The Regulation of the cGMP-binding cGMP Phosphodiesterase by Proteins That Are Immunologically Related to gamma  Subunit of the Photoreceptor cGMP Phosphodiesterase*

(Received for publication, December 18, 1996, and in revised form, April 18, 1997)

Amanda Lochhead Dagger , Elina Nekrasova §, Vadim Y. Arshavsky § and Nigel J. Pyne Dagger par

From the Dagger  Department of Physiology and Pharmacology, University of Strathclyde, Glasgow G1 1XW, Scotland and the § Harvard Medical School, Howe Laboratory of Ophthamolgy, Boston, Massachusetts 02114

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The cGMP phosphodiesterase from retinal rods (PDE-6) is an alpha beta gamma 2 heterotetramer. The alpha  and beta  subunits contain catalytic sites for cGMP hydrolysis, whereas the gamma  subunits serve as a protein inhibitor of the enzyme. Visual excitation of photoreceptors enables the activated GTP-bound form of the G-protein transducin to remove the inhibitory action of the gamma  subunit, thereby triggering PDE-6 activation. The type 5 phosphodiesterase (PDE-5) isoform shares a number of similar characteristics with PDE-6, including binding of cGMP to noncatalytic sites, the cyclic nucleotide specificity, and inhibitor sensitivities. Although the functional role of PDE-5 remains unclear, it has been shown to be activated by protein kinase A (PKA) (Burns, F., Rodger, I. W. & Pyne, N. J. (1992) Biochem. J. 283, 487-491). Here we report that both the recombinant gamma  subunit and a peptide corresponding to amino acids 24-46 in this protein inhibited the activation of PDE-5 by PKA. Furthermore, immunoblotting airway smooth muscle membranes with a specific antibody against amino acids 24-46 of the PDE-6 gamma  subunit identified two major immunoreactive small molecular mass proteins of 14 and 18 kDa (p14 and p18). These appear to form a complex with PDE-5, because PDE activity was immunoprecipitated using antibody against the PDE-6 gamma  subunit. p14 and p18 were also substrates for phosphorylation by a unidentified kinase that was stimulated by a pertussis toxin-sensitive G-protein. Phosphorylation of p14/p18 in membranes treated with guanine nucleotides correlated with a concurrent reduction in the activation of PDE-5 by PKA. We suggest that p14 and p18 share an epitope common to PDE-6 gamma  and that this region may interact with PDE-5 to prevent its activation by PKA.


INTRODUCTION

PDEs1 are expressed as a family of distinct isoforms (type 1-7) with each subgroup of isoforms containing multiple spliced variants (1). The different members of the PDE family are also differentially regulated by kinases (2-5), calcium/calmodulin (6), cGMP (7, 8), and G-proteins (9). The integration of these diverse cell signals enables the precise co-ordinated regulation of intracellular cyclic nucleotide levels in response to receptor stimulation.

PDE-5 from smooth muscle and PDE-6 from photoreceptors share a number of common properties. They have two tightly bound subunits containing both catalytic and noncatalytic cGMP binding sites, hydrolyze cGMP better than cAMP, and are both inhibited by zaprinast. The noncatalytic cGMP-binding site in PDE-5 is formed from an N-terminal tandem repeat that is indicative of gene duplication, whereas the catalytic site is located in the C-terminal region of the protein and is conserved in a number of PDE isoforms (1, 10).

PDE-6 is expressed in photoreceptor rod cells, where it serves as an effector in the visual transduction signal cascade (11-13). This involves the photoexcitation of rhodopsin, the GDP-GTP cyclical activation of the G-protein, transducin, and the subsequent stimulation of PDE-6 activity by the GTP-bound transducin. PDE-6 is a heterotetrameric protein composed of catalytic alpha  and beta  subunits and two inhibitory gamma  subunits. The GTP-bound transducin binds to the gamma  subunits and displaces them, thereby activating PDE-6. PDE-6gamma has two additional functions: first, it increases the affinity for cGMP in the PDE-6 noncatalytic site (14, 15), and second, it participates in the activation of transducin GTPase, thereby terminating the activation of PDE activity by the GTP-bound alpha  subunit of transducin (16). Desensitization of PDE-6 activity might also occur via a recently proposed kinase-directed phosphorylation mechanism (17, 18). When PDE-6gamma is removed from PDE-6alpha beta in a complex with transducin, it is apparently phosphorylated by an unidentified kinase. The phosphorylated gamma  subunits appear to render PDE-6 refractory to stimulation by transducin.

