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

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

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

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

Proximity-dependent ligation assay.

<p>Melanopsin-transfected HEK cells were fixed with 4% PFA for 30 min. with or without pretreatment with 200 µM 8-Br cAMP for 30 minutes before fixation. Melanopsin phosphorylation was assayed with the PLA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045387#s4" target="_blank">Materials and Methods</a>. The red fluorescence puncta indicates that the antibody bound to melanopsin’s intracellular C-terminal domain is within 40 nm of the phospho-serine antibody when bound to phosphorylation sites in the intracellular loops. Cells visualized by confocal microscopy. Blue staining indicates DAPI staining of cell nuclei. Images represent Z-stacks of images taken through entire cell.</p

Effect of 8-Br cAMP on mutant and wild type melanopsin calcium signaling.

<p>A) Graph of the peak calcium response of melanopsin and melanopsin mutants as measured by fluorescent calcium assay. In black is the average maximum fluorescence for untreated cells, while 200 µM 8-Br cAMP treated response is shown in grey. Error bars represent standard deviation. B) The average percent 8-Br cAMP induced inhibition in signaling is shown.</p

List of primers used to create single and double PKA mutants.

<p>List of primers used to create single and double PKA mutants.</p

List of PKA phosphorylation sites predicted by GPS2.0.

<p>List of the predicted PKA phosphoryation sites in mouse melanopsin. The score is a measure of the similarity of a peptide sequence centered on a phosphorylatable residue to a known phosphorylation site for a given kinase family.</p

Expression of recombinant mouse retinal TRPM3.

<p><b>A, B</b>) Ca²⁺ signals were recorded from CHO-K1 cells transiently transfected with mouse TRPM3 by Fura-2-based ratiometric imaging (n = 8 PS-responsive CHO cells). Signal from control solution (1) and Pregnenolone Sulfate (PS)-activated rises in calcium (2). The PS signal was abolished by pretreatment with mefenamic acid (MA; 3). In (<b>B</b>), black line is average, grey is SEM. C, D) CHO-K1 cells were transiently transfected with mouse mCherry-tagged TRPM3. PS activates an outwardly rectifying current in mCherry—TRPM3 transfected cells identified with mCherry fluorescence. PS-stimulated currents were measured from 11 cells; inward currents at -100 mV were 83 ± 13 pA (SEM) and outward currents were 2175 ± 128 pA at +100 mV. Non-transfected CHO cells show no measurable response to application of PS. The currents were blocked by MA. * in (<b>C</b>) indicate time when the holding potential was ramped from -100 to +100 mV to determine I–V relationship in (<b>D</b>).</p