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Butterflies rely extensively on colour vision to adapt to the natural world. Most species express a broad range of colour-sensitive Rhodopsin proteins in three types of ommatidia (unit eyes), which [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.08.016 Byline: Elizabeth C. McDonald, Baotong Xie, Michael Workman, Mark Charlton-Perkins, David A. Terrell, Joachim Reischl, Ernst A. Wimmer, Brian A. Gebelein, Tiffany A. Cook Keywords: Orthodenticle; Crx; Otx tail; Retinogenesis; Cell-specific gene expression; Photoreceptors Abstract: Orthodenticle (Otd)-related transcription factors are essential for anterior patterning and brain morphogenesis from Cnidaria to Mammals, and genetically underlie several human retinal pathologies. Despite their key developmental functions, relatively little is known regarding the molecular basis of how these factors regulate downstream effectors in a cell- or tissue-specific manner. Many invertebrate and vertebrate species encode two to three Otd proteins, whereas Drosophila encodes a single Otd protein. In the fly retina, Otd controls rhabdomere morphogenesis of all photoreceptors and regulates distinct Rhodopsin-encoding genes in a photoreceptor subtype-specific manner. Here, we performed a structure-function analysis of Otd during Drosophila eye development using in vivo rescue experiments and in vitro transcriptional regulatory assays. Our findings indicate that Otd requires at least three distinct transcriptional regulatory domains to control photoreceptor-specific rhodopsin gene expression and photoreceptor morphogenesis. Our results also uncover a previously unknown role for Otd in preventing co-expression of sensory receptors in blue vs. green-sensitive R8 photoreceptors. Sequence analysis indicates that many of the transcriptional regulatory domains identified here are conserved in multiple Diptera Otd-related proteins. Thus, these studies provide a basis for identifying shared molecular pathways involved in a wide range of developmental processes. Article History: Received 20 August 2009; Revised 13 August 2010; Accepted 16 August 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.12.017 Byline: Hui-Yuan Wu (a), Muriel Perron (b), Thomas Hollemann (a) Keywords: Rx-L; Homeobox; Transcription factor; Xenopus; Eye; Retina; Retinogenesis; Lipofection; Rhodopsin Abstract: Members of the Rx (retinal homeobox) gene family play vital roles during eye development. In Xenopus, as in most other vertebrates, two Rx-type genes have been described. While Rx1 deficiency led to loss of optic vesicles and impaired the proper development of ventral forebrain structures, a recently isolated second Rx-gene, Rx-L, seems to function in late retinogenesis. Here, we report that the specific suppression of Xenopus Rx-L function impaired the formation of the photoreceptor layer and reduced the expression of photoreceptor specific genes. Overexpression of Xenopus Rx-L induced ectopic expression of photoreceptor specific genes, but did only marginally promote the proliferation of retinal progenitor cells. Targeted overexpression of Rx-L in developing retinoblasts in vivo led to an increased fraction of photoreceptor cells at the expense of amacrine and bipolar cells and revealed that Rx-L acts as a transcription activator. A phylogenic analysis of all reported Rx-type genes revealed that they could be grouped into three categories, including an 'invertebrate Rx' group, a 'classical vertebrate Rx' group, and a 'vertebrate Qrx/Rx-L' group. Taken together, Rx-L, unlike Rx1, is required for the determination of retinal cell types, especially photoreceptors, rather than for proliferation of retinal progenitors. Author Affiliation: (a) Institut fur Physiologische Chemie, Universitat Halle-Wittenberg, D-06114 Halle/Saale, Germany (b) Universite Sud, UMR Centre National de la RechercheScientifique (CNRS) 8080, 91405 Orsay, France Article History: Received 2 May 2008; Revised 2 December 2008; Accepted 15 December 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.06.030 Byline: Jen-Zen Chuang (1), Yu Zhao (1), Ching-Hwa Sung (1)(2) Keywords: CELLBIO; DEVBIO; HUMDISEASE Abstract: The light-sensing organelle of the vertebrate rod photoreceptor, the outer segment (OS), is a modified cilium containing [approximately equal to]1,000 stacked disc membranes that are densely packed with visual pigment rhodopsin. The mammalian OS is renewed every ten days; new discs are assembled at the base of the OS by a poorly understood mechanism. Our results suggest that discs are formed and matured in a process that involves specific phospholipid-directed vesicular membrane targeting. Rhodopsin-laden vesicles in the OS axonemal cytoplasm fuse with nascent discs that are highly specialized with abundant phosphatidylinositol 3-phosphate (PI3P). This membrane coupling is regulated by the FYVE domain-containing protein, SARA, through its direct interaction with PI3P, rhodopsin, and SNARE protein syntaxin 3. Our model, in contrast to the previously proposed evagination model, suggests that the vesicular delivery of rhodopsin in the OS concentrates rhodopsin into discs, and this process directly participates in disc biogenesis. Author Affiliation: (1) Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, Weill Medical College of Cornell University, 1300 York Avenue, NY, NY 10021, USA (2) Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, NY, NY 10021, USA Article History: Received 24 January 2007; Revised 8 May 2007; Accepted 14 June 2007 Article Note: (miscellaneous) Published: August 9, 2007

