Molecular and Cellular Biochemistry 115: 203-211, 1992. ~) 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Phosphorylation of atrial natriuretic factor receptor by serine/threonine protein kinases: evidences for receptor regulation L. Larose, J.-J. Rondeau, H. Ong and A. De L6an Laboratory of Molecular Pharmacology, Clinical Research Institute of Montreal, Department of Pharmacology and Faculty of Pharmacy, University of Montreal, Montreal, Canada Received 6 November 1991, accepted 21 April 1992

Abstract The 130 kDa atrial natriuretic factor receptor (ANF-Ra) purified from bovine adrenal zona glomerulosa is phosphorylated in vitro by serine/threonine protein kinases such as cAMP-, cGMP-dependent and protein kinase C. This phosphorylation is independent of the presence of ANF (99-126) and there is no detectable intrinsic kinase activity associated with the ANF-Ra receptor or with its activated form. In bovine adrenal zona glomerulosa cells, TPA (phorbol ester) induces a marked inhibition of the ANF-stimulated cGMP accumulation as well as of the membrane ANF-sensitive guanylate cyclase catalytic activity without any change in the binding capacity or affinity for 125I-ANF. However, we have demonstrated a significant 3zp incorporation in the ANF-Ra receptor of the TPA-treated cells. The effect of TPA on the zona glomerulosa ANF-Ra receptors was abolished by calphostin C, a specific protein kinase C inhibitor. Altered ANF actions due to blunted response of guanylate cyclase to ANF could be a consequence of the ANF receptor phosphorylation by excessive activity of protein kinase C and might be involved in the pathogenesis of hypertension. (Mol Cell Biochem 115: 203-211, 1992)

Key words." ANF-R1 receptor, phosphorylation, serine/threonine, protein kinases, receptor regulation Abbreviations: ANF - Atrial Natriuretic Factor; ANF-R1 - Atrial Natriuretic Factor Receptor, subtype 1; ATP Adenosine Triphosphate; CaC12- Calcium Chloride; cAMP - Adenosine cyclic 3',5'-Monophosphate acid; cGMP Guanosine cyclic 3'5'-Monophosphate acid; E D C - 1-Ethyl-3-[3-Dimethylaminopropyl] Carbodiimide; EDTA Ethylenediaminetetraacetic Acid; GTP - Guanosine Triphosphate; IBMX - 3-isobutyl-l-methylxanthine; kDa Kilodaltons; MgC12 - Magnesium Chloride; MgAC - Magnesium Acetate; NaCI - Sodium Chloride; PAGE Polyacrylamide Gel Electrophoresis; PKA - cAMP-dependent protein kinase; PKG - cGMP-dependent Protein Kinase; P K C - Calcium/Phospholipid-dependent Protein Kinase; R I A - Radioimmunoassay; SDS - Sodium Dodecyl Sulfate; SHR - Spontaneously Hypertensive Rat; Tris HC1 - Tris (Hydroxymethyl) aminomethane Hydrochloride; TPA - 12-O-Tetradecanoyl-Phorbol-13-Acetate

The atrial natriuretic factor receptor (ANF-R1) is an integral membrane protein constituted of a single

130 kDa subunit including both the hormone binding and guanylate cyclase activities. The ANF-R1 receptor

Address for offprints." A. De L6an, D6partement de Pharmacologic, Facult6 de M6decine, Universit6 de Montr6al, C.P. 6128, Succursale A, Montr6al H3C 3J7, Canada

