Crosslinking and co-immunoprecipitation of β-arrestin 1 and β-arrestin 2 with melanopsin.

<p>β-arrestin 1 and β-arrestin 2 co-precipitate with melanopsin in a light- and phosphorylation-dependent manner. (A) Quantitation of β-arrestin 1 co-precipitation with wildtype (MEL) and phosphonull (PØ) melanopsin samples (top). Binding of β-arrestin 1 to wildtype melanopsin in the light (MEL-L) as compared to the binding to wildtype melanopsin in the dark (MEL-D) and binding of β-arrestin 1 to wildtype melanopsin in the light (MEL-L) as compared to the binding to phosphonull in the light (PØ-L). Representative immunoblot (IB) of β-arrestin 1 co-immunoprecipitation from dark-adapted melanopsin, light-exposed melanopsin and light-exposed phosphonull melanopsin samples (bottom). (B) Quantitation of β-arrestin 2 co-precipitation with wildtype and phosphonull melanopsin samples (top). The relative binding was compared as described above. Representative immunoblot of β-arrestin 2 co-immunoprecipitation from dark-adapted melanopsin, light-exposed melanopsin and light-exposed phosphonull melanopsin samples (bottom). Loading input confirmed by Bradford and anti-1D4 immunobot. Quantitation reported as the average of 4 independent co-immunoprecipitation experiments with standard deviation plotted as error bars. All experiments were carried out in HEK293 cells stably over-expressing β-arrestin 1 or β-arrestin 2 and transiently expressing wildtype melanopsin-1D4 or phosphonull melanopsin-1D4. (C) Addition of the 1D4 epitope does not alter the signaling kinetics of wildtype melanopsin or phosphonull melanopsin in fluorescent calcium imaging responses.</p

Melanopsin interacts with β-arrestin 1 and β-arrestin 2 in a light- and phosphorylation-dependent manner in fixed HEK293 cells.

<p>HEK293 cells heterologously expressing melanopsin and phosphonull melanopsin were assayed by proximity ligation assay (PLA) to investigate the melanopsin/β-arrestin interaction. Cell nuclei labeled with DAPI (blue). Positive PLA interaction indicated by red fluorescent spots. (A) Melanopsin interacts with β-arrestin 1 and β-arrestin 2 in the light, (B) but not in the dark. (C) Phosphonull melanopsin (PØ), lacking C-tail phosphorylation sites, does not interact with either β-arrestin in the light. (D) Histogram showing average number of fluorescent spots per cell in each condition. N = 30 cells. Error bars represent standard error of the mean.</p

Melanopsin interacts with β-arrestin 1 and β-arrestin 2 in the murine retina.

<p>The melanopsin/β-arrestin interaction demonstrated by PLA in mouse retinal sections. Major nuclear layers of the retina defined by DAPI (GCL, Ganglion cell layer; INL, Inner nuclear layer; PRL, Photoreceptor layer). The inserts in A–C represent magnification of the ganglion cell layer. Positive PLA interaction is indicated by red fluorescent spots. (A) A melanopsin/β-arrestin 1 and melanopsin/β-arrestin 2 dependent PLA signal was detected in light-exposed fixed retinas (C57BL/6). (B) Melanopsin does not appear interact with β-arrestin 1 and β-arrestin 2 in dark-adapted fixed retinas. (C) Little PLA signal was detected in light-exposed melanopsin knockout retinas (opn4^LacZ/LacZ). (D) No signal was detected in retinas (C57BL/6) probed with secondary PLA antibodies only. (E) Specificity of melanopsin (C-terminal), β-arrestin 1, and β-arrestin 2 antibodies confirmed by western blot analysis of protein isolated from mouse retinas.</p

Melanopsin co-localizes with β-arrestin 1 and β-arrestin 2 in ipRGCs.

<p>(A–C) Three major nuclear layers of the mouse retina defined by DAPI (GCL, Ganglion cell layer; INL, Inner nuclear layer; PRL, Photoreceptor layer). (A) Visual arrestin staining localized in the PRL outer segments. (B) β-arrestin 1 (β-arr1) and (C) β-arrestin 2 (β-arr2) staining throughout the retina. (D) High magnification of β-arrestin 1 (green) & β-arrestin 2 (red) staining in the GCL cell bodies. (E) Melanopsin expressing cells visualized with anti-β-galactosidase (red) and anti-melanopsin (green) antibodies to prevent cross-reactivity with β-arrestin antibodies during double labeling. (F) Melanopsin co-localizes with β-arrestin 1 and β-arrestin 2 in ipRGC cell bodies. (G) Specificity of melanopsin (N-terminal), β-arrestin 1, and β-arrestin 2 antibodies confirmed by western blot of heterologously expressed protein isolated from HEK293 cells. Melanopsin typically runs as a monomer and dimer with an expected molecular weight of 50 kDa and 100 kDa respectively. β-arrestin 1 and β-arrestin 2 have an expected molecular weight of 48 kDa. 100% of melanopsin expressing cells were positive for β-arrestin 1 (n = 7 cells) and β-arrestin 2 (n = 10 cells) in 14 retinal sections from 4 mice.</p

Over-expression of β-arrestin 1 and β-arrestin 2 increases the rate of melanopsin deactivation.

