Rapid and Reproducible Deactivation of Rhodopsin Requires Multiple Phosphorylation Sites

Abstract

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

Light-Dependent Translocation of Arrestin in the Absence of Rhodopsin Phosphorylation and Transducin Signaling

“…On the other hand, photolyzed rhodopsin may signal arrestin movement through molecular motors in a process mediated by transducin signaling and/or transducin translocation. To determine whether these events underlie arrestin movement, we examined arrestin localization in mice defective in rhodopsin phosphorylation (Chen et al, 1999a;Mendez et al, 2000) and subsequently in mice lacking transducin (Calvert et al, 2000). To confirm that lightdependent arrestin movement is signaled through rhodopsin, we examined arrestin localization in retinas from RPE65 Ϫ/Ϫ mice that are deficient in rhodopsin because of a defect in the visual cycle (Redmond et al, 1998).…”

“…All experimental procedures were performed in compliance with National Institutes of Health guidelines and the Society for Neuroscience Policy on the Use of Animals in Neuroscience Research. CSM/rho Ϫ/Ϫ mice (rhodopsin knock-out mice expressing a mutant rhodopsin in which all of the phosphorylation sites at the C terminus were substituted to Ala, or "completely substituted mutant" rhodopsin) were derived at the University of Southern California as described previously (Mendez et al, 2000); RK Ϫ/Ϫ and Arr Ϫ/Ϫ mice were derived at the California Institute of Technology on a C57BL/6 ϫ 129/SvJ background (Xu et al, 1997;Chen et al, 1999a). Tr ␣ Ϫ/Ϫ mice were derived at Tufts University on a BALB/c ϫ 129/SvJ background (Calvert et al, 2000).…”

“…outer segment of rods in the intact retina, the immunolocalization of arrestin was analyzed in two mouse models that do not exhibit lightdependent rhodopsin phosphorylation: CSM/rho Ϫ/Ϫ mice (Mendez et al, 2000) (see Materials and Methods) and RK Ϫ/Ϫ mice (Chen et al, 1999a).…”

“…Termination of the light signal requires that light-activated rhodopsin is deactivated by incorporation of multiple phosphates at its C terminus by rhodopsin kinase (RK) (Wilden et al, 1986;Chen et al, 1999a;Mendez et al, 2000) and the subsequent binding of arrestin (Arr). High-affinity arrestin binding to phosphorylated photolyzed rhodopsin prevents its further interaction with transducin (Wilden et al, 1986;Wilden 1995;Xu et al, 1997).…”

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

“…In retina, light-activated rhodopsin undergoes deactivation via phosphorylation by rhodopsin kinase at multiple C-terminal sites and the subsequent binding of rod arrestin (32,(69)(70)(71). For rhodopsin to recycle for full light sensitivity, arrestin and the phosphate moieties must be removed.…”