204 coupled guanylate cyclase is currently thought to be activated by ANF binding to the extracellular domain of the receptor leading to conformational changes of the intracellular guanylate cyclase catalytic domain [1, 2]. However, little is known about the molecular mechanisms of regulation of ANF-Ra receptor functions. The diuretic amiloride and the adenosine triphosphate (ATP) nucleotide were first reported as important elements involved in ANF-induced regulation of membrane-bound guanylate cyclase activity [3-7]. Recently, we and others have reported that ATP interacts in an allosteric manner with the ANF-R1 receptor resuiting in reduced ANF binding and enhanced ANFstimulated guanylate cyclase activity [7-10]. Molecular expression of the ANF-coupled guanylate cyclase receptor (ANF-R1) in insect cells [11] have finally demonstrated that adenine nucleotides are absolutely required for maximal guanylate cyclase activation by ANF. Mutagenesis studies [12] have shown that the kinase-like domain of the ANF receptor is required to support the ATP effect. This protein kinase-like domain was already located in the intracellular part of the ANF receptor between the transmembrane and the guanylate cyclase domains [1, 2]. Phosphorylation of membrane receptors is recognized to play a key role in the regulation of receptor function and in modulation of the cellular responsiveness to extracellular signals [13]. Multiple potential sites of phosphorylation by serine/threonine protein kinases could be predicted in the amino acid sequence of ANF-Ra receptor. However, until now only one report has demonstrated the regulation by phosphorylation of a 180 kDa ANF-sensitive membrane guanylate cyclase [14]. The present study was undertaken to determine if the 130 kDa ANF-R1 receptor can be phosphorylated and eventually to gain insights into its regulation. We have therefore examined the ability of specific serine/threonine protein kinases to catalyze the phosphorylation of the ANF-R~ receptor that has been purified to homogeneity by affinity chromatography. We have also investigated if ANF-R1 receptor phosphorylation could explain the mechanism of phorbol ester (TPA)-induced refractoriness of cGMP response to ANF in bovine adrenal zona glomerulosa cells. We have found that the cAMP-dependent (PKA)--, the cGMP-dependent (PKG) and the calcium/phospholipid-dependent protein kinase (PKC) can produce in vitro phosphorylation of pure ANF-Ra receptor protein and that the receptor

is also substrate for protein kinase C in phorbol estertreated cells.

Material and methods Materials Carrier-free 32pi, [y_3zp] ATP were from New England Nuclear. ATP, GTP were purchased from Boehringer Mannheim. ANF was rat ANF (99-126) obtained from Institut Armand Frappier. Triton X-100 and Iodobeads were purchased from Pierce Chemical Co. (Rockford, IL). Phosphatidylserine was from Avanti Polar Lipids Inc. (Alabama). The antiserum to cyclic GMP was kindly provided by Dr Alain B61anger, Laval University Hospital Center, Qu6bec, Canada. a25I-ANF (99-126) was prepared by radioiodination using the solid phase iodo-beads method [15] and purified by HPLC. The specific activity of the monoiodinated peptide was typically 2000 Ci/mmol. Carrier-free Na ~25I was from Amersham Corp. (Oakville, Canada). Serine/ Threonine protein kinases were from the following commercial sources: bovine heart cAMP-dependent protein kinase (PKA), catalytic subunit from Sigma Chemical Co., bovine aorta cGMP-dependent protein kinase (PKG), alpha isoenzyme from Promega and rat brain protein kinase C (PKC) from Calbiochem. Calphostin C was purchased from Kanuya Biomedical Co. (California). All other reagents were from commercial sources.

Preparation of the pure ANF-R1 receptor Bovine adrenal glands were obtained from a local slaughterhouse, Adrenal zona glomerulosa membranes prepared as described previously [16] were used as a source material for ANF-R~ receptor purification. The receptor protein was purified to homogeneity by affinity chromatography using ANF-agarose and steric exclusion HPLC [17].

Protein kinase C purification Protein kinase C from bovine adrenocortical tissue was purified to homogeneity according to a three-step procedure consisting of ion exchange, hydrophobic interaction and substrate-affinity chromatography [18].