PDE-5 is the major cGMP-binding protein in lung (19). It is a homodimer comprised of two identical catalytic subunits, each with the molecular mass of 93 kDa. PDE-5 contains a site (Ser92) that is phosphorylated by protein kinase A (PKA) and protein kinase G (5). The kinase-catalyzed phosphorylation of the PDE-5 is kinetically enhanced by the binding of cGMP to the noncatalytic cGMP-binding site. However, no change in enzyme activity has been observed as a consequence of the phosphorylation of purified bovine PDE-5 (5). To the contrary, we have demonstrated that the catalytic subunit of PKA can catalyze up to a 10-fold increase in the Vmax of a partially purified preparation of guinea pig lung PDE-5 (20, 21). Activation of PDE-5 by phosphorylation may therefore enhance the rate of cGMP signal termination in intact cells, and this proposal is supported by the observation that cGMP elevating agents elicit rapid transient phosphorylation and activation of PDE-5 in vascular smooth muscle cells (22).

Multiple similarities in the structure and function of PDE-5 and PDE-6 point to the possibility that PDE-5 might contain its own gamma  subunits that couple this enzyme to unidentified G-protein-dependent pathway. Here we have characterized the interaction of PDE-5 with recombinant PDE-6gamma and have identified two small molecular mass proteins in airway smooth muscle cell membranes that appear to share an epitope common to PDE-6gamma . This epitope region interacts with PDE-5 to prevent its activation by PKA.


EXPERIMENTAL PROCEDURES

Materials

All biochemicals were from Boehringer Mannheim, whereas general chemicals were from Sigma. [gamma -32P]ATP, [3H]cGMP, and excitation chemiluminescence detection kits were from Amersham International (Bucks, UK). Ophiophagus Hannah snake venom was from Sigma. Reporter horseradish peroxidase-linked anti-rabbit antibody was from the Scottish Antibody Production Unit (Carluke, Scotland). Purified brain protein kinase C containing multiple isoforms was purchased from Calbiochem.

Cell Culture

The preparation of the primary cultures of guinea pig airway smooth muscle (ASM) cells was achieved as described previously (23). The cells were maintained in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum and 10% (v/v) donor horse serum and were passaged twice using trypsin prior to experimentation. Cells were grown to confluence and routinely used at 15-21 days after the initial preparation. Their identity was confirmed to be smooth muscle by the presence of alpha -actin, using a smooth muscle-specific mouse anti-alpha -actin monoclonal antibody (23).

Membrane Preparation

Guinea Pig ASM Cell Membranes

Cells were placed in Dulbecco's modified Eagle's medium containing 1% (v/v) fetal calf serum and 1% (v/v) donor horse serum for 24 h. In some cases, cells were incubated with pertussis toxin (0.1 µg/ml) for 24 h. All fractionation procedures were performed at 4 °C. The medium was removed from the cells and buffer A containing 0.25 M sucrose, 10 mM Tris/HCl, pH 7.4, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 2 mM benzamidine was added. Cells were scraped off the plate and passed twice through a 0.25-mm gauge syringe. Cell membranes were collected by centrifugation at 14,000 × g for 10 min and resuspended in buffer A.

Guinea Pig Lung Membranes

Lung tissue was homogenized in buffer A, using a Turrex homogenizer, and the subsequently obtained homogenate was filtered through cheese cloth and centrifuged at 10,000 × g in a Beckman prep 65 centrifuge for 10 min. The supernatant was removed and recentrifuged at 48,000 × g for 20 min at 4 °C. The subsequently obtained membrane pellet was resuspended in buffer A.

Protein Purification

Partial purification of guinea pig lung PDE-5 from a "high speed" supernatant fraction was achieved as described previously by us (20). Briefly this involved homogenizing guinea pig lung in phosphate buffer containing 20 mM K2HPO4/K2HPO4, pH 6.8, at 4 °C. The homogenate was passed through cheese cloth and centrifuged identically to that described for the preparation of membranes. The high speed (48,000 × g) supernatant was taken for the partial purification of the PDE-5. This was achieved using three chromatographic columns; namely (i) DEAE-Sepharose 6B (PDE was eluted with 0.25 M NaCl in phosphate buffer, pH 6.8); (ii) Affi-gel blue agarose (PDE was eluted with 0.35 M potassium thiocyanate in phosphate buffer, pH 6.8); and (iii) zinc chelate adsorbent matrix (PDE activity passes straight through the column). This yields a preparation of enzyme that is approximately 10-20% pure, based upon maximal isobutylmethylxanthine-stimulated [3H]cGMP binding to the noncatalytic site of PDE-5. We have previously demonstrated that this enzyme preparation contained a single class of cGMP hydrolytic sites, was potently inhibited by zaprinast (IC50 = 0.68 µM using 0.5 µM cGMP), and could be maximally activated up to 10-fold by the catalytic subunit of PKA (20).

Recombinant wild type PDE-6gamma was purified by a combination of cation exchange and reverse-phase chromatography (24) from Escherichia coli strain BL21 DE3 transformed with an expression plasmid containing PDEgamma synthetic gene. The coding sequence for PDEgamma from the fusion protein (24) was subcloned into an expression vector pET-11a (Novagen) by Dr. J. Sondek (Yale University). The purity of PDE6gamma was estimated to be >95%, and the PDE6gamma concentration was determined spectrophotometrically at 280 nm using a molar extinction coefficient of 7100.

