Cloning and Characterization of Human Homologueof Drosophila Retinal Degeneration B: ACandidateGene for Degenerative Retinal DiseasesJING GUO AND FUSHIN X. YU*The Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts

ABSTRACT Mutations in the Drosophila retinaldegeneration B (D-rdgB) gene cause light-enhancedretinal degeneration. Here, we report the isolation of thecDNA encoding human homologue of the D-rdgB andinitial characterization of the gene products. Like D-rdgB,the human rdgB homologue (H-rdgB) is a transmembraneprotein with the N-terminus sharing high homology totwo closely related cytosolic proteins, phosphatidylinosi-tol transfer protein (PITP) a and b, indicating that rdgBlike proteins belong to the family of PITP proteins. UsingNorthern and Western blotting, we demonstrated thatthe rdgB homologue is expressed in rat retina, olfactorybulb, and brain, but not in nonneuronal tissues. In the ratretina, immunoreactivity of the rdgB homologue wasobserved in photoreceptors and throughout the innernuclear and plexiform layers; the strongest staining wasin the inner plexiform layer. In the photoreceptor cells, therdgB homologue was located primarily in the innersegment where sorting and traffic of membranes re-quired for outer segment assembly take place. Thesedata, together with recent findings showing PITPs as animportant component of intracellular membrane trafficapparatus in mammalian cells, suggest that rdgB homo-logue may play a role in photoreceptor membranerenewal and in neurotransmitter release. Furthermore,using somatic hybrid cell hybridization and fluorescencein situ hybridization H-rdgB gene was mapped to humanchromosome 11q13, a region known to contain severalretinopathy loci, including Best disease and Bardet-Biedlsyndrome I. Therefore, H-rdgB gene is an attractivecandidate for several inherited retinal degenerativediseases. Dev. Genet. 20:235–245, 1997.r 1997 Wiley-Liss, Inc.

Keywords: human homologue of retinal degenerationgene B; retinal function; membrane traffic; retinal degen-eration

INTRODUCTIONRetinitis pigmentosa (RP) is a progressive retinal

dystrophy that affects 1 in 3,000 of the general popula-tion and shows a high degree of genetic heterogeneity[Dryja and Li, 1995; Heckenlively, 1988]. Autosomal

dominant, autosomal recessive, X-linked, andmitochon-drial inheritance patterns have been described for RP[Dryja and Li, 1995]. Among the dozens of RP lociknown to exist, only a handful have been identified; theremainder are inferred from linkage studies. Theseidentified loci include rhodopsin, peripherin/RDS, ROM1[for review, see Dryja and Li, 1995], rod cGMP-gatedcation channel a subunit [Dryja et al., 1995], a and bsubunits of cGMP-phosphodiesterase [Gal et al., 1994;Huang et al., 1995], and RP GTPase regulator [Meindlet al., 1996]. RP also occurs as a clinical feature in anumber of genetic syndromes. Some of the causal geneshave been identified, including an ‘‘unconventional’’myosin VIIa in Usher syndrome type IB [Weil et al.,1995], amicrosomal transfer protein in abetalipoprotein-aemia [Shoulders et al., 1993], and a rab transferase inchoroideremia [Seabra et al., 1993]. However, despitethese findings, the causes for the majority of RP casesare still unknown.Drosophila is an ideal experimental model system

that uses advanced genetic techniques for identifyingand characterizing visual phototransduction compo-nents in this species [Pak, 1995; Wu et al., 1995].Several genes essential for the retinal function, includ-ing opsin, phospholipase C, protein kinase C, andretinal degeneration-B (rdgB), have been identified bythe electroretinogram-defective mutants [Hotta andBenzer, 1970; Pak, 1995; Pak et al., 1970]. The Dro-sophila rdgB (D-rdgB) mutant was initially identifiedby defects in the compound eye, in which rdgB mutantflies undergo light-enhanced retinal degeneration [Hottaand Benzer, 1970]. At the amino acid (aa) level, D-rdgBcontains a putative Ca21 binding site and six hydropho-bic segments, likely to be membrane-spanning domains[Vihtelic et al., 1991, 1993]. Interestingly, the amino-terminal 281 aa residues of D-rdgB are .40% identicalto the mammalian phosphatidylinositol transfer pro-teins (PITPs) [Dickeson et al., 1989; Tanaka et al., 1995;

Received for publication 8 November 1996; accepted 12 December1996.

