrane and soluble forms of the guanylate cyclase.
The results of our past and present study clearly indicate that the hormonally dependent guanylate cyclase is both structurally and functionally different from the soluble guanylate cyclase by a variety of criteria (summarized in Table 2). Upon solubilization, the enzyme loses its ANF-dependent guanylate cyclase activity, but retains other features which distinguish it from the soluble guanylate cyclase (Table 2, and ref.
18): lack of stimulation by nitroprusside, azide, tufstin, dithiothreitol, inhibition instead of stimulation by N-ethylmaleimide, no inhibition at low concentrations of Cd2+, a very different kinetics in response to Mn2+ GTP and lack of the anti-180-kDa membrane guanylate cyclase to cross-react with soluble guanylate cyclase. The molecular mass of the receptor coupled guanylate cyclase subunit in the presence or absence of ß- mercaptoethanol remains unchanged, indicating the absence of intermolecular disulfide bonds in the enzyme, which is a distinct structural difference from the soluble enzyme (18). This supports the antibody results (discussed above), showing the structural determinant differences between the guanylate cyclase epitopes of the membrane and soluble guanylate cyclases. In addition, the subunit molecular mass of the purified soluble guanylate cyclase from rat liver and bovine lung ranges from 69,000 to 72,000 (28-31); from the rat brain it is 59,000 (32), and recently soluble rat lung enzyme has been shown to be a dimer of Mr=70,000 and 80,000 (33), indicating a large mass difference from the 180-kDa membrane guanylate cyclase. As noted earlier, the unequivocal evidence for the biochemical distinctness of the soluble and the membrane guanylate cyclase is that the membrane guanylate cyclase antibody neither blocks the guanylate cyclase activity nor shows immunogenic cross-reactivity with the soluble form of the enzyme.
The 180-kDa guanylate cyclase is also biochemically different from the sea urchin sperm membrane guanylate cyclase (34): there is no evidence that sea urchin enzyme binds ANF; the molecular mass of the sperm enzyme is 135,000; most strikingly, in contrast to the 180-kDa membrane guanylate cyclase, the sperm guanylate cyclase activity is elevated upon phosphorylation (34,35). The effector kinase for the sperm guanylate cyclase is not known, however. An intriguing aspect of the phosphorylation characteristics of the guanylate cyclases is that the soluble guanylate cyclase, like 180- kDa guanylate cyclase, is a substrate of protein kinase C but its activity, unlike the 180- kDa enzyme, is elevated upon phosphorylation. It will be most interesting to determine
if the effector kinase of sperm guanylate cyclase is protein kinase C. If it is, then from the evolutionary aspect the sperm guanylate cyclase exhibits hybrid characteristics; unlike soluble guanylate cyclase, but like the mammalian guanylate cyclase, it does not respond to the nitrite-generating compounds. On the other hand, like soluble guanylate cyclase, but unlike mammalian guanylate cyclase, it is activated upon phosphorylation.
Two other laboratories have also independently reported on the purification and characterization of the guanylate cyclase which appear to contain both guanylate cyclase and ANF receptor functions. One is from the rat lung (22) and the other from bovine adrenal cortex (20,21). The subunit molecular mass of both of these proteins is from 120 to 130-kDa. In contrast to the near 1:1 stoichiometry of ANF binding to the 180- kDa, the 130-kDa lung enzyme binds only 14.5% ANF at the noted theoretical value (7). In addition the lung enzyme is stimulated by hemin, and the pI of the lung enzyme is 6 (24). Thus the lung enzyme is apparently biochemically distinct from the 180-kDa enzyme. It will be most interesting to find out why the lung enzyme possesses the characteristic of responding to two ligands — ANF and hemin — in the formation of cyclic GMP. The coupling of bovine adrenocortical 130-kDa guanylate cyclase to the ANF- dependent formation of cyclic GMP has not been established.
In conclusion, historically, there have been two extraordinary developments in the consolidation of the original concept that cyclic GMP is one of the premium second messengers of receptor-mediated signal transductions: first, demonstration of the hormonally dependent guanylate cyclase, which was different from the soluble guanylate cyclase (18); and second, is the purification of the ANF-dependent guanylate cyclase (7). The third dimension to this research is that the guanylate cyclase signal system at the transmembrane level appears to be under the negatively regulatory loop of protein kinase C. The transmembrane linkage between the two major signal pathways appears to be through the phosphorylation of 180-kDa guanylate cyclase.
ACKNOWLEDGMENTS
This research was supported by the NIH (NS-23744) and the NSF (DCB 8800953).
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