Photoreceptor Membrane Proteins Research Articles

Identification of the glycosylation sites of Opsin3 and its glycosylation modification function

Opsin3 (OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain. In recent years, it has been reported that OPN3 is also highly expressed in adipose tissue, and the protein is associated with the production of skin melanin. We found that the N82 site is the glycosylation site of OPN3. SNAP-tagTM has diverse functions and can be applied to a variety of different studies. By constructing a SNAP-tagged OPN3 recombinant protein, the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface® 549 and SNAP-Cell® OregonGreen®, which provides a new method for studying the function of OPN3. It also shows that SNAP-tag does not affect the function of OPN3. Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.

Effect of conditional deletion of cytoplasmic dynein heavy chain DYNC1H1 on postnatal photoreceptors.

Cytoplasmic dynein (dynein 1), a major retrograde motor of eukaryotic cells, is a 1.4 MDa protein complex consisting of a pair of heavy chains (DYNC1H1) and a set of heterodimeric noncatalytic accessory components termed intermediate, light intermediate and light chains. DYNC1H1 (4644 amino acids) is the dynein backbone encoded by a gene consisting of 77 exons. We generated a floxed Dync1h1 allele that excises exons 24 and 25 and truncates DYNC1H1 during Six3Cre-induced homologous recombination. Truncation results in loss of the motor and microtubule-binding domain. Dync1h1F/F;Six3Cre photoreceptors degenerated rapidly within two postnatal weeks. In the postnatal day 6 (P6) Dync1h1F/F;Six3Cre central retina, outer and inner nuclear layers were severely disorganized and lacked a recognizable outer plexiform layer (OPL). Although the gene was effectively silenced by P6, DYNC1H1 remnants persisted and aggregated together with rhodopsin, PDE6 and centrin-2-positive centrosomes in the outer nuclear layer. As photoreceptor degeneration is delayed in the Dync1h1F/F;Six3Cre retina periphery, retinal lamination and outer segment elongation are in part preserved. DYNC1H1 strongly persisted in the inner plexiform layer (IPL) beyond P16 suggesting lack of clearance of the DYNC1H1 polypeptide. This persistence of DYNC1H1 allows horizontal, rod bipolar, amacrine and ganglion cells to survive past P12. The results show that cytoplasmic dynein is essential for retina lamination, nuclear positioning, vesicular trafficking of photoreceptor membrane proteins and inner/outer segment elaboration.

Photoreaction pathways and photointermediates of retinal-binding photoreceptor proteins as revealed by in situ photoirradiation solid-state NMR spectroscopy.

Photoirradiation solid-state NMR spectroscopy is a powerful means to study photoreceptor retinal-binding proteins by the detection of short-lived photointermediates to elucidate the photoreaction cycle and photoactivated structural changes. An in situ photoirradiation solid-state NMR apparatus has been developed for the irradiation of samples with extremely high efficiency to enable observation of photointermediates which are stationary trapped states. Such observation enables elucidation of the photoreaction processes of photoreceptor membrane proteins. Therefore, in situ photoirradiation is particularly useful study the photocycle of retinal-binding proteins such as sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) because functional photointermediates have relatively longer half-lives than other photointermediates. As a result, several photointermediates have been trapped as stationary state and their detailed structures and photoreaction cycles have been revealed using photoirradiation solid-state NMR spectroscopy at low temperature. Photoreaction intermediates of bacteriorhodopsin, which functions to provide light-driven proton pump activity, were difficult to trap because the half-lives of the photointermediates were shorter than those of sensory rhodopsin. Therefore, these photointermediates are trapped in a freeze-trapped state at a very low temperature and the NMR signals were observed using a combination of photoirradiation and dynamic nuclear polarization (DNP) experiments.

Spontaneous stacking of purple membranes during immobilization with physical cross-linked poly(vinyl alcohol) hydrogel with retaining native-like functionality of bacteriorhodopsin

We previously discovered the correlation between light-induced chromophore color change of a photo-receptor membrane protein bacteriorhodopsin (bR) and its two-dimensional crystalline state in the membrane. To apply this phenomenon to a novel optical memory device, it is necessary that bR molecules are immobilized as maintaining their structure and functional properties. In this work, a poly(vinyl alcohol) (PVA) hydrogel with physical cross-linkages (hydrogen bonds between PVA chains) that resulted from repeated freezing-and-thawing (FT) cycles was used as an immobilization medium. To investigate the effects of physically cross-linked PVA gelation on the structure and function of bR in purple membranes (PMs), spectroscopic techniques were employed against PM/PVA immobilized samples prepared with different FT cycle numbers. Visible circular dichroism spectroscopy strongly suggested PM stacking during gelation. X-ray diffraction data also indicated the PM stacking as well as its native-like crystalline lattice even after gelation. Time-resolved absorption spectroscopy showed that bR photocycle behaviors in PM/PVA immobilized samples were almost identical to that in suspension. These results suggested that a physically cross-linked PVA hydrogel is appropriate for immobilizing membrane proteins in terms of maintaining their structure and functionality.