*Correspondence to: Fushin X. Yu, The Schepens Eye ResearchInstitute, 20 Staniford Street, Boston, MA02114.

DEVELOPMENTAL GENETICS 20:235–245 (1997)

r 1997 WILEY-LISS, INC.

Vihtelic et al., 1993]. PITPs are a group of cytosolicproteins (32–36 kDa) ubiquitously expressed in mam-mals [Wirtz, 1991]. Recent studies suggested that PITPs,through regulation of phosphatidylinositol 4,5-bisphos-phate (PIP2) synthesis, are essential for phospholipaseC-dependent signal transduction and intracellularmem-brane traffic [Hay and Martin, 1993; Kauffmann et al.,1995; Liscovitch and Cantley, 1995]. In Drosophilaphotoreceptor cells, rdgB immunoreactivity was concen-trated in the membranes of subrhabdomeric cisternae(SRC) adjacent to the plasma membranes at the basesof photoreceptive microvilli comprising the rhabdomere[Vihtelic et al., 1993]. The SRC is composed of elaborateextension of the endoplasmic reticulum running thelength of the photoreceptor and likely to play a role inrhabdomere maintenance by transporting membraneproteins and/or phospholipids to the rhabdomericmicro-villi [Mutsumoto-Suzuki et al., 1989]. Recently, D-rdgBwas suggested to be part of the mechanism of mem-brane turnover in the photoreceptors of the compoundeyes [Yoon et al., 1996].The potential role of D-rdgB in retinal function and

the photoreceptor degeneration associated with muta-tions of this gene promoted us to search for humanhomologue of the gene. In this work, we report theisolation of cDNAfrom the human retina encoding rdgBhomologue and provide evidence that the rdgB homo-logue may function in intracellular membrane traffic.Furthermore, we located rdgB homologue gene in hu-man chromosome 11q13, a region known to containseveral retinopathy loci, identifying human rdgB homo-logue as a candidate gene for these inherited retinaldiseases.

MATERIALS AND METHODS

cDNALibraries and PCRAssays

Approximately one million clones were plated outfrom an oligo(dT) and random-primed adult retinallibrary (Clontech HL1132a). The library was screenedby filter hybridization using reverse transcription (RT)-polymerase chain reaction (PCR) generated cDNA frag-ment. The RNA used for RT was isolated from theretina of a 65-year-old male with no known retinaldiseases. The 58 extension of cDNA cloning was basedon RT-PCR and genomic DNA sequencing. The 58

primer was degenerate oligonucleotide: GGI CA(AG)TA(CT) ACI (AC)AI AA(AG) AT(ACT) TA(CT) CA, theparentheses represent positions with mixed bases, and‘‘I’’s deoxyinosine that can form base-pair with all fourdeoxynucleotides. The 38 primer was GGTTGTAGATC-TGTTCAC. The PCR was performed with the cDNAworking mixture in a 50 µl reaction volume containing20 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200µM dNTP, specific 58 and 38 primers (10 pmol each), and1 unit of Taq DNA polymerase. Amplification wascarried out in Biometra Personal Cycler programmed35 cycles of 94°C/45 sec, 56°C/2 min, and 72°C/2.5 min.

PCR product (termed PCR-1) was cloned into pCR-IIplasmid. The sequence comparison of human gnomicDNA with D-rdgB cDNA was performed by Bestfit (aGCG program).

DNASequencing

For cDNA, the sequencingwas carried out by dideoxy-chain termination using alkaline-denatured, double-stranded DNA templates (in Bluescript SKII or pCR-IIvectors, 3–4.5 µg) and sequenase vision II (USBiochemi-cal). Sequencing primers included T7, T3, and SKprimers, as well as synthesized oligonucleotides corre-sponding to the already sequenced region of the DNA.Both strands were sequenced. For genomic DNA, 1 µgof a P1 clone 114G23 (see below) was directly sequencedwith Taq DNA polymerase in the presence of 35S-dATPand 30 cycles of 94°C/30 sec, 55°C/30 sec and 72°C/90sec.

