Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1003-1007, February 1990

Neurobiology

Isolation and structure of an arrestin gene from Drosophila (phototransduction/guanine nucleotide-binding protein/vision/desensitization)

DEAN P. SMITH, BIH-HWA SHIEH, AND CHARLES S. ZUKER Howard Hughes Medical Institute and Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, CA 92093

Communicated by Dan L. Lindsley, November 6, 1989 A Drosophila gene encoding a homologue of ABSTRACT vertebrate arrestin was isolated by subtractive hybridization and identified as a member of a set of genes that are preferentially expressed in the visual system. This gene encodes a 364-amino acid protein that displays >40% amino acid sequence identity with human and bovine arrestin. Interestingly, the Drosophila homologue lacks the C-terminal sequences that were postulated to interact with rhodopsin during the quenching of the phototransduction cascade in the vertebrate visual response. These findings are discussed in terms of invertebrate phototransduction. The Drosophila gene was mapped cytogenetically to chromosomal position 36D1-2, near the ninaD locus. However, the arrestin gene does not appear to be the ninaD locus, as sequence analysis of three ethylmethane sulfateinduced ninaD mutant alleles reveals no alteration in amino acid sequence.

photoresponse (10). In addition, a light-activated GTPase activity in the expected quantities and with the expected kinetics has been demonstrated in Drosophila, strongly suggesting the existence of a transducin homologue in this system (11). The mechanisms underlying receptor inactivation are not well understood in any of the known G protein-coupled transduction processes (12). However, a number of quenching mechanisms appear to operate in the visual cascade to turn off the activated intermediates (4, 13). For instance, in vertebrates, transducin, like other G proteins, has an endogenous GTPase activity that hydrolyzes bound GTP to GDP, thus inactivating the molecule. Additionally, rhodopsin, like other G protein-coupled receptors, is phosphorylated by a specific kinase in response to activation. Phosphorylation is thought to be involved in desensitization of the receptor. Interestingly, light-activated phosphorylated rhodopsin interacts stoichiometrically with a protein known as arrestin, 48-kDa protein, or S-antigen, which inhibits the ability of rhodopsin to interact with transducin in vitro (13-15). The existence of a similar biochemical activity has also been postulated for the inactivation of agonist-activated 1adrenergic receptor (16). The identification and availability of an arrestin gene from a system suitable for molecular, biochemical, and physiological manipulation would allow a detailed dissection of its precise role in G protein-coupled transduction processes. We now report the isolation and characterization of a Drosophila gene, Arr,* encoding an arrestin homologue. Arr displays >40% amino acid identity with the bovine and human sequences, except that the C terminus is 30 residues shorter than its mammalian counterparts. Surprisingly, this C-terminal sequence corresponds to the region of arrestin that is believed to interact with rhodopsin (17). These findings are discussed in terms of vertebrate and invertebrate phototransduction. The Drosophila arrestin gene has been independently cloned by Benzer and co-workers (18).

Phototransduction is the process that converts the energy of an absorbed photon into a change of the ionic permeabilities of the photoreceptor cell membrane. This light-induced change in ionic conductances gives rise to the receptor potential and synaptic activity of the photoreceptor cell. The mechanism of visual excitation in vertebrate photoreceptors is the best understood of all sensory transduction processes (1-4). Light activation of rhodopsin is the first step in the visual response. In the vertebrate, photoactivated rhodopsin molecules activate a guanine nucleotide-binding (G) protein, transducin, which in turn activates a cGMP phosphodiesterase. The reduction of intracellular levels of cGMP leads to the transient closure of a cGMP-gated cation-selective channel and hyperpolarization of the photoreceptor cell. Unlike vertebrates, the microvillar photoreceptors of invertebrates depolarize in response to light and thus open their cation-selective channels. The identity of the intracellular transmitter(s) that mediates excitation in invertebrates has eluded firm identification. Similarly, the enzyme cascade that triggers the visual response has not been defined. However, there is a large body of physiological, genetic, and biochemical work that has strongly implicated calcium and inositol phospholipid metabolism in excitation of dipteran and Limulus photoreceptors (5-7). It is believed that photoactivated rhodopsin interacts with a G protein, which in turn activates a phospholipase C. Phospholipase C would then catalyze the generation of the second messenger inositol 1,4,5-trisphosphate and the subsequent mobilization of calcium from intracellular storage sites. The transient increase in calcium levels (or inositol 1,4,5-trisphosphate) would then lead to the opening of a cation-selective channel and the generation of a depolarizing receptor potential. Strong support for this model was recently provided by the demonstration that the Drosophila no-receptor potential A (norpA) gene encodes a phospholipase C that is abundantly expressed in the adult retina (8, 9); severe mutations of this gene completely abolish the

