Expression of the protein tyrosine phosphatase, phosphatase of regenerating liver 1, in the outer segments of primate cone photoreceptors

Molecular Brain Research 77 (2000) 95–103 www.elsevier.com / locate / bres

Research report

Expression of the protein tyrosine phosphatase, phosphatase of regenerating liver 1, in the outer segments of primate cone photoreceptors Timur O. Yarovinsky a , Dennis W. Rickman a , Robert H. Diamond b , Rebecca Taub c , Gregory S. Hageman a , Catherine Bowes Rickman a , * a

Department of Ophthalmology and Visual Sciences, University of Iowa College of Medicine, 200 Hawkins Blvd., Iowa City, IA 52242 -1091, USA b Department of Medicine and Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA c Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Accepted 15 February 2000

Abstract Foveal cone photoreceptors are morphologically distinct and, presumably, express unique transcripts. We have identified a cDNA clone encoding the protein tyrosine phosphatase (PTP), phosphatase of regenerating liver 1 (PRL-1) in a screen for genes that are enriched in monkey fovea. PRL-1 was originally isolated as an immediate early gene in regenerating liver [R.H. Diamond, D.E. Cressman, T.M. Laz, C.S. Abrams, R. Taub, PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth, Mol. Cell Biol. 14 (1994) 3752–3762]. On cDNA Southern blots of human and monkey retina, radiolabeled PRL-1 cDNA hybridized to a single mRNA species of about 2.5 kb that was most intense in fovea-enriched samples. The monkey PRL-1 deduced amino acid sequence is identical to human, rat and mouse PRL-1. Affinity-purified antibodies directed against PRL-1 preferentially labeled cone photoreceptor cells and a subpopulation of bipolar cells in monkey retina. Immunoreactivity in cones was confined to red and green, but not to blue, cones and was restricted to the outer segments. Immunolocalization also revealed that PRL-1 protein expression was non-nuclear, suggesting that its function in the retina may be unrelated to its role in other tissues where it is expressed primarily in nuclei. Although both foveal and extrafoveal cones were PRL-1 reactive, the high abundance of PRL-1 mRNAs detected in monkey fovea correlates with the high concentration of cones in the fovea. The PRL-1 gene is located on chromosome 6q within an interval that also contains the genes that cause two hereditary retinal dystrophies. These studies demonstrate novel expression of the PRL-1 gene in the neural retina and suggest the phosphatase activity of PRL-1 may modulate normal cone photoreceptor cell function.  2000 Elsevier Science B.V. All rights reserved. Themes: Sensory systems Topics: Retina and photoreceptors Keywords: Retina; Fovea; Photoreceptor; Cone photoreceptor; Protein tyrosine phosphatase; PRL-1

1. Introduction Retinal photoreceptor cells perform a variety of housekeeping and metabolic functions in support of their highly specialized role as phototransducers. The fovea, a morphologically and functionally distinct subregion of the primate retina comprised primarily of cone photoreceptor cells, is specialized for fine visual acuity. Foveal cone *Corresponding author. Tel.: 11-319-335-4630; fax: 11-319-3354347. E-mail address: [email protected] (C. Bowes Rickman)

photoreceptors are morphologically and biochemically distinct as compared to extrafoveal cones and, as such, likely express unique transcripts [37]. As part of an extensive screen for genes that are preferentially expressed or enriched in the monkey fovea, we isolated a fragment of the monkey PRL-1 gene. Phosphatase of regenerating liver, PRL-1, is an immediate-early gene in regenerating liver and mitogen stimulated fibroblasts [12], and it is associated with cellular differentiation, but not proliferation, in the intestine [13]. In these tissues PRL-1 protein is almost exclusively localized within the nucleus [12,13]. PRL-1 is also expressed at significant levels in skeletal

