Differential expression of two distinct functional isoforms of melanopsin (Opn4) in the mammalian retina

Abstract

Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

Figure 1.

A, Alignment of mouse, rat and human Opn4 deduced amino acid sequences. Residues that are identical in two out the three sequences are shaded. The seven probable transmembrane domains are marked by red lines above the sequence and numbered using roman numerals. The characteristic features of an opsin are shown boxed: lysine (K) to form a Schiff's base at position 337; tyrosine (Y), a possible counterion at position 145; aspartate, arginine, and tyrosine (DRY) tripeptide for transducin binding at position 166–168; and cysteines (C) at positions 142 and 220 for disulfide bridge formation (numbers correspond to the mouse sequence). The intron–exon boundaries are delineated by vertical blue lines and are numbered. The epitopes of the N-terminal antibody (PAS8331) and OPN4L are shown boxed. Accession numbers are as follows: Homo sapiens NM_033282, Rattus norvegicus NM_138860, Mus musculus NM_013887. B, Alignment of amino acids encoded by rat Opn4 exons 9 and 10 with those of the newly identified mouse Opn4S showing that the mouse isoform exon 9 is 8 aa shorter than the rat sequence. Boxes show the epitopes of Opn4S and the C-terminal rat antibody (for more details, see Discussion). C, Amplification of Opn4L and Opn4S coding regions from adult retina cDNA. Products are 1566 bp and 1401 bp. M, Marker (1 kb ladder, Invitrogen); lane 1, no template control for Opn4L primers; lane 2, no template control for Opn4S primers; lane 3, Opn4L; lane 4, Opn4S.

Figure 2.

Schematic diagram of the genomic structure of mouse Opn4. The mouse Opn4 gene consists of 10 exons that span ∼9.6 kb of genomic DNA. Exons are shown as boxes and introns as lines; all are to scale except for exons 1–7 and regions of intronic DNA larger than 1 kb that are represented as slashed lines. Intron and exon sizes are marked. The start and stop codons in each gene are also indicated, as are the polyadenylation signals. The gene gives rise to two splice variants, the mOpn4L isoform generated by retention of intron 9 and mOpn4S by splicing to exon 10. The products generated by these two events are shown.

Figure 3.

3′ RACE products. 3′ RACE with the mouse-specific primer mOpn4 8F generated two fragments of 857 bp and 930 bp. The nucleotide sequence of each fragment is shown with the deduced amino acid sequence below. Exon boundaries are delineated with vertical blue lines, potential polyadenylation signals are underlined and potential protein kinase C sites are boxed. A, The 857 bp fragment consists of 425 bp of coding sequence (104 bp of exon 8 and 321 bp of exon 9) and 432 bp of 3′ UTR contiguous to exon 9 and corresponds to Opn4L. B, The 930 bp fragment is composed of 260 bp of coding sequence split across 3 exons (104 bp of exon 8, 117 bp of exon 9 and 39 bp of exon 10). The remaining 670 bp of 3′ UTR is contiguous to the newly identified exon 10. This product corresponds to Opn4S.

Figure 4.

Opn4 localization in RGC-5 cells. RGC-5 cells were transfected with Opn4L (A) and Opn4S (B) and fixed after 24 h. Melanopsin expression was detected using a rabbit anti-Opn4 antibody targeted to the N terminus of the protein (PA8331), and immunofluorescence signal was observed mainly on the plasma membrane (arrows). Scale bar, 10 μm.

Figure 5.

Heterologous expression of Opn4L and Opn4S suggests that both variants can form a sensory photopigment. Representative whole-cell patch-clamp recording from Neuro-2A cells transfected with Opn4L (A) or Opn4S (B), in the presence of 9-cis-retinal. Monochromatic light stimuli 420 nm and 480 nm (presented for 10 s at 8 × 10¹⁴ photons · cm⁻² · s⁻¹) evoke a stimulus-dependent inward current. Holding potential, −50 mV. No differences in amplitude of responses, kinetics, or spectral sensitivity were observed between Opn4L and Opn4S.

Figure 6.

Western blot analysis of isoform-specific expression of melanopsin in retina. A, Western blot analysis using Opn4S antibody. Lane 1, Wild-type Neuro-2A cells; lane 2, Opn4S-transfected cells; lane 3, Opn4L-transfected cells. R, Tau-lacZ^+/− retina single load; R2, Tau-lacZ^+/− retina double load. B, Western blot analysis using Opn4L antibody, gel loading as previous figure. β-Actin was used to confirm equal loading of the gel.

Figure 7.

Single immunolabeling of the mouse retina with anti-Opn4L and anti-Opn4S antibodies. Single labeling of whole eye sections with anti-Opn4L (A–D) and anti-Opn4S (E–H) antibodies (red) and DAPI counterstain (blue) shows that both isoforms are expressed in a subset of RGCs. Higher levels of labeling were observed for Opn4S compared with Opn4L. When visible, the majority of Opn4L cells have dendrites localized near the inner nuclear layer (INL) or are bistratified with processes in the INL and ganglion cell layer. A number of Opn4L cells were also identified whose processes were confined to the vicinity of the ganglion cell layer. All Opn4S-positive cells have dendrites located in the INL and are often bistratified with processes also seen in the ganglion cell layer.

Figure 8.

Immunolabeling of the mouse retina shows a differential pattern of expression for Opn4L and Opn4S. A, Double labeling with Opn4L (green) and Opn4S (red) identified two subsets of pRGCs, those expressing both Opn4L and Opn4S and a second subset of cells expressing only Opn4L. B, Double labeling with β-gal (green) and Opn4S (red) shows a 100% overlap of expression, with all cells positive for both β-gal and Opn4S. C, Labeling with β-gal (green) and Opn4L (red) reveals a subset of Opn4L-positive cells that lack detectable β-gal expression. For all images, DAPI is blue.