DNA and RNA Isolations. RNA was isolated from adult heads, bodies, or whole flies from Oregon R strain at different developmental stages as described (19). Poly(A)+ RNA was isolated by affinity chromatography on oligo(dT)-cellulose columns (20). Drosophila genomic DNA was isolated by homogenization of whole flies in grind buffer (80 mM NaCl/ 160 mM sucrose/58 mM EDTA/0.5% sodium dodecyl sulfate/124 mM Tris, pH 9.2). Nucleic acids were precipitated in ethanol after addition of 8 M potassium acetate to a final concentration of 1 M. Subtractive hybridizations were carried out as described (21). Blotting and Hybridization of DNA and RNA. Poly(A)+ RNA was size-fractionated in a formaldehyde agarose gel and

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: G protein, guanine nucleotide-binding protein. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M30177). 1003

MATERIALS AND METHODS

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Proc. Natl. Acad. Sci. USA 87 (1990)

Neurobiology: Smith et al.

blotted onto nylon membrane or nitrocellulose paper as described by Maniatis et al. (22). DNA probes were labeled by random oligonucleotide priming (Amersham multiprime system). RNA ladders (BRL) were used as size markers. Hybridizations were carried out at 650C in 750 mM NaCI/100 mM NaH2PO4 (pH 6.8)/75 mM sodium citrate/0.04% bovine serum albumin/0.04% polyvinylpyrrolidone-40/0.04% Ficoll/0.5% sodium dodecyl sulfate. Filters were washed in 0.2x SSC (lx SSC is 150 mM NaCl/15 mM sodium citrate)/ 0.5% sodium dodecyl sulfate at 650C. The cDNA library was made from poly(A)+ RNA isolated from the heads of adult flies (0-24 hr after eclosion). DNA Sequencing and Primer Extension. DNA sequencing was carried out according to the chain termination procedure of Sanger et al. (23) by using Sequenase kits (United States Biochemical) with M13mp18 and -mpl9 as vectors. Primerextension analysis was performed on 1 jig of RNA from wild-type (Canton S) RNA using a 32P-labeled 19-mer oligonucleotide complementary to positions +43 to +24 (see Fig. 5). Extension was carried out with avian myeloblastosis virus reverse transcriptase (BRL) as described by Maniatis et al. (22). In Situ Hybridization to Polytene Chromosomes and Tissue Sections. Polytene chromosome squashes (Canton S strain) were prepared as described (19). Hybridization with biotinylated DNA probes were carried out according to LangerSofer et al. (24) with the following modifications: DNA was nick-translated by using Bio-16 dUTP (Enzo Biochemicals), and hybrids were detected by using a Detek-I-HRP detection kit (Enzo Biochemicals). Preparation of 8-,um frozen sections and hybridization of 3H-labeled probes were as described by Hafen et al. (25), except that the Pronase treatment was omitted in the pretreatment of the sections. Analysis of Arrestin Gene in ninaD Alleles. The Arr gene from flies homozygous for three different ninaD mutant alleles (ninaL963, ninaD246, and ninaDTH382) was amplified by the polymerase chain reaction (26). Briefly, oligonucleotides corresponding to nucleotides +23 to +40 and complementary to nucleotides 2026 to 2009 (see Fig. 5), each with attached EcoRI linkers flanking the sequence, were used to prime the amplification of Arr from genomic DNA. The amplification cycle consisted of denaturation for 2 min at 94°C, hybridization for 2 min at 42°C, and synthesis for 10 ;min at 72°C. The amplified DNA was digested with EcoRI and subcloned into M13mpl8 and -mpl9 single-stranded vectors. At least three independent clones were analyzed for each mutant to avoid errors occurring during the amplification of DNA.

