Original Article
Ubiquitous and bifunctional 180 kDa atrial natriuretic factor-dependent guanylate cyclase
Ravi B. Marala and Rameshwar K. Sharma
Section of Regulatory Biology, Department of Brain and Vascular Research Cleveland Clinic Research Institute, Cleveland Clinic Foundation 9500 Euclid Avenue, Cleveland, Ohio 44195-5068, USA
Received 15 December 1989; accepted 26 March 1990
Key words: 180 kDa ANF receptor, atrial natriuretic factor, cyclic GMP, guanylate cyclase
Summary
Original studies with rat adrenocortical carcinoma identified a 180 kDa cell-surface protein which contained both guanylate cyclase and atrial natriuretic factor (ANF) receptor, representing a potentially new type of bifunctional receptor protein. It is both a receptor and a guanylate cyclase. This critical conclusion of bifunctionality was based on the observation that the pure 180 kDa protein, whose purity was established by protein staining of the denatured gels, contained both the ligand binding and guanylate cyclase activities. Utilizing the antibody to 180 kDa membrane guanylate cyclase (180 kDa mGC), we now (i) report the complete purification of 180 kDa mGC from rat testes; (ii) demonstrate by affinity cross-linking studies that the homogeneous 180 kDa protein isolated from rat testes and adrenal gland binds ANF and (iii) show that bovine aortic endothelial cell membranes contain the 180 kDa mGC that is ANF-dependent in the produc- tion of cyclic GMP. These results validate the conclusion of the bifunctionality, ubiquity, and the general linkage to the ANF-dependent generation of cyclic GMP signal of this protein.
Introduction
In our initial studies with rat adrenocortical carci- noma, we isolated a 180kDa protein (180 kDa mGC) whose purity was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein-staining, isoelectric focuss- ing and immunological studies [1, 2]. Because this protein could bind ANF stoichiometrically and contained guanylate cyclase-activity, we concluded that this protein is apparently bifunctional [1]. It is both an ANF receptor and a guanylate cyclase. Similar conclusions have been independently drawn by other laboratories for a rat lung [3] and a bovine adrenocortical 120-140 kDa protein [4, 5], and for a recently cloned and expressed rat brain protein with a predicted Mr of 115,852 [6]. These
studies, therefore, establish the existence of a new class of surface receptor protein/s that contains both ligand binding and guanylate cyclase activ- ities, and is/are the potential ANF signal trans- ducers.
In order to assess the basic signal transducer role of 180 kDa mGC in the ANF-dependent cyclic GMP formation, we have herein applied a combi- nation of immunological and cross-linking tech- niques to address the following pertinent ques- tions: Is this bifunctional protein widely distributed in mammalian tissues, or is it merely an oncogenic expression typical of the rat adrenocortical neo- plastic cells? If its occurrence is indeed wide, is it always coupled to the ANF-dependent generation of cyclic GMP signal? The conclusions are that the 180 kDa mGC is indeed ubiquitous, bifunctional
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and biologically coupled to the ANF-dependent cyclic GMP production.
Materials and methods
Membranes isolated from indicated tissues were solubilized as described by Nambi et al. [7]. The ANF-coupled membrane guanylate cylase was pu- rified to the GTP-affinity step (GTP-affinity-step) as described by Paul et al. [1]. The complete puri- fication of 180 kDa mGC was achieved by immu- noaffinity chromatography in which the affinity li- gand was rabbit anti-180 kDa mGC antibody raised against rat adrenocortical carcinoma 180 kDa mGC [8].
Rabbit anti-180 kDa mGC antibody was pre- pared by immunizing the rabbits with purified rat adrenocortical carcinoma 180kDa mGC (1; for complete characteristics of the antibody see ref. 8).
These immunoglobulin fractions from immune and normal serum pools were used for all experiments.
Western-blot analysis was performed as de- scribed by Towbin et al. [9] by subjecting the GTP- affinity-step fraction to SDS-PAGE [10] followed by electrophoretic transfer of the proteins to nitro- cellulose membrane. The protein-binding sites were blocked by incubating the membrane with 3% bovine serum albumin for 2 h at room temperature with anti-180 kDa rat adrenocortical carcinoma membrane guanylate cyclase polyclonal antibody (1: 200 dilution), followed by 2h incubation at room temperature with peroxidase-conjugated goat anti-rabbit IgG at a 1 : 1500 dilution. The color was developed by soaking the blot in peroxidase color reagent: 600 µg 4-chloro-1-naphthol/0.015% H2O2/20 mM Tris, pH 7.5. The color reaction was terminated by extensive washing with water.
In affinity cross-linking studies, the immuno- affinity-purified apparently homogeneous enzyme
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(2 µg) was incubated at 4℃ for 20h in ANF binding buffer: 125 mM NaCl, 25 mM N-2-hydroxyethyl-pi- perazine-N’-2-ethanesulfonic acid (HEPES), 1 mM Bacitracin, 0.1 mM PMSF, 0.2g/dl bovine serum albumin with 0.2 uCi [125]]ANF, in the absence or presence of 1 uM non-radioactive ANF in a total volume of 100 ul. To the reaction mixture 11 ul of 1 mM disuccinimidyl suberate (DSS) in DMSO was added, incubated on ice-bath for 15 min and the incubation mixture was quenched with 56 pl 1 M Tris, 0.2M EDTA (pH 6.8). The samples were boiled with SDS-PAGE sample buffer, resolved on 7.5% SDS-PAGE and autoradiographed.
