A G protein-coupled receptor phosphatase required for rhodopsin function.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors are phosphorylated by kinases that mediate agonist-dependent receptor deactivation. Although many receptor kinases have been isolated, the corresponding phosphatases, necessary for restoring the ground state of the receptor, have not been identified. Drosophila RDGC (retinal degeneration C) is a phosphatase required for rhodopsin dephosphorylation in vivo. Loss of RDGC caused severe defects in the termination of the light response as well as extensive light-dependent retinal degeneration. These phenotypes resulted from the hyperphosphorylation of rhodopsin because expression of a truncated rhodopsin lacking the phosphorylation sites restored normal photoreceptor function. These results suggest the existence of a family of receptor phosphatases involved in the regulation of G protein-coupled signaling cascades.

Spectral tuning of rhodopsin and metarhodopsin in vivo.

Color vision is dependent upon the expression of spectrally distinct forms of rhodopsin in different photoreceptor cells. To identify the structural features of rhodopsin that regulate spectral sensitivity and absorption in vivo, we have constructed a series of chimeric Drosophila rhodopsin molecules, derived from a blue- and a violet-sensitive rhodopsin, and used P element-mediated germline transformation to generate transgenic flies that express the modified pigments in the R1-R6 photoreceptor cells of the compound eye. Our analysis of these animals indicates that multiple regions of the opsin protein are involved in regulating rhodopsin spectral sensitivity and that the native and photoactivated forms of rhodopsin can be tuned independently of each other. These results demonstrate the feasibility of designing receptor molecules with specifically modified activated states.

Identification of a novel Drosophila opsin reveals specific patterning of the R7 and R8 photoreceptor cells.

The function of the compound eye is dependent upon a developmental program that specifies different cell fates and directs the expression of spectrally distinct opsins in different photoreceptor cells. Rh5 is a novel Drosophila opsin gene that encodes a biologically active visual pigment that is expressed in a subset of R8 photoreceptor cells. Rh5 expression in the R8 cell of an individual ommatidium is strictly coordinated with the expression of Rh3, in the overlying R7 cell. In sevenless mutant files, which lack R7 photoreceptor cells, the expression of the Rh5 protein in R8 cells is disrupted, providing evidence for a specific developmental signal between the R7 and R8 cells that is responsible for the paired expression of opsin genes.

Blue- and green-absorbing visual pigments of Drosophila: ectopic expression and physiological characterization of the R8 photoreceptor cell-specific Rh5 and Rh6 rhodopsins.

Color discrimination requires the input of different photoreceptor cells that are sensitive to different wavelengths of light. The Drosophila visual system contains multiple classes of photoreceptor cells that differ in anatomical location, synaptic connections, and spectral sensitivity. The Rh5 and Rh6 opsins are expressed in nonoverlapping sets of R8 cells and are the only Drosophila visual pigments that remain uncharacterized. In this study, we ectopically expressed Rh5 and Rh6 in the major class of photoreceptor cells (R1-R6) and show them to be biologically active in their new environment. The expression of either Rh5 or Rh6 in "blind" ninaE(17) mutant flies, which lack the gene encoding the visual pigment of the R1-R6 cells, fully rescues the light response. Electrophysiological analysis showed that the maximal spectral sensitivity of the R1-R6 cells is shifted to 437 or 508 nm when Rh5 or Rh6, respectively, is expressed in these cells. These spectral sensitivities are in excellent agreement with intracellular recordings of the R8p and R8y cells measured in Calliphora and Musca. Spectrophotometric analyses of Rh5 and Rh6 in vivo by microspectrophotometry, and of detergent-extracted pigments in vitro, showed that Rh5 is reversibly photoconverted to a stable metarhodopsin (lambda(max) = 494 nm), whereas Rh6 appears to be photoconverted to a metarhodopsin (lambda(max) = 468 nm) that is less thermally stable. Phylogenetically, Rh5 belongs to a group of short-wavelength-absorbing invertebrate visual pigments, whereas Rh6 is related to a group of long-wavelength-absorbing pigments and is the first member of this class to be functionally characterized.

5-Hydroxytryptamine2 (5-HT2) structure-function relationships of the nitro and amino phenylpiperazines on intact human platelets.

