Allosteric activation of cGMP-specific,cGMP-binding phosphodiesterase (PDE5) by cGMP

The effects of cGMP binding on the catalytic activity of cGMP-specific, cGMP-binding phosphodiesterase (PDE5) are unclear because cGMP interacts with both allosteric and catalytic sites specifically. We studied the effects of cGMP on the hydrolysis of a fluorescent substrate analogue, 2'-O-anthraniloyl cGMP, by PDE5 partially purified from rat cerebella. The preparation contained PDE5 as the major cGMP-PDE activity and was not contaminated with cAMP- or cGMP-dependent protein kinases. The Hill coefficients for hydrolysis of the analogue substrate were around 1.0 in the presence of cGMP at concentrations <0.3 microM, while they increased to 1.5 at cGMP concentrations >1 microM, suggesting allosteric activation by cGMP at concentrations close to the bulk binding constant of the enzyme. Consistent with an allosteric activation, increasing concentrations of cGMP enhanced the hydrolysis rate of fixed concentrations of 2'-O-anthraniloyl cGMP, which overcame competition between the two substrates. Such activation was not observed with cAMP, cyclic inosine 3',5'-monophosphate, or 2'-O-monobutyl cGMP, indicating specificity of cGMP. These results demonstrate that cGMP is a specific and allosteric activator of PDE5, and suggest that in cells containing PDE5, such as cerebellar Purkinje cells, intracellular cGMP concentrations may be regulated autonomously through effects of cGMP on PDE5.     

The GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) is a sensor and a sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.     

Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum

Inhibition of cGMP-specific phosphodiesterase type V (PDE5) has been shown to improve penile erection in patients with erectile dysfunction. We report here the cloning of three PDE5 isoforms from human penile tissues. Two of the isoforms were identical to PDE5A1 and PDE5A2, respectively, which had been isolated from nonpenile tissues. The third isoform was novel and hence called PDE5A3. The deduced amino acid sequence of PDE5A3 was the same as the C-terminal 823-residue sequence of PDE5A1 and PDE5A2. While PDE5A1 and A2 isoforms were expressed in all tissues examined, the A3 isoform was confined to tissues with a smooth muscle or cardiac muscle component. When expressed in COS-7 cells, PDE5A1, A2, and A3 isoforms had similar cGMP-catalytic activities with K(m) of 6.2, 5.75, and 6.06 microM, respectively. Their cGMP-catalytic activities were inhibited by zaprinast with IC(50) values of 3.2 microM, 1.3 microM, and 1.6 microM, respectively, and by sildenafil with IC(50) of 28, 14, and 13 nM, respectively.     

Expression of an active, monomeric catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (PDE5)

Phosphodiesterases (PDEs) comprise a superfamily of phosphohydrolases that degrade 3',5'-cyclic nucleotides. All known mammalian PDEs are dimeric, but the functional significance of dimerization is unknown. A deletion mutant of cGMP-binding cGMP-specific PDE (PDE5), encoding the 357 carboxyl-terminal amino acids including the catalytic domain, has been generated, expressed, and purified. The K(m) of the catalytic fragment for cGMP (5.5 +/- 0. 51 microM) compares well with those of the native bovine lung PDE5 (5.6 microM) and full-length wild type recombinant PDE5 (2 +/- 0.4 microM). The catalytic fragment and full-length PDE5 have similar IC(50) values for the inhibitors 3-isobutyl-1-methylxanthine (20 microM) and sildenafil (Viagra(TM))(4 nM). Based on measured values for Stokes radius (29 A) and sedimentation coefficient (2.9 S), the PDE5 catalytic fragment has a calculated molecular mass of 35 kDa, which agrees well with that predicted by amino acid content (43.3 kDa) and with that estimated using SDS-polyacrylamide gel electrophoresis (39 kDa). The combined data indicate that the recombinant PDE5 catalytic fragment is monomeric, and retains the essential catalytic features of the dimeric, full-length enzyme. Therefore, the catalytic activity of PDE5 holoenzyme requires neither interaction between the catalytic and regulatory domains nor interactions between subunits of the dimer.     

The cGMP-binding, cGMP-specific phosphodiesterase (PDE5): intestinal cell expression, regulation and role in fluid secretion

The expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase, PDE5, was studied in intestinal cells. Both PDE5A1 and PDE5A2 splice forms were cloned from the cDNA prepared from human colonic T84 cells, and PDE5 activity was dependent on increases in intracellular cGMP levels which correlated with increased phosphorylation of the enzyme. PDE5 expression was monitored in different regions of the gastrointestinal tract and nearly 50% of the phosphodiesterase activity in the duodenum, jejunum, ileum and colon was inhibited by sildenafil citrate. Administration of the stable toxin to intestinal loops resulted in activation of PDE5. Inhibition of PDE5 by sildenafil citrate led to fluid accumulation in loops, suggesting a possible explanation for the side effect of diarrhoea observed in individuals administered sildenafil citrate. Our results therefore represent the first study on the expression and regulation of PDE5 in intestinal tissue, and indicate that mechanisms to control its activity may have important consequences in intestinal physiology.     

Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC(50)=0.6 microM) and sildenafil (IC(50)=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.     

The gamma subunit of the rod photoreceptor cGMP phosphodiesterase can modulate the proteolysis of two cGMP binding cGMP-specific phosphodiesterases (PDE6 and PDE5) by caspase-3

We have investigated whether the proteolysis of members of the cGMP binding phosphodiesterases (PDE6, PDE5A1, and PDE10A2) by caspase-3 is modulated by the gamma inhibitor subunit of PDE6. We show here that purified caspase-3 proteolyses PDE6, an enzyme composed of two nonidentical catalytic subunits (termed alpha and beta) with molecular mass of 88 and 84 kDa. The proteolysis of PDE6 produced a single fragment with a molecular mass of 78 kDa. This corresponds to the possible cleavage of the caspase-3 consensus DFVD site (amino acids: 164-168) in the alpha subunit and leads to a 50% decrease in the cGMP hydrolysing activity of the enzyme. The addition of rod PDEgamma to the incubation completely blocked the cleavage of PDE6 by caspase-3. In contrast, rod PDEgamma converted PDE5A1 (molecular mass of 98 kDa) to a better substrate for caspase-3. This resulted in the formation of four major fragments with molecular mass of 82-83, 67, 43, and 34 kDa. In addition, caspase-3 induced an approximately 80% reduction in the activity of a partially purified preparation of PDE5A1 in the presence of rod PDEgamma. Caspase-3 also cleaved PDE10A2 (molecular mass of 95 kDa) to a single 48-kDa fragment. This was consistent with cleavage of the DLFD site (amino acids: 312-315) in PDE10A2. In contrast with both PDE6 and PDE5A1, rod PDEgamma was without effect on this enzyme. These data show that rod PDEgamma interacts with at least two members of the cGMP binding PDE family (PDE5A1 and PDE6) and can exert differential effects on the cleavage of these enzymes by caspase-3.     

A photoaffinity probe covalently modifies the catalytic site of the cGMP-binding cGMP-specific phosphodiesterase (PDE-5)

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors or the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-¹²⁵I,-4-azido]-benzyl)-IBMX, which is referred to as 8(¹²⁵IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(¹²⁵IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photo-affinity labeling was progressively blocked by cGMP at concentrations higher than 10 μM, whereas cAMP or 5′-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, clMP, and β-phenyl-1,N ²-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC₅₀ of PET-cGMP for inhibition of photoaffinity labeling was 10 μM, which compared favorably with an IC₅₀ of 5 μM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.     

Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells.

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.     

Expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) in human colonic epithelial cells: role in the induction of cellular refractoriness to the heat-stable enterotoxin peptide

Stable toxin (ST) peptides are the causative agents for a severe form of watery diarrhea. These peptides bind to a membrane-associated form of guanylyl cyclase, guanylyl cyclase C. The result is an accumulation of cyclic guanosine monophosphate (cGMP) in the intestinal cell, regulating protein kinase activity and the phosphorylation of a number of proteins involved in ion transport across the intestine. Using the human T84 colonic cell line as a model system, we show that cGMP accumulation in these cells after ST application is regulated by the activity of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5). The presence of human PDE5 in this cell line was confirmed by Western blot analysis, using an antibody raised to the bovine enzyme, and by the observation that cGMP hydrolytic activity detected in T84 cell lysates was almost completely inhibited by low concentrations of zaprinast, a specific inhibitor of PDE5. An increase in activity of PDE5 was observed in T84 cell lysates on exposure to the ST peptide and prolonged exposure of T84 cells to the ST peptide led to the induction of cellular refractoriness in these cells, which was largely contributed in terms of an increased rate of degradation of cGMP in desensitized cells as a result of PDE5 activation. This activation was correlated with an increase in the affinity of the enzyme for the substrate cGMP, as well as an increased affinity for zaprinast. We provide evidence for the first time that cGMP levels in the human colonocyte are regulated by the cGMP-hydrolytic activity of PDE5 and suggest that the expression and regulation of PDE5 in the intestine could therefore be important in controlling cGMP-mediated signaling in this tissue.     