205 In vitro phosphorylation The pure ANF-R1 receptor (= 3 pmol) was phosphorylated at 30° C for 30 min in a final volume of 100 tzl. The phosphorylation of the receptor by the protein kinase A was carried out in 20mM Tris HCI, pH7.4, 10mM MgAc, 50 units of protein kinase A and 0.02 mM [y_32p] ATP (12.5 Ci/mmol). For the phosphorylation by the protein kinase G, the reaction mixture was constituted of 40mM Tris HC1, pH7.4, 20mM MgAc, 20 units protein kinase G, 10/zM of cGMP and 0.02 mM [,/_3zp] ATP (12.5Ci/mmol). The protein kinase C purified from the bovine adrenocortical tissue or obtained from commercial source was used at 2 x 10 -4 units to phosphorylate the receptor. The typical buffer for the phosphorylation by protein kinase C was constituted by 40mM Tris HC1, pH 7.4, 10mM CaClz, 75 mM MgAC, 0.1mg/ml phosphatidylserine, 10 txM TPA and 0.02mM [y_32p] ATP (12.5 Ci/mmol). The ANF-R1 receptor phosphorylation was stopped by the addition of an equal volume of Laemmli sample buffer [19] and the samples were boiled for 3 min and then subjected to electrophoresis on 5 or 7.5% SDSPAGE according to the method of Laemmli [19].

and resuspended in fresh phosphate-free RPMI with carrier-free 32pi at lmCi/ml. The cells were incubated for four hours in the same conditions as above. During the last 30 min of the labeling period, TPA (1 tzM) was added to the cell suspension. When calphostin C (0.5 t~M) was used, it was added 30 min before TPA and kept present until the end of the labeling period. At the end of the incubation, cells were washed twice with buffered saline and membranes were prepared as previously described [16]. Buffers used for membrane preparation were supplemented with sodium fluoride (50 mM), sodium pyrophosphate (20 mM) and sodium orthovanadate (100/zM) as phosphatase inhibitors. The membranes were immediately used to evaluate 125IANF binding and guanylate cyclase activity or solubilized at 4°C for 60min in 50mM Tris HC1, pH7.4, 100mM NaC1, 20% glycerol, 0.1mM EDTA, 1% Triton X-100 with usual protease and phosphatase inhibitors. After centrifugation (40,000 g, 60min, 4° C) the solubilized proteins were submitted to immunoprecipitation with anti ANF-R1 antibody (overnight, 4 ° C) and protein A-Sepharose adsorption (2 h, 4° C). The immunoprecipitated proteins were resuspended in Laemmli sample buffer [19] and characterized by electrophoresis and autoradiography.

Anti ANF-R1 receptor antibodies 125I-ANF binding assay The synthetic peptide K R V N R K R I E L T R K V L Y corresponding to the residues 535-549 of the 130 kDa rat ANF-R1 receptor with a tyrosine residue added to the carboxy terminus was coupled to bovine serum albumin with 1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide (EDC). Rabbits were immunized and the sera tested for immunoreactivity by RIA using iodinated peptide as tracer and peptide or native purified bovine ANF-R1 receptor as competitors. Specificity of the antibody was also confirmed by Western blot using affinity purified bovine ANF-R1 receptor protein as antigen.

Whole cell phosphorylation Cells were used immediately after collagenase dispersion or after two resting days in F12 medium supplemented with 10% horse serum, 2% fetal bovine serum, 1% penicillin, 1% streptomycin and 2.5/xg/ml fungizone. Cells were washed twice with phosphate-free RPMI and preincubated in the same medium for 60 min at 37°C under 95% 02-5% COz. Cells were detached

The adrenal zona glomerulosa membranes (20/xg/ml) were incubated with 10pM 125I-ANF (99-126) for 90min at 25°C in 50mM Tris HC1, pH7.4, 5raM MnCI2, 0.1mM EDTA and 0.1% bovine serum albumin. Bound lzSI-ANF was separated from free ligand by filtration on Whatman GF/C filters precoated with 1% polyethylenimine. The bound radiolabeled hormone was measured in a gamma counter (LKB 1272, Clinica Gamma). Nonspecific binding was determined in the presence of 0.1/zM unlabeled ANF (99-126). Competition binding curves were analyzed by a nonlinear leastsquares curve-fitting program [20]. Data were expressed as the average of triplicate determinations of the ANF bound in pmol/assay.

