Photoreceptor Phosphodiesterase Research Articles

Cone photoreceptor phosphodiesterase PDE6H inhibition regulates cancer cell growth and metabolism, replicating the dark retina response

BackgroundPDE6H encodes PDE6γ′, the inhibitory subunit of the cGMP-specific phosphodiesterase 6 in cone photoreceptors. Inhibition of PDE6, which has been widely studied for its role in light transduction, increases cGMP levels. The purpose of this study is to characterise the role of PDE6H in cancer cell growth.MethodsFrom an siRNA screen for 487 genes involved in metabolism, PDE6H was identified as a controller of cell cycle progression in HCT116 cells. Role of PDE6H in cancer cell growth and metabolism was studied through the effects of its depletion on levels of cell cycle controllers, mTOR effectors, metabolite levels, and metabolic energy assays. Effect of PDE6H deletion on tumour growth was also studied in a xenograft model.ResultsPDE6H knockout resulted in an increase of intracellular cGMP levels, as well as changes to the levels of nucleotides and key energy metabolism intermediates. PDE6H knockdown induced G1 cell cycle arrest and cell death and reduced mTORC1 signalling in cancer cell lines. Both knockdown and knockout of PDE6H resulted in the suppression of mitochondrial function. HCT116 xenografts revealed that PDE6H deletion, as well as treatment with the PDE5/6 inhibitor sildenafil, slowed down tumour growth and improved survival, while sildenafil treatment did not have an additive effect on slowing the growth of PDE6γ′-deficient tumours.ConclusionsOur results indicate that the changes in cGMP and purine pools, as well as mitochondrial function which is observed upon PDE6γ′ depletion, are independent of the PKG pathway. We show that in HCT116, PDE6H deletion replicates many effects of the dark retina response and identify PDE6H as a new target in preventing cancer cell proliferation and tumour growth.

Unique interface and dynamics of the complex of HSP90 with a specialized cochaperone AIPL1

Photoreceptor phosphodiesterase PDE6 is central for visual signal transduction. Maturation of PDE6 depends on a specialized chaperone complex of HSP90 with aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Disruption of PDE6 maturation underlies a severe form of retina degeneration. Here, we report a 3.9Å cryoelectron microscopy (cryo-EM) structure of the complex of HSP90 with AIPL1. This structure reveals a unique interaction of the FK506-binding protein (FKBP)-like domain of AIPL1 with HSP90 at its dimer interface. Unusually, the N terminus AIPL1 inserts into the HSP90 lumen in a manner that was observed previously for HSP90 clients. Deletion of the 7 N-terminal residues of AIPL1 decreased its ability to cochaperone PDE6. Multi-body refinement of the cryo-EM data indicated large swing-like movements of AIPL1-FKBP. Modeling the complex of HSP90 with AIPL1 using crosslinking constraints indicated proximity of the mobile tetratricopeptide repeat (TPR) domain with the C-terminal domain of HSP90. Our study establishes a framework for future structural studies of PDE6 maturation.

Reproducibility of the Rod Photoreceptor Response Depends Critically on the Concentration of the Phosphodiesterase Effector Enzyme.

The high sensitivity of night vision requires that rod photoreceptors reliably and reproducibly signal the absorption of single photons, a process that depends on tight regulation of intracellular cGMP concentration through the phototransduction cascade. Here in the mouse (Mus musculus), we studied a single-site D167A mutation of the gene for the α subunit of rod photoreceptor phosphodiesterase (PDEA), made with the aim of removing a noncatalytic binding site for cGMP. This mutation unexpectedly eliminated nearly all PDEA expression and reduced expression of the β subunit (PDEB) to ∼5%-10% of WT. The remaining PDE had nearly normal specific activity; degeneration was slow, with 50%-60% of rods remaining after 6 months. Responses were larger and more sensitive than normal but slower in rise and decay, probably from slower dark turnover of cGMP. Remarkably, responses became much less reproducible than WT, with response variance increasing for amplitude by over 10-fold, and for latency and time-to-peak by >100-fold. We hypothesize that the increase in variance is the result of greater variability in the dark-resting concentration of cGMP, produced by spatial and temporal nonuniformity in spontaneous PDE activity. This variability decreased as stimuli were made brighter, presumably because of greater spatial uniformity of phototransduction and the approach to saturation. We conclude that the constancy of the rod response depends critically on PDE expression to maintain adequate spontaneous PDE activity, so that the concentration of second messenger is relatively uniform throughout the outer segment.SIGNIFICANCE STATEMENT Rod photoreceptors in the vertebrate retina reliably signal the absorption of single photons of light by generating responses that are remarkably reproducible in amplitude and waveform. We show that this reproducibility depends critically on the concentration of the effector enzyme phosphodiesterase (PDE), which metabolizes the second messenger cGMP and generates rod light responses. In rods with the D167A mutation of the α subunit of PDE, only 5%-10% of PDE is expressed. Single-photon responses then become much more variable than in WT rods. We think this variability is caused by spatial and temporal inhomogeneity in the concentration of cGMP in darkness, so that photons absorbed in different parts of the cell produce responses of greatly varying amplitude and waveform.