The γ-subunit of the rod photoreceptor cGMP-binding cGMP-specific PDE is expressed in mouse lung

The type 6 phosphodiesterase (PDE-6) from retinal rod photoreceptors is an αβγ[in2] heterotetramer. The α-and β-subunits contain catalytic sites for cGMP hydrolysis, whereas the γ-subunits (Pγ) serve as a protein inhibitor of the enzyme. Pγ is believed to be expressed only in photoreceptors. Using RT-PCR, we have amplified the complete coding sequence for Pγ from mouse lung RNA. The expression of Pγ in this tissue may be related to its ability to interact the type 5 phosphodiesterase (PDE-5), which is the predominant cGMP binding protein in lung. We therefore suggest that Pγ may have a wider signaling role in mammalian cells than previousl y appreciated.     

Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B

Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low k(cat) despite a tight K(m). Cyclic AMP elevation is known to inhibit all pathways of platelet activation, and thus regulation of PDE3 activity is significant. Although cGMP elevation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular details of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A, we constructed a molecular model of the catalytic domain of PDE3A based on homology to the recently determined X-ray crystal structure of PDE4B. Based on the excellent fit of this model structure, we mutated nine amino acids in the putative catalytic cleft of PDE3A to alanine using site-directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A, F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, and had k(cat)/K(m) less than 10% of the wild-type PDE3A using cAMP as substrate. Mutants N845A, F972A, and F1004A showed a 3- to 12-fold increase of K(m) for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9- to 200-fold increase of K(i) for cGMP in comparison to the wild-type PDE3A. Studies of these mutants and our previous study identified two groups of amino acids: E866 and F1004 contribute commonly to both cAMP and cGMP interactions while N845, E971, and F972 residues are unique for cAMP and the residues Y751, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues differently from cAMP.     

Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse.

The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.     

Allosteric sites of phosphodiesterase-5 (PDE5). A potential role in negative feedback regulation of cGMP signaling in corpus cavernosum.

To date, relative cellular levels of cGMP and cGMP-binding proteins have not been considered important in the regulation of smooth muscle or any other tissue. In rabbit penile corpus cavernosum, intracellular cGMP was determined to be 18 +/- 4 nM, whereas the cGMP-binding sites of types Ialpha and Ibeta cGMP-dependent protein kinase (PKG) and cGMP-binding cGMP-specific phosphodiesterase (PDE5) were 58 +/- 14 nM and 188 +/- 6 nM, respectively, as estimated by two different methods for each protein. Thus, total cGMP-binding sites (246 nM) greatly exceed total cGMP. Given this excess of cGMP-binding sites and the high affinities of PKG and PDE5 for cGMP, it is likely that a large portion of intracellular cGMP is associated with these proteins, which could provide a dynamic reservoir for cGMP. Phosphorylation of PDE5 by PKG is known to increase the affinity of PDE5 allosteric sites for cGMP, suggesting the potential for regulation of a reservoir of cGMP bound to this protein. Enhanced binding of cGMP by phosphorylated PDE5 could reduce the amount of cGMP available for activation of PKG, contributing to feedback inhibition of smooth muscle relaxation or other processes. This introduces a new concept for cyclic nucleotide signaling.     

Binding of cGMP to GAF domains in amphibian rod photoreceptor cGMP phosphodiesterase (PDE). Identification of GAF domains in PDE alphabeta subunits and distinct domains in the PDE gamma subunit involved in stimulation of cGMP binding to GAF domains

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Palphabeta) and two identical regulatory subunits (Pgamma). Biochemical studies have shown that amphibian Palphabeta has high affinity, cGMP-specific, non-catalytic binding sites and that Pgamma stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pgamma-depleted membrane-bound PDE (20-40% Pgamma still present), a single type of cGMP-binding site with a relatively low affinity (K(d) approximately 100 nm) was observed, and addition of Pgamma increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pgamma reduced its ability to stimulate cGMP binding. Among these, the site involved in Pgamma phosphorylation by Cdk5 (positions 20-23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pgamma inhibition of the cGMP hydrolytic activity of Palphabeta. In addition, the Pgamma concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Palphabeta heterodimer contains two spatially and functionally distinct types of Pgamma-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Palphabeta-Pgamma interaction in which Pgamma, by binding to one of the two sites in Palphabeta, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.     

Direct allosteric regulation between the GAF domain and catalytic domain of photoreceptor phosphodiesterase PDE6

Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory gamma subunit (Pgamma), known to directly inhibit PDE6 catalysis, was increased approximately 2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of Pgamma to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of Pgamma1-60, Pgamma lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. Pgamma1-60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although Pgamma-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction.     

Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains

Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.