Cyclic GMP determination Whole cells (1 x 106 cells/ml) in F12, 0.1% bovine serum albumin were exposed to ANF (99-126) for 30 min at 37° C. The release of cGMP in the media was evaluat-

206 ed by radioimmunoassay, using the second antibody precipitation technique [21]. When the particulate guanylate cyclase activity was evaluated, the adrenal zona glomerulosa membranes (10 t~g) were incubated during 10min at 37°C in 50mM Tris-HC1 pH7.6 with 10mM theophylline, 2 mM IBMX, 10 mM creatine phosphate, 10 units of creatine kinase, 1 mM GTP, 4 mM MnClz with or without ANF (99-126) and following separation on alumina column, the cGMP producted was evaluated by RIA [22].

Results and discussion Incubation of pure bovine adrenal ANF-R1 receptor with serine/threonine protein kinases (PKA, PKG and PKC) under phosphorylation conditions lead to 32p incorporation in the 130kDa ANF-R~ receptor protein (Fig. 1). In bovine adrenocortical tissue, protein kinase C is expressed as a single form which is different from the four chromatographically resolved isoenzymes in rat brain [23]. For this reason, we have purified the protein kinase C from bovine adrenocortical tissue and used it to phosphorylate pure bovine adrenal ANF-R~ receptor. Rat brain and bovine adrenocortical source of protein kinase C catalyzed the phosphorylation of the pure ANF-Ra receptor with the same level of 32p incorporation (data not shown). The phosphorylated ANF-R~ receptor does not exhibit the commonly observed shift in mobility on SDSPAGE as compared to the migration of the native pure receptor revealed by silver stain (Fig. 1). The presence of ANF (10-8-10 -7 M) in the phosphorylation assay or in preincubation with the pure ANF-R 1 receptor prior to the addition of the protein kinase does not influence ANF-R1 receptor phosphorylation (data not shown). The purified ANF-R~ receptor by itself or in the presence of ANF does not display any autophosphorylation activity. However, this point still remains unsettled because purification of the ANF-Ra receptor could result in loss of intrinsic kinase activity or of any influence of ANF on ANF-R~ receptor autophosphorylation. This is suggested by a parallel situation for guanylate cyclase activity following receptor purification. In fact, it was several times reported that purification of the ANF receptor always resulted in loss of guanylate cyclase activation by ANF, while its ability to bind ANF was retained [17, 24-26]. Receptor purification might induce conformational changes in the ANF receptor protein or loss of cofactors required for adequate rood-

ulation by ANF of the receptor activities, eventhough the receptor can still bind ANF. However, since the pure ANF-R1 receptor protein is a substrate for serine/ threonine protein kinases, phosphorylation of ANF-R1 receptor might represent an important mechanism of regulation of receptor responsiveness. To know more about the possible regulatory mechanisms of ANF-R1 receptor activities in intact cells, we have explored the effects of the acute TPA treatment of bovine adrenal zona glomerulosa cells on the ANF binding, guanylate cyclase activity and receptor phosphorylation. Treatment of intact cells with TPA, a phorbol ester which strongly activates protein kinase C, was several times reported to largely impair the cellular cGMP system. Indeed, in adrenocortical carcinoma cells, TPA inhibits the a2-adrenergic receptor-dependent cGMP production [27]. However, in these cells, it is not absolutely clear if these neurotransmitter receptors are directly coupled to the particulate guanylate cyclase [27, 28]. Interestingly, in rat vascular smooth muscle cells (A10), in a human renal cell line (SK-NEP-1) and also in rat adrenocortical carcinoma cells where the particulate guanylate cyclase activity is directly associated to the ANF receptor, TPA also attenuates the ANF-stimulated cGMP production in intact cells [2932] as well as in membrane preparation [31, 32]. In bovine adrenal zona glomerulosa cells, TPA treatment significantly alters the ANF-stimulated cGMP release without any effects on its basal level (Table 1). This effect of TPA on ANF stimulation is associated with altered activity of the particulate guanylate cyclase since the ANF-stimulated production of the cyclic GMP in zona glomerulosa membranes is almost totally inhibited after TPA treatment (Fig. 2). However, these alterations in the ANF-stimulated guanylate cyclase activity following TPA are not associated with any modification in the ANF receptor binding capacity or affinity (Fig. 3). Preincubation with calphostin C, a specific inhibitor of protein kinase C [33], before exposure of the ceils to phorbol ester completely prevents the inhibitory effect of TPA on the ANF-stimulated release of cGMP (Table 2). The present results suggest that the inhibition of the ANF-R1 receptor guanylate cyclase activity induced by TPA may be due to protein kinase C-mediated phosphorylation events. This is well supported by several reports on different cellular systems where phorbol esters [29-31], hormones associated with activation of protein kinase C [34, 35] and direct protein kinase C addition [36] inhibit the ANF-sensitive guanylate cy-