Northern Blot Analysis

Total RNA was isolated from rat tissues by theguanidine thiocyanate/phenolmethod [Chomczynsk andSachh, 1987] and separated on a 1% agarose gel andblotted onto GeneScreen Plus membrane (NEN). Hy-bridization was carried out with a portion of rat rdgBhomologue cDNA. This cDNA fragment (correspondingto 2594–3186 nucleotide sequence shown in Fig. 1) isgenerated by RT-PCR using primers derived from hu-man rdgB cDNA sequence. The nucleotide sequence ofrat rdgB cDNA in this region displays 96% sequenceidentity to that of human rdgB homologue. The probefor hybridization were prepared using a random primerlabeling kit (United States Biochem). The hybridizationwas carried out overnight and washing was done understringent conditions (final wash with 0.13 SSPE/0.1%SDS at 65°C).

Chromosome Location

Two approaches, somatic hybrid cell hybridizationand fluorescence in situ hybridization (FISH), wereused for chromosome mapping. In a somatic hybrid cellhybridization experiment, PCR-1 cDNA was used toprobe polychromosomal cell hybrid blots of DNA thatwas from hybrid cell lines (rodent background) contain-ing human chromosomes and cut with restriction en-zyme Eco R1. Control panels are human and mouseDNA digested with the same enzyme. For FISH experi-ments, PCR-1 cDNA was used as a probe to screen ahuman P1 genomic library constructed from the humanlymphoblastoid cell line GM1416B [Pierce et al., 1992;Shepherd et al., 1994]. Two clones, 114G23 and 33M19,were isolated. Clone 114G23 and D11Z1 were used forprobing human rdgB gene and chromosome 11 centro-mere and labeled by nick translation with digoxigenindUTP or biotin, respectively. The labeled probes werehybridized to normal metaphase chromosomes derivedfrom PHAstimulated peripheral blood lymphocytes.

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Fig. 1. Domain structure and deduced amino acid sequence ofhuman rdgB homologue. A. Diagram of H-rdgB homologue domainstructure. Two domains at N-terminal (N), PITP and acidic, aremarked. Numbers, membrane spinning sequence; I-L, inner loop; O-L,Outer loop. B. The nucleotide (nt) and its predicted aa sequences ofH-rdgB. Translation initiation methionine is at nt and aa positions 1,stop codon at nt 3633, polyadenylation signal at nt 3978 (boldface),and possible polyadenylation site starting 3999. There is an AUUUA

motif (underlined) within the 38 untranslated region (UTR), which hasbeen correlated with short-lived transcripts [Wennborg et al., 1995].Atthe aa level, six putative transmembrane (TM) domains, beginning ataa 479, 555, 688, 996, 1022, 1130, and 1166, are overlined. Thepotential N-linked glycosylation sites, located at aa positions 127, 344,915, and 976, are indicated by stars (*) above the N residues. The aasequence (aa 56–64) used for designing degenerate primers wasunderlined. Open arrow (aa 271): end of PITP domain.

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In Situ Hybridization

In situ hybridization was performed as described [Yuet al., 1995]. A rat rdgB homologue cDNA fragment wasused as a template for the synthesis of a-35S-UTP-labeled cRNA probes. The size of the probes wasadjusted to ,150 bp by treating cRNAs with 0.2 Msodium carbonate, pH 10.2 for 42 min at 60°C. Cryosec-tions (6 µm) of adult mouse eye were fixed with 4%paraformaldehyde, treated with 1 µg/ml proteinase Kand then acetylated. RNAprobe (0.2 µg/ml per kilobase)was added to 60 µl hybridization solution containing0.3 M NaCl, 10 mM Tris-HCI (pH 7.6), 5 mM EDTA,0.02% (wt/vol) Ficoll 400, 0.02% (wt/vol) polyvinylpyrol-lidine, 0.02% (wt/vol) BSA, 50% deionized formamide,10% dextran sulfate, 10 mM dethiothretiol, and 0.1mg/ml yeast tRNA. Hybridization was carried out in ahumid chamber overnight at 42°C. After removal ofcover-parafilm, sections were washed, treated with 10µg/ml RNase A, and washed again to remove unboundprobes. The sections were exposed to Kodak NTB-2Emulsion, developed, stained with hematoxylin andeosin, viewed, and photographed under brightfield anddarkfield illumination.

