Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor

Abstract

Opsins form a family of light-activated, retinal-dependent, G protein-coupled receptors (GPCRs) that serve a multitude of visual and nonvisual functions. Opsin 3 (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 (HEMs)-the skin cells that produce melanin. The melanin pigment is a critical defense against ultraviolet radiation (UVR), and its production is mediated by the Gαs-coupled melanocortin 1 receptor (MC1R). The physiological function and light sensitivity of OPN3 in melanocytes are yet to be determined. Here, we show that in HEMs, OPN3 acts as a negative regulator of melanin production by modulating the signaling of MC1R. OPN3 negatively regulates the cyclic adenosine monophosphate (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 can form a physical complex. Remarkably, OPN3 can bind retinal, but does not mediate light-induced signaling in melanocytes. Our results identify a function for OPN3 in the regulation of the melanogenic pathway in epidermal melanocytes; we have revealed a light-independent function for the poorly characterized OPN3 and a pathway that greatly expands our understanding of melanocyte and skin physiology.

Keywords: encephalopsin; melanocortin 1 receptor; melanocytes; opsin3; pigmentation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Human OPN3 does not mediate light-induced Ca2+ responses in HEMs. (A) Two-dimensional model of human OPN3 (hOPN3). hOPN3, similar to OPN2, has seven predicted transmembrane domains followed by a short intracellular helix (purple cylinders) and a unique C terminus (amino acids 340–402, green) containing the epitope for the anti-OPN3 antibody (Ab) (blue outline) (58). Each circle represents one amino acid. The conserved lysine K299 (red) is involved in retinal binding, which forms a Schiff base with counter-ion D117 (black). (B) OPN3 mRNA levels in HEMs expressing CTRL or OPN3-targeting miRNA (OPN3-1 or OPN3-2). A qPCR analysis of OPN3 relative (Rel.) to actin mRNA levels was performed. OPN3-1 and OPN3-2 miRNA-expressing HEMs have decreased OPN3 mRNA levels by ∼70% and ∼60%, respectively, compared with CTRL miRNA-transduced HEMs (n = 3 independent experiments, mean ± SEM; *P < 0.05). (C) Specificity of the anti-OPN3 antibody. OPN3 tagged with HA at the C terminus (OPN3-cHA) or N terminus (OPN3-nHA), N-terminal HA-tagged OPN3 mutant missing the last 10 amino acids (OPN3-nHA-ΔC), or empty vector (Mock) were expressed in HEK293 cells. Immunoblots using anti-HA or anti-OPN3 antibodies show the same size band (∼42 kDa) corresponding to OPN3 (calculated molecular mass of 45 kDa) for both full-length constructs but not for the ΔC mutant, which resulted in an ∼40-kDa band using anti-HA antibody and no band using anti-OPN3 antibody (representative of n = 4 independent experiments). WB, Western blot. (D) OPN3 protein levels in HEMs expressing CTRL or OPN3-targeting miRNA (OPN3-1 or OPN3-2). (Inset) Densitometric analysis of HEMs expressing CTRL, OPN3-1, or OPN3-2 miRNA and immunoblotted with anti-OPN3 or anti-integrin α5 antibody. Bars represent OPN3 protein level normalized to integrin α5 (n = 3 independent experiments, mean ± SEM; *P < 0.05). (E and F) Light-induced Ca2+ signaling in HEMs is not dependent on OPN3 expression. (E) Fluorescent Ca2+ imaging of HEMs expressing CTRL, OPN3-1, or OPN3-2 miRNA and stimulated with 200 mJ/cm2 UVR (λmax = 360 nm), blue (λmax = 450 nm), or green (λmax = 550 nm) light, normalized to the maximal Fluo4-AM Ca2+ response obtained with ionomycin (Iono). Each trace represents the average of 10–20 cells from one coverslip. (F) Average amplitude of Ca2+ responses of HEMs under the conditions shown in E (n = 5 independent experiments for each bar, mean ± SEM). max, maximum.

Fig. 2.

