Visual pigment homologies revealed by DNA hybridization.

A bovine rhodopsin complementary DNA probe was used to detect homologous visual pigment genes in a variety of species. Under stringent DNA hybridization conditions, genomic DNA from most vertebrate species carried a single homologous fragment. Additional homologies were detected in some vertebrates by reducing the hybridization stringency. Homologous fragments were also detected in DNA isolated from invertebrate species, a unicellular alga, and an archaebacterium; many of these fragments were homologous to a Drosophila opsin probe. These results suggest that photosensory pigments in a wide variety of species arose from a common precursor.

Pineal opsin: a nonvisual opsin expressed in chick pineal.

Pineal opsin (P-opsin), an opsin from chick that is highly expressed in pineal but is not detectable in retina, was cloned by the polymerase chain reaction. It is likely that the P-opsin lineage diverged from the retinal opsins early in opsin evolution. The amino acid sequence of P-opsin is 42 to 46 percent identical to that of the retinal opsins. P-opsin is a seven-membrane spanning, G protein-linked receptor with a Schiff-base lysine in the seventh membrane span and a Schiff-base counterion in the third membrane span. The primary sequence of P-opsin suggests that it will be maximally sensitive to approximately 500-nanometer light and produce a slow and prolonged phototransduction response consistent with the nonvisual function of pineal photoreception.

Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice.

Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E. coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.

Circadian regulation of iodopsin gene expression in embryonic photoreceptors in retinal cell culture.

A circadian clock regulates a number of diverse physiological functions in the vertebrate eye. In this study, we show that mRNA for the red-sensitive cone pigment, iodopsin, fluctuates with a circadian rhythm in chicken retina. Transcript levels increase in the late afternoon just prior to the time of cone disc shedding. Furthermore, iodopsin mRNA levels are regulated similarly by a circadian oscillator in primary cultures of dispersed embryonic chick retina. Nuclear run-on experiments show that the circadian regulation of iodopsin transcript abundance occurs at the level of gene transcription. Our results provide a demonstration of clock-regulated gene expression in a vertebrate preparation maintained in cell culture.

The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.

Mice express S and M opsins that form visual pigments for the detection of light and visual signaling in cones. Here, we show that S opsin transcription is higher than that of M opsin, which supports ultraviolet (UV) sensitivity greater than midwavelength sensitivity. Surprisingly, most cones coexpress both S and M opsins in a common cone cell type throughout the retina. All cones express M opsin, but the levels are graded from dorsal to ventral. The levels of S opsin are relatively constant. However, in the far dorsal retina, S opsin is repressed stochastically, such that some cones express M opsin only. These observations indicate that two different mechanisms control M and S opsin expression. We suggest that a common cone type is patterned across the retinal surface to produce phenotypic cone subtypes.

Molecular determinants of visual pigment function.

Mutagenesis studies and comparisons of natural variants of rhodopsin and related visual pigments have led to new insights concerning photoreceptor function. The studies identify domains important for receptor folding, the residues that set the wavelength of absorption for the ligand 11-cis retinal, and residues, that when mutated, trigger the cell death of photoreceptors.

The bovine guanylate cyclase GC-E gene and 5' flanking region.

The gene encoding the bovine guanylate cyclase isoform E (GC-E) was isolated as a single 18 kb genomic clone and shown to have 20 exons and 19 introns. Comparison of the structure of the GC-E gene with structures of other membrane guanylate cyclase genes indicates that the GC-E is most closely related to the subfamily of sensory guanylate cyclases. Comparison of the GC-E structure with that of the more distantly related guanylate cyclase isoform A (GC-A) gene shows the most divergence in the extracellular and C-terminal regions, but general conservation of introns and exons in the intracellular kinase-like and catalytic domains. RT-PCR from several bovine tissues shows that GC-E is expressed only in the retina. Consistent with this pattern of expression, elements for the retinal-specific transcription factors RET-1, RET-2 and Talpha-1 are located in the 5' flanking promoter region.

Distinctive properties of the purified Na-Ca exchanger from rod outer segments.