Small GTPases Rab8a and Rab11a Are Dispensable for Rhodopsin Transport in Mouse Photoreceptors.

Rab11a and Rab8a are ubiquitous small GTPases shown as required for rhodopsin transport in Xenopus laevis and zebrafish photoreceptors by dominant negative (dn) disruption of function. Here, we generated retina-specific Rab11a (retRab11a) and Rab8a (retRab8a) single and double knockout mice to explore the consequences in mouse photoreceptors. Rhodopsin and other outer segment (OS) membrane proteins targeted correctly to OS and electroretinogram (ERG) responses in all three mutant mouse lines were indistinguishable from wild-type (WT). Further, AAV (adeno-associated virus)-mediated expression of dnRab11b in retRab11a-/- retina, or expression of dnRab8b in retRab8a-/- retina did not cause OS protein mislocalization. Finally, a retRab8a-/- retina injected at one month of age with AAVs expressing dnRab11a, dnRab11b, dnRab8b, and dnRab10 (four dn viruses on Rab8a-/- background) and harvested three months later exhibited normal OS protein localization. In contrast to results obtained with dnRab GTPases in Xenopus and zebrafish, mouse Rab11a and Rab8a are dispensable for proper rhodopsin and outer segment membrane protein targeting. Absence of phenotype after expression of four dn Rab GTPases in a Rab8a-/- retina suggests that Rab8b and Rab11b paralogs maybe dispensable as well. Our data thus demonstrate significant interspecies variation in photoreceptor membrane protein and rhodopsin trafficking.

2P247 in situ光照射固体NMRによる光受容膜タンパク質ppR/pHtrIIの光励起過程におけるtransducerタンパク質膜貫通領域の構造変化の観測(18A.光生物:視覚・光受容,ポスター,日本生物物理学会年会第51回(2013年度))

2P247 in situ光照射固体NMRによる光受容膜タンパク質ppR/pHtrIIの光励起過程におけるtransducerタンパク質膜貫通領域の構造変化の観測(18A.光生物:視覚・光受容,ポスター,日本生物物理学会年会第51回(2013年度))

1F1558 In-situ光照射固体NMRによる光受容膜蛋白質の光活性状態の解明(光生物-視覚,光受容I,口頭発表,日本生物物理学会第50回年会(2012年度))

1F1558 In-situ光照射固体NMRによる光受容膜蛋白質の光活性状態の解明(光生物-視覚,光受容I,口頭発表,日本生物物理学会第50回年会(2012年度))

Sphingolipids and Retinal Degeneration

Too much dihydrosphingosine 1-phosphate enhances degradation of photoreceptor membrane proteins and leads to retinal degeneration in flies.

Crystallization and crystal properties of squid rhodopsin.

Rhodopsin, a photoreceptor membrane protein in the retina, is a prototypical member of the G-protein-coupled receptor family. In this study, rhodopsin from the retina of the squid Todarodes pacificus was treated with V8 protease to remove the C-terminal extension. Truncated rhodopsin was selectively extracted from the microvillar membranes using alkyl glucoside in the presence of zinc ions and was then crystallized by the sitting-drop vapour-diffusion method. Of the various crystals obtained, hexagonal crystals grown in the presence of octylglucoside and ammonium sulfate diffracted to 2.8 A resolution. The diffraction data suggested that the crystal belongs to space group P6(2), with unit-cell parameters a = b = 122.1, c = 158.6 A. Preliminary crystallographic analysis, together with linear dichroism results, suggested that the rhodopsin dimers are packed in such a manner that their transmembrane helices are aligned nearly parallel to the c axis.

S1H02光受容膜蛋白質の立体構造に基づく機能発現機構

S1H02光受容膜蛋白質の立体構造に基づく機能発現機構

Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms

The proteins which perform and regulate key steps in phototransduction are assumed to be localized in the rhabdomeric membrane of invertebrate photoreceptor cells. We have employed antibodies raised against rhabdoms purified from blowfly eyes in order to isolate copy deoxyribonucleic acid (cDNA) clones encoding proteins that are required in the phototransduction machinery. By immunoscreening a Calliphora retinal cDNA library, we obtained clones of genes coding for five different proteins. As revealed by partial cDNA sequence analysis, three of these genes represent the Calliphora homologs of Drosophila trp, inaC and InaD, while the other two displayed no homology to known genes. Northern blot analysis confirmed that trp, inaC and InaD transcript s were present in RNA isolated from the retina, but not in RNA isolated from brain or thorax. Specific antibodies directed against rp, inaC and InaD protein were raised using recombinantly expressed proteins or synthetic peptides. Western blot analyses revealed that trp, inaC and InaD protein are specifically associated with the rhabdomeral protoreceptor membrane. Extraction of membranes with buffers of different ionic strengths suggested that the trp gene product is an integral membrane protein, whilst the inaC and InaD gene products are peripherally bound membrane proteins. This demonstrates that the immunoscreening approach used here can be successfully applied to isolate genes that code for either integral or peripheral photoreceptor membrane proteins.