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muscle and brain, but its cellular localization in these tissues has not been resolved [12,39,47]. Phosphorylation and dephosphorylation of proteins at tyrosine residues mediates a number of cellular processes, including cellular signal transduction, growth, differentiation and metabolism. These processes are regulated by the interaction of tyrosine-specific kinases (PTKs) and phosphatases (PTPs). PTPs are a structurally diverse superfamily of enzymes that are broadly classified into cell surfaceassociated (receptor) and intracellular (non-receptor) molecules [10,45]. During the past several years, a number of structurally diverse PTPases have been identified [48]. PTPases have been found to be of importance in the regulation of a wide variety of cellular processes including growth, differentiation, hormone signaling, metabolic responses, and cellular motility and adhesion. PTPases can play either positive or negative roles in regulating these processes [34,49]. Some PTPases exclusively act on phosphotyrosine residues, while other ‘dual-specificity’ PTPases also act on phosphoserine and phosphothreonine substrates. Still other PTPases appear to act on lipid or RNA non-protein substrates [11,23,31,46]. PRL-1 is the first identified member of a family of low-molecular weight, nuclear PTPs [12,30]. The product of the PRL-1 gene is a 20-kDa evolutionarily-conserved PTP that shares homology with other PTPs within the catalytic domain [12]. Herein we present data on the expression, regional distribution and subcellular localization of PRL-1 mRNA and protein in primate cone photoreceptors, and suggest a unique role for PRL-1 in these cells. Further elucidation of the role of cone photoreceptor-associated PRL-1 will not only provide insight into the function of PRL-1, but will likely expand our understanding of fundamental regulatory processes in the retina.

2. Methods

2.1. Animals and tissue preparation Adult cynomolgus monkey (Macaca fascicularis) were euthanized with an overdose of barbiturate administered intravenously. The eyes were enucleated immediately, and the anterior segments were excised just anterior to the pars plana. For RNA isolation, the posterior pole was pinned to a wax plate following the placement of four incisions directed towards, but not passing through, the macula. The vitreous was mechanically removed. Trephine punches (2 mm diameter), centered over the fovea (‘fovea’ macular RNA sample), and 6 mm diameter punches from the equatorial retina (‘peripheral’ retina RNA) were removed and frozen immediately in liquid nitrogen. For immunofluorescence analyses, the posterior eye cups were immediately immersed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS; pH 7.4), for 2 h, at room temperature. The eyecups were cryoprotected, overnight, in 0.1 M PBS containing 25% sucrose at 48C. All

procedures for the care and handling of animals complied with federal regulations and The University of Iowa policies. Retinas from normal human donor eyes were collected within 2–4 h of death. RNA was isolated in the same manner as the monkey retinas except that the macula punches (‘macular’ retina RNA) were 6 mm in diameter. Human donor eyes were obtained following informed consent and approval by the institutional review boards at The University of Iowa and Saint Louis University.

2.2. Preparation of RNA and cDNA Total RNA from monkey and human tissues was isolated by isopyknic centrifugation as previously described [6] except that cesium trifluoroacetate (Amersham Pharmacia, Piscataway, NJ, USA) was used instead of CsCl for the density gradient, according to the manufacturer’s instructions. Total RNA was incubated in the presence of RNase-free DNase I (Roche Molecular Biochemicals, Indianapolis, IN, USA) and the ribonuclease inhibitor, RNasin  (Promega, Madison, WI, USA), extracted with phenol–chloroform and recovered by ethanol precipitation. Full-length, high fidelity cDNAs were synthesized using RNA isolated from various regions of monkey and human retinas and a SMART PCR cDNA synthesis kit (Clontech, Palo Alto, CA, USA). A 1-mg amount of RNA per firststrand cDNA synthesis reaction was reverse transcribed using superscript II RNase H 2 reverse transcriptase (Life Technologies, Rockville, MD, USA). The first-strand cDNA reactions were diluted to 50 ml with TE 10-1 (10 mM Tris–HCl, pH 8.0, 1 mM EDTA). A 1-ml sample of the diluted first-strand cDNA from each reverse transcription (RT) reaction per 100 ml PCR reaction was used to generate PCR-amplified cDNAs using supplied primers. PCR amplification was performed in a PTC-100 thermal cycler (MJ Research, Watertown, MA, USA). Optimal PCR amplification conditions to obtain a pool of representative cDNAs were determined following the cDNA synthesis. Ten-microliter aliquots of amplified cDNA samples were removed after 15, 18, 21 and 24 cycles and were separated in 13 TAE (40 mM Tris–acetate, 1 mM EDTA, pH 8.3) 1.2% agarose gels and transferred onto Hybond N 1 nylon membranes (Amersham). The membranes were probed with human a-tubulin to control for over-amplification. The optimal number of cycles was determined to be one less than that which yielded the maximum full-length a-tubulin products in a single discrete band. Following amplification the cDNAs were purified using a QIA-quickE PCR purification kit (Qiagen, Valencia, CA, USA) and quantitated.

