Compartment-specific Phosphorylation of Phosducin in Rods Underlies Adaptation to Various Levels of Illumination

Abstract

Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the G␤␥ subunits of heterotrimeric G proteins in its dephosphorylated state. Light promotes dephosphorylation of phosducin; thus, it was proposed that phosducin plays a role in the light adaptation of G protein-mediated visual signaling. Different functions, such as regulation of protein levels and subcellular localization of heterotrimeric G proteins, transcriptional regulation, and modulation of synaptic transmission have also been proposed. Although the molecular basis of phosducin interaction with G proteins is well understood, the physiological significance of light-dependent phosphorylation of phosducin remains largely hypothetical. In this study we quantitatively analyzed light dependence, time course, and subcellular localization of two principal light-regulated phosphorylation sites of phosducin, serine 54 and 71. To obtain physiologically relevant data, our experimental model exploited free-running mice and rats subjected to controlled illumination. We found that in the dark-adapted rods, phosducin phosphorylated at serine 54 is compartmentalized predominantly in the ellipsoid and outer segment compartments. In contrast, phosducin phosphorylated at serine 71 is present in all cellular compartments. The degree of phosducin phosphorylation in the dark appeared to be less than 40%. Dim light within rod operational range triggers massive reversible dephosphorylation of both sites, whereas saturating light dramatically increases phosphorylation of serine 71 in rod outer segment. These results support the role of phosducin in regulating signaling in the rod outer segment compartment and suggest distinct functions for phosphorylation sites 54 and 71. Phosducin (Pdc)2 was originally identified in the retina as an abundant 33-kDa cytosolic phosphoprotein phosphorylated in the dark and dephosphorylated in the light (1). Within the retina Pdc is expressed in both rod and cone photoreceptors (2, 3). The most distinguished feature of Pdc is its ability to form a specific complex with the ␤␥ subunits of visual heterotrimeric G protein, transducin (4, 5), and other heterotrimeric G proteins (6 -8). Affinity of Pdc toward G␤␥ is down-regulated by multiple phosphorylation and probably consequent binding of 14-3-3 protein (9 -10). Although the identity of Pdc kinase and phosphatase in photoreceptors remains unknown, the analysis of Pdc phosphorylation in vitro and ex vivo revealed that Pdc possesses multiple cAMP-dependent protein kinase and Ca 2ϩ / calmodulin-dependent protein kinase II phosphorylation sites (10 -12) and identified protein phosphatase 2A (PP2A) as the putative Pdc phosphatase (13).Despite the obvious progress in understanding the molecular basis of Pdc/G␤␥ interactions, the role of Pdc and its light-dependent phosphorylation in photoreceptors is poorly understood. Originally it was proposed that, upon activation by light, Pdc scavenges transducin ␤␥ subunits from phototransduction and by doing so reduces photoreceptor light s...

Dopamine and full‐field illumination activate D1 and D2‐D5‐type receptors in adult rat retinal ganglion cells

“…The staining pattern in vertical sections matched that found previously in rat retina with this (Song et al, 2007; Chen et al, 2008) and other (Kim et al, 2005) anti-transducin antibodies.…”

Retinophilin Is a Light-Regulated Phosphoprotein Required to Suppress Photoreceptor Dark Noise in Drosophila

“…The second dark-phosphorylated protein found in vertebrate photoreceptors is phosducin. In a phosphorylated state, phosducin also shows reduced binding of Gβγ protein subunits (Lee et al, 2004; Song et al, 2007). While the phosphorylation of both types of proteins alter the binding affinity for G protein subunits and may provide a means of adapting the photoreceptor to different light conditions, recent data suggests that phosducin also directly affects G protein levels (Krispel et al, 2007).…”

Interaction of Transducin with Uncoordinated 119 Protein (UNC119)

“…Phosducin assists light-dependent translocation of transducin from the OS to the IS by sequestering G␤ 1 ␥ 1 (22). However, in the IS, phosducin is phosphorylated in the dark and releases G␤ 1 ␥ 1 (23,50). Thus, phosphorylation of phosducin serves as a trigger for the formation of heterotrimeric G t in the IS (2,23).…”