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was ~11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 X [10.sup.7] [s.sup.-1], about 860-told higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.007 Byline: R.D. Burke (a), L.M. Angerer (b), M.R. Elphick (c), G.W. Humphrey (d), S. Yaguchi (a), T. Kiyama (e), S. Liang (e), X. Mu (e), C. Agca (f), W.H. Klein (e), B.P. Brandhorst (h), M. Rowe (c), K. Wilson (j), A.M. Churcher (g), J.S. Taylor (g), N. Chen (h), G. Murray (h), D. Wang (g), D. Mellott (g), R. Olinski (i), F. Hallbook (i), M.C. Thorndyke (j) Keywords: Genomics; Neurobiology; Neural development; Deuterostome; Evolution; Echinoderm Abstract: The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein-coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins. Author Affiliation: (a) Department of Biology, Dept of Biochemistry/Microbiology, University of Victoria, Victoria, POB 3020, STN CSC, Victoria, BC, Canada V8W 3N5 (b) National Institute for Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA (c) School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK (d) Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, National Institutes of Health, Bethesda, MD, USA (e) University of Texas, M. D. Anderson Cancer Center, Biochemistry and Molecular Biology, Houston, TX, USA (f) Department of Cell Biology and Anatomy, Louisiana State University, Health Sciences Center, New Orleans, LA, USA (g) Department of Biology, University or Victoria, Victoria, BC, Canada (h) Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada (i) Department Neuroscience, Uppsala University, Uppsala, Sweden (j) Royal Swedish Academy of Sciences, Kristineberg Marine Research Station, Fiskebackskil, 450 34, Sweden Article History: Received 1 June 2006; Revised 29 July 2006; Accepted 2 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.09.051 Byline: Hideto Koso, Yasuo Ouchi, Yoko Tabata, Yutaka Aoki, Shinya Satoh, Ken-ichi Arai, Sumiko Watanabe Keywords: Retina; Mouse; Progenitor cells; FACS; SSEA-1; Proliferation; Differentiation Abstract: Identification and expansion of retinal progenitor cells are critical issues from both scientific and clinical aspects. Here, we identified SSEA-1 (CD15) as a novel surface antigen that can be used to define immature retinal progenitor cells. SSEA-1-expressing retinal cells were found in the peripheral region of the early embryonic mouse retina, and then their number dramatically disappeared along with retinal development. FACS analysis showed that the cells strongly positive for SSEA-1 co-expressed Ki67 proliferation antigen in all the developmental stages examined. The SSEA-1-expressing cells formed larger colonies than the non-expressing ones in retinal re-aggregation cultures. Moreover, late onset of rhodopsin expression was observed in SSEA-1-positive progenitor cells, supporting the idea that these cells have an intrinsically immature character. Differential expression of Wnt signal-related genes between SSEA-1-positive and -negative subpopulations of retina cells was revealed, and the expression of constitutively active forms of Wnt signaling molecules resulted in a greater number of SSEA-1-positive cells. In light of all of the data taken together, we propose SSEA-1 to be a surface marker to define a regionally restricted immature subset of progenitor cells of mouse neural retina, with SSEA-1 expression by them positively regulated by Wnt signals. Author Affiliation: Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Article History: Received 2 August 2005; Revised 16 September 2005; Accepted 30 September 2005

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.