2.3. Cloning monkey PRL-1 2.3.1. Suppression subtractive hybridization An 867 base pair (bp) cDNA clone, SF28, corresponding to the 39 untranslated region of the PRL-1 gene

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was isolated in a screen for genes that are differentially expressed in the primate fovea. The subtraction and screening protocol used is described briefly below and in detail elsewhere (Bowes Rickman et al., in preparation). Subtraction of monkey foveal and peripheral retina cDNA was performed with the PCR-Select cDNA subtraction kit (Clontech) using the suppression subtractive hybridization (SSH) method described by Diatchenko et al. [14]. Tester cDNA consisted of either foveal or peripheral retina cDNA and driver cDNA consisted of either peripheral retina cDNA (when tester was fovea) or foveal cDNA (when tester was peripheral retina). Subtracted foveal PCR products were subcloned in pCR2.1 using a TA Cloning kit (Invitrogen, Carlsbad, CA, USA). The cloned subtracted foveal cDNAs were differentially screened by hybridizing dot blots with a cDNA probe made from the ‘forward’subtracted tester (foveal) and the ‘reverse’-subtracted driver (peripheral retina) cDNA. Clones that showed differential binding to the tester (foveal) probe were then sequenced and used as probes on cDNA Southern blots to confirm foveal specificity.

2.3.2. Reverse transcription polymerase chain reaction Monkey foveal cDNA was used as a template to generate a 1.7-kb fragment of monkey PRL-1 and human macula cDNA was used to generate a 0.6-kb amplicon spanning the PRL-1 coding region. Primers were based on sequences of human PTPCAAX1 (GenBank accession number U48296 [5]), PTP4 A1 cDNA (GenBank accession number NM-003463 [35]) and monkey SF28 clone (GenBank accession number AF190930). cDNA amplicons of interest were amplified in 100 ml reactions consisting of 1 ng monkey foveal cDNA, 0.2 mM dNTPs (Clontech), 0.2 mM each primer, 1X cloned Pfu DNA polymerase reaction buffer (20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 20 mM MgSO 4 , and 0.1% Triton  X-100) and 2.5 U PfuTurboE (Stratagene), for 35 cycles, at 958C, for 1 min; 558C for 45 sec; 728C for 2 min and a final incubation at 728C for 10 min. The resulting PCR products were then purified and sequenced directly or after subcloning in pCR Blunt II TOPO vector (Invitrogen). 2.4. cDNA Southern blot analysis Due to the paucity of RNA in the primate fovea, analysis of the relative expression of mRNAs was performed by cDNA Southern blot analysis. Although the fovea and peripheral retina SMART cDNA pools do not contain an exact representation of the starting mRNAs because of nonrandom amplification by PCR, each individual fragment is expected to be amplified identically between two cDNA preparations. Therefore, as demonstrated previously [15,17], the comparison of the abundance of any fragment between the amplified cDNA pools on cDNA Southern blots accurately represents their relative abundance in the original mRNAs. Equal amounts of human and monkey retinal cDNAs

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(0.5 mg / lane) were separated in 1.2% agarose gels using 13 TAE buffer, denatured and transferred to nylon membranes (Hybond N 1 , Amersham). The filters were hybridized overnight under stringent conditions in 0.25 M Na 2 HPO 4 , 0.25 M NaH 2 PO 4 , 1 mM EDTA, 7% SDS and 10% BSA, at 658C, to PCR-derived inserts of PRL-1 cDNA (monkey foveal derived clone, SF28, spanning nucleotides 1868–2730 of PTP4 A1 ) or to a human atubulin cDNA (527 bp, Clontech) that were [ 32 P]dCTPlabeled using Ready-to-GoE labelling beads (Amersham). Following hybridization, blots were washed twice in 23 SSC, 0.1% SDS at 658C, for 10 min, twice in 0.23 SSC, 0.2% SDS, at 658C, for 20 min and exposed to film (Hyperfilm MP, Amersham).

2.5. Sequence analysis DNA sequencing was performed by using dye terminator cycle sequencing chemistry with AmpliTaq DNA polymerase FS enzyme (PE Applied Biosystems, Foster City, CA, USA). The reactions were run and analyzed with a PE Applied Biosystems Model 373 or Model 377 fluorescent automated sequencer at The University of Iowa DNA Facility. Sequence analyses and alignment were performed with MacVectorE and AssemblyLIGN (Oxford Molecular Group), SeqWeb interface to Wisconsin GCG Package and BLAST searches of the GenBank and EMBL nucleic acid databases [1].