The Na-Ca exchanger of rod outer segments plays an important role in the regulation of Ca levels in photoreceptor cells. While this transporter shares functional properties with other Na-Ca exchangers, it has several unique features. The purified ROS exchanger migrates as a single band at 220 kDa in SDS-polyacrylamide gels, indicating that the unit size of its polypeptide is larger than other known Na-Ca exchangers (and most transporters). A specific antiserum to the ROS exchanger does not bind to the Na-Ca exchangers found in sarcolemmal vesicles or brain synaptic plasma membranes. Similarly, polyclonal antiserum specific for the cardiac exchanger does not react with ROS or brain proteins. The ROS exchanger requires K for transport activity. By incorporating the purified exchanger into proteoliposomes and measuring the sequestration of K, the actual transport of K is demonstrated. A stoichiometry of 4Na:1Ca,1K for the exchanger of ROS has been measured.

Dynamic processes of visual transduction.

Rhodopsin is one of those rare macromolecules whose inherent chromophore, 11-cis retinaldehyde, allows one to naturally observe triggered macromolecular changes on the timescale of picoseconds to minutes. Investigations of these molecular processes have been carried out with laser monochromatic light under conditions where the photon flux used for photolysis was carefully measured. The formation of bleaching intermediates has been examined as a function of fluence. Under conditions where the formation of intermediates is unaffected by photon reversal the following observations hold: Upon the absorption of a photon, the initial photochemical event results in production of metastable bathorhodopsin within 6 psec. Artificial rhodopsin regenerated with 9-cis retinal forms a distinct bathorhodopsin which must reflect distortions at the active site differing from those generated with 11-cis retinal. Bathorhodopsin thermally decays through lumirhodopsin and meta I-rhodopsin, to meta II-rhodopsin through a series of coupled equilibria. The final meta I-meta II equilibrium is stable for seconds. The process provides a unique model for utilization of energy to drive (trigger) a biological cascade of events.

Molecular biology of the visual pigments.

With the identification and structural characterization of several visual pigments has come a new era of investigation. The above comparisons of amino acids sequences predict specific functional domains that may be tested to tell us how visual pigments function to absorb light and transform this "signal" to trigger a neural response. The details of how rod and cone pigments differ are now known for human pigments. The striking similarities between vertebrate and invertebrate pigments are remarkable for pigments that have been subject to divergence for over 500 million years. There are yet challenges ahead of us. The true tertiary structure of visual pigments must be obtained from a 3-dimensional crystal structure. The predictions for functional domains of interaction with the GTP binding protein must be confirmed or redefined. A rigorous definition of the chromophore environment and the properties that control the wavelength of absorption of 11-cis retinal chromophore are certainly still on the drawing boards. Specific genetic alteration through in vitro mutagenesis promises much insight, but the technology for expressing these membrane proteins in functional form has yet to be achieved. We may expect, however, these problems will be addressed and in the next few years facts should replace what are now speculations. Finally, it is a delightful observation that nature has capitalized on a general biochemical mechanism for control of second messengers in the cytoplasm of cells. Protein structural data deduced from genetic information now document the hypothesis that the structure and function of receptors for the catecholamines and that of visual pigments are similar. The receptors for serotonin, leukotrienes, prostaglandins, histamine and acetylcholine (muscarinic) are expected to belong to this same family. The lessons learned about visual pigments can be applied broadly to a general set of membrane receptors.

Transient expression of thyroid hormone nuclear receptor TRbeta2 sets S opsin patterning during cone photoreceptor genesis.

Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR beta 2, the nuclear thyroid hormone receptor beta isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR beta 2 acts, we compared the spatiotemporal expression of TR beta 2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR beta 2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR beta 2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR beta 2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR beta 2. The mechanism by which TR beta 2 functions was probed in transgenic animals with TR beta 2 ablated, TR beta 2 that is DNA binding defective, and TR beta 2 that is ligand binding defective. These studies show that TR beta 2 is necessary for dorsal repression, but not ventral activation of S opsin. TR beta 2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR beta 2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.

Relationships of G-protein-coupled receptors. A survey with the photoreceptor opsin subfamily.

Relationships among the G-protein-coupled receptors were evaluated using several distance matrices with the neighbor-joining method of Saitou and Nei (1987). The relationships generated vary depending upon alignment, length, or region of sequence compared, and the distance matrix used to score similarity. To provide a statistical level of confidence, bootstrap resampling was applied to the analysis of a selection of G-protein-coupled receptors and the subfamily photoreceptor opsins. A general consensus indicates that the opsins behave as a discrete subfamily among the superfamily of G-protein-coupled receptors. Their relationship to other subfamilies remains unresolved. Within the opsin subfamily, the retinochromelike opsins segregate as a discrete group, but are more closely related to the invertebrate than vertebrate opsins. Among vertebrate opsins, the long wavelength cone opsins, the blue/violet opsins, and the rod opsins (including a class of green cone opsins) form distinct subgroups, but their relationships to one another remain unresolved. For this superfamily of receptors, the confidence levels for many branch pairings are low. The application of methods complimentary to those used in this preliminary study will be necessary to resolve questions about appropriate pairing and evolutionary relationships.