207

Fig. 1. In vitro phosphorylation of pure ANF-R1 receptor (130kDa) by cAMP-dependent (PK A), cGMP-dependent (PK G) and calcium,

phospholipids-dependent (PK C) protein kinases and silver stain SDS-PAGE migration of the ANF-RI receptor. The phosphorylation assays were conducted as detailed in material and methods section. The phosphorylated samples were analyzed by autoradiography of dried 7.5% (PK A, PK G) or 5% (PK C) SDS-PAGE. R: ANF-R1receptor, K: protein kinase, 1: protein kinase alone, 2:ANF-R1 receptor and protein kinase. Silver stain, the first lane is the molecular weight markers (St) and the second lane is a sample of the ANF-R1 receptor affinity purified.

clase activity. In agreement with this hypothesis, preloading with 32p and treatment with TPA of intact zona glomerulosa cells stimulated 32p incorporation into immunoprecipitable ANF-R1 receptor (Fig. 4). Occasionally, in the absence of TPA, the 32p labeled zona glomerulosa cells present immunoprecipitable radioactive ANF-R1 receptor, indicating that the receptor can be constitutively phosphorylated. In those instances, TPA failed to markedly increase the specific 32p incorporation in the ANF-R1 receptor protein. Usually following immunoprecipation of the 32p labeled cells with the polyclonal anti ANF-R1 receptor antibody three additional bands other than the receptor itself were seen (Fig. 4) and these bands shown more 32p incorporation when the cells were treated with TPA. One of these bands could be the rabbit IgGs provided by the serum itself (50 kD). The presence of the two other bands (60, 97 kD) cannot be attributed to ANF-R~ receptor degradation since protease inhibitors were always present from the moment where the cells were lysed. One possible explanation might be that proteins associated to the ANF-R1 receptor follow along the

immunoprecipation procedure. However, further experiments are required to support this hypothesis. Protein kinase C has been reported to phosphorylate membrane receptors and to modulate their responsiveness [13, 37]. The phosphorylation by protein kinase C of ANF-R 1 receptor could be proposed as a potential mechanism for the inhibitory effect of phorbol ester on membrane ANF-sensitive guanylate cyclase. Interestingly, it is possible to note that phosphorylation has opposite effects on catalytic activity among different forms or species of guanylate cyclase. Indeed, rat brain soluble guanylate cyclase was shown to be phosphorylated by PKC and this resulted in increased enzyme activity [38]. In sea urchin spermatozoa, the activity of the membrane bound guanylate cyclase was increased after phosphorylation [39, 40]. In contrast, in bovine adrenal zona glomerulosa cells, we have reported here that phosphorylation by PKC of ANF-R1 receptor resuited in the inhibition of its ANF-sensitive guanylate cyclase activity. These observations are in agreement with the fact that functional properties of cellular proteins and enzymes are often regulated by phosphoryla-

208

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Fig. 2. Effect of T P A on ANF-stimulated guanylate cyclase activity.

