Figure 1.
Phylogenetic positions of iguana opsins.
The tree was inferred by the neighbor-joining method based on a comparison of the alignment of conserved regions, using other GPCRs as an outgroup. The bootstrap probabilities are indicated at each branch node. The names of evolutionarily and functionally classified groups are shown to the right of each cluster. The scale bar indicates 0.1 substitutions per site. Accession numbers of the sequence data from the DDBJ/EMBL/GenBank database are as follows: human Mel-1a melatonin-R (receptor), U14108; human M1 muscarinic acetylcholine-R (receptor), Y00508; human OPN4, AF147788; human OPN5, BC126198; human rgr, U15790; human peropsin, AF012270; human OPN3, AF140242; zebrafish parietopsin, XM_003201434; clawed frog parietopsin, NM_001045791; lizard parietopsin, DQ100320; iguana parietopsin, AB626970; lamprey parapinopsin, AB116380; clawed frog parapinopsin, AB159672; anole parapinopsin, AB626968; iguana parapinopsin, AB626969; catfish parapinopsin, AF028014; trout parapinopsin, AB159673; marine lamprey P-opsin, U90671; zebrafish VAL, AB035277; toad pinopsin, AF200433; iguana pinopsin, AB626971; chicken pinopsin, U15762; zebrafish red, NM_131175; human red, AH005298; human green, AH005296; zebrafish UV, NM_131319; human blue, AH003620; zebrafish blue, NM_131192; chicken blue, M92037; zebrafish green, NP_571329; chicken green, M88178; zebrafish rhodopsin, NM_131084; human rhodopsin, U49742.
Figure 2.
Opsin expression in various tissues of green iguana.
RT-PCR analyses indicate that the iguana parapinopsin gene is expressed in the parietal eye, and the iguana UV cone pigment gene is detected only in the eye. The iguana parietopsin gene is expressed in the parietal eye. The iguana pinopsin gene is detected in the pineal organ but not in the parietal eye. The GAPDH gene was used as an internal standard.
Figure 3.
The difference absorption spectra of iguana parapinopsin and parietopsin.
(A) The detergent-extract of the HEK 293 cells expressing parapinopsin was irradiated with UV light at pH 6.5. The difference spectrum of before minus after the irradiation shows that an absorption maximum of the non-irradiated parapinopsin is in the UV region. (Inset) The relative difference absorption spectrum of lamprey parapinopsin. (B) The detergent extract of iguana parietopsin-expressing cells was irradiated with green light in the presence of 50 mM hydroxylamine at pH 6.5. The difference spectrum of before minus after irradiation shows that an absorption maximum of non-irradiated parietopsin is at 522 nm.
Figure 4.
The specificity of the antibodies to parapinopsin and parietopsin.
The anti-iguana parapinopsin antibody (anti-PP) labels the cells expressing iguana parapinopsin (A, PP cells) but not those expressing iguana parietopsin (B, PRT cells). The anti-iguana parietopsin antibody (anti-PRT) labels PRT cells (C) but not PP cells (D). (E) Immunoblot profiles showing that anti-PP and anti-PRT antibodies specifically recognize parapinopsin and parietopsin expressed in cultured cells. Monoclonal antibody rho 1D4 stains iguana parapinopsin (arrowhead) and parietopsin (asterisk) tagged with rho 1D4 epitope, in SDS-extracts form PP and PRT cells (lanes 3 and 4) but not from mock transfected cells (lane 2). Anti-PP specifically stained ∼38 k peptide (lane 6, arrowhead), which is the same as the band stained by rho 1D4 (lane 3), in PP cells but not in mock cells (lane 5) or PRT cells (lane 7). Anti-PRT antibodies recognize specifically ∼39 k peptide (lane 10, asterisk), which is identical to the stained band by rho 1D4 (lane 4) in PRT cells, but not in mock cells (lane 8) or PP cells (lane 9). M indicates Molecular weight standard markers (lane 1) (Bio-Rad Laboratories).
Figure 5.
Colocalization of parapinopsin and parietopsin in the photoreceptor cells of the iguana parietal eye.
(A) A Nomarski image of the parietal eye of the iguana. L, lens; OS, outer segments of the photoreceptor cells; Pg, pigment granule-rich region. (B and C) Immunofluorescence labeling of the photoreceptor outer segments in the parietal eye with the anti-iguana parapinopsin antibody (B, magenta) and the anti-iguana parietopsin antibody (C, green). (D) A merged image showing the colocalization of iguana parapinopsin and parietopsin in the photoreceptor cells. E, F and G are high magnifications of the boxed regions in B, C and D, respectively. The arrowheads show the outer segments of photoreceptor cells in the parietal eye. The scale bars indicate 100 µm in A, B, C and D and 50 µm in E, F and G.