The bovine photoreceptor outer segment guanylate cyclase: purification, kinetic properties, and molecular size.

A simple protocol was developed to isolate the integral membrane guanylate cyclase from bleached bovine photoreceptor outer segments. Hypotonic and hypertonic washes strip the photoreceptor outer segment membranes of peripheral proteins. The guanylate cyclase activity is solubilized by dodecyl-b-D-maltoside in a low salt concentration buffer. Phosphatidylcholine, glycerol, and dithiothreitol are used to stabilize the activity during chromatography. GTP-affinity chromatography achieves a 250-fold increase in specific activity over that of membranes stripped of peripheral proteins. From 100 retinas, the protocol yields 100-140 mg of purified guanylate cyclase composed of a 115-kDa subunit. The molar ratio of the guanylate cyclase to rhodopsin is estimated to be 1:440. A significant portion of the freshly solubilized enzyme behaves as a monomer with a Stokes radius of 48.7 A, whereas the purified protein forms homooligomers ranging from dimers to tetramers. These properties are similar to those of ANP and guanylin receptors, indicating that the photoreceptor protein shares characteristics of the membrane receptor guanylate cyclase family. For the physiological substrate MgGTP, the Km and Vmax are 1.07 +/- 0.20 mM and 3262 +/- 514 nmol cGMP min-1 mg-1, respectively, generating a turnover rate of approximately 3.9 nmol cGMP s-1 at physiological substrate concentrations. The relatively high Km suggests that in vivo changes in GTP concentration might modulate the rate of cGMP synthesis. These properties indicate that the photoreceptor membrane guanylate cyclase can sustain a rate of cGMP synthesis comparable to the dark-adapted (basal) rate of cGMP degradation by the cGMP phosphodiesterase.

The photoreceptor guanylate cyclase is an autophosphorylating protein kinase.

The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of four structural motifs: an extracellular ligand binding domain, a transmembrane domain, an intracellular protein kinase-like domain, and an intracellular catalytic domain. Purified preparations of the photoreceptor guanylate cyclase have allowed us to explore the function of the protein kinase-like domain. ATP enhances the guanylate cyclase activity 2-fold in membranes stripped of peripheral proteins. The stimulation can be mimicked by ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)), AMPPNP (5'-adenylyl beta,gamma-imidodiphosphate), and ADP, but not AMP. While this effect is lost by solubilizing guanylate cyclase, ATP binds the purified, solubilized enzyme in a site distinct from the catalytic GTP site as shown by specific labeling with 8-N3[alpha-32P]ATP. The enzyme has a protein kinase activity that is Mg2+-dependent and autophosphorylates serine residues. Myelin basic protein serves as a substrate for the kinase and enables further characterization of the kinase properties. The Km for ATP is 81 microM. The kinase activity is unaffected by calcium, cyclic nucleotides, and phorbol 12-myristate 13-acetate/L-alpha-phosphatidylserine/Ca2+ and is inhibited by high concentrations of staurosporine. These properties are distinct from other Ser/Thr kinases identified in rod outer segment preparations including protein kinase A, protein kinase C, and rhodopsin kinase. The observations offer the first biochemical evidence that a member of the receptor guanylate cyclase family has intrinsic protein kinase activity.

Methylation of proteins in photoreceptor rod outer segments.

A set of proteins in bovine rod outer segments is specifically methylated by S-adenosyl-L-methionine. The reaction can be demonstrated in the intact retina as well as in fragmented preparations of isolated rod outer segments. The apparent molecular weights of these proteins are 88,000, 61,000, and a subset between 21,000 and 26,000. The Mr = 88,000 protein is shown to be the alpha subunit of the rod outer segment cGMP phosphodiesterase by peptide mapping, two-dimensional gel electrophoresis, the ionic strength dependence of its interaction with the membrane, and immunoprecipitation by antiserum raised against purified phosphodiesterase. For each of these proteins, the incorporated methyl groups are hydrolyzed in alkali to yield methanol, indicating that the proteins are carboxymethylated.