Melanopsin is an atypical vertebrate visual pigment expressed in a subset of retinal ganglion cells in the mammalian retina. Melanopsin-based phototransduction is involved in non-image forming light responses including circadian photo-entrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep in vertebrates. Phosphorylation is the most common post-translational modification of proteins. The addition of a phosphate group to either a serine, threonine or tyrosine can cause a variety of changes in a protein. These include activation or inhibition of protein function, changes in protein structure and changes in the protein's interactions with other molecules. This work explores some of the ways in which melanopsin is regulated by phosphorylation. I have determined that mouse melanopsin is phosphorylated in a light-dependent manner and that this phosphorylation is involved in the deactivation of the signaling response. This phosphorylation was demonstrated in vitro, in heterologous expression systems and in vivo. I have also shown that the G protein coupled receptor kinase 2 (GRK2) is the best candidate kinase for phosphorylation in a heterologous expression system. I characterized the members of the GRK family expressed in melanopsin-expressing retinal ganglion cells to determine the endogenous kinase candidates. I then identified six sites in the cytoplasmic carboxy tail that are important for this phosphorylation by mutational analysis. The importance of these sites was also shown by characterization of the naturally diverse zebrafish melanopsins. Additionally, I demonstrated that protein kinase A (PKA) mediated phosphorylation of melanopsin occurs in both a heterologous expression system and in vivo. These phosphorylations were localized to the intracellular loops of melanopsin by mutational and functional analysis. Phosphorylation of these sites was shown to be involved in inhibiting melanopsin signaling in HEK293 cells. I hypothesize that phosphorylation of melanopsin by PKA is a form of circadian regulation of melanopsin function