RESULTS AND DISCUSSION Isolation of A36, a Drosophila Arrestin Gene. To isolate genes that are likely to encode proteins involved in the phototransduction process, we used a subtractive hybridization protocol to identify genes that are preferentially expressed in the compound eyes of Drosophila melanogaster (21). Our strategy was based on the availability of mutant Drosophila strains carrying a complete loss of the adult compound eye (eyes absent: eya) (27) and the finding that a large number of the components of the visual transduction cascade appear to be encoded by visual system-specific transcripts rather than by ubiquitously expressed genes (19, 21, 28-30). Genomic clones selected in our screen were used as probes for in situ hybridization to salivary gland chromosomes to determine their cytogenetic map positions. Two of 120 eye-specific clones we isolated (Fig. 1) contained overlapping sequences that encoded a very abundant 1.6 kilobase (kb) transcript (Fig. 2). To determine the specific sites of expression of this gene we determined the spatial distribution of its mRNA by in situ hybridizations to tissue sections of the wild-type head. Fig. 3 shows that RNAs homologous to A36 are specifically localized in the photoreceptor cells, both in

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FIG. 1. Restriction map of A36 and structure of the RNA it encodes. (Upper) Map of A36, indicating EcoRI and BamHI restriction sites. (Lower) In expanded view, the region of the 3.0-kb fragment that encodes the RNA. The structure of the RNA was deduced by comparison of the cDNA and genomic sequences. Inverted triangles indicate the location and size of introns.

the retina and the ocelli. To determine the developmental profile of expression of A36, poly(A)+ RNA was isolated from wild-type Oregon R flies at different times during development, fractionated on an agarose-formaldehyde gel, and hybridized to a labeled 0.85-kb BamHI-EcoRI fragment of A36. Fig. 2 shows that the 1.6-kb RNA species accumulates only by late pupation (8 days) and reaches maximum levels after eclosion (10 days). Interestingly, this is the time when most genes involved in phototransduction are first transcribed (19, 21, 28-30). Clone A36 was mapped by in situ hybridization to polytene chromosomes to position 36D1-2 (Fig. 4), within the cytogenetic location of the ninaD gene (31). The ninaD locus is one of eight known complementation groups that drastically affect rhodopsin levels in Drosophila (ninaA ... ninaH, neither-inactivation-nor-afterpotential) (10, 31, 32). ninaA encodes a photoreceptor cell-specific prolyl-cis-trans isomerase that may be involved in rhodopsin folding (21, 33); ninaC encodes a cytoskeletal component of the photoreceptor rhabdomeres (30, 32), and ninaE is the structural gene for the rhodopsin expressed in the major class of photoreceptor cells (19, 34). Both ninaB and ninaD encode proteins likely to be involved in chromophore metabolism; ninaB mutant flies can be rescued by supplementing food with retinal, whereas ninaD flies can be rescued by supplementing food with many vitamin A derivatives (31). ..

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wild-type adults. We also isolated RNA from the heads of ninaL946 flies. (Upper) The RNAs (3 jig per lane) were size fractionated, blotted, and hybridized as described. A36 hybridizes to a 1.6-kb transcript (arrow) that is first detectable at day 8, during late pupation. (Lower) The blot was also hybridized to an actin probe to control for integrity of the RNA. An RNA ladder (BRL) was used to size markers.

Neurobiology: Smith et al.

1005

Proc. Natl. Acad. Sci. USA 87 (1990)

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FIG. 3. Spatial localization of arrestin RNA by in situ hybridization to tissue sections of adult heads. Frozen sections of adult fly heads were hybridized to a nick-translated Arr cDNA (exposure, 21 day). (A) Bright field view; (B) darkfield view. re, Retina; la, lamina ganglionaris; oc, ocelli; br, brain. Note that Arr is expressed specifically in photoreceptors in both the compound eye and ocelli.