PDE Assay

The assay of PDE activity was by the two-step radiotracer method (25). Assays were performed at 0.5 µM [3H]cGMP. All PDE activity measurements were done under conditions of linear rate product formation and where less than 10% of the substrate was utilized during the assay.

Protein Phosphorylation

Partially purified guinea pig PDE-5 and membranes were separately incubated with an "activation mixture" containing final concentrations of 25 µM ATP, 25 mM Hepes, pH 7.4, 5 mM MgCl2, and the catalytic subunit of PKA (10 units). Incubations were performed at 37 °C for 20 min. Samples were then withdrawn for PDE assay. In experiments where the effect of either PDE-6gamma or a peptide corresponding to amino acids 24-46 of PDE-6gamma was investigated, we combined each with the PDE prior to the addition of the activation mixture and kinase.

In studies investigating the phosphorylation of PDE-6gamma peptide (amino acids 24-46), we combined the peptide with the activation mixture and the catalytic subunit of PKA (10 units). In studies using purified protein kinase C (PKC), we added 0.75 mM CaCl2, 50 µg/ml phosphatidylserine, and 0.05 units of PKC to the activation mixture. Each reaction was initiated by adding 1 µCi of [gamma -32P]ATP. Incubations were performed at 37 °C for 60 min and terminated by the addition of 75 mM orthophosphoric acid. Samples were spotted onto P81 cellulose discs and washed in 3 × 250 ml acetic acid (1%, v/v) washes. The amount of 32P-labeled peptide was quantified by liquid scintillation counting.

In experiments where ASM membranes were utilized to assess p14 and p18 phosphorylation, the catalytic subunit of PKA was omitted, and the activation mixture was supplemented with [gamma -32P]ATP (10 µCi/assay). Incubations were performed at 37 °C for 20 min. At the termination of the incubation, membranes were then harvested by centrifugation at 14,000 × g for 10 min at 4 °C and taken for immunoprecipitation with anti-PDE-6gamma antibody.

In some experiments, ASM membranes were incubated with varying concentrations of GppNHp (1-100 µM) and ATP (0.005-50 µM) in 10 mM Tris/HCl, pH 7.4, and 5 mM MgCl2. This was performed at 37 °C for 10 min. GppNHp was prepared in 60 mM NaCl, 5 mM Hepes, pH 7.4, and 5 mM MgCl2. At the termination of the incubation, membranes were then harvested by centrifugation at 14,000 × g for 10 min at 4 °C and taken either for immunoblotting with anti-PDE-6gamma or for activation with PKA.

Immunoprecipitation

ASM membranes were solubilized in 10 mM K2HPO4/K2HPO4 and 0.15 M NaCl, pH 6.8 (PBS) containing 1% (w/v) deoxycholate and 0.1% (w/v) SDS for 60 min at 4 °C. The material was harvested and centrifuged at 14,000 × g for 10 min at 4 °C, and 250 µl of supernatant was taken and immunoprecipitated with the anti-PDE-6gamma antibody. After agitation for 1-2 h at 4 °C, 50 µl of protein A-Sepharose was added for 30 min at 4 °C. The subsequently obtained protein A-Sepharose immune complex was collected by centrifugation at 14,000 × g for 5 min at 4 °C, washed three times in PBS, and then taken either for PDE assay or for analysis of [32P]phosphate incorporation into p14 and p18.

Immunoblotting

Nitrocellulose sheets were blocked in 5% gelatin in PBS at 37 °C for 1 h and then probed with anti-PDE-6gamma antibody in PBS containing 1% gelatin (w/v) plus 0.05% (v/v) Nonidet P-40 at 37 °C for 12 h. After this time, the nitrocellulose sheets were washed in PBS plus 0.05% (v/v) Nonidet P-40. Detection of immunoreactivity was achieved by incubating nitrocellulose sheets for 2 h at 37 °C with a reporter horseradish peroxidase-linked anti-rabbit antibody in PBS containing 1% gelatin (w/v) plus 0.05% (v/v) Nonidet P-40. After washing the blots as described above to remove excess reporter antibody, immunoreactive bands were detected using an excitation chemiluminescence detection kit.


