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Cone Phosphodiesterase-6?' Subunit Augments Cone PDE6 Holoenzyme Assembly and Stability in a Mouse Model Lacking Both Rod and Cone PDE6 Catalytic Subunits.

ABSTRACT: Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6? and PDE6? and two identical inhibitory PDE6? subunits, while cone PDE6 is composed of two identical PDE6?' catalytic subunits and two identical cone-specific PDE6?' inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contributes to the functional differences between rods and cones. In this study, we generated an rd10/cpfl1 mouse model lacking rod PDE6? and cone PDE6?' subunits. Both rod and cone photoreceptor cells are degenerated with age and all PDE6 subunits degrade in rd10/cpfl1 mice. We expressed cone PDE6?' in both rods and cones of rd10/cpfl1 mice by adeno-associated virus (AAV)-mediated delivery driven by the ubiquitous, constitutive small chicken ?-actin promoter. We show that expression of PDE6?' rescues rod function in rd10/cpfl1 mice, and the restoration of rod light sensitivity is attained through restoration of endogenous rod PDE6? and formation of a functional PDE6?'? complex. However, improved photopic cone responses were achieved only after supplementation of both cone PDE6?' and PDE6?' subunits but not by PDE6?' treatment alone. We observed a two fold increase of PDE6?' levels in the eyes injected with both PDE6?' plus PDE6?' relative to eyes receiving PDE6?' alone. Despite the presence of both PDE6?' and PDE6?, the majority of PDE6?' formed functional complexes with PDE6?', suggesting that PDE6?' has a higher association affinity for PDE6?' than for PDE6?. These results suggest that the presence of PDE6?' augments cone PDE6 assembly and enhances its stability. Our finding has important implication for gene therapy of PDE6?'-associated achromatopsia.

SUBMITTER: Deng WT

PROVIDER: S-EPMC6046437 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Cone Phosphodiesterase-6γ' Subunit Augments Cone PDE6 Holoenzyme Assembly and Stability in a Mouse Model Lacking Both Rod and Cone PDE6 Catalytic Subunits.

Deng Wen-Tao WT Kolandaivelu Saravanan S Dinculescu Astra A Li Jie J Zhu Ping P Chiodo Vince A VA Ramamurthy Visvanathan V Hauswirth William W WW

Frontiers in molecular neuroscience 20180709

Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6α and PDE6β and two identical inhibitory PDE6γ subunits, while cone PDE6 is composed of two identical PDE6α' catalytic subunits and two identical cone-specific PDE6γ' inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contribu ...[more]

PMID: 30038560

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Project description:Light-activated cyclic GMP-phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells which regulates the level of the internal transmitter cyclic GMP. PDE consists of catalytic P alpha and P beta subunits, and two copies of inhibitory P gamma subunit. The two P gamma subunits block the enzyme's activity in the dark and are removed by the alpha-subunit of transducin (alpha 1) upon light-activation of photoreceptor cells. Here we have examined the role of various regions of P gamma, the N-terminal, the central cationic and the C-terminal regions, in interaction with the catalytic subunits of PDE. N-Terminal truncation of P gamma (12-87-P gamma) did not change the potency of PDE inhibition, and thus we conclude that the P gamma N-terminal region is not critical for P gamma-P alpha beta interaction. The central region, 24-46-P gamma, participates in interaction with the catalytic P alpha beta subunits. A synthetic peptide corresponding to this site inhibited approximately 50% of trypsin-activated PDE (tPDE) (Ki approximately 15 microM) and competed with P gamma for inhibition of tPDE. We demonstrated, by using h.p.l.c. gel filtration, that 125I-Tyr-24-46-P gamma peptide bound with high affinity to tPDE, but not to P alpha beta gamma 2. The C-terminal region of 46-87-P gamma was found to be the major region involved in inhibition of PDE. It fully inhibited tPDE with a Ki of approximately 0.8 microM. It also bound to tPDE, but not P alpha beta gamma 2, in h.p.l.c. gel-filtration experiments. In addition, P gamma was cross-linked by p-phenylenedimaleimide to both P alpha and P beta, as was shown by using subunit-specific anti-P alpha, -P beta and -P gamma antibodies. Cys68 of P gamma, which presumably participates in cross-linking, is located near the P gamma C-terminus. These data provide evidence for two regions of P gamma that interact with, and inhibit, P alpha beta. The central region, 24-46 P gamma, is important in binding, but inhibits PDE only weakly, whereas the C-terminal region is most important for PDE inhibition. These results help to explain the well-known fact that P gamma trypsin-activation and C-terminal truncation both lead to PDE activation. Furthermore, our findings on the mechanism of PDE inhibition of P gamma are relevant for understanding the mechanism of PDE activation by transducin.

Dataset Information

Cone Phosphodiesterase-6?' Subunit Augments Cone PDE6 Holoenzyme Assembly and Stability in a Mouse Model Lacking Both Rod and Cone PDE6 Catalytic Subunits.

Publications

Cone Phosphodiesterase-6γ' Subunit Augments Cone PDE6 Holoenzyme Assembly and Stability in a Mouse Model Lacking Both Rod and Cone PDE6 Catalytic Subunits.

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