Phosducin Facilitates Light-driven Transducin Translocation in Rod Photoreceptors

Abstract

Phosducin is a photoreceptor-specific protein known to interact with the ␤␥ subunits of G proteins. In pursuit of the function of phosducin, we tested the hypothesis that it regulates the light-driven translocation of G protein transducin from the outer segments of rod photoreceptors to other compartments of the rod cell. Transducin translocation has been previously shown to contribute to rod adaptation to bright illumination, yet the molecular mechanisms underlying the translocation phenomenon remain unknown. In this study we provide two major lines of evidence in support of the role of phosducin in transducin translocation. First, we have demonstrated that transducin ␤␥ subunits interact with phosducin along their entire intracellular translocation route, as evident from their co-precipitation in serial tangential sections from light-adapted but not darkadapted retinas. Second, we generated a phosducin knockout mouse and found that the degree of lightdriven transducin translocation in the rods of these mice was significantly reduced as compared with that observed in the rods of wild type animals. In knockout animals the translocation of transducin ␤␥ subunits was affected to a larger degree than the translocation of the ␣ subunit. We also found that the amount of phosducin in rods is sufficient to interact with practically all of the transducin present in these cells and that the subcellular distribution of phosducin is consistent with that of a soluble protein evenly distributed throughout the entire rod cytoplasm. Together, these data indicate that phosducin binding to transducin ␤␥ subunits facilitates transducin translocation. We suggest that the mechanism of phosducin action is based on the reduction of transducin affinity to the membranes of rod outer segments, achieved by keeping the transducin ␤␥ subunits apart from the ␣ subunit. This increased solubility of transducin would make it more susceptible to translocation from the outer segments.Vertebrate photoreceptors are highly specialized neurons responsible for the reception and primary processing of visual information. The outer segment compartment of the photoreceptor contains large amounts of the proteins involved in light detection and in the generation of the visual signal (for review, see Refs. 1-4). Photons entering the outer segment are absorbed by rhodopsin, which triggers sequential activation of many molecules of the photoreceptor-specific heterotrimeric G protein, transducin. Transducin activation consists of GTP binding to its ␣ subunit followed by dissociation of the ␣ subunit from the transducin ␤␥ subunits and the subsequent stimulation of the downstream effector, cGMP phosphodiesterase. Signaling persists until GTP is hydrolyzed and transducin subunits re-associate into a heterotrimer. In dark-adapted rods most of the transducin is located in the outer segments. However, prolonged exposures to bright light cause massive transducin translocation from rod outer segments to other subcellular compartments of the rod (5-10). In a recent st...

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

“…First, for the sequestration model to be valid Pdc expression levels in photoreceptors would need to equal the very high expression level of transducin found in these cells, otherwise there would not be sufficient Pdc to sequester G t βγ. This was found to be the case, both G t and Pdc are expressed at ∼ 1 copy per 10 rhodopsins [19,35]. Second, the structure of the Pdc-G t βγ complex, solved by X-ray crystallography, was consistent with a G t βγ sequestration function for Pdc [36,37].…”

“…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.…”

“…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]. Thus, in the low light regimes in which rods function, there appears to be insufficient Pdc in the outer segments to sequester much G t βγ.…”

“…At the level of Gβγ, the binding of Pdc [11][12][13][14] and PhLP1 [15,16] to Gβγ has been proposed to control the amount of Gβγ available for interaction with effectors or with Gα-GDP. However, this Gβγ sequestration model for Pdc and PhLP1 function has been brought into serious question by recent findings [17][18][19]. This article will focus on current understanding of the roles of Pdc and PhLP1 in Gβγ signaling and on the other possible functions of Pdc family members.…”