RESULTS

PDE-6gamma Inhibits PKA-activated PDE-5 Activity

To explore the hypothesis that PDE-5 can be regulated by small proteins homologous to PDE-6gamma , we have tested the ability of recombinant PDE-6gamma to modulate the activity of the partially purified PDE-5. As shown in Fig. 1A, the incubation of PDE-6gamma with PDE-5 at 4 °C for 30 min prevented the subsequent activation of PDE-5 by PKA. A doubling in kinase activity does not reduce the inhibitory effect of PDE-6gamma , suggesting that the kinase does not compete with PDE-6gamma for PDE-5. It is well documented that PDE-6gamma interacts with PDE-6 via a central polycationic region whose role is to increase the affinity between PDE-6gamma and PDE-6 catalytic subunits (26-28). To establish whether this region of PDE-6gamma is responsible for preventing PDE-5 activation by PKA, we studied the effect of a peptide, whose sequence corresponded to amino acids 24-46 in PDE-6gamma on the activation of PDE-5 by PKA. As shown in Fig. 1A, the incubation of PDE-5 with micromolar quantities of the polycationic peptide at 4 °C for 30 min prevented the subsequent activation of PDE-5 by PKA.


Fig. 1. PDE-6gamma and a peptide corresponding to amino acids 24-46 of PDE-6gamma inhibit PKA-activated PDE-5 activity. A, the partially purified PDE-5 (total protein, 30 µg/assay) was activated using PKA (10 or 20 units) in a 150-µl final volume of activation mixture (see "Experimental Procedures"). After incubation for 20 min at 37 °C, aliquots were removed for PDE activity assay. Recombinant PDE-6gamma (0.1 µM) or peptide (amino acids (aa) 24-46 of PDE-6gamma (1 µM) was added to the PDE at 4 °C for 30 min prior to activation with PKA. B, recombinant PDE-6gamma was added to the PDE (basal PDE activity, 5pmol/min/ml) at 4 °C for 30 min prior to activation with PKA (10 units). The final concentration of PDE-6gamma was 3 pM to 0.3 µM. C, activated PDE-5 was separated from PKA on a Sephacryl S200 column. PDE-6gamma was added (final concentration, 1 µM) to isolated PDE-5 and taken for assay. PDE activities are expressed as the means ± S.D. for assays in triplicate from a representative experiment performed three times.
[View Larger Version of this Image (9K GIF file)]

The effect of PDE-6gamma was also concentration-dependent (Fig. 1B), with an approximately 80% inhibition of the PKA-dependent activation of PDE-5 being evident at 0.3 µM PDE-6gamma . The addition of PDE-6gamma to the activated PDE-5 was without effect (Fig. 1C), suggesting that PDE-6gamma elicits its effect by preventing the activation process.

It was also necessary to show whether PDE-6gamma is required to be phosphorylated to inhibit PDE-5. This concern was raised after we found that PDE-6gamma can in fact serve as a substrate for PKA (see Fig. 8). A definitive answer came from experiments with the peptide. The peptide does not contain putative sites for the phosphorylation by PKA and indeed was not phosphorylated by this kinase (Fig. 2). The peptide does contain a phosphorylation site for PKC at Thr35 (29) and is vigorously phosphorylated by this kinase (Fig. 2). We conclude from these studies that the phosphorylation of PDE-6gamma subunit by PKA has no influence upon its interaction with PDE-5.


Fig. 8. The effect of GppNHp on the phosphorylation of p14 and p18. An autoradiogram of 32P phosphorylation of p14 and p18 in isolated ASM cell membranes and the enhanced phosphorylation of these proteins in response to GppNHp (100 µM) are shown. Left-hand lane, PKA-phosphorylated recombinant PDE6gamma (gamma R); middle lane, untreated membranes; right-hand lane, GppNHp-treated membranes. p14 and p18 were immunoprecipitated with anti-PDE6gamma according to "Experimental Procedures." PDE6gamma was phosphorylated by the catalytic subunit of PKA as per the method for activation of PDE-5. These are representative results from three different membrane preparations.
[View Larger Version of this Image (23K GIF file)]


Fig. 2. Peptide corresponding to the sequence 24-46 in PDE-6gamma was phosphorylated by PKC but not by PKA. Purified PDE-6gamma peptide (final concentration, 600 µM) was subjected to phosphorylation by PKC and PKA according to "Experimental Procedures." 32P incorporation into peptide is expressed as means ± S.D. for assays in triplicate from a representative experiment performed three times.
[View Larger Version of this Image (15K GIF file)]

Finally we needed to show that the effect of PDE-6gamma and the polycationic peptide on the regulation of PDE-5 by PKA was not due to inhibition of the kinase. This was established by demonstrating that neither PDE-6gamma nor the polycationic peptide inhibited the PKA-catalyzed phosphorylation of an exogenous substrate, myelin basic protein (Fig. 3).


