Maxim Sokolov

Arrestin Translocation Is Induced at a Critical Threshold of Visual Signaling and Is Superstoichiometric to Bleached Rhodopsin

Light induces massive translocation of major signaling proteins between the subcellular compartments of photoreceptors. Among them is visual arrestin responsible for quenching photoactivated rhodopsin, which moves into photoreceptor outer segments during illumination. Here, for the first time, we determined the light dependency of arrestin translocation, which revealed two key features of this phenomenon. First, arrestin translocation is triggered when the light intensity approaches a critical threshold corresponding to the upper limits of the normal range of rod responsiveness. Second, the amount of arrestin entering rod outer segments under these conditions is superstoichiometric to the amount of photoactivated rhodopsin, exceeding it by at least 30-fold. We further showed that it is not the absolute amount of excited rhodopsin but rather the extent of downstream cascade activity that triggers translocation. Finally, we demonstrated that the total amount of arrestin in the rod cell is nearly 10-fold higher than previously thought and therefore sufficient to inactivate the entire pool of rhodopsin at any level of illumination. Thus, arrestin movement to the outer segment leads to an increase in the free arrestin concentration and thereby may serve as a powerful mechanism of light adaptation.

Transducin translocation in rods is triggered by saturation of the GTPase-activating complex.

Light causes massive translocation of G-protein transducin from the light-sensitive outer segment compartment of the rod photoreceptor cell. Remarkably, significant translocation is observed only when the light intensity exceeds a critical threshold level. We addressed the nature of this threshold using a series of mutant mice and found that the threshold can be shifted to either a lower or higher light intensity, dependent on whether the ability of the GTPase-activating complex to inactivate GTP-bound transducin is decreased or increased. We also demonstrated that the threshold is not dependent on cellular signaling downstream from transducin. Finally, we showed that the extent of transducin α subunit translocation is affected by the hydrophobicity of its acyl modification. This implies that interactions with membranes impose a limitation on transducin translocation. Our data suggest that transducin translocation is triggered when the cell exhausts its capacity to activate transducin GTPase, and a portion of transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. Overall, the threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less-sensitive energy-saving mode beneficial in bright light, when vision is dominated by cones.

Flow of energy in the outer retina in darkness and in light

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor’s synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

Targeting of RGS7/Gβ5 to the Dendritic Tips of ON-Bipolar Cells Is Independent of Its Association with Membrane Anchor R7BP

Complexes of regulator of G-protein signaling (RGS) proteins with G-protein β5 (Gβ5) subunits are essential components of signaling pathways that regulate the temporal characteristics of light-evoked responses in vertebrate retinal photoreceptors and ON-bipolar cells. Recent studies have found that RGS/Gβ5 complexes bind to a new family of adapter proteins, R9AP (RGS9 anchor protein) and R7 family binding protein (R7BP), that in case of the RGS9/Gβ5 complex were shown to determine its precise subcellular targeting to either the outer segment of photoreceptors or postsynaptic structures of striatal neurons, respectively. In this study, we establish that another trimeric complex consisting of RGS7, Gβ5, and R7BP subunits is specifically targeted to the dendritic tips of retinal bipolar cells. However, examination of the mechanisms of complex targeting in vivo surprisingly revealed that the delivery of RGS7/Gβ5 to the dendrites of ON-bipolar cells occurs independently of its association with R7BP. These findings provide a new mechanism for adapter-independent targeting of RGS/Gβ5 complexes.

Localization and differential interaction of R7 RGS proteins with their membrane anchors R7BP and R9AP in neurons of vertebrate retina

G protein signaling in the retina is crucially regulated by the R7 family of regulators of G protein signaling (RGS) proteins, which act to stimulate the rate of G protein inactivation. Recent findings indicate that R7 RGS proteins form complexes with two newly identified membrane anchors: RGS9 Anchor Protein (R9AP) and R7 Binding Protein (R7BP), which play essential roles in modulating the expression and localization of R7 RGS proteins. Here we demonstrate that the four R7 RGS proteins: RGS6, RGS7, RGS9 and RGS11 differentially associate with two membrane anchors. R9AP was found to form complexes with RGS9 and RGS11 which were substantially enriched in the photoreceptors. In contrast, complexes of R7BP with R7 RGS proteins were predominantly localized to the synaptic projections of retina neurons, suggesting their involvement in regulation of synaptic transmission between retina neurons. Furthermore, studies of knockout mice revealed that R9AP is necessary for the expression of only RGS9 but not for RGS6, 7 or 11. Together these data suggest that R7 RGS proteins in the retina are present as macromolecular complexes with their membrane anchors that could differentially regulate their function in various retina neurons.

Phosducin Regulates the Expression of Transducin βγ Subunits in Rod Photoreceptors and Does Not Contribute to Phototransduction Adaptation

For over a decade, phosducins interaction with the βγ subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd−/−) rods. Pd−/− rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 32% lower in Pd−/− rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin βγ-subunit expression, and thus available heterotrimer in Pd−/− rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods.

Disruption of the Chaperonin Containing TCP-1 Function Affects Protein Networks Essential for Rod Outer Segment Morphogenesis and Survival

Type II Chaperonin Containing TCP-1 (CCT, also known as TCP-1 Ring Complex, TRiC) is a multi-subunit molecular machine thought to assist in the folding of ∼10% of newly translated cytosolic proteins in eukaryotes. A number of proteins folded by CCT have been identified in yeast and cultured mammalian cells, however, the function of this chaperonin in vivo has never been addressed. Here we demonstrate that suppressing the CCT activity in mouse photoreceptors by transgenic expression of a dominant-negative mutant of the CCT cofactor, phosducin-like protein (PhLP), results in the malformation of the outer segment, a cellular compartment responsible for light detection, and triggers rapid retinal degeneration. Investigation of the underlying causes by quantitative proteomics identified distinct protein networks, encompassing ∼200 proteins, which were significantly affected by the chaperonin deficiency. Notably among those were several essential proteins crucially engaged in structural support and visual signaling of the outer segment such as peripherin 2, Rom1, rhodopsin, transducin, and PDE6. These data for the first time demonstrate that normal CCT function is ultimately required for the morphogenesis and survival of sensory neurons of the retina, and suggest the chaperonin CCT deficiency as a potential, yet unexplored, cause of neurodegenerative diseases.

Current understanding of usher syndrome type II.

Usher syndrome is the most common deafness-blindness caused by genetic mutations. To date, three genes have been identified underlying the most prevalent form of Usher syndrome, the type II form (USH2). The proteins encoded by these genes are demonstrated to form a complex in vivo. This complex is localized mainly at the periciliary membrane complex in photoreceptors and the ankle-link of the stereocilia in hair cells. Many proteins have been found to interact with USH2 proteins in vitro, suggesting that they are potential additional components of this USH2 complex and that the genes encoding these proteins may be the candidate USH2 genes. However, further investigations are critical to establish their existence in the USH2 complex in vivo. Based on the predicted functional domains in USH2 proteins, their cellular localizations in photoreceptors and hair cells, the observed phenotypes in USH2 mutant mice, and the known knowledge about diseases similar to USH2, putative biological functions of the USH2 complex have been proposed. Finally, therapeutic approaches for this group of diseases are now being actively explored.

Serine/threonine kinase akt activation regulates the activity of retinal serine/threonine phosphatases, PHLPP and PHLPPL

J. Neurochem. (2010) 113, 477–488.

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

Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the Gβγ 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.