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

Abstract

Opsins form a family of light-activated, retinal-dependent G-protein coupled receptors (GPCRs) that serve a multitude of visual and non-visual functions. Opsin3 (OPN3 or encephalopsin), initially identified in the brain, remains one of the few members of the mammalian opsin family with unknown function and ambiguous light-absorption properties. We recently discovered that OPN3 is highly expressed in human epidermal melanocytes-the skin cells that produce melanin. The melanin pigment is a critical defense against ultraviolet radiation and its production is mediated by the Gs-coupled melanocortin-1 receptor (MC1R). The physiological function and light-sensitivity of OPN3 in melanocytes is yet to be determined.Here we show that in human epidermal melanocytes OPN3 acts as a negative regulator of melanin production by interacting with MC1R and modulating its cAMP signaling. OPN3 negatively regulates the cAMP response evoked by MC1R via activation of the Gαi subunit of G-proteins, thus decreasing cellular melanin levels. In addition to their functional relationship, OPN3 and MC1R colocalize at both the plasma membrane and in intracellular structures and form a physical complex. Remarkably, OPN3 can bind retinal, but does not mediate light-induced signaling in melanocytes. Our results identify a novel function for OPN3 in the regulation of the melanogenic pathway in epidermal melanocytes. Our results reveal a light-independent function for the poorly characterized OPN3 and a novel pathway that greatly expands our understanding of melanocyte and skin physiology. Key words:Opsin3 | Encephalopsin | Melanocortin 1 Receptor | Pigmentation | Human Epidermal Melanocyte SignificanceOur data reveals a novel function for the non-visual opsin OPN3 in regulating the pigmentation of human melanocytes by interacting with and modulating the activity of MC1R.

Endogenous Opsin 3 (OPN3) Protein Expression in the Adult Brain Using a Novel OPN3-mCherry Knock-In Mouse Model

“…This was a major concern of ours even before generating the OPN3-mCh mouse model and we took precautions to the best of our ability to ensure that OPN3-mCh maintained endogenous localization. In our previous publication, we identified that OPN3 subcellular localization in melanocytes and other heterologous cell types is at the plasma membrane, as expected of a GPCR, but also in internal vesicles within the cytoplasm; OPN3-mCh significantly colocalized with endogenous OPN3 (using the now discontinued OPN3 antibody) in primary human melanocytes (Ozdeslik et al, 2019). In addition to retaining its subcellular localization, we found that OPN3-mCh retained its regulatory activity in melanocytes, suggesting that OPN3-mCh is functional despite fusion to a fluorescent protein (Ozdeslik et al, 2019).…”

“…In our previous publication, we identified that OPN3 subcellular localization in melanocytes and other heterologous cell types is at the plasma membrane, as expected of a GPCR, but also in internal vesicles within the cytoplasm; OPN3-mCh significantly colocalized with endogenous OPN3 (using the now discontinued OPN3 antibody) in primary human melanocytes (Ozdeslik et al, 2019). In addition to retaining its subcellular localization, we found that OPN3-mCh retained its regulatory activity in melanocytes, suggesting that OPN3-mCh is functional despite fusion to a fluorescent protein (Ozdeslik et al, 2019). Although there is a possibility that OPN3 processing may lead to altered subcellular localization of OPN3-mCh, we believe this is unlikely, as tagging other GPCRs at the direct C terminus with fluorescent proteins and using similar knock-in methods did not alter subcellular localization (as in the MC4R-GFP mouse; Siljee et al, 2018).…”

“…Indeed, it has been shown using DREADDs and endogenous receptors that astrocytes of the hippocampal stratum radiatum (as opposed to the striatum) have differential Gi-coupled GPCR signaling that may result from variation in gene expression (Chai et al, 2017). Because OPN3 in a variety of species, including human, has been shown to couple to the G-protein a-subunit Gi (Koyanagi et al, 2013;Sugihara et al, 2016;Ozdeslik et al, 2019), OPN3 could exert specific Gi-based signaling or regulation specific to the HCF, explaining its strong expression in astrocytes in the HCF versus other regions. OPN3, as a member of the opsin family of light-receptive GPCRs, may also participate in light-evoked processes in the brain.…”

“…The fluorescent tag mCh was chosen over other red fluorophores (such as tdTomato) based on availability of reliable antibodies against mCh, in the event that fluorescent expression on its own was too dim to detect. We used the same OPN3 fusion protein construction (mCh attached to the C terminus of OPN3 via a flexible linker) that was previously demonstrated not to interfere with localization or function of the OPN3 protein in melanocytes (Ozdeslik et al, 2019). After confirming successful knock-in of mCh at the direct C terminus of Opn3, heterozygous mice were backcrossed and bred to create mice homozygous for OPN3-mCh.…”

“…We next sought to confirm the fidelity of OPN3-mCh expression to endogenous OPN3 expression in the brain. In previous publications we used an OPN3 antibody to detect endogenous OPN3 (Ozdeslik et al, 2019), but the discontinuation of this antibody and lack of a suitable replacement led us to pursue an alternative approach to confirm that OPN3-mCh in the knock-in mouse can successfully be detected in areas where endogenous OPN3 is known to be expressed. We crossed the previously generated OPN3 b galactosidase (LacZ)-reporter mouse (OPN3 LacZ/LacZ ; Buhr et al, 2015) with our OPN3-mCh knock-in mouse to create OPN3-mCh/ LacZ mice.…”