Absorption Characteristics of Vertebrate Non-Visual Opsin, Opn3

Abstract

Most animals possess multiple opsins which sense light for visual and non-visual functions. Here, we show spectral characteristics of non-visual opsins, vertebrate Opn3s, which are widely distributed among vertebrates. We successfully expressed zebrafish Opn3 in mammalian cultured cells and measured its absorption spectrum spectroscopically. When incubated with 11-cis retinal, zebrafish Opn3 formed a blue-sensitive photopigment with an absorption maximum around 465 nm. The Opn3 converts to an all-trans retinal-bearing photoproduct with an absorption spectrum similar to the dark state following brief blue-light irradiation. The photoproduct experienced a remarkable blue-shift, with changes in position of the isosbestic point, during further irradiation. We then used a cAMP-dependent luciferase reporter assay to investigate light-dependent cAMP responses in cultured cells expressing zebrafish, pufferfish, anole and chicken Opn3. The wild type opsins did not produce responses, but cells expressing chimera mutants (WT Opn3s in which the third intracellular loops were replaced with the third intracellular loop of a Gs-coupled jellyfish opsin) displayed light-dependent changes in cAMP. The results suggest that Opn3 is capable of activating G protein(s) in a light-dependent manner. Finally, we used this assay to measure the relative wavelength-dependent response of cells expressing Opn3 chimeras to multiple quantally-matched stimuli. The inferred spectral sensitivity curve of zebrafish Opn3 accurately matched the measured absorption spectrum. We were unable to estimate the spectral sensitivity curve of mouse or anole Opn3, but, like zebrafish Opn3, the chicken and pufferfish Opn3-JiL3 chimeras also formed blue-sensitive pigments. These findings suggest that vertebrate Opn3s may form blue-sensitive G protein-coupled pigments. Further, we suggest that the method described here, combining a cAMP-dependent luciferase reporter assay with chimeric opsins possessing the third intracellular loop of jellyfish opsin, is a versatile approach for estimating absorption spectra of opsins with unknown signaling cascades or for which absorption spectra are difficult to obtain.

The human non-visual opsin OPN3 regulates pigmentation of epidermal melanocytes through interaction with MC1R

“…1B These findings raised an interesting question: does human OPN3 form a photopigment? Spectroscopic analyses of recombinant OPN3 homologues indicated that they can bind 11-cis retinal and have an absorption maximum at ~470 nm [24,25]. To determine if human OPN3 and retinal form a photopigment, we expressed C-terminal truncated, 1D4-tagged human OPN3 (OPN3ΔC-c1D4) [28] in HEK293 GnTIcells.…”

“…1D and 1E). Moreover, when we tested whether purified human OPN3 can respond to visible light, similar to its homologues [24,25], we found no evidence of visible light absorption in the OPN3 UV-VIS spectrum (Fig. 2, Supp.…”

“…OPN3 has a unique, long carboxy (C)-terminus with no sequence homology to any known GPCR. Recent studies have determined the photoreceptive properties for several OPN3 homologs [24][25][26]: zebrafish, pufferfish, and chicken OPN3 absorb blue light (λmax=465 nm) [25]; mosquito OPN3 forms a bistable photopigment with 13-cis, 11-cis and 9-cis retinal, absorbs blue-green light (λmax=490 nm), and activates G-proteins Gi/o in a light-dependent manner [24,26]. However, the light sensitivity, G-protein coupling, and function of human OPN3 remain unknown.…”

Two Opsin 3-Related Proteins in the Chicken Retina and Brain: A TMT-Type Opsin 3 Is a Blue-Light Sensor in Retinal Horizontal Cells, Hypothalamus, and Cerebellum

“…It could be that chicken Opn3 protein mediates other roles, such as regulating apoptosis or immune modulation [45,46]. A recent study has shown that a chimeric chicken Opn3, in which the third intracellular loop was replaced with that of a jellyfish opsin, forms a blue-sensitive pigment [47]. Given that zebrafish Opn3 has a an absorption maximum at around 465 nm when it is expressed in mammalian cultured cells [47], the intact chicken Opn3 may retain the similar property.…”

A new path in defining light parameters for hair growth: Discovery and modulation of photoreceptors in human hair follicle

“…OPN3 expression was found in retina, as well as in the cells outside of visual system, which suggest that OPN3 might function as a photoreceptor in various tissues . Interestingly, a homolog of the vertebrate OPN3 maintained photosensitive properties in vitro after transfection in mammalian cells, suggesting that OPN3 homologs might have same ability to bind the non‐conventional isomer 13‐cis retinal to form an active photopigment .…”