The C-terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction

Abstract

27Melanopsin, an atypical vertebrate visual pigment, mediates non-image forming light responses 28 including circadian photoentrainment and pupillary light reflexes, and contrast detection for image 29formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), are 30 characterized by sluggish activation and deactivation of their light responses. The molecular determinants 31 of mouse melanopsin's deactivation have been characterized (i.e. C-terminal phosphorylation and β-32 arrestin binding), but a detailed analysis of melanopsin's activation is lacking. We propose that an 33 extended 3 rd cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the 34 inactive conformation which is stabilized by ionic interaction of these two regions. This model is 35supported by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy of 36 melanopsin, the results of which suggests a high degree of steric freedom at the 3 rd cytoplasmic loop, 37which is increased upon C-terminus truncation, supporting the idea that these two regions are close in 3-38 dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, 39 calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and 40 proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin's activation rate. 41Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into 42 alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo 43 melanopsin-which has substitutions of various potential phosphorylation sites and a substitution of the 44 conserved tyrosine-indicates that substitution of these potential phosphorylation sites and the tyrosine 45 residue result in dramatically slower activation kinetics, a finding that also supports the role of 46 phosphorylation in signaling activation. We therefore propose that melanopsin's C-terminus is proximal 47 to intracellular loop 3 and C-terminal phosphorylation permits the ionic interaction between these two 48 regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling. 49 50 STATEMENT OF SIGNIFICANCE 51 Melanopsin Structure and Activation 3 Melanopsin is an important visual pigment in the mammalian retina that mediates non-image 52forming responses such as circadian photoentrainment and pupil constriction, and supports contrast 53 detection for image formation. In this study, we detail two critical structural features of mouse 54 melanopsin-its 3 rd cytoplasmic loop and C-terminus-that are important in the activation of 55 melanopsin's light responses. Furthermore, we propose that these two regions directly participate in 56 coupling mouse melanopsin to its G-protein. These findings contribute to further understanding of GPCR-57 G-protein coupling, and given recent findings suggesting flexibility of melanopsin signal trans...

Functional Optimization of Light-Activatable Opto-GPCRs: Illuminating the Importance of the Proximal C-terminus in G-protein Specificity

“…The fully truncated Mela(NPxxY)-mGluR6 variant was much less effective in G-protein activation, assumingly due to the lack of the amphipathic H8 shown to stabilize conformational states in rhodopsin, and potentially other opsins (Krishna et al, 2002). Valdez-Lopez and colleagues (Valdez-Lopez et al, 2020) suggested ionic interactions between the proximal melanopsin C-terminus (C367 - Y382) and IL3, specifically within the helical intracellular extensions of TM5 (R259, R262, R266, R277) and in the IL3 loop region (R280, Q281, W282, Q283, R284, L285), forming a stable conformation critical for initiating G-protein signaling. However, in our study, additionally replacing IL3 of melanopsin by that of mGluR6 significantly reduced the capacity of the chimera to activate the G-protein, hinting towards conformational disturbances.…”

“…Thus, the second aim of this study was to determine which intracellular domains were crucial to replace to induce the desired downstream signaling. For this, we engineered a systematic assortment of chimeras between the structurally and functionally best known receptors, namely bovine rhodopsin (Gt and Gi selective (Terakita et al, 2002)) and the beta2-adrenoceptor (b2AR) (primarily Gs, secondarily Gi selective (Hasseldine et al, 2003; Rasmussen, DeVree, et al, 2011; Xiao, 2001)), as well as a set of chimeras between the not yet crystallized melanopsin (mainly Gq, partially Gi/o selective (Bailes & Lucas, 2013)) and mGluR6 (mainly Go selective, minimally Gi selective (Tian & Kammermeier, 2006)), two proteins with potentially different G-protein activation mechanisms (Seven et al, 2021; Spoida et al, 2016; Valdez-Lopez et al, 2020). We found that exchanging the proximal C-terminus was simultaneously crucial and sufficient to shift G-protein tropism in the melanopsin-mGluR6 chimera, whereas it was the known TM5-IL3-TM6 domain in the bRhod-b2AR chimera that induced the desired signaling.…”