OPN3 requires the K299 residue for retinal binding. (A) Schematic representation of OPN3 variants. We purified 1D4-tagged OPN3 with a partially truncated C terminus variant (OPN3ΔC-c1D4) or a K299G mutation of this variant known to inhibit retinal binding [OPN3(K299G)ΔC-c1D4]. (B) OPN3ΔC-c1D4 and OPN3(K299G)ΔC-c1D4 maintain the cellular localization of full-length OPN3. Confocal images of HeLa cells coexpressing OPN3-cMCh and OPN3ΔC-c1D4 or OPN3(K299G)ΔC-c1D4 and immunostained with anti-1D4 antibody show that OPN3ΔC variants have similar cellular localization as OPN3-cMCh. Quantitative analysis of OPN3-cMCh colocalization with OPN3ΔC-c1D4 or OPN3(K299)ΔC-c1D4, measured as percent overlap between the two fluorescent signals, shows significant colocalization (bar graph) (n = 30 cells from three independent experiments). (Calibration bar: 10 μm.) (C) UV-visible absorption spectra of OPN3ΔC and OPN3(K299G)ΔC. Absorption spectra of purified OPN3ΔC-c1D4 and OPN3(K299G)ΔC-c1D4 incubated with 11-cis retinal were measured in the dark (black) and after hydroxylamine (NH2OH) + SDS treatment (red). Absorption spectra measured in the dark have similar protein peaks at λmax = 280 nm for the two OPN3 variants. NH2OH + SDS treatment of OPN3ΔC-c1D4, but not OPN3(K299G)ΔC-c1D4, led to a peak at λmax = 360 nm corresponding to retinal oxime. (Insets) Retinal oxime peak of OPN3ΔC-c1D4 was ∼10-fold smaller than the protein peak. Traces are representative of n = 3 independent experiments.

Fig. 3.

OPN3 expression inversely correlates with cellular melanin concentration. (A) Effect of OPN3 levels on melanin. HEMs expressing OPN3-1 or OPN3-2 miRNA have significantly higher melanin levels compared with CTRL miRNA-expressing cells (Left), while MNT-1 cells expressing OPN3-MCh have reduced melanin compared with CTRL MCh-expressing cells (Right). (Insets) Representative pellets from each condition reflecting melanin levels (n = 3 independent experiments, mean ± SEM). Rel., relative. (B) Endogenous OPN3 and MC1R mRNA levels in HEM and MNT-1. mRNA levels of OPN3 and MC1R in HEMs and MNT-1 cells were measured by qPCR relative to actin. MNT-1 cells have similar MC1R levels as HEMs, but significantly lower OPN3 expression (n = 3 independent experiments, mean ± SEM; *P < 0.05, **P < 0.01).

Fig. 4.

