Melanopsin: An opsin in melanophores, brain, and eye

Approximately 10⁵ cultured Xenopus laevis dermal melanophores were washed twice with 1× calcium- and magnesium-free Dulbecco’s phosphate buffer and lysed with lysis buffer [1% IGEPAL CA-630 (Sigma)/0.2 M NaBH₃CN/1 mM phenylmethylsulfonyl fluoride/aprotinin (0.1 trypsin inhibitor unit/ml)/5 mM EDTA/0.086 M NaC₂H₃O₂, pH 5.0, 4°C]. A homogenate of one early postmetamorphic adult eye was also extracted as above. Lysates were centrifuged and the supernatants subjected to SDS/PAGE analysis and subsequent electroblotting onto a poly(vinylidene difluoride) membrane. The blot was probed with a 1:2,000 dilution of antisera (CERN 886) raised against bovine rhodopsin and detected by enhanced chemiluminescence.

A X. laevis dermal melanophore oligo(dT) cDNA library was screened with a mixture of ³²P-labeled probes. Probes were synthesized by random priming of TA-cloned (Invitrogen) fragments of X. laevis rhodopsin (7) (759 bp) and violet opsin (8) (279 bp) cDNAs. Phage DNA was isolated from positive clones and sequenced in both directions by using λgt10 universal primers. Internal sequencing primers were designed from newly acquired sequence data until both strands of the insert were completely sequenced. The insert sequence was confirmed by PCR amplification and sequencing of the entire melanopsin ORF from fresh melanophore cDNA. All sequences were assembled by using the seqman program within the DNAstar software suite (Lasergene, Madison, WI). Sequences were compared with the GenBank database by using the blast algorithm (9).

Methods are detailed elsewhere (10). Briefly, adult and tadpole (stages 56 and 57) serial sections were incubated for 16–20 h with sense or antisense ³⁵S-labeled RNA probes representing bases 957–1,553 of the melanopsin ORF, which correspond to the unique cytoplasmic tail. After incubation, unhybridized transcripts were digested with RNase A and sections were washed at high stringency in 0.1× SSC at 65°C. Emulsion-dipped sections were then exposed for 6 weeks. Frog staging (11) and neuroanatomy (12) were according to established criteria.

Photosensitive melanophores contain a protein that is immunoreactive to an antiserum raised against bovine rhodopsin (Fig. 1). The immunoreactive band has a molecular mass approximately 50% greater than the major 35-kDa rhodopsin band from whole Xenopus eye extract. The discrepancy between the predicted molecular mass of rhodopsin (40 kDa) and that observed after SDS/PAGE is a phenomenon commonly found among opsins and other integral membrane proteins (13). A minor protein band from whole Xenopus eye extract had the same molecular weight as that observed from the melanophore extract.

A 2.2-kb cDNA clone was isolated by screening a X. laevis dermal melanophore cDNA library. This clone contains a 1.6-kb ORF encoding a 534-amino acid protein that is more homologous to known opsins than other families of G protein-coupled receptors. We have named this opsin melanopsin because of its isolation from a melanophore cDNA library.

Melanopsin shares structural similarities with all known opsins including an extracellular amino terminus and seven transmembrane domains, as determined by tmpred software (14) (Fig. 2 A and B). Other similarities include a lysine (Lys-294) in the seventh transmembrane domain to serve as a site for Schiff’s base linkage of the chromophore (16) and a pair of cysteines (Cys-100 and Cys-178) in the second and third extracellular loops to stabilize the tertiary structure by disulfide bridge formation (17). There is a notable absence of N-glycosylation sites typically found in the extracellular amino terminus of most opsins.

Despite melanopsin’s vertebrate origin, it is more homologous to invertebrate opsins. It has a deduced amino acid sequence that shares 39% identity with octopus rhodopsin and approximately 30% identity with typical vertebrate opsins (Table 1). This homology to the invertebrate opsins is reflected in several domains thought to have functional significance (Fig. 3). In particular, melanopsin has an aromatic residue (Tyr-103) in the third transmembrane helix that may be a component of a larger complex involved in stabilizing the Schiff’s base (18). The corresponding vertebrate residue is acidic (19, 20). Also, like invertebrate opsins, melanopsin has an insertion (Asn-225 to Gly-233) in the third cytoplasmic loop, albeit the similarity to invertebrate opsins at the amino acid level is limited. Such an insertion is absent in the vertebrate opsins. This third cytoplasmic loop determines the family of G protein that is activated by heptahelical receptors (21, 22). Melanopsin’s conspicuously long cytoplasmic tail contains 14 potential phosphorylation sites, suggesting that its activation state may be regulated by kinases (23).

Phylogenetic analysis indicates that melanopsin and the invertebrate opsins diverged from a common ancestor (Fig. 2C). Bootstrapping demonstrates that this node is well resolved (485 of 500 trees). The alignment and distance matrix was generated with clustal w 1.6 (24), and the subsequent phylogenetic tree was constructed by using the neighbor joining method (25). Placement of the vertebrate opsin groups agrees with published trees (26, 27).

Because of its initial isolation from a melanophore cDNA library, the presence of melanopsin message in dermal melanophores was predicted (Fig. 4 A–C). Unexpectedly, melanopsin message is also localized to several structures within the brain. Transcripts are present in the ventral part of the magnocellular preoptic nucleus (Fig. 4 D–F) and the suprachiasmatic nucleus (SCN; Fig. 4 G–I). In addition, we have identified melanopsin message in at least three ocular sites: iris, retinal pigment epithelium (RPE), and inner retina (Fig. 5). Melanopsin is most highly expressed within the iris; a relatively low level of mRNA expression is observed in the RPE. Perhaps most interesting is the high level of expression within nonphotoreceptor cells of the retina. These retinal cells are located in the outermost lamina of the inner nuclear layer where horizontal cells are typically found.