Role of Rhodopsin and Arrestin Phosphorylation in Retinal Degeneration of Drosophila

Abstract

Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. We employed eGFP-tagged arrestin 2 (Arr2) to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. We demonstrate that during degeneration of norpAP24 photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpAP24 is due to a failure to activate CaMKII and RDGC because of a loss of light-dependent Ca2+ entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. We investigated how reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration. To uncover the consequence underlying a lack of CaMKII activation, we characterized ala1 flies in which CaMKII was suppressed by an inhibitory peptide, and showed that morphology of rhabdomeres was not affected. In contrast, we found that expression of phosphorylation deficient Rh1s, which either lack the C-terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpAP24 photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Taken together, we conclude that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration.

The effect of phosphorylation on arrestin–rhodopsin interaction in the squid visual system

“…Our binding studies show that phosphorylation of squid arrestin on these two serines partially inhibits its binding to metarhodopsin or phosphorylated metarhodopsin. This effect is quite different from that seen for Drosophila arrestin 2 or mammalian arrestins 2 and 3, where phosphorylation does not affect their interactions with receptors (Lin et al 1997(Lin et al , 2002Kiselev et al 2000;Kristaponyte et al 2012). Structural studies of mammalian arrestins have not fully resolved the C-terminus, but enzymatic digestion and mutagenesis studies, as well as more recent electron paramagnetic resonance spectra of spin-labeled arrestin 1 have shown large movements of the C-tail associated with binding to phosphorylated metarhodopsin (Palczewski et al 1991;Vishnivetskiy et al 2000;Hanson et al 2006;Kim et al 2012).…”

“…; Kristaponyte et al . ). Structural studies of mammalian arrestins have not fully resolved the C‐terminus, but enzymatic digestion and mutagenesis studies, as well as more recent electron paramagnetic resonance spectra of spin‐labeled arrestin 1 have shown large movements of the C‐tail associated with binding to phosphorylated metarhodopsin (Palczewski et al .…”

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

“…MRh1, in turn, is recycled to Rh1 by a process that requires (1) an orange photon, (2) Ca 2+ dependent activation of Retinal Degeneration C (RDGC) to dephosphorylate Rh1- and (3) Ca 2+ -dependent Arr2 release (E, left) [ 31 – 34 , 45 ]. A reduced Ca 2+ influx would impair Rh1 dephosphorylation and Arr2 release causing endocytosis of Rh1-Arr2 complex [ 33 , 47 , 51 , 52 ]. A 10 min transient exposure to blue light followed by orange light, which is typically required for Rh1 cycling, induces Rh1 (green) internalization in ppr mutant (-/-, lacking RFP) but not in control (-/+, marked by RFP) PRs in mosaic eyes.…”

Arrestins in Apoptosis

“…A naturally occurring splice variant of arrestin-1 p44 lacking a part of the arrestin-1 C-tail and thus incapable of interacting with AP-2 but competent to quench phototransduction prevents the death of photoreceptors expressing constitutively active rhodopsin (Moaven et al 2013). This pathway is evolutionarily conserved, since the same tight association of arrestin with activated rhodopsin induces apoptotic death of Drosophila photoreceptors (Alloway et al 2000; Kiselev et al 2000; Kristaponyte et al 2012). …”