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.

Melanopsin Carboxy-terminus phosphorylation plasticity and bulk negative charge, not strict site specificity, achieves phototransduction deactivation.

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin's Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and ß-arrestin binding are centered around these critical phosphorylation sites (S388-S395). The degree of functional versatility described in this study, along with ipRGC biophysical heterogeneity and the possible use of multiple signal transduction cascades, might contribute to the diverse ipRGC light responses for use in non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.

Sequence, Structure, and Expression of Opsins in the Monochromatic Stomatopod Squilla empusa.

Most stomatopod crustaceans have complex retinas in their compound eyes, with up to 16 spectral types of photoreceptors, but members of the superfamily Squilloidea have much simpler retinas, thought to contain a single photoreceptor spectral class. In the Atlantic stomatopod Squilla empusa, microspectrophotometry shows that all photoreceptors absorb light maximally at 517 nm, indicating that a single visual pigment is present in all photoreceptors in the retina. However, six distinct, but partial, long wavelength sensitive (LWS) opsin transcripts, which encode the protein component of the visual pigment, have been previously isolated through RT-PCR. In order to investigate the spectral and functional differences among S. empusa's opsins, we used RT-PCR to complete the 3' end of sequences for five of the six expressed opsins. The extended sequences spanned from the first transmembrane (TM1) helix to the 3' end of the coding region. Using homology-based modeling, we predicted the three-dimensional structure of the amino acid translation of the S. empusa opsins. Based on these analyses, S. empusa LWS opsins share a high sequence identity in TM regions and in amino acids within 15 Å of the chromophore-binding lysine on TM helix 7 (TM7), suggesting that these opsins produce spectrally similar visual pigments in agreement with previous results. However, we propose that these spectrally similar opsins differ functionally, as there are non-conservative amino acid substitutions found in intracellular loop 2 (ICL2) and TM5/ICL3, which are critical regions for G-protein binding, and substitutions in extracellular regions suggest different chromophore attachment affinities. In situ hybridization of two of the opsins (Se5 and Se6) revealed strong co-expression in all photoreceptors in both midband and peripheral regions of the retina as well as in selected ocular and cerebral ganglion neuropils. These data suggest the expression of multiple opsins-likely spectrally identical, but functionally different-in multiple types of neuronal cells in S. empusa. This suggests that the multiple opsins characteristic of other stomatopod species may have similar functional specialization.