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S.H. Tsang, M.L. Woodruff, C.K. Yamashita, D.B. Farber, G.L. Fain; A Possible Phosphorylation Switch for PDE6 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3746.
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© ARVO (1962-2015); The Authors (2016-present)
Biochemical evidence indicates that threonines 22 and 35 of the γ subunit of PDE6(PDE6γ) can be phosphorylated, but it remains unclear if this process plays any role in rod physiology. We have tested this possibility by recording light responses of mice whose PDE6γ lacks one or more of the phosphorylation sites.
Suction electrode recordings were made from rods of mice containing PDE6γ with either T22A, T35A, or both mutations.
T22 phosphorylation was not detected in T22A retinal extract. Rods from T22A mice had nearly the same circulating current and sensitivity as WT rods but decayed more rapidly. The best–fitting single–exponential fit to the declining phase of the just–saturating response decreased from 187 ± 15 ms for WT to 137 ± 16 ms for T22A mice, and the integration time was reduced from 175 ± 13 ms to 139 ± 21 ms. The effect of the T35A mutation was quite different. The circulating current was significantly smaller than WT mice (7.5 ±0.7 vs. 11.9 ±0.4 pA) and the rods were desensitized: flash sensitivity (in pA photon –1µm –2) was 0.25 ±0.02 for WT but only 0.10 ±0.02 for T35A and the intensity at half saturation was 29 ±2 photons µm –2for WT but 76 ±10 for T35A rods. In contrast to T22A with its accelerated time course of decay, the kinetics of the responses of T35A rods were dramatically slowed. The best–fitting single exponential fit to the declining phase of the just–saturating response increased to 654 ±56 ms and the integration time, to 325 ±28 ms. There was also an increase in the dominant time constant of decay (Td) from 166 ±7 ms in WT rods to 476 ±43 ms in T35A rods. Remarkably, removal of both phosphorylation sites in the double T22A/T35A animals produced a rod response that decayed more rapidly than T35A but more slowly than T22A and was more nearly like WT in sensitivity and wave form than either T22A or T35A alone.
Our results emphasize the central role played by the PDE6γ in rod physiology, and show that removal of the phosphorylation sites of PDE6γ can have several effects on the rod light response, notably an increase or decrease in the time course of decay. Since we have manipulated PDE6γ and no other part of the transduction cascade, it is likely that T22A and T35A alter response decay by changing the interaction between GNAT1 and the RGS9 complex that determines the rate of hydrolysis of GTP. Our studies raise the possibility that phosphorylation of the PDE6γ at T22 and T35 may play a role in regulating response wave form in a normal rod, and that the two phosphorylation sites may have opposite effects, capable together of modulating turn off in either direction over a wide range
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