Fig. 3. PDE-6gamma and peptide (amino acids 24-46) do not affect PKA activity. Purified PDE-6gamma (0.1 µM) and peptide (1 µM) were separately incubated with PKA (10 units) in an activation mixture containing 1 µg of myelin basic protein and 1 µCi of [32P]ATP. Resolved 32P-myelin basic protein on SDS-polyacrylamide gel electrophoresis was quantified by Cerenkov counting. The data are representative of three identical experiments. aa, amino acids.
[View Larger Version of this Image (15K GIF file)]

Airway Smooth Muscle Membranes Contain Two Proteins Immunoreactive with Anti-PDE-6gamma Antibodies

We next attempted to establish whether ASM cell membranes express proteins that contain a similar peptide region to that found in PDE-6gamma (amino acids 24-46), because these may serve as inhibitors of PDE-5. Immunoblotting cultured airway smooth muscle membranes with rabbit antibodies raised against amino acids 24-46 of bovine PDE-6gamma identified two immunoreactive staining polypeptides in the low molecular mass range (Fig. 4A). These polypeptides each have an Mr of 14 and 18 kDa (termed here p14 and p18) and migrated close to recombinant PDE-6gamma (see Figs. 8 and 10).


Fig. 4. Detection of proteins that are immunologically similar to PDE-6gamma in airway smooth muscle membranes. A, an immunoblot of airway smooth muscle membrane proteins resolved on SDS-polyacrylamide gel electrophoresis using anti-PDE-6gamma antibody. Molecular mass markers are shown. p14 and p18 are denoted with arrows. B, an immunoblot showing that the immunizing PDE-6gamma peptide (amino acids 24-46, 100 µg/ml) inclusion with antibody reduces the immunodetection of p14 and p18. Nitrocellulose sheets were incubated with the same amount of antibody in the presence and the absence of peptide. After washing, both sheets were added to the same reaction vessel and incubated with ECL reactants. p14 and p18 are denoted with arrows. C, a histogram showing the immunoprecipitation of PDE activity with anti-PDE-6gamma antibody. These are representative results from three different membrane preparations.
[View Larger Version of this Image (21K GIF file)]


Fig. 10. The effect of combined GppNHp and ATP upon the immunoreactivity of p14 and p18 in washed isolated ASM cell membranes. The immunoblot shows the effect of separate and combined treatments of membranes with GppNHp (100 µM) and ATP (5-500 nM) on the immunoreactivity of p14 and p18. Membranes were pretreated with GppNHp and ATP as described under "Experimental Procedures." Recombinant PDE6gamma (gamma R) was also electrophoresed along side the ASM samples. These are representative results from three different membrane preparations.
[View Larger Version of this Image (17K GIF file)]

We also show that the detection of p14 and p18 was reduced when the immunizing peptide was combined with antibody (Fig. 4B). p14 and p18 appear to form a stable complex with PDE-5 in membranes. This was indicated from experiments in which the anti-PDE-6gamma antibody immunoprecipitated PDE activity from detergent solubilized membranes (Fig. 4C).

Activation of Membrane-bound PDE-5 by PKA

The finding that a peptide corresponding to amino acids 24-46 of PDE-6gamma can modulate the activation of PDE-5 and that an antibody raised to this region identified two low molecular mass proteins suggests that these proteins may function like PDE-6gamma . It also indicates that a G-protein-dependent mechanism similar to that regulating PDE-6gamma may be present in ASM cell membranes. To test this hypothesis we needed to show that PDE-5 could be activated by PKA in isolated membranes and that this could be modified by p14 and p18 in a G-protein-dependent manner. In this regard, Fig. 5 shows that PDE-5 in guinea pig lung and ASM membranes was markedly activated by PKA in the presence of ATP. To assess the role of G-proteins, we have tested the effect of GppNHp (a nonhydrolyzable analogue of GTP) on the activation of PDE-5 by PKA. The addition of GppNHp (1-100 µM) to membranes prevented activation in a concentration-dependent manner (Fig. 6). The inhibition was not due to direct activation of PKA or binding of this guanine nucleotide to the PDE, because GppNHp was without effect on the activation of partially purified PDE-5 by PKA (data not shown). Further, GppNHp did not modulate membrane PDE-5 activity in the absence of PKA (data not shown).


Fig. 5. Characterization of PDE-5 in isolated cell membranes and the activation by PKA. Membrane-bound PDE was activated by PKA as described under "Experimental Procedures." The histogram shows the effect of the catalytic subunit of PKA upon cGMP PDE activity in guinea pig lung and ASM cell membrane. The identity of the PDE activity was confirmed as type 5, based upon a high potency inhibition by zaprinast (IC50 = 1 µM, n = 4). The results are expressed as the means ± S.D. for three different membrane preparations.
[View Larger Version of this Image (17K GIF file)]


Fig. 6. GppNHp inhibits PKA-activated PDE-5 activity in isolated ASM cell membranes. Membranes were pretreated with GppNHp and taken for incubation with PKA. The graph shows the concentration-dependent inhibition of PKA-stimulated PDE-5 activity by GppNHp (1-100 µM) in isolated ASM cell membranes. The results are expressed as the means ± S.D. for three different membrane preparations.
[View Larger Version of this Image (16K GIF file)]

p14 and p18 and PKA-dependent Activation of PDE in ASM Membranes Can Be Modulated by GppNHp

We show in Fig. 7A that pertussis toxin treatment of ASM cells, which was used to inactivate Gi and Go, reversed the inhibition induced by GppNHp on the PKA-dependent activation of PDE-5. When samples are prepared from membranes treated with GppNHp, a reduction in the immunoreactivity of p14 and p18 was also detected on immunoblots (Fig. 7B). This reduction was prevented in cells pretreated with pertussis toxin (Fig. 7B) and therefore correlated with the reversal of the inhibitory effect of GppNHp on the PKA-dependent activation of PDE-5.