2.6. Western analysis Isolated monkey retinas were homogenized at 48C in 50 mM Tris, 250 mM sucrose, 1 mM EDTA, pH 7.3, containing 0.5% Triton X-100 and CompleteE protease inhibitors (Roche Biochemicals, Indianapolis, IN, USA). The homogenate was centrifuged at 10 000 g for 15 min, at 48C, and the protein concentration in the supernatant was determined by Bradford assay. Protein samples (20 mg / lane) were denatured in SDS loading buffer and separated electrophoretically on precast 10–20% gradient SDS–polyacrylamide gels (Amresco, Solon, OH, USA) using the Laemmli buffer system [21]. Proteins were transferred electrophoretically to PVDF membranes, incubated with anti-PRL-1 antibodies (diluted 1:250) in Trisbuffered saline (10 mM Tris–HCl, 150 mM NaCl, pH 7.5) containing 0.1% Tween-20 (TTBS) for 2 h, washed and incubated with the appropriate biotinylated goat anti-rabbit antibody (diluted 1:150): the reaction product was visualized using a Vectastain Elite ABC kit (Vector Labs., Burlingame, CA, USA) and 0.05% diaminobenzidine tetrahydrochloride (DAB). Blots were treated in parallel without PRL-1 antibody as a negative control.

2.7. Immunofluorescence and immunohistochemistry An anti-PRL-1 polyclonal antibody was generated

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against bacterially-expressed, purified, denatured PRL-1 protein and affinity purified, as described previously [12]. Rabbit antisera directed against human red / green opsin (JH492) and blue opsin (JH455) were a generous gift from Dr. Jeremy Nathans (John Hopkins University, Baltimore, MD, USA) [8,53]. Retina punches (6-mm diameter) were embedded in Tissue Tek O.C.T. Compound (Sakura Finetek USA, Torrance, CA, USA) and sectioned, transversely, on a cryostat (7 mm). Sections were mounted onto Superfrost Plus glass slides and stored at 2208C until processing. Sections were pretreated in a blocking solution of PBS containing 0.5% Triton X-100 and 10% normal goat serum (antibody buffer) for 30 min, then incubated overnight with anti-PRL-1 affinity purified antibodies (diluted 1:500) in 0.1 M PBS with 10% normal goat serum. After thorough washing, sections were incubated for 1 h in affinity purified goat anti-rabbit secondary antibody conjugated to Cy3 (diluted 1:100; Chemicon, Temecula, CA, USA), thoroughly washed again and coverslipped. In some experiments tissue was initially permeabilized by incubation in 100% methanol for 30 min. PRL-1-stained sections processed for double labeling studies were incubated with affinity purified goat antirabbit secondary antibody conjugated to Cy3 (diluted 1:50) and subsequently stained with either JH492 or JH455 antisera (each diluted 1:5000) for 30 min at room temperature, washed and incubated with affinity purified goat anti-rabbit secondary antibody conjugated to FITC (diluted 1:50). Negative controls excluded the cone opsin primary antibodies and on these sections there was no detectable FITC staining. As an additional negative control, double labeling experiments were also performed using the following order of antibodies: JH455 with goat anti-rabbit conjugated to Cy3, and JH492 with goat anti-rabbit conjugated to FITC. No double labeled cones were observed (data not shown). Immunolabeled sections were examined using a Nikon Optiphot flourescence microscope and photographed. Slides were scanned using a Nikon LS 2000 slide scanner and processed (cropped, adjusted for brightness and contrast and labeled) using Adobe PHOTOSHOPE (Version 4.01, Adobe Systems, Mountain View, CA, USA). Confocal immunofluorescence imaging was performed with a BioRad MRC-1024 laser scanning confocal microscope equipped with a krypton–argon mixed gas laser at The University of Iowa Central Microscopy Research Facility.