Research examines the molecular mechanism underlying the molecular properties between the rhodopsin and cone pigments. Results indicate that differences in the meta II decay rate between the chicken cone pigments and rhodopsin is due to differences in the amino acid residues at 189 and 122.

Nuclear magnetic resonance analysis reveal that 11-cis-locked cyclopropyl retinal adopts a twist chair conformation, involving the seven-membered ring, in solution. Data indicate that the compound exists as two diastereomeric populations. The native retinal does not exhibit twist preference around C6/C7 bond.

The conformational changes and dynamics of the cytoplasmic loops during activation of rhodopsin are analyzed using time-resolved fluorescence depolarization technique. Two accessible and reactive sulfhydryl groups of the native cysteines C140 and C316 measurements show marked differences with a large decrease in motional freedom in M(sub)II.

Results point out that the rhodopsin chromophore ring exhibits preference to 6-s-cis orientation in the dark-adapted protein, different from the twisted 6-s-trans conformation revealed by (sup)2H nuclear magnetic resonance measurements. The major portion of retinylidene in rhodopsin shows the former conformation.

In retinal rods, light-induced isomerization of 11-cis-retinal to all-trans-retinal within rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP. Consequently, cyclic nucleotide-gated (CNG) ion channels close, generating the first electrical response to light. After isomerization, all-trans-retinal dissociates from rhodopsin. We now show that all-trans-retinal directly and markedly inhibits cloned rod CNG channels in excised patches. 11-cis-retinal and all-trans-retinol also inhibited the channels, but at somewhat higher concentrations. Single-channel analysis suggests that all-trans-retinal reduces average open probability of rod CNG channels by inactivating channels for seconds at a time. At physiological cGMP levels, all-trans-retinal inhibited in the nano-molar range. Our results suggest that all-trans-retinal may be a potent regulator of the channel in rods during the response to bright light, when there is a large surge in the concentration of all-trans-retinal.

[[[alpha-.sup.15]N]Lysine-labeled rhodopsin, prepared by expression of a synthetic gene in HEK293 cells, was investigated both by conventional and transverse relaxation optimized spectroscopy-type heteronuclear single quantum correlation spectroscopy. Whereas rhodopsin contains 11 lysines, 8 in cytoplasmic loops and 1 each in the C-terminal peptide sequence and the intradiscal and transmembrane domains, only a single sharp peak was observed in dodecyl maltoside micelles. This result did not change when dodecyl maltoside was replaced by octyl glucoside or octyl glucoside-phospholipid-mixed micelles. Additional signals of much lower and variable intensity appeared at temperatures above 20 [degrees] C and under denaturing conditions. Application of the transverse relaxation optimized spectroscopy sequence resulted in sharpening of resonances but also losses of signal intensity. The single peak observed has been assigned to the C-terminal Lys-339 from the following lines of evidence. First, the signal is observed in HNCO spectra of rhodopsin, containing the labeled [[sup.13]C]Ser-338/[[sup.15]N]Lys-339 dipeptide. Second, addition of a monoclonal anti-rhodopsin antibody that binds to the C-terminal 8 aa of rhodopsin caused disappearance of the peak. Third, truncated rhodopsin lacking the C-terminal sequence Asp-330-Ala-348 showed no signal, whereas the enzymatically produced peptide fragment containing the above sequence showed the single peak. The results indicate motion in the backbone amide groups of rhodopsin at time scales depending on their location in the sequence. At the C terminus, conformational averaging occurs at the nanosecond time scale but varies from microsecond to millisecond in other parts of the primary sequence. The motions reflecting conformational exchange may be general for membrane proteins containing transmembrane helical bundles.

Nuclear magnetic resonance analyses of wild-type alpha2A adrenergic receptor intracellular loop and a mutant peptide, in a micellar environment, show upto 12 residues in the transmembrane 3 and 4 helices flanking the loop. Further, a novel cytoplasmic helix in the alpha2A loop is detected.

