Phosphorylation of phosducin accelerates rod recovery from transducin translocation

Abstract

Purpose: In rods saturated by light, the G protein transducin undergoes translocation from the outer segment compartment, which results in the uncoupling of transducin from its innate receptor, rhodopsin. We measured the kinetics of recovery from this adaptive cellular response, while also investigating the role of phosducin, a phosphoprotein binding transducin βγ subunits in its de-phosphorylated state, in regulating this process.

Methods: Mice were exposed to a moderate rod-saturating light triggering transducin translocation, and then allowed to recover in the dark while free running. The kinetics of the return of the transducin subunits to the outer segments were compared in transgenic mouse models expressing full-length phosducin, and phosducin lacking phosphorylation sites serine 54 and 71, using Western blot analysis of serial tangential sections of the retina.

Results: In mice expressing normal phosducin, transducin α and βγ subunits returned to the outer segments with a half-time (t(1/2)) of ∼24 and 29 minutes, respectively. In the phosducin phosphorylation mutants, the transducin α subunit moved four times slower, with t(1/2) ∼95 minutes, while the movement of transducin βγ was less affected.

Conclusions: We demonstrate that the recovery of rod photoreceptors from the ambient saturating levels of illumination, in terms of the return of the light-dispersed transducin subunits to the rod outer segments, occurs six times faster than reported previously. Our data also support the notion that the accumulation of transducin α subunit in the outer segment is driven by its re-binding to the transducin βγ dimer, because this process is accelerated significantly by phosducin phosphorylation.

Figure 1.

(A) Transgenic construct used for the generation of Pdc-FLAG and Pdc^S54A/S71A-FLAG mutant mice. Partial amino acid sequence alignment of human and mouse phosducin showing the positions of serine 54 and 71 (*), which were substituted with alanine in the Pdc^S54A/S71A-FLAG mutant. (B) Representative Western blot shows the levels of epitope-tagged or endogenous phosducin in whole retina extracts of Pdc-FLAG, Pdc^S54A/S71A-FLAG, and wild-type (129Sv) mice. (C) Freshly dissected retinas from dark-adapted mice of the indicated genotypes were incubated with 10 nM okadaic acid in the dark for 20 minutes, and the phosphorylation of phosducin at serines 54 and 71 was assayed by Western blotting, using pan-specific (Pdc-pan) and phospho-specific antibodies against Pdc phosphorylated at serine 54 (Pdc54p) and serine 71 (Pdc71p). (D) Phosducin was pulled-down by its FLAG tag from retinal extracts, and Gβ₁ protein in the pull-downs was detected by Western blotting using an anti-Gβ₁ antibody. Pdc-pan antibody blots show equal levels of the FLAG-tagged Pdc in the extracts.

Figure 2.

(A) Schematic illustration of how a flat-mounted fragment of retina was sectioned. Inset: cellular compartments of an individual mouse rod expressing GFP (unpublished image obtained in this laboratory) and their distribution in serial sections of the retina. (B) Representative blots showing the distribution of protein markers of different rod cellular compartments (β-tubulin, COX I, and rom1), and arrestin, and transducin α and β subunits throughout a set of serial tangential sections (1–14) of the retinas of a mouse dark-adapted for 840 minutes (DARK) following 10 minutes of light conditioning with 600 lux (LIGHT). (C) Quantification of arrestin (triangles), and transducin α (circles) and β (squares) subunits distribution in blots shown in (B). The fluorescence value of a specific band in each section on the corresponding blot was plotted as a percentage of the total fluorescence value of all bands in all sections on the blots, dark-adapted mouse (closed symbols), light-conditioned mouse (open symbols).

Figure 3.

(A) Data for the transducin α subunits from Table 1 for Pdc-FLAG mice (white circles) and Pdc^S54A/S71A-FLAG mice (black circles) were fitted with exponential rise to maximum function y = y_o + . The half-time ( ) of transducin α return to the rod outer segment was 24 and 95 minutes in Pdc-FLAG mice and in Pdc^S54A/S71A-FLAG mice, respectively. (B) Similar analysis as in (A) revealed t_1/2 = 29 minutes for the transducin β movement in Pdc-FLAG mice. Movement of this subunit in the Pdc^S54A/S71A-FLAG strain did not obey exponential kinetics (dashed line) and its t_1/2 could not be determined. (C) Fitting of the arrestin data from Table 1 was performed using an exponential decay function . Arrestin moves from the outer segment with t_1/2 = 44 and 38 minutes in Pdc-FLAG and Pdc^S54A/S71A-FLAG mice, respectively. For all data a t-test was applied: no asterisk indicates a P value > 0.1; (**) indicates a P value < 0.05.

Figure 4.

(A) Representative Western blot showing the levels of epitope-tagged (*) and endogenous (**) phosducin in the whole retina extracts of Pdc-FLAG mice back-crossed on the wild-type background (Pdc-FLAG × WT) and Pdc^S54A/S71A-FLAG mice. The combined level of phosducin (* and **) in Pdc-FLAG × WT retinas was 248 ± 17% (SEM, n = 4) of that of the mutant phosducin (*) expressed in Pdc^S54A/S71A-FLAG retinas. (B) Subcellular localization of transducin α subunit (green), as determined by immunofluorescence microscopy, in Pdc-FLAG × WT and Pdc^S54A/S71A-FLAG mice dark-adapted for 60 minutes following 10 minutes of light conditioning with 600 lux light. Retinal layers are abbreviated as OS (outer segment), IS (inner segment), and ONL (outer nuclear layer).

Figure 5.

Subcellular localization of transducin α subunit (red) in cross-sections of retina from wild-type mice dark-adapted for 0 minutes, 30 minutes, and 12 hours, following 10 minutes of light conditioning with 140 lux and 3300 lux light. Photoreceptor nuclei are shown in blue. OPL, outer plexiform layer.