The C-terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction
Juan C. Valdez-Lopez, Stephen T. Petr, Matthew P. Donohue, Robin J. Bailey, Meheret Gebreeziabher, Evan G. Cameron, Julia B. Wolf, Veronika A. Szalai, Phyllis R. Robinson
Melanopsin, an atypical vertebrate visual pigment, mediates non-image forming light responses including circadian photoentrainment and pupillary light reflexes, and contrast detection for image formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells are characterized by sluggish activation and deactivation of their light responses. The molecular determinants of mouse melanopsins deactivation have been characterized, but a detailed analysis of melanopsins activation is lacking. We propose that an extended 3rd cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the inactive conformation which is stabilized by ionic interaction of these two regions. This model is supported by site-directed spin labeling and electron paramagnetic resonance spectroscopy of melanopsin, the results of which suggests a high degree of steric freedom at the 3rd cytoplasmic loop, which is increased upon C-terminus truncation, supporting the idea that these two regions are close in 3-dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsins activation rate. Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into alanines also delayed the activation rate. We therefore propose that melanopsins C-terminus is proximal to intracellular loop 3 and C-terminal phosphorylation permits the ionic interaction between these two regions, thus forming a stable structural conformation that is critical for initiating G- protein signaling.