Fig. 7. The effect of pertussis toxin on the GppNHp-induced inhibition of PKA-activated PDE-5 and on the decrease in p14 and p18 immunoreactivity. A, histogram showing the effect of GppNHp (100 µM) on the PKA (10 units)-dependent activation of PDE-5 in guinea pig ASM membranes isolated from cells treated with and without pertussis toxin (Ptox, 0.1 µg/ml, 24 h). B, immunoblot showing the decrease in the immunoreactivity of the 14- and 18-kDa proteins in response to GppNHp (100 µM) in guinea pig ASM membranes isolated from cells treated with and without pertussis toxin (p-tox, 0.1 µg/ml, 24 h). These are representative results from three different membrane preparations.
[View Larger Version of this Image (19K GIF file)]

The corresponding appearance of p14 and p18 in the supernatant of GppNHp-treated membranes was not detected, indicating that the reduced immunoreactivity of p14 and p18 was not due to their displacement from the membrane but rather a consequence of a post-translational modification. This may involve phosphorylation of the antibody recognition epitope supported by endogenous ATP. Alternatively, GppNHp may activate a protease that cleaves p14/p18 to remove the epitope. However, phosphorylation in this region is consistent with the following observations. In Fig. 8 we show that p14 and p18 were phosphorylated by an endogenous kinase when radiolabeled ATP is added, and this could be increased by the addition of GppNHp to membranes. In Fig. 9 we show that the addition of ATP (0.5-50 µM) to membranes also induced a reduction in the immunoreactivity of p14 and p18. However, when membranes were washed with buffer A to remove endogenous nucleotides, GppNHp and ATP were only effective when added together (Fig. 10).


Fig. 9. The effect of ATP upon the immunoreactivity of p14 and p18 in isolated ASM cell membranes. The immunoblot shows the effect of either GppNHp (100 µM) or ATP (0.5-50 µM) on the immunoreactivity of p14 and p18 in unwashed guinea pig ASM cell membranes. Membranes were pretreated with GppNHp and ATP as described under "Experimental Procedures." These are representative results from three different membrane preparations.
[View Larger Version of this Image (15K GIF file)]

In Fig. 11, we show that the addition of PDE inhibitors (isobutylmethylxanthine and theophylline, 100 µM) at a concentration that is known to completely inhibit PDE-5 activity negated the effect of GppNHp on the phosphorylation and immunoreactivity of p14 and p18. These observations indicate that PDE-5, p14 and p18, unidentified G-protein, and kinase may be linked in a signal transduction cascade, because their ligand bindings appear to modulate their interactions.


Fig. 11. Catalytic site PDE inhibitors block the GppNHp-induced decrease in the immunoreactivity and stimulated phosphorylation of p14 and p18 in isolated ASM cell membranes. A, immunoblot showing the effect of theophylline (100 µM) and isobutylmethylxanthine (IBMX, 100 µM) on the GppNHp (100 µM)-induced reduction in the immunoreactivity of p14 and p18. B, autoradiograph showing the effect of isobutylmethylxanthine (100 µM) on the GppNHp (100 µM)-induced phosphorylation of p14 and p18. p14 and p18 were immunoprecipitated with anti-PDE6gamma antibody according to "Experimental Procedures." These are representative results from three different membrane preparations.
[View Larger Version of this Image (43K GIF file)]


DISCUSSION

The major observations of this study are: (i) recombinant PDE-6gamma and a peptide corresponding to amino acids 24-46 of PDE-6gamma prevent the PKA-dependent activation of PDE-5 and (ii) airway smooth muscle cells express two small molecular mass proteins (p14 and p18) that are immunologically related to the inhibitory gamma  subunit of the photoreceptor cGMP phosphodiesterase. p14 and p18 appear to interact with PDE-5, a major cGMP hydrolyzing enzyme that is present in airway smooth muscle. Phosphorylation of membrane-bound p14/p18 is stimulated by guanine nucleotides, and this correlates with a concurrent reduction in the ability of PKA to activate PDE-5. Phosphorylation of p14/p18 appears to be regulated by a pertussis toxin-sensitive G-protein-dependent kinase. At present we do not know if an additional G-protein is responsible for regulating PDE-5 independently of p14/p18, although our correlative data tend to favor the idea that p14/p18 is responsible for linking a single G-protein to PDE-5. Further evidence for an interaction between PDE-5 and p14/p18 is the formation of a stable complex between these proteins in cell membranes. Experiments with isobutylmethylxanthine and theophylline show that ligand binding to the catalytic site of PDE-5 appears to affect the phosphorylation of p14/p18 by the G-protein-dependent kinase. Ligand binding to catalytic sites is known to increase binding of cGMP to noncatalytic sites in PDE-5, suggesting that interaction of p14/p18 with PDE-5 may be governed by this occupancy. It follows that the association of p14/p18 with PDE-5 may affect the ability of the former to be phosphorylated by the G-protein-dependent kinase.