Nitro and amino phenylpiperazines were synthesized to study the agonist and antagonist activities of the phenylpiperazines at the human platelet 5-hydroxytryptamine2 (5-HT2) receptor. Amplification of ADP-induced aggregation and binding competition experiments with [3H]ketanserin were used to evaluate receptor interactions in this system. All the monosubstituted phenylpiperazines were antagonists despite the wide variation in electronic and hydrophobic properties. The parent compound, unsubstituted phenylpiperazine (PP), had the lowest affinity for the [3H]ketanserin binding site. The intensely electron-withdrawing NO2 substituent increased binding affinity at all ring positions and this activity correlated with antagonist potency in platelet aggregation studies (rank order: 4-NO2-PP greater than 3-NO2-PP greater than 2-NO2-PP). NH2 substitution decreased binding affinity at the 4- and 2-positions compared with the analogous NO2 substituted compounds; however, evaluation of NH2-PP antagonist potency in aggregation studies was complicated due to slow association with the receptor. To compare the activities of the phenylpiperazines at other 5-HT sites, binding competition experiments were performed using [3H]5-HT in bovine brain membranes. The rank order of the affinities for the NO2 substituted compounds was distinctly different from that determined with platelets, reflecting the heterogeneous composition of 5-HT receptor subtypes in the brain membrane preparations. The platelet aggregation experiments demonstrated that marked alteration of the electronic and hydrophobic properties of phenylpiperazine by ring substitution did not impart 5-HT2 agonist activity. By contrast, 5-HT2 antagonist activity appeared to be enhanced markedly by electron-withdrawing resonance effects which decreased the electron density at the 1'-piperazine nitrogen. This enhancement appeared to be specific for the 5-HT2 receptor subtype.

Inactivation of cytochrome P-450 by 2-isopropyl-4-pentenamide and other xenobiotics leads to heme-derived protein adducts.

When cytochrome P-450 in phenobarbital-induced rat liver microsomes was destroyed by 2-isopropyl-4-pentenamide (AIA) in vitro, 50% of the degraded heme was recovered as heme-derived products irreversibly bound to microsomal proteins. In contrast, less than 50% of the degraded heme was accounted for as N-alkylated porphyrins. Furthermore, 64% of the irreversibly bound products was bound specifically to a 54-kD form of cytochrome P-450. Several other compounds which have been reported to destroy cytochrome P-450 by forming N-alkylated porphyrins also produced heme-derived protein adducts. These findings indicate that the formation of heme-derived protein adducts may represent an important pathway for the irreversible degradation of cytochrome P-450 by many xenobiotics.

Carbon tetrachloride and 2-isopropyl-4-pentenamide-induced inactivation of cytochrome P-450 leads to heme-derived protein adducts.

When CCl4 was incubated with rat liver microsomes from phenobarbital-treated rats in an aerobic or anaerobic atmosphere, over 69% of the heme moiety of cytochrome P-450 was destroyed. At least 45% of the degraded heme under both reaction conditions was accounted for as heme-derived products irreversibly bound to microsomal proteins. Furthermore, 33% of the irreversibly bound products were bound specifically to a 54-kDa form of cytochrome P-450. A structurally different compound, 2-isopropyl-4-pentenamide, also destroyed the heme moiety of cytochrome P-450 and produced heme-derived adducts of microsomal proteins that accounted for 28% of the destroyed heme. These results represent a novel mechanism for the destruction of cytochromes P-450 by xenobiotics.

Agonist and antagonist activities of arylpiperazines at human platelet serotonin2 receptors.

A series of arylpiperazines was examined for structure-function relationships at the human platelet serotonin (5-HT) receptor. Amplification of ADP-induced aggregation was used to measure 5-HT receptor activation. The platelet serotonergic agonists 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), 5-HT and 5-methoxytryptamine (5-MeOT) and the antagonist ketanserin were used for comparison of potency and amplitude of response. All arylpiperazines, including the parent compound phenylpiperazine (PP) showed antagonist activity. The monosubstituted phenylpiperazines acted only as antagonists, and electron-withdrawing substituents markedly enhanced activity. Modification of PP by addition of another phenyl ring or benz-fusion also enhanced antagonist activity. Benz-fusion at the b face of PP (1-NP) yielded greater antagonist potency than benz-fusion at the c face (2-NP). The latter modification, however, also conferred a variable agonist activity with a very weak response. In contrast, the heteroaromatic piperazines consistently demonstrated concentration dependent mixed antagonist-agonist activity. These compounds were weak agonists compared with 5-HT, 5-MeOT and DOI, although the amplitude of the quipazine response was similar to DOI. This study demonstrates that the arylpiperazines, which are variably selective for the multiple brain 5-HT receptors, are all antagonists on the platelet 5-HT receptor. The antagonist activity is markedly increased by ring monosubstitution or aryl modification. Compared with the monosubstituted analogues, antagonist activity is decreased by heteroaromatic modification or by the addition of an N-aminophenethyl group to the 4-position nitrogen. Weak agonist activity can be conferred by heteroaromatic modification.

