Exchange of Cone for Rod Phosphodiesterase 6 Catalytic Subunits in Rod Photoreceptors Mimics in Part Features of Light Adaptation.

Despite the expression of homologous phototransduction components, the molecular basis for differences in light-evoked responses between rod and cone photoreceptors remains unclear. We examined the role of cGMP phosphodiesterase (PDE6) in this difference by expressing cone PDE6 (PDE6C) in rd1/rd1 rods lacking rod PDE6 (PDE6AB) using transgenic mice. The expression of PDE6C rescues retinal degeneration observed in rd1/rd1 rods. Double-transgenic rods (PDE6C++) were compared with rd1/+ rods based on similar PDE6 expression. PDE6C increased the basal PDE activity and speeded the rate-limiting step for phototransduction deactivation, causing rod photoresponses to appear light adapted, with reduced dark current and sensitivity and faster response kinetics. When PDE6C++ and rd1/+ rods were exposed to similar background light, rd1/+ rods displayed greater desensitization. These results indicate an increased spontaneous activity and faster deactivation of PDE6C compared with PDE6AB in darkness, but that background light increases steady PDE6C activity to a lesser extent. In addition to accelerating the recovery of the photoresponse, faster PDE6C deactivation may blunt the rise in background-induced steady PDE6C activity. Therefore, higher basal PDE6C activity and faster deactivation together partially account for faster and less sensitive cone photoresponses in darkness, whereas a reduced rise of steady PDE6C activity in background light may allow cones to avoid saturation. SIGNIFICANCE STATEMENT: Cones are the primary photoreceptors responsible for most of our visual experience. Cone light responses are less sensitive and display speeded responses compared with rods. Despite the fact that rods and cones use a G-protein signaling cascade with similar organization, the mechanistic basis for these differences remains unclear. Here, we examined the role of distinct isoforms of PDE6, the effector enzyme in phototransduction, in these differences. We developed a transgenic mouse model that expresses cone PDE6 in rods and show that the cone PDE6 isoform is partially responsible for the difference in sensitivity and response kinetics between rods and cones.

Structural basis of phosphodiesterase 6 inhibition by the C-terminal region of the gamma-subunit.

The inhibitory interaction of phosphodiesterase-6 (PDE6) with its gamma-subunit (Pgamma) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3-isobutyl-1-methylxanthine and the Pgamma-inhibitory peptide Pgamma(70-87) have been determined at 2.9 and 3.0 A, respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pgamma and suggest the conformational change of Pgamma on transducin activation. Two variable H- and M-loops of PDE5/6cd form a distinct interface that contributes to the Pgamma-binding site. This allows the Pgamma C-terminus to fit into the opening of the catalytic pocket, blocking cGMP access to the active site. Our analysis suggests that disruption of the H-M loop interface and Pgamma-binding site is a molecular cause of retinal degeneration in atrd3 mice. Comparison of the two PDE5/6cd structures shows an overlap between the sildenafil and Pgamma(70-87)-binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.

Rod phosphodiesterase-6 PDE6A and PDE6B subunits are enzymatically equivalent.

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the phototransduction cascade in rods and cones. The catalytic core of rod PDE6 is a unique heterodimer of PDE6A and PDE6B catalytic subunits. The functional significance of rod PDE6 heterodimerization and conserved differences between PDE6AB and cone PDE6C and the individual properties of PDE6A and PDE6B are unknown. To address these outstanding questions, we expressed chimeric homodimeric enzymes, enhanced GFP (EGFP)-PDE6C-A and EGFP-PDE6C-B, containing the PDE6A and PDE6B catalytic domains, respectively, in transgenic Xenopus laevis. Similar to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments and concentrated at the disc rims. PDE6C, PDE6C-A, and PDE6C-B were isolated following selective immunoprecipitation of the EGFP fusion proteins. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with similar K(m) (20-23 µM) and k(cat) (4200-5100 s(-1)) values. Likewise, the K(i) values for PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone- and rod-specific PDE6 γ-subunits (Pγ) were comparable. Recombinant cone transducin-α (Gα(t2)) and native rod Gα(t1) fully and potently activated PDE6C, PDE6C-A, and PDE6C-B. In contrast, the half-maximal activation of bovine rod PDE6 required markedly higher concentrations of Gα(t2) or Gα(t1). Our results suggest that PDE6A and PDE6B are enzymatically equivalent. Furthermore, PDE6A and PDE6B are similar to PDE6C with respect to catalytic properties and the interaction with Pγ but differ in the interaction with transducin. This study significantly limits the range of mechanisms by which conserved differences between PDE6A, PDE6B, and PDE6C may contribute to remarkable differences in rod and cone physiology.

Intrinsically disordered gamma-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure.