The identity of the G-protein can be considered on the basis of pertussis toxin sensitivity and may be ascribed to Gialpha , because Goalpha is not expressed in these cells (30). The G-protein can either bind to p14 and p18 to increase their susceptibility to phosphorylation by an unidentified kinase or increase the activity of this kinase directly. The role of a G-protein in regulating PDE-5 indicates the possibility that this signal transduction pathway may originate from the occupancy of a receptor with extracellular ligand. The proposed model for the regulation of PDE-5 by G-protein is distinct from that explaining the regulation of PDE-6 by its own gamma  and by transducin. In the latter case, gamma  inhibits PDE-6 activity whereas G-protein reverses this inhibition. The model proposed for PDE-5 bears some limited analogy with another regulatory mechanism that was recently suggested to modulate activation of PDE-6 in amphibian rod outer segments (17, 18). In this case, when PDE-6gamma is complexed with the GTP-bound transducin it is phosphorylated by an unidentified kinase. The phosphorylated gamma  prevents subsequent activation of PDE-6 by GTP-bound transducin.

Two functional regions in the gamma  subunit of PDE-6, a polycationic (amino acids 24-46) and a C-terminal domain, interact with both transducin and PDE-6 catalytic subunits (31-34). The C-terminal domain is essential for both the inhibitory action against PDE-6 (26, 31, 32, 35) and for stimulating transducin GTPase (36). The role of the polycationic domain is to provide a second site for interaction, which serves to increase the affinity of PDE-6gamma for PDE-6alpha beta and transducin. However, the peptide corresponding to this region was shown to inhibit the activation of PDE-5 by PKA. The PDE-6gamma antibody was raised to this polycationic region, suggesting that p14 and p18 may also contain a similar domain. On the other hand, PDE-5 contains a region that has some homology with the sites in the PDE-6 catalytic subunits that interact with the polycationic region of PDE-6gamma (28). In contrast, PDE-5 does not have a region homologous to the site on PDE-6 that was shown to interact with the C terminus of PDE-6gamma . Because the polycationic region in PDE-6gamma has not been shown to have any catalytic activity on PDE-6, this suggests either that a similar region in p14 and p18 subserves a different function that may be to modulate the activation of PDE-5 by PKA or that this proposed function is conferred to a different domain(s) in p14 and p18. Our finding that the polycationic peptide prevents the PKA-dependent activation of partially purified PDE-5 favors the former proposal.

In conclusion, p14 and p18 are PDE-5-associated proteins that appear to be affected by G-protein and kinase-directed regulation. Along with PDE-6gamma they may represent a novel class of proteins that differentially modulate the function of various cyclic nucleotide phosphodiesterases.


FOOTNOTES

*   This work was supported by a British Lung Foundation grant (to N. J. P.) and National Institutes of Health Grant EY 10336 (to V. Y. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Recipient of a Jules and Doris Stein Professorship from the Research to Prevent Blindness, Inc.
par    To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, University of Strathclyde, 204 George St., Glasgow, G1 1XW, Scotland. Tel.: 44-41-552-4400, Ext. 2659; Fax: 44-41-552-2562.
1   The abbreviations used are: PDE, phosphodiesterase; PKA, protein kinase A; PKC, protein kinase C; ASM, airway smooth muscle; PBS, phosphate-buffered saline.