Guanylate cyclase activity was determined in
particulate fractions, solubilized-membranes and GTP-affinity-step fractions [11]. The indicated fractions (10-20 µg protein) were preincubated with or without 180 kDa mGC antibody for 1h at 0°C. The assay mixture contained 10 mM theophyl- line, 15 mM phosphocreatine, 20 µg creatine ki- nase, 1 mM CaCl2 and 50 mM Tris-HCI (pH 7.5) in a total volume of 100 ul. The samples were in- cubated with or without 1 µM ANF for 10 min at 0°C. The reaction was initiated by the addition of 4 mM MnCl2 and 1 mM GTP and the reaction con- tinued at 37º C for 10 min. Incubations were termi- nated by the addition of 0.9 ml ice-cold 50 mM sodium acetate buffer (pH 6.2), followed by heat-
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ing in a boiling water bath for 3 min. The amount of cyclic GMP formed was determined as described earlier [11] and expressed as pmol or nmol cyclic GMP/mg protein/min.
Results and discussion
We have recently reported the development of an immunoaffinity column technique for the direct purification of 180 kDa mGC from its tissue sources [8]. With this technique we were able to isolate a 180 kDa protein from rat adrenal gland [8] that was biochemically and immunologically indistinguish- able from the 180 kDa mGC [1], originally purified by standard biochemical techniques from rat adre- nocortical carcinoma. We have now applied this technique to purify a 180kDa protein from rat testes which by the following protein-staining and immunological criteria is homogeneous and is iden-
tical to both the original tumor and adrenal gland 180 kDa mGC.
SDS-PAGE of the testes protein shows a single band with a molecular mass of 180 kDa as evi- denced by Coomassie Blue staining, silver staining, and the radioautographic analysis of the radioiod- inated protein (Fig. 1: Lanes 2-4). The migration pattern of this protein is indistinguishable from those of the tumor and adrenal gland 180 kDa mGCs (Fig. 1: Lanes 5 and 6). Western-blot analy- sis of the GTP-affinity-step enzyme from all three tissue sources (adrenocortical carcinoma, adrenal gland, testes) shows a single 180 kDa immunoreac- tive band that is again indistinguishable from each other (Fig. 2A: Lanes 1-3). It is noteworthy that the GTP-affinity-step enzyme [1] consists of a very complex mixture of proteins (Fig. 2B: Lanes 1-3), but only one 180 kDa protein is detected by the 180 kDa mGC antibody. These results establish that the tumor, rat adrenal and testes 180 kDa pro- teins are biochemically and immunologically iden- tical.
Western blot studies indicate that the 180 kDa mGC protein is also present in the aortic endo- thelial cells (Fig. 2A: Lane 4). Previously with this technique we have established the presence of this protein in GTP-affinity-step fraction of rat renal glomerular cells [12] and mouse testes [13] and have provided immuno-cytochemical evidence for the preponderance of 180 kDa mGC in outer segments of photoreceptors in chicken and rat retina [14] and rat neural tissues (lumbar region of the spinal cord and Purkinje cells and post synaptic regions of the cerebellum) [15]. Together, these results demon- strate ubiquity of the 180 kDa mGC in mammalian tissues.
In our original studies the bifunctional character- istic (coexistence of ANF receptor and guanylate cyclase activities) of the 180 kDa protein was estab- lished on the basis that the apparently homogene- ous protein contained both the guanylate cyclase and ANF-binding activities [1]; the binding of ANF was stoichiometric. The 180 kDa mGC specific an- tibody probe was crucial in arriving at this conclu- sion of protein bifunctionality: The highly specific antibody not only exclusively recognized the 180 kDa protein but also almost completely
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blocked its guanylate cyclase activity. But, surpris- ingly, the antibody did not block the ANF binding to the 180 kDa mGC. For this reason a cautionary note was added in the publication [1]: ‘Although the antibody to the 180kDa guanylate cyclase blocks guanylate cyclase activity, it does not inhibit the binding of ANF to the protein. This indicates that either the antibody is solely against the guany- late cyclase epitope of the protein or that there are two tightly coupled 180 kDa proteins which are inseparable by the present techniques’. To answer this question in the present study, the immunoaf- finity-purified 180 kDa guanylate cyclase was scru- tinized for ANF receptor activity. The homogene- ous 180 kDa mGC derived from two tissue sources - rat adrenal and testes - was affinity cross-linked with [125I]ANF through DSS. SDS-PAGE under reducing conditions revealed the radioactive label in a single 180 kDa protein (Fig. 3). Because the labeling was abolished upon inclusion of the nonra- dioactive ANF (1 µM) in the reaction mixture (Fig. 3: compare lanes 1 and 2), the 180 kDa protein represented a true ANF receptor. Thus by two independent ANF-binding techniques - direct and affinity cross-linking - it is established that the
180 kDa protein is both an ANF receptor and a guanylate cyclase.
Our previous studies have demonstrated the bi- ological coupling of 180 kDa mGC with ANF-de- pendent formation of cyclic GMP in rat and mouse testes [13], rat ranal glomerular cells [12] and adre- nal gland [8]. We now show that similarity in the case of bovine aortic endothelial cells: ANF stim- ulates crude membrane guanylate cyclase and the hormonal stimulation is blocked by the 180 kDa mGC antibody (Fig. 4), indicating the hormonal dependence of the 180 kDa enzyme.
From this and other referenced studies, we con- clude that the bifunctional 180 kDa mGC is one of the key general components of the ANF-depend- ent cyclic GMP signal transduction.
Acknowledgements
This work was supported by the National Institutes of Health grant NS-23744 and National Science Foundation grant DCB-83-00500.
References
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Address for offprints: R.K. Sharma, Section of Regulatory Biol- ogy, Department of Brain and Vascular Research, Cleveland Clinic Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-5068, USA