Results demonstrate that the second extracellular loop 2 of rhodopsin renders the chromophore-binding domain inaccessible to solvent. The glutamine 181 makedly affects the retinylidene Schiff base of the active metarhodopsin II.

Results show that the conformational equilibria of rhodopsin is regulated by charged ions, they are accessible to pH titration and exhibit susceptibility to salts. Data indicate that the protein domains involved in the G protein-coupled receptors are stable and highly conserved.

The halorhodopsin from Natronobacterium pharaonis exhibits a blue-shift in its absorption in the absence of anions in the medium, which is restored upon anion addition, suggesting that the protein loses anion from around the chromophore. The anion binding affinity and the binding kinetics of halorhodopsin under various conditions are discussed.

Research describes mutations in the retinal binding pocket of rhodopsin that will render the protein to retain its normal functional properties under ambient lighting conditions. Results demonstrate a triple mutant that exhibits a large blue-wavelength shift with an absorbance maxima at wavelength 453.

A method to reevaluate the detailed structure and orientation of the retinyl chromophore of rhodopsin, with respect to normal membrane, is presented. Data show that the protein does not change the geometry of the chromophore from its minimum energy configuration.

Research has been conducted on the heterotrimeric G-protein alpha-subunit N-terminal regions. The authors discuss the role of the N-terminal transducin-alpha domain in G-protein function.

The FTIR spectra for the photoreactions of halorhodopsin from Halobacterium salinarum at 170 and 250 K showed no difference at the visible spectra nor at the FTIR bands of the chromophore. Halorhodopsin is a membrane protein that works as a light-driven chloride pump in H. salinarum. It was observed that Asp141 is perturbed at the L intermediate at 250 K but not at 170 K. The photoproducts have been termed La at 170 K and Lb at 250 K. Both have dissimilar spectra in the different shifts of water O-H stretching bands. These findings indicate that the photocycle of halorhodopsin has two states with different protein and internal water molecuel structures.

The basal activities of the rhodopsin receptor are activated by the G protein-coupled receptor in its multi-state conformation. Amino acid substitution of G protein-coupled receptor mutations in specific sites indicated that the receptor can be activated without endogenous stimulus. In addition, the three cytoplasmic loops of the receptor41 is related to the inactive state of rhodopsin, wherein displacement of one or more of these loops result in the lower affinity for the G protein.

Met(super 257) in the TM helix 6 of opsin has been replaced with each of the remaining 19 amino acids. The 18 mutant opsins were expressed in COS-cell membranes with the exception of M257R. The 18 mutant opsins were also active except M257L and mutants M257Y and M257N showed high levels of activity. Mutants with substitutions for both Met(super 257) and Glu(super 113) in TM helix 3 were more active than single-site mutants.

Byline: Juan J. Perez (1), Marta Filizola (1,2), Maria CaritenA!aFarina (2) Abstract: The only results available at present about the structural features of Gaprotein coupled receptors are the low resolution electron projection maps obtained from microscopy studies carried out on twoadimensional crystals of rhodopsin. These studies support previous suggestions that these integral proteins are constituted by seven transmembrane domains. The low resolution electron density map of rhodopsin can be used to extract information about helix relative positions and tilt. This information, together with a reliable procedure to assess the residues involved in each of the transmembrane regions, can be used to construct a model of rhodopsin at atomic resolution. We have developed an algorithm that can be used to generate such a model in a completely automated fashion. The steps involved are: (i) locate the centers of the helices according to the low resolution electron density map (ii) compute the tilt of each helix based on the elliptical shape observed by each helix in the map (iii) define a local coordinate system for each of the helices (iv) bring them together in an antiparallel orientation (v) rotate each helix through the helical axis in such a way that its hydrophobic moment points in the same direction as the bisector formed between three consecutive helices in the bundle (vi) rotate each helix through an axis perpendicular to the helical one to assign a proper tilt (vii) translate each of the helix to its center deduced from the projection map. A major advantage of the procedure presented is its generality and consequently can be used to obtain a model of any Gaprotein coupled receptor with the only assumption that the shape of the bundle is the same as found in rhodopsin. This avoids uncertainties found in other procedures that construct models of Gaprotein coupled receptors based on sequence homology using rhodopsin as template. Author Affiliation: (1) Dept. d'Enginyeria Quimica, UPC, ETS d'Enginyers Industrials, Av. Diagonal 647, 08028, Barcelona, Spain (2) Centro di Ricerca Interdipartimentale di Scienze Computazionali e Biotecnologiche (CRISCEB), Seconda Universita degli Studi di Napoli, Via Costantinopoli 16, 80138, Napoli, Italy Article History: Registration Date: 29/09/2004

