Purification of visual arrestin from squid photoreceptors and characterization of arrestin interaction with rhodopsin and rhodopsin kinase.
Invertebrate visual signal transduction involves photoisomerization of rhodopsin, activating a guanine nucleotide binding protein (G protein) of the G(q) class, iG(q), which stimulates a phospholipase C, increasing intracellular Ca2+. Arrestin binding to photoactivated rhodopsin is a key mechanism of desensitization. We have previously reported the cloning of a retina-specific arrestin cDNA from Loligo pealei displaying 56-64% sequence similarity to other reported arrestin sequences. Here, we report the purification of the 55-kDa squid visual arrestin. Purified squid visual arrestin is able to inhibit light-activated GTPase activity dose-dependently in arrestin-depleted rhabdomeric membranes and associate with the membrane in a light-dependent manner. Membrane association can be partially inhibited by inositol 1,2,3,4,5,6-hexakisphosphate (IP6), a soluble analog of the membrane lipid phosphatidylinositol 3,4,5-triphosphate. In reconstitution assays, we demonstrate arrestin phosphorylation by squid rhodopsin kinase, a novel function among the G protein-coupled receptor kinase family. Phosphorylation of purified arrestin requires squid rhodopsin kinase, membranes, light-activation, and the presence of Ca2+. This is the first large-scale purification of an invertebrate arrestin and biochemical demonstration of arrestin function in the invertebrate visual system.
Swardfager
Walter
W
Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada.
Mitchell
Jane
J
D000818:Animals; D019393:Arrestin / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D049832:Decapodiformes / Q000166:cytology / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism / Q000528:radiation effects; D008563:Membrane Lipids / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017956:Photoreceptor Cells, Invertebrate / Q000166:cytology / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D014785:Vision, Ocular / Q000187:drug effects / Q000502:physiology
D019393:Arrestin; D008563:Membrane Lipids; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; C474322:visual arrestin protein, Loligo pealei; D012243:Rhodopsin; D051606:G-Protein-Coupled Receptor Kinase 1; D020558:GTP Phosphohydrolases