C-terminal phosphorylation regulates the kinetics of a subset of melanopsin-mediated behaviors in mice

Abstract

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and mediate several non-image-forming visual functions, including circadian photoentrainment and the pupillary light reflex (PLR). ipRGCs act as autonomous photoreceptors via the intrinsic melanopsin-based phototransduction pathway and as a relay for rod/cone input via synaptically driven responses. Under low light intensities, where only synaptically driven rod/cone input activates ipRGCs, the duration of the ipRGC response will be determined by the termination kinetics of the rod/cone circuits. Little is known, however, about the termination kinetics of the intrinsic melanopsin-based phototransduction pathway and its contribution to several melanopsin-mediated behaviors. Here, we show that C-terminal phosphorylation of melanopsin determines the recovery kinetics of the intrinsic melanopsin-based photoresponse in ipRGCs, the duration of the PLR, and the speed of reentrainment. In contrast, circadian phase alignment and direct effects of light on activity (masking) are not influenced by C-terminal phosphorylation of melanopsin. Electrophysiological measurements demonstrate that expression of a virally encoded melanopsin lacking all C-terminal phosphorylation sites (C terminus phosphonull) leads to a prolonged intrinsic light response. In addition, mice expressing the C terminus phosphonull in ipRGCs reentrain faster to a delayed light/dark cycle compared with mice expressing virally encoded WT melanopsin; however, the phase angle of entrainment and masking were indistinguishable. Importantly, a sustained PLR in the phosphonull animals is only observed at brighter light intensities that activate melanopsin phototransduction, but not at dimmer light intensities that activate only the rod/cone pathway. Taken together, our results highlight how the kinetics of the melanopsin photoresponse differentially regulate distinct light-mediated behaviors.

Melanopsin Carboxy-terminus phosphorylation plasticity and bulk negative charge, not strict site specificity, achieves phototransduction deactivation

“…In M1-type ipRGCs, light-activated melanopsin is proposed to couple to Gαq/11 [9,10,11], which activates PLCβ4 that ultimately results in a depolarization of the cell. Previous studies suggest that melanopsin signaling desensitization is initiated by G protein-coupled Receptor Kinase (GRK)-mediated C-terminal phosphorylation [12,13,14,15] which stimulates βarrestin binding [16,17]. However, there is evidence suggesting that GRK2-mediated phosphorylation is not critical in ipRGCs [18], and other studies also suggest that melanopsin phosphorylation can be mediated by alternate kinases such as Protein Kinase C [19], S6K1 [19], and Protein Kinase A [20].…”

Regulation of Melanopsin Signaling: Key Interactions of the Nonvisual Photopigment

“…The issue is of interest considering the necessity of different responses of melanopsin in different processes. Such a diversity was recently shown in mice where the absence of all C‐tail phosphorylation sites affected the recovery kinetics of the intrinsic melanopsin‐based photoresponse in ipRGCs, the length of PLR and the pace of re‐entrainment but not circadian phase alignment or negative masking .…”

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

“…Mouse melanopsin’s C-terminus possesses 38 serine and threonine residues (Figure 1B) that may serve as potential phosphorylation sites. It is well established that melanopsin C-terminal phosphorylation is critical for signaling deactivation and the lifetime of melanopsin-driven behaviors such as pupil constriction and jet-lag photoentrainment (28, 29, 31, 32). Given the enrichment of serine and threonine residues at the proximal region (from residues H351-T385) of mouse melanopsin’s C-terminus, we tested if phosphorylation of these sites contributes to regulation of phototransduction activation, in addition to deactivation.…”

“…Given the enrichment of serine and threonine residues at the proximal region (from residues H351-T385) of mouse melanopsin’s C-terminus, we tested if phosphorylation of these sites contributes to regulation of phototransduction activation, in addition to deactivation. To test this idea, we performed calcium imaging of transiently-transfected HEK293 cells expressing melanopsin C-terminal phosphorylation mutants: phosphonull melanopsin (28, 31), a mutant with all 38 C-terminal serine and threonine residues mutated to alanine residues, and phosphonull + Y382A melanopsin, a mutant with an evolutionarily conserved tyrosine residue (Figure 5A) mutated to an alanine residue in addition to the phosphonull C-terminus mutations. Calcium imaging of these mutants (Figure 5B) suggests that melanopsin C-terminal phosphorylation contributes to signaling activation, due to both mutants displaying slower rates of activation (Figure 5C) compared to wildtype melanopsin.…”

“…GPCR regions important for G-protein binding selectivity include the 2 nd and 3 rd intracellular loops, and cytoplasmic extensions of transmembrane helices 5 and 6, which form critical contacts with helices 4 and 5 on Gα (26). GPCR C-termini can regulate G-protein activation as well as serve as substrates for GPCR Kinase (GRK) to mediate arrestin binding (for melanopsin C-terminal phosphorylation and deactivation, see 27, 28, 29, 30, 31, 32, 33). Structural analysis of rhodopsin—G-protein complexes reveals contacts between helix 8 on rhodopsin’s C-terminus and the C-terminal helix 5 on Gαi (34) or transducin (35), and contacts have been found on rhodopsin’s C-terminus with Gβ when in complex with Gαi (36).…”