Preparation of Polyclonal Antibodies

The polyclonal antibodies were raised against recom-binant polypeptides of human rdgB homologue. ThePCR-1 cDNA fragment was subcloned into pET28cplasmid and the recombinant protein was expressed inE. coliBL21(DE3)pLysS in the presence of isopropylthio-b-D-galactoside. This recombinant protein had appar-ent molecular weight ,80 kDa (aa residues 58–771plus fused plasmid-encoded aa residues). The presenceof six histidine residues (His-tag) fused to N-terminusof the rdgB fragment allows affinity purification byNi21

column from inclusion bodies. Purified recombinantpolypeptides (1.2 mg/ml PBS) was mixed with an equalvolume of complete Freund’sAdjuvant and injected intotwo rabbits (1 ml each) at multiple subcutaneous sites.The animals were boosted with the same amountantigen mixed with incomplete Freund’s Adjuvant. Thesera of second boost and thereafter were collected andassayed for immunoreactivity by immunoblot analysiswith rat retinal extract described below.Affinity purification of rdgB homologue specific anti-

bodies was performed by incubation of the antiserumwith 80 kDa H-rdgB fragment blotted nitrocellulose[Harlow and Lane, 1988]. The inclusion bodies contain-ing 80 kDa recombinant polypeptides were subjected toa 7% SDS-PAGE and transferred onto nitrocellulose.The 80 kDa band was visualized by staining of thenitrocellulose with ponceau S and excised. The excisedstrip, after air dry, was blocked with 1% BSA and thenincubated with anti-rdgB homologue serum. The boundantibodies was eluted in 0.1 glycine solution (pH 2.3)and neutralized with 0.5 M Tris/HCl (pH 7.4).

Immunoblot Analysis

Rat tissues were homogenized in the RIPA buffer[Harlow and Lane, 1988] containing 5 µg/ml pepstatin,5 µg/ml leupeptin, 1 µg/ml aprotinin, and 0.25 mMphenylmethylsulfonyl fluoride. The homogenate wascentrifuged at 10,000 3 g to remove insoluble debris.Protein concentration was determined using PierceMicro BCA Protein Assay kit. Samples (20 µg protein)were adjusted to 13 Laemmli buffer and separated on7% SDS-polyacrylamide gels. Proteins were trans-ferred to a nitrocellulose membrane. The membranewas blocked for 1 hr with 1% goat serum, 5% BSA inTris-buffered saline with 0.5% Tween-20. Blots wereincubated with 1:2,000 dilution of polyclonal antibodiesraised against human rdgB for 1 hr at room tempera-ture, followed by incubation of horseradish peroxidase-coupled secondary antibodies. The immunoreactivitywas detected by enhanced chemiluminescence.

Immunohistochemical Analysis

Eye cups were fixed in 4% paraformaldehyde in 0.1 Msodium phosphate buffer (pH 7.4) containing 1 mMCaCl2. The fixed eye cups were embedded in 3% gelatinand 30% egg albumen and cryostat sectioned (10 µm).The sections on gelatin-coated slides were incubatedwith 2% normal goat serum and 1% (wt/vol) bovineserum albumin (BSA) in PBS for 10 min, and thenincubated with affinity purified antibody for 1 hr atroom temperature in a moist chamber. After rinsingwith PBS three times, the slides were incubated for 1 hrat room temperature with fluorescein isothiocyanate-conjugated goat antirabbit IgG (Jackson Laboratories,West Grove, PA). After washing, coverslips weremounted with a medium containing 0.1 M Tris, pH 8.5,1/4 (vol/vol) glycerol, 10% (wt/vol) Mowiol, and 2.5%(wt/vol) 1,4-diazobicyclo-[2.2.2]-octane. Negative con-trols include incubation of tissue sections with second-ary antibodies alone and with serum depleted of rdgBhomologue specific antibodies. The fluorescence stain-ing was viewed with a confocal microscope.

