Protein Phosphatase 2A and Clathrin-Mediated Endocytosis Facilitate Robust Melanopsin Light Responses and Resensitization.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the visual pigment melanopsin regulate non-image-forming visual tasks, such as circadian photoentrainment and pupil constriction, as well as contrast detection for image formation. Sustained ipRGC function throughout the day is, therefore, of great importance. Melanopsin is a bistable rhabdomeric-type (R-type) visual pigment, which is thought to use light to regenerate its chromophore from all-trans-retinal back to 11-cis-retinal and does not depend on constant chromophore supply to the extent required by visual pigment in rod and cone photoreceptors. Like the majority of photopigments and G-protein-coupled receptors (GPCRs), melanopsin deactivation requires C-terminal phosphorylation and subsequent ß-arrestin binding. We hypothesize that melanopsin utilizes canonical GPCR resensitization mechanisms, including dephosphorylation and endocytosis, during the light, and together, they provide a mechanism for prolonged light responses. Methods: Here, we examined expression of protein phosphatases from a variety of subfamilies by RT-PCR and immunohistochemical analyses of the mouse retina. The expression of protein phosphatase 2A (PP2A) in ipRGCs was assessed. We also examine the role of phosphatase and endocytic activity in sustaining melanopsin signaling using transiently-transfected HEK293 cells. Results: Our analyses suggest that melanopsin-mediated light responses can be rapidly and extensively enhanced by PP2A activity. Light-activated melanopsin undergoes endocytosis in a clathrin-dependent manner. This endocytic activity enhances light responses upon repeated stimulation, implicating a role for endocytic activity in resensitization. Conclusions: Thus, we propose that melanopsin phototransduction is maintained by utilizing canonical GPCR resensitization mechanisms rather than reliance on chromophore replenishment from supporting cells.

The C-Terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction.

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 ß-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.