Abstract
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 β-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.
Highlights
Visual pigments are light-sensitive molecules comprised of specialized G protein-coupled receptors (GPCRs), or opsins, which are covalently linked to a retinaldehyde molecule, typically 11-cis-retinal in the mammalian retina
Of the proximal twelve sites, our model suggests that the nine residues S381, S384, T385, S388, T389, S391, S392, S394, S395 are positioned optimally for arrestin interaction (Fig 8)
Given our results reported here, we propose that melanopsin’s C-terminus undergoes phosphorylation modification on a larger number of serine and threonine residues, centered primarily on a cluster of six sites, P-II (S388, T389, S391, S392, S394, S395), and additional phosphorylations of residues immediately surrounding it (P-I or P-III)
Summary
Visual pigments are light-sensitive molecules comprised of specialized G protein-coupled receptors (GPCRs), or opsins, which are covalently linked to a retinaldehyde molecule, typically 11-cis-retinal in the mammalian retina. We hypothesize that the proper function of mouse melanopsin’s C-terminus during deactivation requires a larger bulk negative charge due to more serine and threonine phosphorylations than in opsins expressed in rod and cone photoreceptors This requirement for extra phosphorylation sites in the C-terminus may contribute to melanopsin’s sustained and slow light response. We found that a bulk negative charge on melanopsin’s C-terminus is centered at P-II (The second cluster of putative C-terminal phosphorylation sites, see Fig 1) on the proximal region, but neighboring serine and threonine residues are necessary and capable of contributing to melanopsin deactivation as well. This suggests a unique level of flexibility in the capability of melanopsin’s. C-terminus to regulate signaling deactivation through phosphorylation, and this property paves the way to understanding the unique signaling kinetics of the ipRGC
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