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FIG. 4. In situ hybridization to salivary gland chromosome squashes. A36 was biotinylated as described and used as a hybridization probe to determine its chromosomal location. Shown is the 36 region of the second chromosome of Drosophila melanogaster (Canton S). The arrow indicates the site of hybridization at 36D1-2; no other sites of hybridization were observed.

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A36 Encodes an Arrestin Homologue. We used the 0.85-kb BamHI-EcoRI fragment of A36 (see Fig. 1) to screen a Drosophila head cDNA library and isolated several cDNA clones. Using M13 dideoxynucleoside triphosphate sequencing we have determined the nucleotide sequence of one of those cDNA clones and of the 3.5-kb genomic fragment of A36 (Fig. 1). Fig. 5 shows the nucleotide sequence and the deduced amino acid sequence of the A36 gene. The Drosophila arrestin gene is divided into four exons by three introns of 421, 233, and 60 nucleotides, respectively. The structure of the RNA (Fig. 1) and the position of the start of transcription

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were determined by comparison of genomic and cDNA sequences and by performing primer-extension analysis. Comparison of the deduced amino acid sequence of A36 with previously sequenced genes and proteins revealed very high homology to the bovine retinal arrestin protein (Fig. 6). These two proteins display >40% amino acid identity throughout their length (shaded residues in Fig. 6); conserved substitutions account for >17% of the remaining amino acid residues (unshaded boxes in Fig. 6). Arrestin, also known as S-antigen or 48 kDa-protein, is a very abundant soluble protein found in the vertebrate retinal and pineal photoreceptors (35, 36). Human and bovine S-antigen are very immunogenic and have been linked to vision-threatening ocular autoimmune disorders (37, 38). Recently, Kuhn and

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Neurobiology: Smith et al.

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Proc. Natl. Acad. Sci. USA 87 (1990)

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co-workers (14, 15) showed that photoactivated rhodopsin is phosphorylated by rhodopsin kinase and that arrestin binds to light-activated, phosphorylated rhodopsin, thereby inhibiting or "arresting" its ability to interact with transducin. Indeed, the existence of an activity involved in deactivating ligand-activated receptors after removal of the stimulus had long been postulated (39). Interestingly, the predicted amino acid sequence of both human and bovine arrestins revealed regions of amino acid sequence homology with the a subunit of transducin, suggesting that these proteins may compete for a common binding site on rhodopsin (17, 40-42). Therefore, phosphorylation of rhodopsin and subsequent arrestin binding have been postulated to be the mechanism responsible for terminating the active state of rhodopsin. More recently, Benovic et al. (16) have shown that inactivation of activated ,3-adrenergic receptor, another G protein-coupled receptor, can also be significantly enhanced by retinal arrestin and, thus, postulated the existence of a similar protein in nonretinal tissue. While significant differences exist between vertebrate and invertebrate phototransduction cascades, the initial events are very similar; therefore, mechanisms involving the regulation of these initial steps might be conserved. The identification of a highly conserved Drosophila homologue of arrestin supports this conjecture. Vertebrate and invertebrate rhodopsins are expected to interact with at least three cytoplasmic proteins: a G protein, rhodopsin kinase, and

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arrestin (4). It is interesting to note that vertebrate and invertebrate arrestins are far more conserved when compared with each other (42% amino acid identity; see Fig. 6) than rhodopsins (-22% identity between the major Drosophila opsin and bovine rhodopsin) (29). These findings suggest that arrestin recognizes a structural motif in rhodopsin rather than primary sequence. In this regard, the major difference between the vertebrate and invertebrate arrestin molecules is the lack of the C-terminal 30 residues in the Drosophila protein, which contains the domain of homology with a-transducin in the human and bovine proteins. Thus, if termination of the active state of rhodopsin is similar in both phototransduction cascades and is carried out by competition between arrestin and transducin, it is not likely to be mediated exclusively by interactions in this C-terminal domain. Finally, Zuckerman and Cheasty have postulated that arrestin functions at the level of the cGMP phosphodiesterase in the vertebrate visual cascade (43, 44). However, the finding of a highly conserved arrestin in a visual system that most likely lacks cGMP phosphodiesterase as the effector molecule may argue against such a mechanism. Arrestin Is Not Encoded by the ninaD Locus. To determine whether Drosophila arrestin is encoded by the ninaD locus, we used the polymerase chain reaction to amplify the entire arrestin genomic sequence from three independent ninaD ethylmethane sulfate-induced alleles, ninaD246, ninaD263, and ninaDTH382 (31). Thus, if arrestin were encoded by the