OPN3 modulates MC1R signaling. (A) OPN3 inhibits the MC1R-evoked cAMP response via a PTX-sensitive mechanism in MNT-1 cells. (A, i) OPN3 inhibits the α-MSH–induced cAMP responses of MC1R. MNT-1 cells expressing the FRET-based cAMP indicator Epac H187 and OPN3-cMCh or MCh alone (CTRL) were stimulated with α-MSH (1 μM). The cAMP response of individual cells was monitored as the ratio of CFP and YFP fluorescence intensities and represented as a function of time. FSK and IBMX were added to elicit a maximal cAMP response, used for normalization. α-MSH elicits a significant cAMP response in cells expressing MCh (CTRL, red trace), but not in cells expressing OPN3-cMCh (dark blue trace) (n = 5–10 cells, mean ± SEM). A.U., arbitrary units. (A, ii) OPN3 specifically attenuates the MC1R-mediated cAMP response. Prostaglandin (PGD; 5 μM)-mediated activation of the endogenous prostaglandin E2 receptor leads to an increase in cellular cAMP (red trace) that is not attenuated in the presence of OPN3-cMCh (dark blue trace) (n = 5–10 cells, mean ± SEM). (A, iii) OPN3-mediated attenuation of MC1R signaling is PTX-sensitive. MNT-1 cells treated with PTX (200 ng/mL, 4 h), which specifically inhibits the Gαi subunit of G proteins, and stimulated with α-MSH exhibited a similar cAMP response both in cells expressing MCh (red trace) and OPN3-cMCh (dark blue trace) (n = 5–10 cells, mean ± SEM). (A, iv) OPN3 inhibits MC1R signaling specifically and in a Gαi-dependent manner. Average normalized amplitudes of α-MSH– or PGD-induced cAMP responses in the presence or absence of PTX show that OPN3 expression significantly reduces the amplitude of cAMP responses to α-MSH, but not to PGD, and this effect is prevented by blocking Gαi activation with PTX (n = 3 independent experiments per condition, mean ± SEM; *P < 0.05). Norm. max, normalized maximum. (B) OPN3 inhibits MC1R-evoked cAMP responses independent of its retinal-binding ability. (B, i) OPN3-mediated inhibition of MC1R-induced cAMP responses does not require retinal binding. MNT-1 cells expressing OPN3(K299G)-cMCh (light blue trace) and stimulated with α-MSH (1 μM) exhibited significantly lower cAMP responses compared with MCh-expressing cells (red trace) (n = 5–10 cells, mean ± SEM). (B, ii) OPN3(K299G)-mediated attenuation of MC1R signaling is PTX-sensitive. MNT-1 cells treated with the Gαi inhibitor PTX (200 ng/mL, 4 h) and stimulated with α-MSH exhibited a similar cAMP response both in cells expressing MCh (red trace) and OPN3(K299G)-cMCh (light blue trace) (n = 5–10 cells, mean ± SEM). (B, iii) OPN3(K299G) inhibits MC1R signaling in a Gαi-dependent manner. Average normalized amplitudes of α-MSH–induced cAMP responses of MNT-1 cells expressing either OPN3(K299G)-cMCh or MCh. OPN3(K299G)-cMCh expression significantly reduces the amplitude of cAMP responses (n = 3 independent experiments per condition, mean ± SEM; *P < 0.05). (C) OPN3 inhibits MC1R-evoked cAMP responses in immortalized Hermes 2b HEMs independent of retinal binding. (C, i) OPN3 inhibits the α-MSH–induced cAMP responses of MC1R in Hermes 2b melanocytes in a retinal-independent manner. Hermes 2b cells expressing the red fluorescent cAMP indicator R-FlincA and OPN3-cYFP, OPN3(K299G)-cYFP, or YFP alone (CTRL) were stimulated with α-MSH (1 μM). α-MSH elicits a significant cAMP response in cells expressing YFP (CTRL, yellow trace), but not in cells expressing OPN3(K299G)-cYFP (light green trace) or OPN3-cYFP (dark green trace) (n = 5–10 cells per condition, mean ± SEM). ΔF/F, change in fluorescence over baseline fluorescence. (C, ii) OPN3 and OPN3(K299G) inhibit MC1R signaling in Hermes 2b cells. Average normalized amplitudes of α-MSH–induced cAMP responses of Hermes 2b cells expressing OPN3-cYFP or OPN3(K299G)-cYFP are significantly lower than for YFP alone (CTRL) (n = 2 independent experiments per condition, mean ± SEM; *P < 0.01).

Fig. 5.

OPN3 modulates MITF and TYR expression. (A) Reduced expression of OPN3 leads to increased MITF and TYR protein levels in HEMs. Representative Western blots of HEMs expressing CTRL or OPN3-targeted miRNA show increased MITF and TYR expression in cells with reduced levels of OPN3 compared with CTRL miRNA. MITF or TYR protein levels measured relative (Rel.) to β-actin were approximately twofold higher for MITF and approximately fivefold higher for TYR in HEMs with reduced OPN3 expression, compared with CTRL miRNA-expressing cells (n = 3 independent experiments for each condition, mean ± SEM). (B) Increased expression of OPN3 leads to reduced MITF and TYR levels in MNT-1 cells. Representative Western blots of MNT-1 cells stably expressing MCh (CTRL) or OPN3-cMCh show decreased MITF and TYR expression in OPN3-cMCh–expressing cells, compared with CTRL. MITF and TYR levels measured relative to β-actin were reduced by ∼30% for MITF and by ∼20% for TYR in cells expressing OPN3-cMCh, as compared CTRL (n = 3 independent experiments for each condition, mean ± SEM).

