Light-dependent translocation of arrestin in rod photoreceptors is signaled through a phospholipase C cascade and requires ATP

Abstract

Partitioning of cellular components is a critical mechanism by which cells can regulate their activity. In rod photoreceptors, light induces a large-scale translocation of arrestin from the inner segments to the outer segments. The purpose of this project is to elucidate the signaling pathway necessary to initiate arrestin translocation to the outer segments and the mechanism for arrestin translocation.Mouse retinal organotypic cultures and eyes from transgenic Xenopus tadpoles expressing a fusion of GFP and rod arrestin were treated with both activators and inhibitors of proteins in the phosphoinositide pathway. Confocal microscopy was used to image the effects of the pharmacological agents on arrestin translocation in rod photoreceptors. Retinas were also depleted of ATP using potassium cyanide to assess the requirement for ATP in arrestin translocation.In this study, we demonstrate that components of the G-protein-linked phospholipase C (PLC) pathway play a role in initiating arrestin translocation. Our results show that arrestin translocation can be stimulated by activators of PLC and protein kinase C (PKC), and by cholera toxin in the absence of light. Arrestin translocation to the outer segments is significantly reduced by inhibitors of PLC and PKC. Importantly, we find that treatment with potassium cyanide inhibits arrestin translocation in response to light.Collectively, our results suggest that arrestin translocation is initiated by a G-protein-coupled cascade through PLC and PKC signaling. Furthermore, our results demonstrate that at least the initiation of arrestin translocation requires energy input.

Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

“…Despite these studies, no definitive answer has been provided about the link between light and sector RP, and it is not clear whether the direct action of light on Rh, or other unknown light-sensitive factors located in the retina, may cause this particular phenotype. The detailed molecular basis of light-dependent translocation of proteins from the inner segment to the outer segment in photoreceptor cells, such as arrestin (61), or from the outer segment to the inner segment, like Gt (62), is still largely undeciphered, but appears to be a finely regulated machine, and a small change in one of the partners could significantly affect the function of the others. Here, we show that N55K presents a different behavior upon illumination by forming a photointermediate unable to completely finish the retinal release process from its binding pocket.…”

Loss of Retinoschisin (RS1) Cell Surface Protein in Maturing Mouse Rod Photoreceptors Elevates the Luminance Threshold for Light-Driven Translocation of Transducin But Not Arrestin

“…This is consistent with independent pathways for at least the activation of transducin and arrestin translocation. Photoactivation of rhodopsin is requisite for both, but the level of activation required and downstream signaling mechanisms leading to activation, as well as binding partners that facilitate diffusion, are probably different (Slepak and Hurley, 2008;Elsaesser et al, 2010;Orisme et al, 2010;Satoh et al, 2010;Kerov and Artemyev, 2011). Transducin translocation is triggered at intensity levels beyond rod saturation and occurs by diffusion that depends on the concentration gradient of free G␣ t1 , which in turn is set by the rate of G␣ t1 activation and inactivation.…”

Regulation of Arrestin Translocation by Ca2+ and Myosin III in Drosophila Photoreceptors

“…The mechanism is unclear; however, intriguingly a recent study has implicated roles for PLC and protein kinase C possibly stimulating release of arrestin from its cytosolic sink (Orisme et al, 2010). Such regulated-sink models have the advantage of simplicity: directed translocation requires no more than diffusion coupled with regulated binding, can rapidly transport virtually unlimited quantities of protein, and per se consumes essentially no energy (Nair et al, 2005;Slepak and Hurley, 2008) (but see Orisme et al, 2010). While it can be conveniently studied in photoreceptors with their distinctive polarized morphologies and high concentrations of transduction machinery, translocation according to the same general principles may represent a general and elegant solution to the problem of directed movements of signaling proteins.…”

“…There is also evidence indicating light-regulated acceleration of translocation in vertebrate rods . The mechanism is unclear; however, intriguingly a recent study has implicated roles for PLC and protein kinase C possibly stimulating release of arrestin from its cytosolic sink (Orisme et al, 2010). Such regulated-sink models have the advantage of simplicity: directed translocation requires no more than diffusion coupled with regulated binding, can rapidly transport virtually unlimited quantities of protein, and per se consumes essentially no energy (Nair et al, 2005;Slepak and Hurley, 2008) (but see Orisme et al, 2010).…”

“…Although the possible role of active transport by molecular motors remains debated (Lee and Montell, 2004;Calvert et al, 2006;Strissel et al, 2006;Orisme et al, 2010), recent evidence in both vertebrate rods and Drosophila microvillar photoreceptors supports an essentially passive diffusional model of arrestin translocation, down gradients established by light-regulated "sinks" (Nair et al, 2005;Slepak and Hurley, 2008;Satoh et al, 2010). We recently provided evidence that metarhodopsin (M) is the major light-activated sink in fly rhabdomeres by showing that the dominant arrestin isoform (Arr2) translocated in a 1:1 stoichiometric relationship to the number of rhodopsin photoisomerizations (Satoh et al, 2010).…”