RESULTS

Cloning and Characterization of Human rdgBHomologue cDNA

In an attempt to isolate the gene encoding humanhomologue of D-rdgB, the reported cDNA sequence ofD-rdgB [Vihtelic et al., 1991] was used to scan theGenbank database for homologous sequences. As wellas obtaining matches for mammalian phosphatidylino-sitol transfer proteins (PITPs) [de Vries et al., 1994;Dickeson et al., 1989; Tanaka et al., 1995], we noted amatch with an anonymous 280 base pair human braincDNA entry (Genbank accession number Z44552, re-leased Nov 6, 1994). This match, which was significant,led us to reason that Z44552 was a small piece of cDNAencoding human homologue of D-rdgB protein. In order

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to characterize this homologue, we set out to isolatefulllength cDNAs for this rdgB-like gene. A humanretinal cDNA library was screened with a probe consist-ing of PCR amplified Z44552. Six positive clones se-quenced covered 1999 bp of 38 end plus a poly(A) tail. Toclone the 58 sequence, a degenerate primer was de-signed from aa sequence GQYT(K/H)KIYH, which wasfound in the D-rdgB and all soluble mammalian PITPproteins; together with a 38 primer designed from thecloned cDNA sequence, a 2019 bp cDNA, correspondingto the sequence encoding aa residues 56–769 (Fig. 1),was generated from human retinal RNA by RT-PCR.The cDNA sequence encoding aa residues 1–56 shownin Figure 1 was derived from genomic DNAsequence bycomparing sequence of human rdgB gene with that ofD-rdgB cDNA. Each of the junctions of three cDNAfragments was verified by RT-PCR from human retinalcDNA templates and subsequent sequencing.The human rdgB cDNA has 3998 bp with an open

reading frame of 3732 bp, consistent with a conceptualtranslation product of 1244 amino acids with a calcu-lated molecular mass 135.6 kDa and pI 5 6.04 (Fig. 1).The deduced aa sequence from cloned cDNA is homolo-gous to D-rdgB in overall structure as well as aasequence. Like D-rdgB, human rdgB homologue (H-rdgB) can be divided into four regions: a N-terminaldomain with PITP activity, an acidic region (208 aaresidues with PI 5 4.98), transmembrane region con-taining six tentative membrane spinning sequences,and a small C-terminal region. Table 1 shows theresults of sequence comparison of the H-rdgB PITPdomain with D-rdgB and human cytosolic PITP mol-ecules. Both rdgB proteins from Drosophila and fromhuman display .40% identity and ,60% similarity inaa sequence to human cytosolic PITP, indicating thatthey aremembers of the PITP family. Sequence compari-son (Table 1) also revealed that the 64% identity in aasequence between H-rdgB and D-rdgB in PITP domainis substantially higher than that between H-rdgB andcytosolic PITPa (44.3%) or b (42.1%) and that humanPITP proteins have similar homology to both humanand Drosophila rdgB gene products. These imply thatH-rdgB and human PITP may have evolved fromdifferent genes. Whereas the non-PITP regions of H-

rdgB and D-rdgB share high homology (ranging from25.4% in the acidic region to 45.4% in the transmem-brane region of sequence identity), they show no signifi-cant homology to any known proteins deposited in thedatabase compared by BLAST programs. Taken to-gether, we concluded that the cloned cDNA encodes ahuman homologue of D-rdgB.Four putative asparagine-linked glycosylation sites

(Fig. 1) and numerous potential phosphorylation sites(by protein kinases A, B, C, and tyrosine kinases, notshown) were identified in the deduced aa sequence ofH-rdgB, suggesting possiblemeans of functional regula-tion.

Chromosome Location of Human rdgB Gene

Using somatic hybrid cell hybridization, human rdgBspecific hybridization band was found in the somaticcell hybrid lines containing human chromosome 11(data not shown). In FISH experiments, hybridizationwith a P1 clone, 114G23 containing the entire humanrdgB homologue gene, resulted initially in specificlabeling of the long arm of a group C chromosome. Asecond experiment was conducted in which a biotin-labeled probe specific for the centromere of chromosome11 (D11Z1) was co-hybridizedwith clone 114G23, result-ing in the specific labeling of the centromere (red,arrowhead) and 114G23 (green, arrow) in the long armof chromosome 11 (Fig. 2A). Measurements of 10 specifi-cally hybridized chromosomes 11 demonstrated that114G23 is located at a position that is 20% of thedistance from the centromere to the telomere of chromo-some arm 11q, an area that corresponds to chromosome11q13 (Fig. 2B), consistent with the recently reportedlocation of an expression sequence tag (EST) thatshares significant homology D-rdgB and is part ofH-rdgB [Banfi et al., 1996].