I 0

t 1

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Fig. 3. Effect of TPA on the competition curve of A N F for the binding

Bovine adrenal zona glomerulosa cells were first exposed to TPA (1/zM, 30°), then the membranes were prepared and challenged with A N F in terms of cGMP produced as described in material and methods. The results are from a typical of 3 experiments.

of 125I-ANF to zona glomerulosa membranes. Bovine adrenal zona glomerulosa cells were first exposed to T P A (1/zM, 30°), then the membranes were prepared and assayed for 125I-ANF binding as described in material and methods. The data are from a typical of 3 experiments.

tion and dephosphorylation, however, more informations are still required to totally understand the phenomenon. The fact that protein kinase C directly phosphorylates the 130 kDa ANF-R1 receptor in vitro and also supports the phosphorylation of ANF-R~ receptor in intact cells is suggestive of a cross interaction between these two intracellular signalling pathways. Another intracellular cross interaction could be also proposed between the cAMP mediated cellular signalling and the

ANF guanylate cyclase system since the pure bovine ANF-R1 receptor is well phosphorylated in vitro by the presence of the cAMP-dependent protein kinase. Moreover, a cGMP-mediated mechanism could be also suggested in the autoregulation of the ANF-stimulated guanylate cyclase activity because the pure ANF-R~ receptor is also substrate of the cGMP-dependent protein kinase. However, as these two serine/threonine protein kinases are concerned, in spite of potential sites

Table 1. Effect of T P A on subsequent ANF-stimulated cGMP released in isolated bovine adrenal zona glomerulosa cells

Table2. Effect of calphostin C on ANF-stimulated cGMP released in isol~ated bovine adrenal zona glomerulosa cells exposed or not to TPA

cGMP released (fmol/30'/1 × 106 cells)

cGMP released (pmol/30'/1 × 106 cells)

BASAL

A N F (100 nM)

BASAL

A N F (100 nM)

46.4 + 6.0 4 1 . 7 + 4.6

226.6 + 19.1 170.7+ 9.9*

47.2 + 7.4 40.9_+ 6.9

238.0 + 12.3 208.8 + 26.2

Control TPA

Cells were exposed to T P A (1 ~ M , 30'), washed and then challenged by ANF. cGMP released in the media was evaluated as described in material and methods section. The results are the mean + SEM (n = 3). * Statistically different from control, at least p < 0.05.

Control TPA

Cells were exposed to calphostin C (0.5/zM) 30' prior the addition of TPA (1/~M, 30') and then washed and challenged by ANF. cGMP released in the media was evaluated as described in material and methods section. The results are the mean + SEM (n = 3).

209

97-,

these animals, the protein kinase C activity was seen to be increased [44]. Besides, in SHR animals, protein kinase C activity [45] and phosphoinositide metabolism [46] which generates the natural protein kinase C activator were also found to be increased in essential human hypertension. Impaired ANF physiological functions due to the blunted response of guanylate cyclase to ANF as a consequence of the ANF receptor phosphorylation by activated protein kinase C may be a potential mechanism which plays an important role in the pathogenesis of hypertension.

66--

Acknowledgements

43--

The authors are grateful to Isabelle Blain for her secretarial assistance and Normand McNicoll for his helpfull discussion. This research has been supported by grants from the Canadian Heart and Stroke Foundation and the Medical Research Council of Canada.

1

2

200--

-ANF-R1 116--

References

Fig. 4. TPA-induced phosphorylation of bovine adrenal ANF-R1 receptor. Bovine adrenal zona glomerulosa cells preloaded with 3zp were exposed to TPA (1/~M, 30'), then membranes were prepared and solubilized. The soluble fraction was immunoprecipitated with the anti ANF-R1 antibody, submitted to SDS-PAGE and autoradiographied as detailed in material and methods. 1: control, 2: TPAtreated.

of phosphorylation in the ANF-R1 receptor amino acid sequence, cellular data are still missing to confirm their relative importance in the regulation of the ANF-R1 functions. Further studies are required to assert the physiological importance of phosphorylation on the membrane ANF-sensitive guanylate cyclase activity. Our present study demonstrates that an alteration in transducing the stimulatory signal of ANF in guanylate cyclase activity can be associated with a protein kinase C-mediated phosphorylation of the ANF-RI receptor. Interestingly, in spontaneously hypertensive rats (SHR) [41--43], a similar resistance in vascular smooth muscle cell response to ANF was also reported and in

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