Small Molecule Inhibition of PDE6D-RAS Interaction Suppresses the Growth of Acute Lymphoblastic Leukemia Cell Lines

Small Molecule Inhibition of PDE6D-RAS Interaction Suppresses the Growth of Acute Lymphoblastic Leukemia Cell Lines

Photoreceptor phosphodiesterase (PDE6): activation and inactivation mechanisms during visual transduction in rods and cones.

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes recent advances in the structural characterization of the rod and cone PDE6 catalytic and regulatory subunits in the context of previous biochemical studies of the enzymological properties and allosteric regulation of PDE6. Emphasis is given to recent advances in understanding the structural and conformational changes underlying the mechanism by which the activated transducin α-subunit binds to-and relieves inhibition of-PDE6 catalysis that is controlled by its intrinsically disordered, inhibitory γ-subunit. The role of the regulator of G-protein signaling 9-1 (RGS9-1) in regulating the lifetime of the transducin-PDE6 is also briefly covered. The therapeutic potential of pharmacological compounds acting as inhibitors or activators targeting PDE6 is discussed in the context of inherited retinal diseases resulting from mutations in rod and cone PDE6 genes as well as other inherited defects that arise from excessive cGMP accumulation in retinal photoreceptor cells.

Photoreceptor Phosphodiesterase (PDE6): Structure, Regulatory Mechanisms, and Implications for Treatment of Retinal Diseases.

The photoreceptor phosphodiesterase (PDE6) is a member of large family of Class I phosphodiesterases responsible for hydrolyzing the second messengers cAMP and cGMP. PDE6 consists of two catalytic subunits and two inhibitory subunits that form a tetrameric protein. PDE6 is a peripheral membrane protein that is localized to the signal-transducing compartment of rod and cone photoreceptors. As the central effector enzyme of the G-protein coupled visual transduction pathway, activation of PDE6 catalysis causes a rapid decrease in cGMP levels that results in closure of cGMP-gated ion channels in the photoreceptor plasma membrane. Because of its importance in the phototransduction pathway, mutations in PDE6 genes result in various retinal diseases that currently lack therapeutic treatment strategies due to inadequate knowledge of the structure, function, and regulation of this enzyme. This review focuses on recent progress in understanding the structure of the regulatory and catalytic domains of the PDE6 holoenzyme, the central role of the multi-functional inhibitory γ-subunit, the mechanism of activation by the heterotrimeric G protein, transducin, and future directions for pharmacological interventions to treat retinal degenerative diseases arising from mutations in the PDE6 genes.

Structural Analysis of the Regulatory GAF Domains of cGMP Phosphodiesterase Elucidates the Allosteric Communication Pathway

Regulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6. Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas rod PDE6 consists of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3 Å resolution, in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory γ-subunit (Pγ). Ligand-induced changes in cross-linked residues implicate multiple conformational changes in the GAFa and GAFb domains in forming an allosteric communication network. Molecular dynamics simulations of cone GAFab revealed differences in conformational dynamics of the two subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of Pγ to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled Pγ identified the central polycationic region of Pγ interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of several retinal degenerative diseases.