<p>(A) Fluorescent calcium imaging responses in HEK293 cells transiently transfected with wildtype melanopsin: wildtype HEK293 (solid squares) and stable cell lines over-expressing either β-arrestin 1 (open circles), or β-arrestin 2 (solid triangles). Increasing the cellular concentration of β-arrestin increases the rate of melanopsin deactivation. The melanopsin mutant lacking C-tail phosphorylation sites (phosphonull) (cross) was also assayed in wildtype HEK293 and deactivates at a slower rate due to the absence of C-tail phosphorylation. (B) The deactivation portion of each kinetic curve was fit to an exponential function and the rate constant for each condition was calculated and is indicated in bold. (C) Comparison of deactivation rate constants for wildtype melanopsin expressed in wildtype HEK293 cells and stable cell lines over-expressing either β-arrestin 1, or β-arrestin 2. This analysis reveals that over-expression of β-arrestin 1 and β-arrestin 2 increases the rate of melanopsin deactivation by 1.5 times. Rate measurements reported as average of 4 independent calcium assays with standard error of the mean reported as error bars. */** denotes statistically significant difference {p<0.05}. (D) Fluorescent calcium imaging responses of the melanopsin mutant lacking C-tail phosphorylation sites (phosphonull) transiently transfected in wildtype HEK293 cells (star) and stable cell lines over-expressing either β-arrestin 1 (solid circle), or β-arrestin 2 (open triangle). (E) Over-expression of β-arrestin does not change the deactivation kinetics of phosphonull melanopsin. An exponential fit of the deactivation portion of these curves reveals little to no difference in the deactivation rate constants indicated in bold. Untransfected cells do not yield a calcium response (solid diamond). All calcium imaging measurements were carried out in triplicate with standard deviation plotted as error bars. Each kinetic curve is representative of 4 independent experiments. (F) Western blot analysis confirming over-expressing β-arrestin 1 and β-arrestin 2 stable cell lines (10–15× higher than endogenous β-arrestin levels).</p

Dopamine agonist induces phosphorylation <i>in vivo</i>.

<p>PLA was performed on mouse retinal section following treatment with the D1 agonist A68930. Retinal sections (16 mm) were taken from dark-adapted wild type C57/B6 mice (panels labeled Dark, or Dark + D1 agonist) or dark-adapted melanopsin knockout mice (opn4 ^LacZ/LacZ) (panel labeled Melanopsin knock out + D1 agonisit). Before fixation retina were treated with the dopamine D1 agonist A68930 (labled +D1 agonist) or left untreated (Dark). Outer nuclear layer (ONL): Inner nuclear layer (INL) and Ganglion cell layer (GCL).</p

Quantification of the number of fluorescent puncta per cell induced by 8-Br cAMP treatment.

<p>HEK cells were transfected with wild type or mutant melanopsin. After 48-hours, cells were treated with 200 µM 8-Br cAMP for 30-minutes, fixed in 4% PFA for 30 minutes, and assayed with the PLA. Cells were imaged by confocal microscopy and the number of fluorescent spots per cell were counted. Expression of melanopsin was confirmed by functional calcium assay. Error bars represent standard deviation of 50 cells counted for each condition pooled from two separate transfections.</p

3-dimenstional model of mouse melanopsin highlighting the predicted PKA phosphorylation sites found in intracellular loops.

<p>The sites in the C-tail are not depicted. Model constructed by LOMETS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045387#pone.0045387-Wu1" target="_blank">[30]</a> modeling server, and sites identified by Group-based Prediction System (GPS 2.0) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045387#pone.0045387-Xue2" target="_blank">[20]</a>.</p

Modified mammalian tree highlighting presence of putative homologous PKA phosphorylation sites.

<p>Tree based on (Tree of Life Web Project. 1997. Eutheria. Placental Mammals. Version 01 January 1997 <a href="http://tolweb.org/Eutheria/15997/1997.01.01" target="_blank">http://tolweb.org/Eutheria/15997/1997.01.01</a><i>in</i> The Tree of Life Web Project,<a href="http://tolweb.org/" target="_blank">http://tolweb.org/</a><i>).</i> Conserved PKA phosphorylation sites are indicated on the right.</p

Effect of 8-Br cAMP on light induced calcium mobilization in melanopsin-transfected cells.

<p>A) Time course of the calcium response of HEK-293 cells in the presence and absence of 8-Br cAMP. HEK-293 cells were transiently transfected with DNA for wild-type melanopsin. Some cells were treated with 200 µM 8-Br cAMP. Melanopsin signaling was monitored by measuring intracellular calcium levels as described in Methods. B) HEK-293 cells transfected with melanopsin were treated with varying concentrations of 8-Br cAMP to show a concentration dependent decrease in melanopsin signaling. The peak response of the time course is plotted. C) Pre-treatment of transfected cells with the specific PKA inhibitor KT5720 removed the effect of 8-Br cAMP treatment also in a concentration dependent manner. Error bars represent standard deviation.</p