Photopigments make up a specialized class of G-protein coupled receptors (GPCRs) capable of detecting light. In mammals, photopigments expressed in rods and cones relay photic information to numerous processing centers in the brain, including the visual cortex. On the receptor level, photopigments propagate light information through intracellular G-proteins and downstream effector channels. The resulting photoresponse is regulated in a two step manner that includes receptor phosphorylation and arrestin association. These processes sequentially attenuate and terminate cellular signaling contributing to an overall adaptational response. In many tissues, binding of β-arrestin 1 and 2 (Barr1/Barr2) completely quench receptor signaling and direct receptor endocytosis. This process does not occur in rods or cones however, which express specialized visual arrestins that terminate the photoresponse but do not initiate endocytotic pathways.
The unique photopigment, melanopsin, is restricted to a small subset of retinal ganglion cells (RGCs) within the mammalian retina. These intrinsically photosensitive cells (ipRGCs) regulate numerous non-visual functions including sleep, circadian photoentrainment and pupil constriction. IpRGCs exhibit attenuated response characteristics following intense and prolonged light exposures indicative of an adaptational response. This notion is supported by the phosphorylation dependent reduction of melanopsin signaling in vitro and ubiquitous expression of β-arrestin in the retina. These observations along with the conspicuous absence of visual arrestin in ipRGCs suggest melanopsin is deactivated in a β-arrestin dependent manner. The overall goal of my research is to identify the role of Barr1 and Barr2 in the melanopsin signaling pathway and elucidate the molecular mechanisms that contribute to ipRGC adaptation. To that end, I have begun to identify the arrestins present in ipRGCs by immunohistochemistry. Double labeling experiments utilizing melanopsin and β-arrestin antibodies confirm co-expression of these proteins in mouse ipRGCs. Using an in vitro calcium imaging assay I can monitor the affect of Barr1 and Barr2 on melanopsin signaling. Preliminary studies demonstrate increasing concentrations of Barr1 significantly inhibit the melanopsin calcium response. By comparing the inhibitory effects of Barr1 and Barr2 we can begin to understand how these proteins influence the melanopsin signaling. I am also currently developing a fluorescent-based in vitro binding assay which will be used to determine the relative binding affinities of Barr1 and Barr2 with melanopsin.