The molecular architecture of photoreceptor phosphodiesterase 6 (PDE6) with activated G protein elucidates the mechanism of visual excitation

Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαβ) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gtα) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαβ in its nonactivated state. Following reconstitution of PDE6 and activated Gtα with liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα-binding sites per holoenzyme. Each Gtα interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.

Allosteric Regulation of Rod Photoreceptor Phosphodiesterase 6 (PDE6) Elucidated by Chemical Cross-Linking and Quantitative Mass Spectrometry

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαβγ2) is composed of a catalytic heterodimer (Pαβ) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pβ) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαβ, Pαβ–cGMP, Pαβγ2, and Pαβγ2–cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαβ catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.

Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases.

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein-coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo-electron microscopy to determine the structure of the full-length PDE6αβ2γ complex. The final density map resolved at 3.4 Å reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6γ subunits with the PDE6αβ heterodimer. Comparison of the PDE6αβ2γ complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.

Mutations in the gene PDE6C encoding the catalytic subunit of the cone photoreceptor phosphodiesterase in patients with achromatopsia.

Biallelic PDE6C mutations are a known cause for rod monochromacy, better known as autosomal recessive achromatopsia (ACHM), and early-onset cone photoreceptor dysfunction. PDE6C encodes the catalytic α'-subunit of the cone photoreceptor phosphodiesterase, thereby constituting an essential part of the phototransduction cascade. Here, we present the results of a study comprising 176 genetically preselected patients who remained unsolved after Sanger sequencing of the most frequent genes accounting for ACHM, and were subsequently screened for exonic and splice site variants in PDE6C applying a targeted next generation sequencing approach. We were able to identify potentially pathogenic biallelic variants in 15 index cases. The mutation spectrum comprises 18 different alleles, 15 of which are novel. Our study significantly contributes to the mutation spectrum of PDE6C and allows for a realistic estimate of the prevalence of PDE6C mutations in ACHM since our entire ACHM cohort comprises 1,074 independent families.

Mechanistic insights into the role of prenyl-binding protein PrBP/\u03b4 in membrane dissociation of phosphodiesterase 6

Isoprenylated proteins are associated with membranes and their inter-compartmental distribution is regulated by solubilization factors, which incorporate lipid moieties in hydrophobic cavities and thereby facilitate free diffusion during trafficking. Here we report the crystal structure of a solubilization factor, the prenyl-binding protein (PrBP/δ), at 1.81 Å resolution in its ligand-free apo-form. Apo-PrBP/δ harbors a preshaped, deep hydrophobic cavity, capacitating apo-PrBP/δ to readily bind its prenylated cargo. To investigate the molecular mechanism of cargo solubilization we analyzed the PrBP/δ-induced membrane dissociation of rod photoreceptor phosphodiesterase (PDE6). The results suggest that PrBP/δ exclusively interacts with the soluble fraction of PDE6. Depletion of soluble species in turn leads to dissociation of membrane-bound PDE6, as both are in equilibrium. This “solubilization by depletion” mechanism of PrBP/δ differs from the extraction of prenylated proteins by the similar folded solubilization factor RhoGDI, which interacts with membrane bound cargo via an N-terminal structural element lacking in PrBP/δ.

Abstract LB-B07: Photoreceptor phosphodiesterase 6H expression regulates HCT116 proliferation and cell cycle progression