3. Results The PRL-1 gene fragment was isolated as a 867-bp cDNA clone in a screen for genes expressed in the primate fovea. One clone, designated SF28, shared 99% identity with the 39 untranslated region of human protein tyrosine

Fig. 1. Structure of the human PRL-1 cDNA, PTP4A1, and retinaspecific expression of PRL-1 mRNA. (A) Diagram of the PTP4A1 cDNA and positions of cDNA clones relevant to this work. The 521-base open reading frame is represented by the open box. The fragments are positioned with respect to the PTP4A1 cDNA structure based on their homology alignment. (B) cDNA Southern analysis of PRL-1 cDNA levels in primate retina. Equal amounts (0.5 mg / lane) of full-length, high fidelity SMART cDNA pools derived from monkey fovea (mF), monkey peripheral retina (mP), human macula (hM) and human peripheral retina (hP) were sequentially hybridized with 32 P-labeled cDNA probes for PRL-1 (monkey fovea-derived clone, SF28, spanning nucleotides 1868– 2730 of PTP4 A1 accession number NM-003463) and human a-tubulin. The control hybridization to a-tubulin cDNA demonstrates that relatively equal amounts of cDNA were electrophoresed from each primate retinal cDNA pool.

phosphatase type IVA (PTP4 A1 ) member 1, encoded by the PRL-1 gene (Fig. 1) [35]. Using primers derived from the human PTP4 A1 sequence a 1.7-kb cDNA was amplified from monkey foveaderived cDNA and a 0.6-kb amplicon was generated from human macula cDNA (Fig. 1A). Nucleic acid sequence alignment revealed that the 1.7-kb fragment of monkey PRL-1 is 98% homologous to the human PTP4 A1 sequence and 87% with the rat PRL-1 cDNA. The human macula cDNA is identical to the PTP4 A1 sequence. The predicted amino acid sequence of monkey PRL-1 cDNA shares 100% identity with the human, rat and mouse PRL-1 proteins.

3.1. PRL-1 expression Relative expression of PRL-1 transcripts in the primate retina was estimated by hybridization of SF28 to pools of cDNA derived from central and peripheral regions of human and monkey retina (Fig. 1B). On cDNA Southern blots of primate retinal cDNA the radiolabeled SF28 probe intensely hybridized to a single band of 2.5 kb in the monkey fovea-derived sample and weakly to a single band in the human macula-derived, and monkey and human peripheral retina-derived samples, suggesting that the level of PRL-1 gene expression was significantly higher in the monkey fovea. Affinity purified antibodies directed against PRL-1 [12],

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Fig. 2. Immunoblot of monkey retina reacted with anti-PRL-1 antibodies. Lanes: 15reaction to a 26-kDa band was detected with an anti-PRL-1 antibody (visualized with biotinylated goat anti-rabbit antibodies, Vectastain Elite ABC kit and DAB); 25negative control, incubation with secondary antibody only.

reacted specifically with a 26-kDa protein in monkey neural retina homogenate (Fig. 2).

3.2. Immunohistochemical localization of PRL-1 In the monkey, specific PRL-1 immunostaining was localized to a subset of cone photoreceptor cells and to numerous bipolar cells in all retinal regions (Fig. 3A). Reactivity with cones was primarily confined to the outer segments (Fig. 3A and B). Diffuse staining was detectable in cone cell bodies and within the myoid on some sections, but inner segments, nuclei and pedicles were unstained. In the monkey fovea, all cones appeared to be immunostained, although the characteristic cone morphology and the resolution of the immunostaining pattern were difficult to discern due to the high density of cones in this region (Fig. 3B). The specificity of cone staining was confirmed by preadsorption of the primary antibody (diluted 1:500) with an excess of bacterially-expressed PRL-1 protein (8 mg / ml) and subsequent elimination of immunostaining (Fig. 4A and B). Specific immunostaining of bipolar cells was achieved at an antibody dilution of 1:1000, and elimination of staining was achieved following preadsorption of the antibody with 8 mg / ml bacterially-expressed PRL-1 protein (data not shown). In peripheral retina, PRL-1 antibody appeared not to react with all cones. In order to determine whether PRL-1 was associated with specific cone subtypes, double-label immunofluorescence studies were conducted using wellcharacterized antibodies directed against specific cone opsins [7]. JH492 antibody, which reacts with red / green

