Molecular determinants of melanopsin signaling and deactivation

The unique visual pigment melanopsin is expressed in a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) within the mammalian retina. The axons of ipRGCs primarily project to brain nuclei that regulate non-image forming visual functions including circadian photoentrainment, pupillary light reflex, and sleep. Unlike rods and cones which hyperpolarize in response to light, ipRGCs depolarize likely through the activation of a Gq-mediated phototransduction cascade. IpRGCs exhibit very poor temporal resolution with sluggish on/off kinetics and a high threshold of activation. Despite this, ipRGCs respond robustly to prolonged illumination and are capable of firing sustained action potentials beyond the lifetime of the stimulus. These response characteristics raise questions about how melanopsin signaling is regulated within the retina. Typically, visual pigment signaling is deactivated by receptor phosphorylation and arrestin binding which act to attenuate and inhibit G-protein binding, respectively. In this work, I investigate the role of arrestin in melanopsin signaling and identify a region within the C-terminal tail of melanopsin that is required for proper activation and deactivation. Using a combination of in vitro and in vivo approaches, I provide evidence for a light- and phosphorylation-dependent deactivation of melanopsin by Beta-arrestin. This work is the first description of a visual pigment deactivation mechanism mediated by Beta-arrestin and is consistent with canonical deactivation mechanism described for other non-visual G-protein coupled receptors (GPCRs). Additionally, I demonstrate that removal or mutation of a region within the proximal portion of melanopsin's C-tail delays the activation and deactivation of the photoresponse. I also show that this region is conserved within the melanopsin family implying important functional significance. Based on these results, I hypothesize that a 9 th helix in the C-tail of melanopsin serves two purposes; 1) to stabilize ICL3 and form a portion of the G-protein binding domain and 2) to tether the C-tail close to the membrane surface, facilitating receptor phosphorylation and arrestin binding.