REFERENCES

  1. Beavo, J. A., Conti, M. C., and Heaslip, R. J. (1994) Mol. Pharmacol. 46, 399-405 [Abstract]
  2. Harrison, S. A., Reifsnyder, D. H., Gallis, B., Cadd, G. G., and Beavo, J. A. (1986) Mol. Pharmacol. 25, 506-514
  3. Macphee, C. H., Reifsnyder, D. H., Moore, T. A., Lerea, K. M., and Beavo, J. A. (1988) J. Biol. Chem. 263, 10353-10358 [Abstract/Free Full Text]
  4. Sharma, R. K., and Wang, J. H. (1986) J. Biol. Chem. 261, 1322-1328 [Abstract/Free Full Text]
  5. Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978 [Abstract/Free Full Text]
  6. Wu, Z., Sharma, R. K., and Wang, J. H. (1992) in The Biology of Cyclic Nucleotide Phosphodiesterase (Strada, S. J., and Hidaka, H., eds), pp. 29-44, Raven Press, New York
  7. Mumby, M. C., Martins, T. J., Chang, M. L., and Beavo, J. A. (1982) J. Biol. Chem. 257, 13283-13290 [Abstract/Free Full Text]
  8. Pyne, N. J., Cooper, M., and Houslay, M. D. (1986) Biochem. J. 234, 325-334 [Medline] [Order article via Infotrieve]
  9. Fung, B. K.-K., Young, J. M., Yamanae, H. K., and Griswold-Prenner, I. (1990) Biochemistry 29, 2657-2664 [Medline] [Order article via Infotrieve]
  10. McAllister-Lucas, L. M., Sonnenburg, W. K., Kadlecek, A., Seger, D., Trong, H. L., Colbran, J. L., Thomas, M. K., Walsh, K. A., Francis, S. H., Corbin, J. D., and Beavo, J. A. (1993) J. Biol. Chem. 268, 22863-22873 [Abstract/Free Full Text]
  11. Stryer, L. (1991) J. Biol. Chem. 266, 10711-10714 [Free Full Text]
  12. Hurly, J. (1992) J. Bioenerg. Biomembr. 24, 219-226 [Medline] [Order article via Infotrieve]
  13. Chabre, M., and Deterre, P. (1989) Eur. J. Biochem. 179, 255-266 [Medline] [Order article via Infotrieve]
  14. Yamazaki, A., Batucca, P., Ting, A., and Bitensky, M. W. (1982) Proc. Natl. Acad. Sci. 79, 3702-3706 [Abstract]
  15. Cote, R. H., Bownds, M. D., and Arshavsky, V. Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4845-4849 [Abstract]
  16. Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417 [CrossRef][Medline] [Order article via Infotrieve]
  17. Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williams, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023 [Abstract/Free Full Text]
  18. Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024-15029 [Abstract/Free Full Text]
  19. Francis, S., and Corbin, J. D. (1988) Methods Enzymol. 159, 722-729 [Medline] [Order article via Infotrieve]
  20. Burns, F., Rodger, I. W., and Pyne, N. J. (1992) Biochem. J. 283, 487-491 [CrossRef][Medline] [Order article via Infotrieve]
  21. Burns, F., and Pyne, N. J. (1992) Biochem. Biophys. Res. Commun. 189, 1389-1396 [Medline] [Order article via Infotrieve]
  22. Wyatt, T. M., Francis, S. H., McAllister-Lucas, L. M., and Corbin, J. D. (1994) FASEB. J. 8, 372 (abstr.)
  23. Pyne, S., and Pyne, N. J. (1993) Biochem. Pharmacol. 45, 593-603 [Medline] [Order article via Infotrieve]
  24. Brown, R. L., and Stryer, L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4922-4926 [Abstract]
  25. Thompson, W. J., and Appleman, M. M. (1971) Biochemistry 10, 311-316 [Medline] [Order article via Infotrieve]
  26. Brown, R. L. (1992) Biochemistry 31, 5918-5925 [Medline] [Order article via Infotrieve]
  27. Artemyev, N. O., and Hamm, H. E. (1992) Biochem. J. 283, 273-279 [Medline] [Order article via Infotrieve]
  28. Natochin, M., and Artemyev, N. O. (1996) J. Biol. Chem. 271, 19964-19969 [Abstract/Free Full Text]
  29. Udovichenko, I. P., Cunnick, J., Gonzales, K., and Takemoto, D. J. (1994) J. Biol. Chem. 269, 9850-9856 [Abstract/Free Full Text]
  30. Grady, M., Stevens, P., and Pyne, N. J. (1993) Biochem. Biophys. Acta 1176, 313-320 [Medline] [Order article via Infotrieve]
  31. Lipkin, V. M., Dumler, I. L., Muradov, K. G., Artemyev, N. O., and Etingof, R. M. (1988) FEBS Lett. 234, 287-290 [CrossRef][Medline] [Order article via Infotrieve]
  32. Artemyev, N. O., Busman, M., and Hamm, H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412 [Abstract/Free Full Text]
  33. Morrison, D. F., Rider, M. A., and Takemoto, D. J. (1987) FEBS Lett. 222, 266-270 [CrossRef][Medline] [Order article via Infotrieve]
  34. Takemoto, D. J., Hurt, D., Oppert, B., and Cunnick, J. (1992) Biochem. J. 281, 637-643 [Medline] [Order article via Infotrieve]
  35. Skiba, N. P., Artemyev, N. O., and Hamm, H. E. (1995) J. Biol. Chem. 270, 13210-13215 [Abstract/Free Full Text]
  36. Slepak, V. Z., Artemyev, N. O., Zhu, Y., Dumke, C. L., Sabacan, L., Sondek, J., Hamm, H. E., Bownds, M. D., and Arshavsky, V. Y. (1995) J. Biol. Chem. 270, 14319-14324 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.