Expression of rdgB Homologuein Mammalian Tissues

To investigate the expression of rdgB homologue inmammalian tissues, Northern and Western blot analy-ses were carried out on samples prepared from a varietyof rat tissues. Northern blotting of a rat rdgB homo-logue (R-rdgB) cDNA fragment (Fig. 3) showed that theR-rdgB messages, coincident with the 28s rRNA band(,4.2 kilobases in size), were detected in the brain,retina, and olfactory bulb, but not in other tissues,including kidney and liver. Detection of rdgB homo-logue transcripts in rat brain is consistent with the factthat several ESTs with identical sequence were identi-fied by the WashU-Merck EST project from a humanbrain cDNA library. For Western blotting, antibodiesagainst human rdgB recombinant protein (correspond-ing to aa residues 56–769) were raised in the rabbit. Asexpected, no immunoreactivity was seen in liver, kid-ney and intestine extracts (Fig. 4). A single band ofpolypeptides with an apparent molecular weight of

TABLE 1. Pairwise Comparison of AminoAcidSequences of rdgB PITP Domains and Cytosolic

PITP Proteins

Sequencesa Length%

Identity%

Similarity GapsH-rdgB 3 D-rdgB 281 63.636 76.030 1H-rdgB 3 H-PITPa 281 44.351 58.996 5H-rdgB 3 H-PITPb 271 42.083 61.250 6H-PITPa 3 H-PITPb 271 77.778 88.519 5D-rdgB 3 H-PITPa 281 41.353 63.534 6

aH 5 human, D 5 Drosophila.

RDGB HOMOLOGUE IN THE RETINA 239

,160 kDa was observed in the retina and olfactorybulb; in the brain, there were the major band of ,160kDa and two minor bands around 40 kDa. These twominor bands may correspond to cytosolic PITP isoformsa and b since out of 713 aa residues in the recombinantprotein used as antigen 214 were from PITP domain.Alternatively, they might be the degradated products ofthe 160 kDa protein. The apparent molecular weight ofdetected rdgB homologue in rat retina (,160 kDa) islarger than that predicted from deduced AA sequence(135.6 kDa), implying possible translational modifica-tion (e.g., glycosylation).

Cellular Localization of rdgB Homologuein the Retina

To evaluate rdgB homologue distribution in rat retina,in situ hybridization and immunohistochemistry wereperformed. In situ hybridization (Fig. 5) revealed thatrdgB homologue mRNA was present in cells fromretinal pigment epithelium to ganglion cell layer, butnot in the choroid layer. The immunofluorescence wascarried out with affinity purified antihuman rdgB anti-body. Strong staining throughout the inner nuclear andplexiform layers is consistent with that of neuronalelements, including that of amacrine and bipolar cells.In photoreceptor cells, the immunoreactivity of rdgBhomologue was seen primarily in the inner segment; nospecific staining was observed in the outer segment(Fig. 6). The confocal images of the inner segmentstaining of rat photoreceptor cells showed two patterns:a continuous distribution of low level diffuse stainingand strong staining within a portion of a few cells

scattered over the region with diffuse staining. Thestrong rdgB homologue staining was within the innersegment or occasionally in the distal side of the outernuclear layer adjacent to the inner segment (Fig. 7).Due to autofluorescence, it is not clear if RPE waspositive for rdgB homologue staining at the presenttime.

DISCUSSIONWe have cloned the cDNA encoding the human

homologue of D-rdgB and determined its tissue expres-sion pattern and its cellular distribution in the retina.The H-rdgB displays high homology in aa sequence toD-rdgB, e.g., the N-terminal 270 aa residues of H-rdgBare 64% identical and 76% similar to the correspondingsequence of D-rdgB [Vihtelic et al., 1991]. Using North-ern andWestern blotting, we demonstrated that HrdgBis expressed in the rat retina, olfactory bulb, and brain.This expression pattern is very similar to that of theD-rdgB gene detected by Western blot [Vihtelic et al.,1993]. Thus considering the remarkable evolutionarydistance between humans and Drosophila, the overallconservation of aa sequence, domain structure, and theexpression pattern implies that the proteins encoded byrdgB genes play a critical role in retinal and neuronalcells.

Family of PITP Proteins

Recent studies have indicated that PITP is an essen-tial component of the polyphosphoinositide synthesis

Fig. 2. Fluorescence in situ hybridization of human 114G23 P1clone to human chromosomes. A. The entire metaphase spread, red(arrowheads): the centromere of chromosome 11 hybridized withD11Z1; green (arrows): human rdgB homologue hybridized with

114G23. B. Ideogram of chromosome 11; (2) indicates the possibleposition in which the hybridization signal was detected in a sample of10 banded chromosomes.