Rhodopsin is a prototypical G protein-coupled receptor that is activated by photoisomerization of its 11-cis-retinal chromophore. Mutant forms of rhodopsin were prepared in which the carboxylic acid counterion was moved relative to the positively charged chromophore Schiff base. Nanosecond time-resolved laser photolysis measurements of wild-type recombinant rhodopsin and two mutant pigments then were used to determine reaction schemes and spectra of their early photolysis intermediates. These results, together with linear dichroism data, yielded detailed structural information concerning chromophore movements during the first microsecond after photolysis. These chromophore structural changes provide a basis for understanding the relative movement of rhodopsin's transmembrane helices 3 and 6 required for activation of rhodopsin. Thus, early structural changes following isomerization of retinal are linked to the activation of this G protein-coupled receptor. Such rapid structural changes lie at the heart of the pharmacologically important signal transduction mechanisms in a large variety of receptors, which use extrinsic activators, but are impossible to study in receptors using diffusible agonist ligands.

The importance of arrestin with a single amino acid substitution in light-activated rhodopsin was determined through utilization of in vitro assays that monitored the rhodopsin-dependent activation of enzyme cGMP phosphodiesterase (PDE). The protein also has a specific site for phosphorylation recognition which interacts with the phosphorylated C-terminus and another site for activation recognition.

An assay for transphosphorylation of rhodopsin using split rhodopsin mutant SR(1-4/5-7), in which the protein was assembled in functional form from two separately expressed fragments, was devised. The use of SR(1-4/5-7) permits the biochemical distinction to be established for phosphorylated forms on the basis of electrophoretic mobility.

Raman microprobe spectroscopy was used to investigate the wavelength mechanism in human green and red cone pigments. Results have shown that the opsin shift from rhodopsin to green pigment was determined by the weakening of the interaction of the rhodopsin chromophore with a protein hydrogen bond donor which lies adjacent to the protonated Schiff base group. Interactions between the hydroxyl group with amino acids and chromophore caused a red shift in the red cone pigment.

The structure of the first and second cytoplasmic loops of Rhodopsin was analyzed by nuclear magnetic resonance (NMR) spectroscopy. The peptides that contained the amino acid sequence of the first and second cytoplasmic loops of the transmembrane protein exhibited similar well-ordered protein conformations. The cytoplasmic loops also stabilized the formation of a helical hairpin for insertion into the membrane. Furthermore, the second cytoplasmic loop exhibited a beta-turn in the middle of the peptide with helical amino and carboxy terminals.

The crystal structure of halorhodopsin is determined directly in its centrosymmetric projection using 6.0-[Angstrom]-resolution electron diffraction intensities, without including any previous phase information from the Fourier transform of electron micrographs. The potential distribution in the projection is assumed a priori to be an assembly of globular densities. By an appropriate dimensional re-scaling, these 'globs' are then assumed to be pseudo-atoms for normalization of the observed structure factors. After this treatment, the structure is determined directly by conventional direct methods, followed by Fourier refinement, leading to a mean phase deviation of only 20 [degrees] (from the values originally found from the image transform) for the 45 most intense reflections.

Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared two sets of rhodopsin mutants in the cytoplasmic CD and EF loops. In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences. In the second set of mutants, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1-3 per mutant. The deletion and replacement mutants in the CD loop showed essentially no phosphorylation, and they appeared to be defective in binding of RK. Of the mutants in the EF loop, that with a deletion of 13 amino acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in [V.sub.max] for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R/R135E showed a 50% reduction in [V.sub.max] relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the [K.sub.m] by 5-fold for E239Q and E247Q/K248L/E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.