Fig. 3. PRL-1 immunoreactivity with peripheral and central monkey retina. (A) PRL-1 antibody reacts primarily with cone photoreceptor outer segments (OS) and presumed rod bipolar cells in the peripheral retina (detection with Cy3-labeled secondary antibody, visualized by confocal immunofluorescence histochemistry). (B) PRL-1 antibody reacts with foveal cone photoreceptor outer segments in the fovea at the edge of the foveola. Apical retinal pigment epithelial cell layer (RPE) labeling in (B) is due to non-specific binding of the secondary antibody; staining in ganglion cells is due to non-specific binding of rabbit immunoglobulins. This staining is not blocked by preadsorbtion with PRL-1 protein. IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar550 mm.

opsins, labeled the majority of cones in monkey retina. Virtually all the red / green opsin-labeled cones were also PRL-1 immunoreactive (Fig. 5). Conversely, ‘blue’ cone photoreceptor cells, identified with the blue opsin antibody JH455, did not react with PRL-1 antibody. PRL-1 antibody immunoreactive somata were also observed in the outer part of the inner nuclear layer (INL). These cells gave rise to processes that were distributed to the outer plexiform layer (OPL) and to the innermost strata of the inner plexiform layer (IPL). Based on this pattern of

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Fig. 4. PRL-1 antibody reaction with cone outer segments in peripheral retina (A) is specific based on lack of binding by anti-PRL-1 antibodies (diluted 1:500) preadsorbed with PRL-1 protein (B). OS, outer segments; IS, inner segments; ONL, outer nuclear layer. Scale bar550 mm.

immunostaining, these cells correspond, predominantly, to rod bipolar cells [2,38].

4. Discussion Screening of a fovea-enriched cDNA library for differentially expressed genes revealed a cDNA clone (SF28 ) for PRL-1 that was preferentially expressed in the primate fovea. Expression of PRL-1 protein in the retina was demonstrated by Western analysis and by immunohisto-

chemistry. Immunofluorescence studies revealed preferential localization of PRL-1 expression to cone photoreceptor cell outer segments. The fact that the highest density of cones in the primate retina occurs in fovea (where cone densities reach 84 000–260 000 cones per mm 2 in the macaque retina [33,36,54]) may explain why elevated levels of PRL-1 cDNA were detected in monkey foveaderived cDNA samples on retina cDNA Southern blots. Conversely, the lower PRL-1 cDNA signal intensity in the human macula sample is likely due to the relative dilution of foveal cones within the 6 mm diameter macular punches that were used to generate the human macular cDNAs. Here, the peak density of cones is restricted to a 0.5-mm diameter region in the macula outside of which the cone density declines exponentially out to 1 mm eccentricity. The 26-kDa protein detected by anti-PRL-1 antibodies on Western blots of retinal homogenates has a slightly higher molecular weight than that predicted for PRL-1, based on its amino acid sequence and reported for bacterially expressed PRL-1, PRL-1 in regenerating liver [12] and mitogen-stimulated NIH 3T3 fibroblasts [13] (21 kDa). This suggests that, in the monkey retina, PRL-1 may undergo alternative post-translational modification. PTPases form a diverse superfamily of enzymes that are characterized by a common catalytic signature sequence (VHCXAGXXR) [16,19,25,41,50]. PRL-1 contains this signature sequence, but has very little homology to other known PTPases outside of the active site [12]. Little is known about its precise substrates and cellular functions at present. However, two PRL-1 homologues (PRL-2 and

Fig. 5. Blue cones do not react with anti-PRL-1 antibody. Sections were sequentially labeled with anti-PRL-1 antibodies and blue-(JH455) or red / green (JH492) antisera. (A, C) Monkey retina reacted with antibodies directed against PRL-1 (detected with Cy3 conjugated secondary antiserum); (B) blue- or short-wavelength sensitive, S, opsin and (D) red / green- or medium wavelength sensitive, M / L, opsin (B, D; detected with FITC conjugated antiserum). Arrows delineate cone outer segments that are not reactive for PRL-1 antibody but are positive for blue-sensitive opsin antibody. OS, outer segments; IS, inner segments. Scale bar550 mm.