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machinery and promotes the synthesis of PIP2 in thecapacity of substrate presenter [Cunningham et al.,1995; Liscovitch and Cantley, 1995]. H-rdgB and cyto-solic PITPs (32–36 kDa) belong to a protein family andare products of different genes. Our study suggests thatthe rdgB homologue and cytosolic PITPs are evolvedfrom different genes andmay belong to different proteinsubfamilies. These subfamilies are likely evolved froman early ancestral gene through gene fusion or deletion.Proteins in the cytosolic PITP subfamily are ubiqui-tously expressed [Wirtz, 1991] and are likely to play ageneral role in controlling cellular concentration ofPIP2 [Cunningham et al., 1995; Liscovitch and Cantley,1995]. However, limited tissue distribution and mem-brane association imply a specialized role for the rdgBsubfamily of proteins. We hypothesize that rdgB pro-teins are adapted to a unique functional need of photo-receptor and neuronal cells.

Functioning in Signal Transduction and/orIntracellular Membrane Traffic

PITP may regulate the rate of IP3 production andgrowth factor signaling by controlling PIP2 synthesis[Cunningham et al., 1995; Liscovitch and Cantley,1995]. In Drosophila photoreceptor cells, light-excitedrhodopsin activates a G-protein [for recent review, seePak, 1995], followed by a phospholipase C (PLC)-dependent signal transduction pathway to generatesecond messengers: IP3 and diacylglycerol. In contrast,phototransduction in vertebrate photoreceptors is basedon G-protein-dependent activation of cGTP phosphodi-esterase [Farber, 1995; Palczewski, 1994; Yau, 1994],and no role has been determined for PLC in vertebratephototransduction [Ferreira and Pak, 1994; Ferreira etal., 1993; Pak, 1995]. One possible role for PLC is toactivate protein kinase C, which in turn phosphorylatesand inactivates metarhodopsin [Newton and Williams,1993]. However, the role of protein kinase C in rhodop-sin phosphorylation in vivo remains controversial; ex-perimental evidence suggested that rhodopsin kinase isresponsible for in vivo rhodopsin phosphorylation andinactivation [Bennett and Sitaramayya, 1988; Ohguroet al., 1994, 1995; Palczewski, 1994]. Thus the effects ofthe rdgB homologue on phototransduction in verte-brates may not be direct.

Fig. 3. Northern blot analysis of rdgB homologue expression in rattissues. The random labeled R-rdgB homologue cDNA fragment washybridized to total 20 µg RNA. A. Ethidium bromide staining showingthe loading. B. rdgB homologue hybridization from rat tissues asmarked on the top. The positions of 28s and 18s in B are marked. Oneband of hybridization, coincident with 28s, was seen in lanes corre-sponding to the neuronal tissues.

Fig. 4. Expression of rdgB homologue in rat tissues detected byWestern blot analysis. Homogenates of rat tissues (20 µg of protein)were separated on 7% SDS-polyacrylamide gels. Proteins were trans-ferred onto nitrocellulose and rdgB homologue was visualized with1:1,000 dilution of rdgB homologue-specific antiserum, followed byhorseradish peroxidase-coupled secondary antibodies and enhancedchemiluminescence detection. Protein standards (std, Bio-Rad broadrange SDS-PAGE standards) of the indicated molecular weighs weremarked. Note: a major (arrow) at ,160 kDa seen in retina, brain andolfactory bulb, two minor bands (arrowhead) smaller than 40 kDapresent only in brain, and no immunoreactivity detected in othertissue extracts.

RDGB HOMOLOGUE IN THE RETINA 241

As a member of PITP family, the rdgB homologuecould play a role in intracellular vesicle traffic. Photore-ceptors have very high rate of membrane turnover.

Phospholipids and proteins used for the outer segmentdisk assembly are synthesized, processed in the innersegment, and transported to, and fused with, a specialregion of connecting cilium. Our immunocytochemistryrevealed that the rdgB homologue was concentrated inthe inner segment of photoreceptors or the inner plexi-form of the retina consisting primarily neuron pro-cesses and termini. It is known that in these neuronalstructures the rate of intracellular membrane traffic isvery high. Thus the detected distribution of rdgBhomologue suggests that it is involved in the process ofouter segmentmembrane renewal ofmammalian photo-receptors and synaptic vesicle trafficking in the neu-rons.