Retinals with fluorine atoms at the odd-numbered side-chain positions past C-7 were synthesized The cis-fluororetinals were converted into the more stable trans-fluororetinals, which were reacted to n-butylamine to form the protonated Schiff base and incubated with bacterio-opsin to form bacteriorhodopsin analogues. The fluorinated retinals defied expectations by providing artificial pigments with near normal absorption properties. This showed that electrostatic interactions between the fluorine atoms and protein groups cannot thwart normal binding.

Raman resonance spectroscopy and circular dichroism (CD) spectroscopy were utilized to determine the role of nonbonded interactions in the torsional dynamics and isomerization mechanism of rhodopsin. The isomerization of the analogs of the 11-cis-retinal pigment involves the elimination of torsional deformation of the chromophore by the removal of the 13-CH3 group. Spectral analysis of 13-CH3 removal in rhodopsin also indicated the elimination of the steep slope of the C11=C12 isomerization coordinate.

The activation of rhodopsin in rod cells involves a collective rearrangement of opsin structures that is assisted by the three and six helices of the helical bundle. The tilting of the helices in the cytoplasmic membrane surface changes the second and third cytoplasmic loops of helices three and six. Hydrogen-bonding interactions of Trp126 are disrupted by the tilting of the helices which increases the polarity of the tryptophans.

Raman spectra studies reveal that the C=N configuration is anti in octopus rhodopsin and bathorhodopsin while more work is necessary for determining the C=N bond configuration in isorhodopsin. Octopus bathorhodopsin is 11-trans and anti about the Schiff base. The conformation of rhodopsin is 11-cis, 12-s-trans and the Schiff base C=N bond is anti. The spectra of octopus rhodopsin are different from those of bovine rhodopsin due to changes in the C14-C15 and C12-C13 stretch motions and suggest large protein perturbations on the C11=C12-C13 moiety due to electrostatic or steric interactions.

Mutation of Gly90, Glu113, Ala292, and Lys296 in the rhodopsin pigment constitutively activates G protein transducin, but the mutants are constitutively phosphorylated by rhodopsin kinase. The phosphorylated mutants bind arrestin in a reaction that prevents the interaction with transducin. Thus the same mutations which activate the transducin also cause phosphorylation by rhodopsin kinase and the inhibition by arrestin binding.

Photoregeneration of bovine rhodopsin involves Schiff base reprotonation and proton release. The metarhodopsin II intermediate (MII) generates the active state catalyzing GDP/GTP exchange in the G-protein (Gt), while Schiff base translocation along with proton uptake mediates the process. Spectral analysis shows that MII photolyzes by fast and slow processes. The slow pathway regenerates rhodopsin in which 11-cis-retinal is attached via a protonated Schiff base. The process of regeneration is stopped by Gt. The results suggest that both Gt and cis-retinal can be simultaneously bound to the receptor.

The role of the structure in the cytoplasmic loop which corrects helices C and D rhodopsin, a part involved in protein-protein interactions during signal transduction, in the reactivity of the successive cysteine residues is demonstrated. This has been done by replacing, one at a time, the amino acids 136-150 by cysteine residues. The results show differences in both the reactivity and accessibility of the different cysteine residues in the CD loop.

The 20 single cysteine substitution mutants in the sequence Y136-M155, which contains the helices C-D interhelical loop, of bovine rhodopsin prepared and modified with a sulfhydryl-specific nitroxide reagent and determined the accessibilities of the attached nitroxide to collisions with paramagnetic probes in solution. The data showed that the rhodopsin polypeptide crosses an aqueous/hydrophobic boundary near V138 and H152.

A study conducted using perforated patch-clamp technique shows that the sustained outward K+ current helps in establishing the resting potential of the apical and/or basolateral membranes and also involves K+ transport across human retinal pigment epithelial cells (RPE). The outwardly rectifying K+ conductance has an activation threshold voltage near -60mV. K+ conductance is active at physiological membrane voltages and participates in various aspects of RPE transport physiology.