The retinal phosphodiesterase (PDE6) inhibitory gamma-subunit (PDEgamma) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic alphabeta-subunits of PDE6 and the alpha-subunit of transducin (alpha(t)). Detailed structural analysis of PDEgamma has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDEgamma, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDEgamma(46-87) when bound to alpha(t) in the transition-state complex. NMR mapping of the interaction between PDEgamma(46-87) and the chimeric PDE5/6 catalytic domain confirmed that C-terminal residues 74-87 of PDEgamma are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to alpha(t), is left free at this stage. These results indicate that the interaction between PDEgamma and alpha(t) during the phototransduction cascade involves the selection of preconfigured transient conformations.

Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.

PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone- and rod-specific PDE6 gamma-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.

Analysis of PDE6 function using chimeric PDE5/6 catalytic domains.

cGMP-phosphodiesterases of the PDE6 family are expressed in retinal photoreceptor cells, where they mediate the phototransduction cascade. A system for expression of PDE6 in vitro is lacking, thus straining progress in understanding the structure-function relationships of the photoreceptor enzyme. Here, we report generation and characterization of bacterially expressed chimeric PDE5/6 catalytic domains which are highly soluble, catalytically active, and sensitive to inhibition by the PDE6 Pgamma subunit. Two flexible PDE6 loops, H and M, impart chimeric PDE5/6 catalytic domains with PDE6-like properties. The replacement of the PDE6 H-loop into the PDE5 catalytic domain increases the catalytic rate and the K(m) value for cGMP hydrolysis, whereas the substitution of the M-loop produces catalytic PDE domains responsive to Pgamma. Multiple PDE6 segments preventing functional expression of the catalytic domain are identified, supporting the requirement for specialized photoreceptor chaperones to assist PDE6 folding in vivo.

Structural determinants of the PDE6 GAF A domain for binding the inhibitory gamma-subunit and noncatalytic cGMP.

Photoreceptor cGMP phosphodiesterases (PDE6 family) are modular enzymes with each catalytic subunit containing two N-terminal regulatory GAF domains, GAF A and GAF B. The GAF A domains contribute to dimerization of the PDE6 catalytic subunits and to binding of the inhibitory Pgamma subunits, and represent candidate sites for noncatalytic binding of cGMP. We performed a mutational analysis of selected residues from the GAF A domain of cone PDEalpha' to identify the cGMP-binding pocket and delineate the Pgamma-binding surface. Results of this analysis establish the noncatalytic cGMP-binding site within the PDE6 GAF A domain and suggest that occupation of the pocket by cGMP is required for high-affinity binding of Pgamma to the proximate contact surface.

CAPN5 gene silencing by short hairpin RNA interference.

BACKGROUND: The purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and protein expression was determined using quantitative RT-PCR and immunoblot analysis. FINDINGS: Two of four shRNA plasmids reduced mutant CAPN5 RNA in a stable cell line. Similar knockdown was observed in SH-SY5Y cells that natively express CAPN5. Lactose dehydrogenase assays showed that down-regulation of CAPN5 was not cytotoxic. CONCLUSIONS: CAPN5 expression can be suppressed by shRNA-based RNA interference. Further testing in ADNIV models will determine the potential of gene silencing as a strategy to treat, delay, or prevent blindness in ADNIV patients.

Atypical retinal degeneration 3 in mice is caused by defective PDE6B pre-mRNA splicing.

Mutations in the key rod phototransduction enzyme phosphodiesterase 6 (PDE6) are known to cause recessive retinitis pigmentosa in humans. Mouse models of mutant PDE6 represent a common approach to understanding the mechanisms of visual disorders related to PDE6 defects. Mutation N605S in the PDE6B subunit is linked to atypical retinal degeneration 3 (atrd3) in mice. We examined PDE6 in atrd3 mice and an atrd3 mutant counterpart of human cone PDE6C expressed in rods of transgenic Xenopus laevis. These animal models revealed remarkably different phenotypes. In contrast to dramatic downregulation of the mutant rod PDE6 protein and activity levels in mice, expression and localization of the cone PDE6C in X. laevis were essentially unaffected by this mutation. Examination of the PDE6B mRNA in atrd3 retina showed that the mutation-carrying exon 14 was spliced-out in the majority of the transcript. Thus, retinal degeneration in atrd3 mice is caused by low levels of PDE6 protein due to defective processing of PDE6B pre-mRNA rather than by deleterious effects of the N605S mutation on PDE6 folding, stability or function.

Unique transducins expressed in long and short photoreceptors of lamprey Petromyzon marinus.

Lampreys represent the most primitive vertebrate class of jawless fish and serve as an evolutionary model of the vertebrate visual system. Transducin-alpha (G alpha(t)) subunits were investigated in lamprey Petromyzon marinus in order to understand the molecular origins of rod and cone photoreceptor G proteins. Two G alpha(t) subunits, G alpha(tL) and G alpha(tS), were identified in the P. marinus retina. G alpha(tL) is equally distant from cone and rod G proteins and is expressed in the lamprey's long photoreceptors. The short photoreceptor G alpha(tS) is a rod-like transducin-alpha that retains several unique features of cone transducins. Thus, the duplication of the ancestral transducin gene giving rise to rod transducins has already occurred in the last common ancestor of the jawed and jawless vertebrates.