Elimination of ascorbic acid-induced membrane lipid peroxidation and serotonin receptor loss by Trolox-C, a water soluble analogue of vitamin E.

Ascorbic acid is commonly used as an antioxidant to prevent the decomposition of ligands in neurotransmitter receptor studies, but may alter biological membranes by initiating lipid peroxidation in the presence of physiologic metal ions. The aim of the present study was to characterize the effect of ascorbic acid-induced lipid peroxidation on an applicable membrane receptor and to examine an appropriate antioxidant system. Ascorbic acid generated significant lipid peroxidation (5.5 to 45 fold increase in malonaldehyde levels) in three diverse tissues having different membrane properties: bovine brain, mouse teratoma, and rat kidney. In membranes from bovine cerebral cortex, ascorbate-induced lipid peroxidation was associated with a 26% decrease in [3H]-serotonin receptor binding (Bmax = 159 +/- 11 from control of 216 +/- 10 fmol/mg protein), with no significant change in KD. Trolox-C, a water soluble analogue of vitamin E, completely blocked the ascorbate-induced loss of serotonin receptor binding in brain membranes, and the combination of Trolox-C and ascorbate prevented [3H]-serotonin decomposition in solution. Trolox-C also prevented ascorbate-induced lipid peroxidation in brain, teratoma, and kidney membranes. Lipid peroxidation may be a significant factor in the ascorbate-induced alteration of brain membranes as reflected by reduced binding to serotonin receptors. The combination of Trolox-C (200 microM) and ascorbic acid (1.0 mM) maintains a protective environment for oxygen sensitive neurotransmitters while blocking the deleterious effects of ascorbic acid on lipid membranes.

Characterization of the primary photointermediates of Drosophila rhodopsin.

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.

Honeybee blue- and ultraviolet-sensitive opsins: cloning, heterologous expression in Drosophila, and physiological characterization.

The honeybee (Apis mellifera) visual system contains three classes of retinal photoreceptor cells that are maximally sensitive to light at 440 nm (blue), 350 nm (ultraviolet), and 540 nm (green). We performed a PCR-based screen to identify the genes encoding the Apis blue- and ultraviolet (UV)-sensitive opsins. We obtained cDNAs that encode proteins having a high degree of sequence and structural similarity to other invertebrate and vertebrate visual pigments. The Apis blue opsin cDNA encodes a protein of 377 amino acids that is most closely related to other invertebrate visual pigments that are thought to be blue-sensitive. The UV opsin cDNA encodes a protein of 371 amino acids that is most closely related to the UV-sensitive Drosophila Rh3 and Rh4 opsins. To test whether these novel Apis opsin genes encode functional visual pigments and to determine their spectral properties, we expressed them in the R1-6 photoreceptor cells of blind ninaE mutant Drosophila, which lack the major opsin of the fly compound eye. We found that the expression of either the Apis blue- or UV-sensitive opsin in transgenic flies rescued the visual defect of ninaE mutants, indicating that both genes encode functional visual pigments. Spectral sensitivity measurements of these flies demonstrated that the blue and UV visual pigments are maximally sensitive to light at 439 and 353 nm, respectively. These maxima are in excellent agreement with those determined previously by single-cell recordings from Apis photoreceptor cells and provide definitive evidence that the genes described here encode visual pigments having blue and UV sensitivity.

Patterning of the R7 and R8 photoreceptor cells of Drosophila: evidence for induced and default cell-fate specification.

Opsin gene expression in the R7 and R8 photoreceptor cells of the Drosophila compound eye is highly coordinated. We have found that the R8 cell specific Rh5 and Rh6 opsins are expressed in non-overlapping sets of R8 cells, in a precise pairwise fashion with Rh3 and Rh4 in the R7 cells of individual ommatidia. Removal of the R7 cells in sevenless, boss or sina mutants, disrupts Rh5 expression and dramatically increases the number of Rh6-expressing R8 cells. This suggests that the expression of Rh5 may be induced by an Rh3-expressing R7 cell, whereas Rh6 expression is most likely a default state of the R8 cell. We found that the paired expression of opsin genes in the R7 and R8 cells occurs in a sevenless and boss independent manner. Furthermore, we found that the generation of both Rh3- and Rh4-expressing R7 cells can occur in the absence of an R8 cell. These results suggest that the specification of opsin expression in the R7 cells may occur autonomously, whereas the R7 photoreceptor cell may be responsible for regulating a binary developmental switch between induced and default cell-fates in the R8 cell.