Rethinking the role of phosducin: Light-regulated binding of phosducin to 14-3-3 in rod inner segments

Abstract

Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity in the rod outer segment through interaction with the heterotrimeric G protein transducin. But, based on histochemistry and Western blot analysis, Pd is found almost entirely in the inner segment in both light and dark, most abundantly near the rod synapse. We report a second small protein, 14-3-3, in the rod with a similar distribution. By immunoprecipitation, phospho-Pd is found to interact with 14-3-3 in material from dark-adapted retina, and this interaction is markedly diminished by light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds to inner segment G protein(s) in the light. From these results and reported functions of 14-3-3, we have constructed a hypothesis for the regulation of light sensitivity at the level of rod synapse. By dissociating the Pd͞14-3-3 complex, light enables both proteins to function in this role.

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

“…First, the localization of Pdc and G t βγ in photoreceptors has been measured more carefully, both by immunohistochemistry [45,46] and by microtome sectioning [19]. These studies have shown that Pdc localization does not mimic that of G t βγ.…”

“…These studies have shown that Pdc localization does not mimic that of G t βγ. Pdc is found throughout the rod photoreceptor cell, from the outer segment to the inner segment and synaptic terminus [19,45,46]. Moreover, its distribution doesn't change in response to light.…”

“…Moreover, its distribution doesn't change in response to light. This leaves ∼ 15 % of the total Pdc in the outer segment in both the light and dark [19,45,46]. In contrast, ∼ 70 % of G t βγ is found in the outer segment in dark-adapted rods, associated with the disc membranes, and G t βγ redistributes like a soluble protein to the inner segment and synaptic region in response to intense light that saturates rod responses, leaving only ∼ 15 % of G t βγ in the outer segment under such conditions [19].…”

“…This inconsistency between the results in vitro and in intact retina could be reconciled by the binding of 14-3-3 proteins. Pdc binds 14-3-3 proteins [46,47], but only when both S54 and S73 are phosphorylated [47]. Moreover, 14-3-3 competes with G t βγ for binding to Pdc [47].…”

Site-specific Phosphorylation of Phosducin in Intact Retina

“…Pdc is found in all compartments of the photoreceptor cell, with a distribution expected for a soluble, cytosolic protein (22,23). This distribution leaves only 10 -15% of the Pdc in the outer segment (22,23), an amount that appears insufficient to sequester G t ␤␥ during an initial light response given that there are approximately equal amounts of G t and Pdc in the entire photoreceptor cell (23)(24)(25) and that G t is localized almost exclusively in the outer segment in the dark (26 -29), whereas Pdc distribution does not change in the light or dark (22,23). This notion was confirmed by data from the Pdc knock-out mouse that exhibited no change in initial photoresponse kinetics when compared with wild-type (23).…”