PDE6 in lamprey Petromyzon marinus: implications for the evolution of the visual effector in vertebrates.

Photoreceptor rod and cone phosphodiesterases comprise the sixth family of cyclic nucleotide phosphodiesterases (PDE6). PDE6s have uniquely evolved as effector enzymes in the vertebrate phototransduction cascade. To understand the evolution of the PDE6 family, we have examined PDE6 in lamprey, an ancient vertebrate group. A single PDE6 catalytic subunit transcript was found in the sea lamprey Petromyzon marinus cDNA library. The lamprey PDE6 sequence showed a high degree of homology with mammalian PDE6 and equally distant relationships with the rod and cone enzymes. In contrast, two different PDE6 inhibitory Pgamma subunits, a cone-type Pgamma1 and a mixed cone/rod-type Pgamma2, have been identified in the lamprey retina. Immunofluorescence analysis demonstrated that Pgamma1 and Pgamma2 are expressed in the long and short photoreceptors of sea lamprey, respectively. The catalytic PDE6 subunit was present in the photoreceptors of both types and colocalized with the Pgamma subunits. Recombinant Pgamma1 and Pgamma2 potently inhibited trypsin-activated lamprey and bovine PDE6 enzymes. Our results point to a high degree of conservation of PDE6 genes during the vertebrate evolution. The apparent duplication of the Pgamma gene in the stem of vertebrate lineage may have been an essential component of the evolution of scotopic vision in early vertebrates.

The inhibitory gamma subunit of the rod cGMP phosphodiesterase binds the catalytic subunits in an extended linear structure.

The unique feature of rod photoreceptor cGMP phosphodiesterase (PDE6) is the presence of inhibitory subunits (Pgamma), which interact with the catalytic heterodimer (Palphabeta) to regulate its activity. This uniqueness results in an extremely high sensitivity and sophisticated modulations of rod visual signaling where the Pgamma/Palphabeta interactions play a critical role. The quaternary organization of the alphabetagammagamma heterotetramer is poorly understood and contradictory patterns of interaction have been previously suggested. Here we provide evidence that supports a specific interaction, by systematically and differentially analyzing the Pgamma-binding regions on Palpha and Pbeta through photolabel transfer from various Pgamma positions throughout the entire molecule. The Pgamma N-terminal Val16-Phe30 region was found to interact with the Palphabeta GAFa domain, whereas its C terminus (Phe73-Ile87) interacted with the Palphabeta catalytic domain. The interactions of Pgamma with these two domains were bridged by its central Ser40-Phe50 region through interactions with GAFb and the linker between GAFb and the catalytic domain, indicating a linear and extended interaction between Pgamma and Palphabeta. Furthermore, a photocross-linked product alphabetagamma(gamma) was specifically generated by the double derivatized Pgamma, in which one photoprobe was located in the polycationic region and the other in the C terminus. Taken together the evidence supports the conclusion that each Pgamma molecule binds Palphabeta in an extended linear interaction and may even interact with both Palpha and Pbeta simultaneously.

Asymmetric interaction between rod cyclic GMP phosphodiesterase gamma subunits and alphabeta subunits.

Rod phosphodiesterase (PDE6) is the central effector enzyme in vertebrate visual transduction. Holo-PDE6 consists of two similar catalytic subunits (Palphabeta) and two identical inhibitory subunits (Pgamma). Palphabeta is the only heterodimer in the PDE superfamily, yet its significance for the function of PDE6 is poorly understood. An unequal interaction of Pgamma with Pbeta as compared with Palpha in the PDE6 complex has not been reported. We investigated the interaction interface between full-length Pgamma and Palphabeta, by differentiating Pgamma interaction with each individual Palphabeta subunit through radiolabel transfer from various positions throughout the entire Pgamma molecule. The efficiency of radiolabel transfer indicates that the close vicinity of serine 40 on Pgamma makes a major contribution to the interaction with Palphabeta. In addition, a striking asymmetry of interaction between the Pgamma polycationic region and the Palphabeta subunits was observed when the stoichiometry of Pgamma versus the Palphabeta dimer was below 2. Preferential photolabeling on Pbeta from Pgamma position 40 and on Palpha from position 30 increased while lowering the Pgamma/Palphabeta ratio, but diminished when the Pgamma/Palphabeta ratio was over 2. Our finding leads to the conclusion that two classes of Pgamma binding sites exist on Palphabeta, each composed of GAF domains in both Palpha and Pbeta, differing from the conventional models suggesting that each Pgamma binds only one of the Palphabeta catalytic subunits. This new model leads to insight into how the unique Palphabeta heterodimer contributes to the sophisticated regulation in visual transduction through interaction with Pgamma.