Arrestin Translocation Is Stoichiometric to Rhodopsin Isomerization and Accelerated by Phototransduction in Drosophila Photoreceptors

Abstract

Upon illumination visual arrestin translocates from photoreceptor cell bodies to rhodopsin and membrane-rich photosensory compartments - vertebrate outer segments or invertebrate rhabdomeres - where it quenches activated rhodopsin. Both the mechanism and function of arrestin translocation are unresolved and controversial. In dark-adapted photoreceptors of the fruitfly Drosophila, confocal immunocytochemistry shows arrestin (Arr2) associated with distributed photoreceptor endomembranes. Immunocytochemistry and live imaging of GFP-tagged Arr2 demonstrate rapid reversible translocation to stimulated rhabdomeres in stoichiometric proportion to rhodopsin photoisomerization. Translocation is very rapid in normal photoreceptors (time constant <10 s), and can also be resolved in the time course of electroretinogram recordings. Genetic elimination of key phototransduction proteins, including phospholipase C (PLC), Gq and the light-sensitive Ca2+ permeable TRP channels, slows translocation by 10-100 fold. Our results indicate that Arr2 translocation in Drosophila photoreceptors is driven by diffusion, but profoundly accelerated by phototransduction and Ca2+ influx.

Arrestin-Rhodopsin Binding Stoichiometry in Isolated Rod Outer Segment Membranes Depends on the Percentage of Activated Receptors

“…The authors (24) concluded that every rhodopsin was bound by its own arrestin, although the ratio of translocated arrestin to receptor was only 0.65 in wild-type mice. Similarly, arrestin translocation in Drosophila rhabdomeres was found to be directly proportional to the amount of active photoreceptors, which could be consistent with a one-to-one binding stoichiometry (25). Finally, it has been recently shown by two separate groups that monomeric Rho*P in nanodiscs is sufficient to bind arrestin (26,27).…”

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

“…One of the best-studied examples is arrestin, which terminates the light response by binding to photo-isomerized rhodopsin (metarhodopsin). In dark-adapted photoreceptors most arrestin localizes to the cell body in both vertebrate and insect photoreceptors, but on illumination translocates to the photosensory compartment (Broekhuyse et al, 1985;Alloway et al, 2000;Kiselev et al, 2000;Lee et al, 2003;Peterson et al, 2003;Calvert et al, 2006;Slepak and Hurley, 2008;Satoh et al, 2010). In fly (Drosophila) photoreceptors the photosensory compartment is represented by the rhabdomere, a lightguiding, rod-like stack of ϳ30,000 densely packed apical microvilli loaded with rhodopsin and proteins of a phototransduction cascade mediated by heterotrimeric Gq protein, phospholipase C (PLC), and Ca 2ϩ -permeable "transient receptor potential" (TRP) channels (for review, see Wang and Montell, 2007;Katz and Minke, 2009;Yau and Hardie, 2009;Hardie, 2012).…”

“…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).…”

“…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). We also showed that Arr2 translocation was very rapid ( ϳ10 s), but profoundly slowed in mutants of various phototransduction proteins including Gq, phospholipase C (PLC) (norpA), and the major Ca 2ϩ -permeable TRP channel (Satoh et al, 2010). Our evidence suggested that Ca 2ϩ influx via the light-sensitive channels was required to accelerate Arr2 translocation, possibly by releasing Arr2 from a Ca 2ϩ -dependent cytosolic sink (Satoh et al, 2010).…”

Protein Gq Modulates Termination of Phototransduction and Prevents Retinal Degeneration

“…In the G␣ q 961 mutant, impaired Ca 2ϩ influx leads to the slow release of Arr2 from Rh1. On the other hand, lack of G q partially inhibits basal Rh1 endocytosis (24) and inhibits the translocation of Arr2 to rhabdomeres (46). These two opposite effects lead to the slow accumulation of stable Arr2-Rh1 complexes and trigger mild retinal degeneration.…”