Human rdgB and Inherited Human RetinalDegeneration

Since membrane renewal is vital for the photorecep-tor cell function, disruption of this orderly flow ofmembrane from inner segment to the outer segment islikely to cause photoreceptor degeneration. With evi-dence suggesting potential functions of rdgB homo-logue in membrane traffic, it is tempting to speculatethat mutations in this gene also may cause humaninherited retinal degeneration. This is further sup-ported by our finding that rdgB homologue is located in

Fig. 5. In situ detection of rdgB homologue transcripts in rat retina.In situ hybridization of 35S-labeled antisense rat rdgB cRNA fragment(2594–3186) to cryostat section of adult mouse eye. Left panel:darkfield microscopy showing distribution of rdgB homologue mRNA.

Right panel: image viewed by brightfield microscopy. RPE, retinalpigment epithelium; ON, outer nuclear layer; OP, out plexiform layer;IN, inner nuclear layer; IP, inner plexiform layer; GC, ganglion cells.Scale bar 5 100 mm.

Fig. 6. Localization of rdgB homologue in the rat retina detected byConfocal microscopy. Cryostat sections (,10 µm) of rat retina wasincubated with affinity purified, rdgB homologue specific antibody(1:6,000 dilution of the original serum). The bound antibodies werevisualized by incubation with 1:250 dilution of fluorescein-conjugatedgoat antirabbit IgG. This is a image of single confocal optical plane(0.25 µm) showing that in photoreceptor cells only inner segment isstained and that not all neuronal cells in retinal are equally labeledwith rdgB homologue specific antibodies. CH, Choroid; RPE, retinalpigment epithelium; OS, outer segment; IS, inner segment; ON, outernuclear layer; IN, inner nuclear layer; IP, inner plexiform layer; GC,ganglion cells. Scale bar 5 50 mm.

Fig. 7. Confocal fluorescence micrograph of inner segments ofphotoreceptor cells labeled with anti-rdgB homologue antibody. Therat retina cryostat sections were stained with affinity purified anti-rdgB homologue antibody (A–G) or with serum depleted anti-rdgBhomologue antibody as control (H).A series of 0.25 µm optical sections,spaced by 1.1 µm, were arranged fromA–F and their stacked image inG. Although a difuse pattern of staining can be seen in the innersegment, a few cells with strong staining at or near the inner segmentare clearly seen in different optical planes. OS, outer segment; IS,inner segment; ON, outer nuclear layer. Scale bar 5 20 mm.

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Fig. 7.

human chromosome 11q13, a region known to containseveral retinopathy loci. Human band 11q13 has beenreported to be a site of numerous aberrant chromo-somal rearrangements [Tedder et al., 1989] and of acluster of genes that are retina-specific or have en-riched expression in the retina [Nichols et al., 1994].Mapping to chromosome 11q13 indicates that rdgBhomologue is a candidate gene for these diseases includ-ing Best disease, Bardet-Biedl syndrome I, vitreoreti-nopathy, and exudative vitreoretinopathy-1 for whichthe causing genes are unknown. During the course ofthis study, the rdgB homologue gene was linked withthe genetic marker D11S13, which showed strong ge-netic linkage with the locus of Bardet-Biedl syndrome[Banfi et al., 1996; Hartley, 1996]. Bardet-Biedl syn-drome is an infrequent, autosomal recessive conditionthat combines mental retardation, postaxial polydac-tylia, obesity, and hypogenitalism with progressiveretinal pigmentary dystrophy [Bek and Rosenberg,1995]. Our study provides further support for rdgBgene as a strong candidate gene and, furthermore,offers a potential biochemical explanation for the dis-ease. The effort is underway to determine if mutationsin rdgB homologue gene cause the Bardet-Biedl syn-drome.

ACKNOWLEDGMENTSThe authors thank Drs. Kathleen Dorey, Audrius

Kazlauskas, and Charles Zucker for their comments onthe manuscript, Mara Lorenzi for the gift of humanretinal RNA, and Qieng Zhang for his technique assis-tance. This study was supported by National Institutesof Health grant EY10869 (FXY), and J.G. is a recipientof Alcon/ARVO 1996 postdoctoral fellowship award.

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