Neurobiology: Smith et aL ninaD gene, sequence alterations would be expected in these mutant DNAs. We determined the complete DNA sequence of all coding exons from the amplified Arr gene from all three alleles in homozygous flies with multiple independent polymerase chain reactions. Analysis revealed no coding differences from wild-type controls (data not shown). In addition, we examined the expression profile of arrestin transcript in ninaL)i46 mutant flies and observed no differences in either its size, abundance, or time ofexpression (Fig. 2). Therefore, we conclude that Drosophila arrestin is probably not encoded by the ninaD locus, and that the 36D chromosomal region contains at least two genes encoding products involved in visual transduction. The existence of chromosomal rearrangements and nearby marked transposable elements (45) should now permit the generation of mutants lacking the arrestin

gene. CONCLUDING REMARKS We have isolated a Drosophila arrestin gene by a protocol involving subtractive hybridization, the generation of eyespecific probes, and subsequent identification of photoreceptor-specific genes. Drosophila arrestin is 364 residues long and displays 42% amino acid identity with the bovine and human proteins. Diptera and mammals are separated by >500 million years of evolution (46). Thus, the amino acid sequence conservation between Drosophila and human arrestin is likely to reflect the presence of common events in both transduction cascades. The availability of an arrestin gene from a system suitable to genetic and molecular manipulation will allow detailed and precise characterization of the role of arrestin in the visual cascade and its possible involvement in other G protein-coupled receptor transduction processes. We thank Suzanne Seveallo for her expert technical assistance. We also thank William Pak for stocks ninaD'46 and ninaD163, and Robert Stephenson for the stock ninaDTH382. We give particular thanks to Seymour Benzer, David Hyde, John Pollock, and Kirk Mecklenburg for communicating their results before publication and for helpful comments. Finally, we thank the members of this laboratory for comments and suggestions. This work was supported by grants from the National Institutes of Health to C.S.Z. D.P.S. is a National Institutes of Health predoctoral trainee. C.S.Z. acknowledges support from the McKnight Endowment Fund for Neuroscience, the Sloan Foundation, and the Pew Scholars Program in Biomedical Sciences. C.S.Z. is an Investigator of the Howard Hughes Medical Institute. 1. Fein, A. & Szuts, E. Z. (1982) Photoreceptors: Their Role in Vision (Cambridge Univ. Press, Cambridge, U.K.), p. 212. 2. Kuhn, H. (1984) in Progress in Retinal Research, eds. Osborne, N. & Chader, J. (Pergamon, Oxford, U.K.), Vol. 3, pp. 124-156. 3. Stieve, H. (1986) The Molecular Mechanism ofPhotoreception Dahlem Konferenzen, 1986, ed. Stieve, H. (Springer, Berlin), pp. 1-10. 4. Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119. 5. Payne, R. (1986) Photobiochem. Photobiophys. 13, 373-397. 6. Fein, A. (1986) Trends Neurosci. 93, 110-114. 7. Devary, O., Heichal, O., Blumenfeld, A., Cassel, D., Suss, E., Barash, S., Rubinstein, C. T., Minke, B. & Selinger, Z. (1987) Proc. Natl. Acad. Sci. USA 84, 6939-6943. 8. Yoshioka, T., Inoue, H. & Hotta, Y. (1985) J. Biochem. 97, 1251-1254. 9. Bloomquist, B., Shortridge, R., Schneuwly, S., Perdew, M., Montell, C., Steller, H., Rubin, G. & Pak, W. (1988) Cell 54, 723-733. 10. Pak, W. L. (1979) in Neurogenetics: GeneticApproaches to the

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