Interaction of arrestin with enolase1 in photoreceptors.

Rod and cone photoreceptors are highly specialized cells in the mammalian retina that capture photons and transduce light energy into a change in membrane potential that is ultimately relayed to the visual cortex. Photons are absorbed in these photoreceptors by opsin-based visual pigments to initiate the phototransduction cascade. The activity of the visual pigment is regulated by the arrestin family of proteins, 45-kDa proteins that sterically occlude access of transducin to the activated visual pigment until the vitamin A-derived chromophore is released and the rhodopsin is regenerated with 11-cis retinal (recently reviewed in Ref. 1). Since arrestin functions to quench phototransduction, one would expect it to be primarily concentrated in photoreceptor outer segments where the visual pigment resides. Instead, the distribution of arrestin is quite dynamic, primarily localizing to the inner segments and perinuclear region of photoreceptors in the dark and then translocating to the outer segments during light adaptation.2–6 This light-dependent change in arrestin distribution has been noted in both rods4,7,8 and cones.5,6,9 The function of arrestin translocation is unclear, although it has been hypothesized to have a role in adapting the photoreceptor's response to light, improving the temporal resolution of the photoresponse in background light.3 Since the translocation occurs on a time scale that is relatively slow, however, an alternative hypothesis for the function of arrestin translocation is that it provides protection for rods against light-induced damage resulting from continuous operation of the phototransduction cascade.10 The mechanism of arrestin translocation has been investigated by various laboratories and revealed to be complex. It was originally proposed that arrestin translocation could be accounted for by a two-partner, diffusion-mediated model in which arrestin binds to activated rhodopsin in the outer segments in the light and microtubule elements in the inner segments in the dark.11,12 The diffusion of arrestin through the connecting cilium is sufficiently fast to account for the translocation of arrestin in response to light.13,14 However, it is clear that arrestin translocation is more complex, with a signaling cascade regulating the initial translocation of arrestin15 and with more molecules of arrestin moving to the outer segments than the number of rhodopsin molecules bleached at threshold levels of light.3 This initial signaling of arrestin translocation appears to be accomplished by a phospholipase C cascade.15 In addition to this involvement of a signaling cascade, arrestin translocation also appears to be facilitated by cytoskeletal elements, with microtubules assisting in the distribution of arrestin to the apical end of the outer segments16,17 and microfilaments facilitating the movement of arrestin from the outer segments to the inner segments.17 Although the evidence supporting arrestin binding to microtubules in vitro is quite strong,18–20 the immunohistochemical data do not completely agree with tubulin/microtubules serving as the binding sink in the inner segments of dark-adapted rods. For example, binding of arrestin to microtubules in dark-adapted rod inner segments would be expected to generate a more linear or cross-hatched distribution of arrestin. This has not been observed in any of the studies of arrestin localization, whether studied by immunostaining2,21 or direct observation of fluorescently tagged arrestin2,13 or whether studied at the confocal2,21 or ultrastructural level.22 In all these studies, the distribution of arrestin is relatively uniform, occupying the available cytoplasmic volume of the inner segments. Since arrestin appears to have a relatively uniform distribution in the cytoplasm of the rod inner segments, we initiated this study to determine whether the localization of arrestin to the inner segments in the dark-adapted state might be through a specific association with a protein or complex other than the microtubule cytoskeletal elements. Using cross-linking agents in dark-adapted retinas, we show that arrestin and enolase1 are cross-linked, suggesting that they interact. This interaction is confirmed in multiple manners, using immunoprecipitation, SPR, and an enolase activity assay. We demonstrate that this interaction is direct between the two molecules, not requiring any additional binding elements.