Structural and Molecular Analysis of Melanopsin Phototransduction Activation, Deactivation, and Re-sensitization

Abstract

The visual pigment melanopsin is expressed in intrinsically photosensitive retinal ganglion cells in the mammalian retina, which regulate non-image forming visual tasks such as circadian photoentrainment and pupil constriction, and image forming visual tasks such as contrast detection. Although these cells comprise a small (1-2%) fraction of the total retinal ganglion cell population, they are remarkably diverse biophysically, morphologically, and functionally. The majority of past molecular analyses of melanopsin function have been devoted to describing its signal transduction activation and deactivation, specifically through computational and experimental tests of the biochemistry of melanopsin'sattachment to its chromophore, and analysis of C-terminal phosphorylation and subsequent binding to ?-arrestin, which contribute to melanopsin activation and deactivation, respectively. In this dissertations, I aim to i. describe melanopsin'sstructural features that mediate signal transduction activation, ii. dissect melanopsin C-terminal phosphorylation in greater detail using biochemical techniques, and iii. provide the first description of melanopsin re-sensitization mechanisms. I explored these aims using heterologous expression of melanopsin in cultured cells to biochemically, functionally, and structurally analyze wildtype or mutated versions of mouse melanopsin. First, I found that dark-adapted melanopsin possesses a cytoplasmic conformation comprised of an interacting 3rd cytoplasmic loop and C-terminus, similar to the conformation found in invertebrate visual pigments. Additionally, the C-terminal conformation and 3rd cytoplasmic loop charge are critical for proper attachment to the G-protein when melanopsin is activated by light. Thus, this cytoplasmic conformation is a critical structural determinant of melanopsin signaling activation. Second, I found that light-dependent melanopsin C-terminal phosphorylation is much less site-specific than previously thought. Melanopsin'sC-terminus can be phosphorylated at several serine and threonine residues around, and including, a cluster of six sites (385-396) to achieve varying degrees of signaling deactivation. Mass spectrometric analysis reveals phosphorylation at various sites on the C-terminus; interestingly at proximal C-terminal residues after short duration light exposure, and distally after exposure to prolonged light. Third, I found that robust melanopsin light responses and re-sensitization are supported through dephosphorylation activity by protein phosphatase 2A, and I also found evidence of melanopsin endocytic activity in the light, which also supports light responses during repeated stimulation. Taken all together, I present these findings as a comprehensive analysis of all primary aspects of melanopsin signal transduction?activation, deactivation, re-sensitization.