Fig. 6.

OPN3 and MC1R are localized to the same cellular compartments. (A) OPN3-cYFP is colocalized with MC1R, but not with EP2. Representative fluorescence confocal images of HeLa cells coexpressing OPN3-cYFP and either MC1R-nHA or EP2-nHA and immunostained with anti-HA antibody are shown. OPN3-cYFP shows significant colocalization with MC1R-nHA, but not with EP2-nHA. As the bar graph illustrates, the percent overlap between the fluorescent signals of OPN3-cYFP and MC1R-nHA is ∼50%, compared with less than 10% for EP2-nHA (n = 20 cells from three independent experiments, mean ± SEM). (B) Anti-OPN3 antibody specifically recognizes OPN3-cFLAG and OPN3-cYFP. Representative fluorescence confocal images of HeLa cells expressing either OPN3-cFLAG or OPN3-cYFP and immunostained with anti-FLAG and anti-OPN3 antibodies are shown. As the bar graph illustrates, the percent overlap between anti-OPN3 fluorescence and anti-FLAG or YFP fluorescent signal is ∼50%, suggesting significant colocalization of anti-OPN3 with both anti-FLAG and YFP signals (n = 20 cells from three independent experiments, mean ± SEM). (C) Anti-MC1R antibody specifically recognizes MC1R-nHA. Representative fluorescence confocal images of HeLa cells expressing MC1R-nHA and coimmunostained with anti-HA and anti-MC1R antibodies are shown. As the bar graph illustrates, the percent overlap between anti-MC1R and anti-HA fluorescent signals is >50%, indicating significant colocalization (n = 25 cells from three independent experiments, mean ± SEM). (D) Endogenously expressed OPN3 and MC1R colocalize in HEMs. Representative fluorescence confocal images of HEMs coimmunostained with anti-OPN3 and anti-MC1R antibodies are shown. As the bar graph illustrates, the percent overlap between anti-OPN3 and anti-MC1R fluorescent signals is ∼50%, indicating significant colocalization (n = 20 cells from three independent experiments, mean ± SEM). (Scale bar: 10 μm.)

Fig. 7.

OPN3 and MC1R can form a molecular complex. (A) OPN3-cFLAG and MC1R-nHA coimmunoprecipitate. HeLa cells expressing OPN3-cFLAG, MC1R-nHA, or both were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody. A band corresponding to MC1R in the cell lysates (Left) was detected in the anti-FLAG immunoprecipitation (IP) only when both OPN3 and MC1R were expressed (Center). The same band was detected by IP with anti-HA antibody (Right). Findings are representative of n = 3 independent experiments. WB, Western blot. (B) Endogenously expressed OPN3 and MC1R coimmunoprecipitate. WB analysis of HeLa cells expressing MC1R-nHA and immunoblotted with anti-MC1R antibody reveal the same size and pattern of bands as detected with the anti-HA antibody in A (Left). HEM lysates coimmunoprecipitated with anti-OPN3 antibody and immunoblotted with anti-MC1R antibody reveal the same band corresponding to MC1R, suggesting that endogenously expressed OPN3 and MC1R can form a complex in HEMs. Findings are representative of n = 3 independent experiments for each condition.

Fig. 8.

OPN3 and MC1R interact functionally and physically to regulate melanin levels in melanocytes. In melanocytes, α-MSH binds to and activates the Gαs-coupled MC1R, leading to stimulation of AC and a transient increase in cAMP levels, which, through a series of molecular steps, leads to activation and up-regulation of the MITF. MITF controls the expression of TYR, the main melanogenic enzyme; higher MITF levels will result in more TYR that will generate a higher amount of melanin. Our data suggest that OPN3, via Gαi activation, reduces the amount of cAMP generated by MC1R activation, causing decreased transcription of MITF and, in consequence, of TYR. OPN3 and MC1R can form a complex, and OPN3 may additionally alter the function of MC1R by enhancing its internalization and lowering the number of receptors available to bind α-MSH at the plasma membrane.