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Mouse cones require an arrestin for normal inactivation of phototransduction.

ABSTRACT: Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.

SUBMITTER: Nikonov SS

PROVIDER: S-EPMC2610856 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Mouse cones require an arrestin for normal inactivation of phototransduction.

Nikonov Sergei S SS Brown Bruce M BM Davis Jason A JA Zuniga Freddi I FI Bragin Alvina A Pugh Edward N EN Craft Cheryl M CM

Neuron 20080801 3

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation ...[more]

PMID: 18701071

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Project description:PurposeCone function and survival are compromised in the guanylate cyclase-1 (GC1) knockout mouse. Disruption of the light-driven translocation of cone arrestin is one of the phenotypes of cone cells in this retina: the cone arrestin in these cells is localized to the outer segments and synaptic terminals, regardless of the state of light adaptation. The purpose of this study was to determine whether the expression of GC1 restores cone arrestin translocation in the cone cells of postnatal GC1 knockout mouse retina.MethodsSubretinal injections of AAV-GC1 were performed on 3-week-old GC1 KO mice. Electroretinographic and immunohistochemical analyses of treated retinas were carried out 5 weeks after injection. GC1 and cone arrestin antibodies were used to identify photoreceptors transduced by the AAV vector and to localize cone arrestin within cone cells, respectively.ResultsTreatment of GC1 knockout retinas with AAV-GC1 restored the light-driven translocation of cone arrestin in transduced cone cells. Staining patterns for cone arrestin in transduced and wild-type cone cells were indistinguishable after dark and light adaptation. In dark-adapted retinas, cone arrestin was distributed throughout the subcellular compartments of the cone cells. In light-adapted retinas, cone arrestin was concentrated in the cone outer segments. Successful restoration of cone arrestin translocation did not translate to a restoration of cone ERG responses, which remained undetectable in the treated retinas.ConclusionsAAV-mediated expression of GC1 in a subpopulation of cone cells in postnatal GC1 knockout retina restores light-driven translocation of cone arrestin in these cells. These findings, which show that fully developed cone cells that have developed in the absence of GC1 can respond to viral-mediated expression of this enzyme, support further analysis of this animal model of Leber congenital amaurosis type 1 (LCA1), a disease that results from null mutations in the gene encoding this enzyme.

Project description:Melanopsin, an atypical vertebrate visual pigment, mediates non-image-forming light responses including circadian photoentrainment and pupillary light reflexes and contrast detection for image formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells are characterized by sluggish activation and deactivation of their light responses. The molecular determinants of mouse melanopsin's deactivation have been characterized (i.e., C-terminal phosphorylation and β-arrestin binding), but a detailed analysis of melanopsin's activation is lacking. We propose that an extended third cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the inactive conformation, which is stabilized by the ionic interaction of these two regions. This model is supported by site-directed spin labeling and electron paramagnetic resonance spectroscopy of melanopsin, the results of which suggests a high degree of steric freedom at the third cytoplasmic loop, which is increased upon C-terminus truncation, supporting the idea that these two regions are close in three-dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin's activation rate. Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo melanopsin-which has substitutions of various potential phosphorylation sites and a substitution of the conserved tyrosine-indicates that substitution of these potential phosphorylation sites and the tyrosine residue result in dramatically slower activation kinetics, a finding that also supports the role of phosphorylation in signaling activation. We therefore propose that melanopsin's C-terminus is proximal to intracellular loop 3, and C-terminal phosphorylation permits the ionic interaction between these two regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling.

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Mouse cones require an arrestin for normal inactivation of phototransduction.

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Mouse cones require an arrestin for normal inactivation of phototransduction.

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