Abstract Phosphodiesterase 6 (PDE6) protein complex, which is responsible for the turnover of the second messenger cyclic guanosine monophosphate (cGMP) and is involved in visual signal transduction of light stimuli, is highly expressed in certain cancer cell lines. Despite having been discovered in the outer segment of rods and cones of the retina, PDE6 protein is also detected in brain, adrenal glands, lungs, and gonads. Genes encoding the catalytic subunits of the enzyme are known to be upregulated in certain breast, non-small cell lung cancer and medulloblastoma cells. PDE6H, which encodes the cone-specific inhibitory gamma subunit of PDE6 protein, is part of the gene signature of achromatopsia and congenital stationary night blindness while its relation to cancer remains unclear. We recently identified PDE6H as a cell cycle controller in HCT116 colorectal cancer cell line, as part of an siRNA screen in which DNA content measurement via flow cytometry was undertaken to monitor cell cycle distribution. The screen was conducted with a custom library of metabolic genes commonly amplified in primary tumours, as well as those known to be involved in the key regulatory points of metabolic pathways. We have shown that PDE6H knockdown and pharmacological inhibition of PDE6 protein hindered the activity of the protein complex, causing an accumulation of cGMP in various colorectal and breast cancer cell lines. This inhibition resulted in a G1 cell cycle arrest and disruption of G1/S checkpoint mechanism. PDE6H knockdown inhibited cell proliferation and cellular oxidative phosphorylation. It also altered mitochondrial morphology and induced apoptosis. Our results suggest that the gamma subunit is important for the function of photoreceptor phosphodiesterase PDE6 complex and PDE6H inhibition can be explored as an anti-proliferative strategy. Citation Format: Ceren Yalaz, Syed Haider, Jianzhou Chen, Remko Prevo, Mick Woodcock, Adrian L. Harris, Ruth J. Muschel. Photoreceptor phosphodiesterase 6H expression regulates HCT116 proliferation and cell cycle progression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B07.

Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations.

Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6aV685M/R562W mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies.

Activation of bovine retinal rod outer segment cGMP-specific phosphodiesterase by the transducin-GTP complex in a physiologically significant range of free calcium ion concentrations

The kinetic behavior of cGMP-specific phosphodiesterase in a totally bleached bovine retinal rod outer segment suspension was studied by the pH-metric method at high and low concentrations of free calcium ions (≈ 100 μM and 10 nM, respectively). The phosphodiesterase was activated by low GTP concentrations (about 1-2 μM) that were comparable with the concentration of G-protein transducin, its GTP-binding alpha-subunit was the intrinsic activator of photoreceptor phosphodiesterase. The results allow the suggestion that besides the earlier described system of RGS proteins, participating in the acceleration of GTP hydrolysis, rod outer segments also contain an additional Ca(2+)-dependent mechanism to inactivate so called "free transducin", i.e. active transducin that has not managed to interact with phosphodiesterase during the time, restricted by duration of photoreceptor response.

Molecular Architecture of Photoreceptor Phosphodiesterase Elucidated by Chemical Cross-Linking and Integrative Modeling

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αβγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αβ), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for differential binding of the γ-subunit to the GAFa domains of the α- and β-subunits are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.

The GAFa domain of phosphodiesterase‐6 contains a rod outer segment localization signal

Photoreceptor phosphodiesterase-6 (PDE6) is a peripheral membrane protein synthesized in the inner segment of photoreceptor cells. Newly synthesized PDE6 is transported to the outer segment (OS) where it serves as a key effector enzyme in the phototransduction cascade. Proper localization of PDE6 in photoreceptors is critically important to the function and survival of photoreceptor cells. The mechanism of PDE6 transport to the OS remains largely unknown. In this study, we investigated potential OS targeting signals of PDE6 by constructing cGMP-binding, cGMP-specific phosphodiesterase-5/PDE6 chimeric proteins and analyzing their localization in rods of transgenic Xenopus laevis. We found that efficient OS localization of chimeric isoprenylated PDE enzymes required the presence of a targeting motif within the PDE6 GAFa domain. Furthermore, the GAFa-dependent localization signal was sufficient to target GAFa fusion protein to the OS. Our results support the idea that effective trafficking of the peripheral membrane proteins to the OS of photoreceptor cells requires a sorting/targeting motif in addition to a membrane-binding signal.

CGMP Accumulation Causes Photoreceptor Degeneration in CNG Channel Deficiency: Evidence of cGMP Cytotoxicity Independently of Enhanced CNG Channel Function