Phosphorylation of the InaD Gene Product, a Photoreceptor Membrane Protein Required for Recovery of Visual Excitation

In an approach directed to isolate and characterize key proteins of the transduction cascade in photoreceptors using the phosphoinositide signaling pathway, we have isolated the Calliphora homolog of the Drosophila InaD gene product, which in Drosophila InaD mutants causes slow deactivation of the light response. By screening a retinal cDNA library with antibodies directed against photoreceptor membrane proteins, we have isolated a cDNA coding for an amino acid sequence of 665 residues (Mr = 73,349). The sequence displays 65.3% identity (77.3% similarity) with the Drosophila InaD gene product. Probing Western blots with monospecific antibodies directed against peptides comprising amino acids 272-542 (anti-InaD-(272-542)) or amino acids 643-655 (anti-InaD-(643-655)) of the InaD gene product revealed that the Calliphora InaD protein is specifically associated with the signal-transducing rhabdomeral photoreceptor membrane from which it can be extracted by high salt buffer containing 1.5 M NaCl. As five out of eight consensus sequences for protein kinase C phosphorylation reside within stretches of 10-16 amino acids that are identical in the Drosophila and Calliphora InaD protein, the InaD gene product is likely to be a target of protein kinase C. Phosphorylation studies with isolated rhabdomeral photoreceptor membranes followed by InaD immunoprecipitation revealed that the InaD protein is a phosphoprotein. In vitro phosphorylation is, at least to some extent, Ca 2+ dependent and activated by phorbol 12-myristate 13-acetate. The inaC-encoded eye-specific form of a protein kinase C (eye-PKC) is co-precipitated by antibodies specific for the InaD protein from detergent extracts of rhabdomeral photoreceptor membranes, suggesting that the InaD protein and eye-PKC are interacting in these membranes. Co-precipitating with the InaD protein and eye-PKC are two other key components of the transduction pathway, namely the trp protein, which is proposed to form a Ca2+ channel, and the norpA-encoded phospholipase C, the primary target enzyme of the transduction pathway. It is proposed that the rise of the intracellular Ca2+ concentration upon visual excitation initiates the phosphorylation of the InaD protein by eye-PKC and thereby modulates its function in the control of the light response.

Fourier transform infrared difference spectroscopy of the nicotinic acetylcholine receptor: evidence for specific protein structural changes upon desensitization

We have previously reported a new method based on Fourier transform infrared spectroscopy for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR) [Baenziger, J. E., Miller, K. W. & Rothschild, K. J. (1992) Biophys. J. 61, 983-992; Baenziger, J. E., Miller, K. W., McCarthy, M. P. & Rothschild, K. J. (1992) Biophys. J. 62, 64-66]. Spectra are recorded using attenuated total reflection both in the presence and absence of agonists. The resulting nAChR "resting-to-desensitized" difference spectra reveal small highly reproducible infrared bands which can arise from vibrations of the agonist and structural changes in the nAChR membrane during the conversion of the receptor from the resting to desensitized state. In this work we have used a combination of different agonists and an antagonist along with isotopic labeling to assign bands in these spectra. nAChR membranes pretreated with the competitive antagonist alpha-bungarotoxin exhibit no bands above the noise level (approximately 10(-5) au) demonstrating that vibrations of the unbound agonist do not contribute to the normal difference spectrum. In contrast, bands in the resting-to-desensitized difference spectra are identified which can be assigned to the bound agonist, providing a means to probe its interaction and orientation in the binding site. Additional difference bands are due to secondary structural changes of the protein, perturbation of tyrosine(s), and changes in carboxyl groups possibly from Asp and/or Glu residues. Remarkably, some of these spectral changes are similar to those detected during the bleaching of the photoreceptor membrane protein rhodopsin.

Cloning of the CDNA for a novel photoreceptor membrane protein (rom-1) identifies a disk rim protein family implicated in human retinopathies

The molecules essential to the continual morphogenesis and shedding of the opsin-containing disks of vertebrate photoreceptors are largely unknown. We describe a 37 kd protein, rom-1, which is 35% identical and structurally similar to peripherin/retinal degeneration slow (rds). Like peripherin, rom-1 is a retina-specific integral membrane protein localized to the photoreceptor disk rim. The two proteins are similarly oriented in the membrane, and each has a highly conserved (1516 residues) cysteine- and proline-rich domain in the disk lumen. Although both rom-1 and peripherin form disulfide-linked dimers, they do not form heterodimers with each other, but appear to associate noncovalently. These results suggest both that rom-1 and peripherin are functionally related members of a new photoreceptor-specific protein family and that rom-1, like peripherin, is likely to be important to outer segment morphogenesis. The association of mutations in RDS with retinitis pigmentosa indicates that ROM1 is a strong candidate gene for human retinopathies.

Biochemical aspects of the visual process XXVIII. Classification of sulfhydryl groups in rhodopsin and other photoreceptor membrane proteins

Reaction of isolated bovine rod outer segment membrane with radioactive N-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule. On the basis of their reactivity towards p-chloromercuribenzoate and p-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11- cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with the p-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and is probably located in the vicinity of the chromophoric center. The difference between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) or N-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of the p-chloromercuri-derivatives.