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

Abstract

Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to G q and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G proteincoupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (K d 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3-to 5-fold (K d 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system.

Molecular mechanism of modulating arrestin conformation by GPCR phosphorylation

“…In addition, some variants of arrestin, such as the p44 splice variant and squid arrestin, have the capacity to bind receptors independent of receptor phosphorylation status39. The p44 splice variant lacks part of the C-tail that stabilizes the three-element interaction and polar core, and is likely already in a pre-activated state capable of binding the receptor (which may or may not include destabilization of the finger-loop lock).…”

“…Although the finger-loop motif is present in squid arrestin sequence, it is unclear how the phosphorylation-independent binding is achieved. It has previously been speculated that this could arise due to a difference in the receptor as the squid rhodopsin contains a cluster of nine negatively charged residues in the C-tail that could mimic the role of phosphates39,40.…”

Expression and light-dependent translocation of β-arrestin in the visual system of the terrestrial slug Limax valentianus

“…Because visual arrestin is known to be phosphorylated in a light-dependent manner in Drosophila and squid (Kahn and Matsumoto, 1997;Alloway and Dolph, 1999;Robinson et al, 2015), there is a possibility that the change in the immunoreactivity of β-arrestin is caused by the change in the binding affinity to the primary antibody resulting from phosphorylation. To test this possibility, the protein samples of the dark-adapted and lightadapted superior tentacles were western blotted with anti-β-arrestin antibody.…”

A combinatorial approach for the synthesis of multi-phosphorylated peptides: new tool for studying phosphorylation patterns in proteins

“…6,7 A number of studies highlighted the importance of the phosphorylation patterns for protein activities such as DNA binding, protein-protein interaction, nuclear import and export, receptor activation and many other functions. [8][9][10] To elucidate the role of each individual phosphorylation pattern in signaling, multiphosphorylated peptides with close to 100% homogeneity should be obtained.…”