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca(2+) influx in rod and cone photoreceptors. cGMP, the native ligand of the photoreceptor CNG channels, has been associated with cytotoxicity when its levels rise above normal due to defects in photoreceptor phosphodiesterase (PDE6) or regulation of retinal guanylyl cyclase (retGC). We found a massive accumulation of cGMP in CNGA3-deficient retina and investigated whether cGMP accumulation plays a role in cone degeneration in CNG channel deficiency. The time course study showed that the retinal cGMP level in Cnga3(-/-);Nrl(-/-) mice with CNGA3 deficiency on a cone-dominant background was sharply increased at postnatal day 8 (P8), peaked around P10-P15, remained high through P30-P60, and returned to near control level at P90. This elevation pattern correlated with photoreceptor apoptotic death, which peaked around P15-P20. In Cnga3(-/-);Gucy2e(-/-) mice lacking retGC1, cone density and expression levels of cone-specific proteins were significantly increased compared with Cnga3(-/-), consistent with a role of cGMP accumulation as the major contributor to cone death caused by CNG channel deficiency. The activity and expression levels of cGMP-dependent protein kinase G (PKG) were significantly increased in Cnga3(-/-);Nrl(-/-) retina compared with Nrl(-/-), suggesting an involvement of PKG regulation in cell death. Our results indicate that cGMP accumulation in photoreceptors can itself exert cytotoxic effect in cones, independently of CNG channel activity and Ca(2+) influx.

Probing the Structure of Photoreceptor Phosphodiesterase (PDE6) and Its Interactions with Transducin by Cross‐linking and Molecular Modeling

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme of visual signaling. Its activity is regulated by the inhibitory subunit (Pγ). Upon light stimulation, activated transducin (Tα*) relieves Pγ inhibition of the PDE6 catalytic dimer (Pαβ), activates PDE6, and causes a drop in cellular cGMP levels. However, the mechanism by which Tα* activates PDE6 is unclear. Although structures for some PDEs have been solved, no high‐resolution structures of PDE6 are available. Using homology modeling, we generated models of the GAF and catalytic domains of the α and β catalytic subunits. Using PDE2 (PDB:3IBJ) as a template, we built a topology model for the full‐length Pαβ catalytic dimer. Chemical cross‐linking of Pαβ identified 18 cross‐links that validated our homology model. We also identified multiple cross‐links between Pγ and Pαβ that support an extended structure for Pγ bound to Pαβ, as well as sites within the N‐terminal region of Pγ which may allosterically regulate cGMP binding to PDE6 GAF domains. Preliminary cross‐linking results of Tα*‐activated PDE6 reveal multiple sites of interaction of Tα* with Pα, Pβ, and Pγ. We conclude that cross‐linking and molecular modeling provide unique insights into PDE6 regulation not available by other approaches. Supported by NIH grant EY‐05798 (RHC), NH Agricultural Experiment Station grant NH00567 (FC), and the Karabelas Faculty Development Fund (FC).

Regulation of photoreceptor phosphodiesterase (PDE6) by the rod photoreceptor‐specific glutamic acid‐rich protein 2 (GARP2)

As the central effector of the visual signaling pathway, photoreceptor phosphodiesterase (PDE6) is precisely regulated to control the sensitivity, speed and amplitude of the light response. This study seeks to define the regulatory role of the glutamic acid‐rich protein 2 (GARP2) that binds to PDE6 in rod photoreceptors. GARP2 is a splice variant of the rod photoreceptor cGMP‐gated channel β‐subunit. Using interaction assays, we identified the inhibitory γ‐subunit (Pγ) of the PDE6 holoenzyme (αβγγ) as the site of interaction with GARP2. Using Pγ truncation mutants, we found that GARP2 binds primarily to the N‐terminal region (a.a 1–30) of Pγ. The stoichiometry of GARP2 binding to PDE6 holoenzyme was determined to be 1.0 ± 0.2 mol GARP2 per mol PDE6 holoenzyme. In addition to suppressing PDE6 basal catalytic activity, GARP2 also accelerates cGMP dissociation from noncatalytic cGMP binding sites in the regulatory domain of PDE6. Together, these results indicate that GARP2 binding to the N‐terminal region of the inhibitory Pγ subunit can lower PDE6 catalytic activity in the dark‐adapted state, effectively reducing photoreceptor dark noise. In addition, GARP2 modulation of cGMP binding affinity to the PDE6 regulatory domains is consistent with a negative feedback role for GARP2 in PDE6 regulation following light activation of the phototransduction cascade. Supported by NIH grant EY‐05798 (to RHC).