Juan Valdez-Lopez

Poster presentation

Structural and Molecular Analysis of Signaling Activation in Melanopsin, an Opsin G-Protein Coupled Receptor

Melanopsin is a unique visual pigment expressed in a small subset of retinal ganglion cells in the mammalian retina, and is involved in non-image forming tasks such as circadian photoentrainment and pupil constriction. Data from both in vitro electrophysiology and calcium imaging assays, suggests that the activation kinetics of the melanopsin phototransduction cascade are sluggish, and the response has a unique sustained property in response to light, unlike the transient rod and cone responses. While several key players of melanopsin’s signaling cascade have been described (such as melanopsin’s coupling to GRKs & β-arrestins), there are additional signaling molecules that remain to be elucidated. Melanopsin’s structural properties are also not well understood, particularly the important regions that couple to signaling molecules. We hypothesize that melanopsin adopts an intracellular conformation closer to rhabdomeric visual pigments found in invertebrates than those found in mammalian rods and cones. We aim to test this hypothesis through mutagenesis of melanopsin’s gene, expression in HEK293 cells, in vitro calcium imaging to measure signaling kinetics, and biophysical testing of melanopsin’s structure using electron paramagnetic resonance (EPR) spectroscopy. Structural modeling of the active and inactive form of melanopsin suggests an intracellular conformation consisting of an extended intracellular loop 3 (ICL3) that is adjacent to the proximal region of the C-terminus, and these structures are part of a stable G-protein binding pocket. This model is supported by analysis of C-terminus mutants using in vitro calcium imaging. Our in vitro evidence also suggests that C-terminus phosphorylation stabilizes this conformation. Our model is further supported through structural analysis of affinity purified melanopsin, singly-spin labeled on ICL3, and analysed using EPR spectroscopy. We present these findings as contributors to melanopsin’s unique signaling properties within the mammalian retina and as unique and novel mechanisms of G protein-coupled receptor signaling regulation.