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PRL-3) have recently been identified [5,57]. All three of these enzymes share a C-terminal prenylation domain [5,57]. An interesting feature of PRL-1 is its high degree of evolutionary conservation. The PRL-1 amino acid sequence is identical in all mammalian species analyzed so far. Moreover, the PRL-1 homologue in C. elegans, which shares 55% amino acid homology with human PRL-1, is 100% identical within the enzymatic domain and C-terminal isoprenylation sequence [35,56]. Indeed, the predicted amino acid sequence for monkey PRL-1 also shares 100% identity with the other mammalian PRL-1 sequences. This high degree of conservation supports the concept that PRL-1 performs a critical function(s) in cellular regulation. In the nervous system, protein tyrosine phosphorylation has been shown to play a key role in neuronal development [40,51], neurite outgrowth [4] and presynaptic and postsynaptic modulation [22]. PRL-1 is the only known PTP expressed both in neurons and oligodendrocytes in brain, and its expression in cerebral cortex has been shown to be induced after reperfusion, following transient forebrain ischemia [47]. Unlike regenerating liver and intestine, where PRL-1 was localized to the nucleus [12,13], the present studies revealed that, in postmitotic, neuroectodermally-derived cells in the adult retina, PRL-1 is restricted to the cytoplasm where it may be membrane associated. This suggests a different functional role in these cells. Unfortunately, developmental expression of PRL-1 protein in neurons has not been analyzed. A key observation of the present studies is finding differential expression of PRL-1 in red- and green-, but not blue-sensitive cone photoreceptors. Additional experiments to determine whether differential PRL-1 expression is related to a specific cone functional subtype are underway. PRL-1 is believed to modulate growth in cells of endodermal origin, but the function of this protein in neurons is still unknown. Furthermore, the intracellular substrate for phosphatase activity of PRL-1 has not been identified. In vitro studies have demonstrated activity of PRL-1 as a tyrosine phosphatase [12], but the possibility of its serine– threonine phosphatase activity in vivo has not been excluded. The fact that PRL-1 may be associated with membranes via isoprenylation, in consort with localization to the outer segments of cone photoreceptors, suggests that PRL-1 may function to modulate membrane channels or participate in the regulation of the phototransduction cascade. Interestingly, it has recently been demonstrated that the rod photoreceptor cyclic nucleotide-gated channel is modulated by phoshorylation of a specific tyrosine residue and that this activity is mediated by specific, though as yet unidentified, PTKs and PTPs located in the rod outer segments [28,29]. Expression of PRL-1 in cone outer segments suggests that a similar mechanism could also modulate the phototransduction cascade in red and green cone photoreceptors.

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This subtype specificity is not unique. Apart from the visual pigment proteins, the type I receptor for inositol 1,4,5-triphosphate (IP3 ) appears to exhibit a cone subtypespecific expression pattern [52]. The type I IP3 receptor has been localized to the plasma membrane and outer face of outer segment discs of red and green cones where it functions in IP3 -mediated Ca 21 flux. Wang et al. [52] postulate that this receptor contributes to the faster recovery of cones following light-activation of the phototransduction cascade by providing a feedback loop for Ca 21 flux. It is proposed that faster recovery from the light response and adaptation is mediated by the IP3 receptor, perhaps by accelerating the fall of cytoplasmic Ca 21 following a light flash and by accelerating an increase in cytoplasmic Ca 21 concentration following the offset of light. These investigators further suggest that this receptor may be absent from blue-sensitive cones due to the closer resemblance of blue-cone- and rod-mediated responses. Rods and blue cones both have longer integration times and a higher sensitivity than red- and green-sensitive cones [3,26,27,32,55,59,60], and they would therefore be illserved by the faster regulation of cytoplasmic Ca 21 that the IP3 receptor provides. Subcellular localization of PRL-1 is thought to be directed by post-translational modifications. Several studies reported that PRL-1 is isoprenylated in vivo [5]. Prenylation of proteins allows them to interact with either membranes or other proteins [58]. Whether a prenylated protein is directed to the nucleus or to the plasma membrane is determined by other signals which have not been defined for PRL-1. The importance of prenylation for protein function is illustrated by the pathology that results from prenylation deficiency in choroideremia. Mutations in the CHM gene encoding the Rab escort protein-1 (REP-1) — an accessory protein required for Rab geranylgeranyl:protein transferase activity — causes Ram / Rab27 protein prenylation deficiencies leading to dysfunction of a specific vesicular transport step in RPE and / or choriocapillaris cells resulting in choroideremia, a form of retinal degeneration [9,18,24,42,43]. The PRL-1 gene has been localized to chromosome 6q12 by fluorescence in situ hybridization [35] and PRL-1 ESTs have been mapped between the D6 S1695 and D6 S421 markers within this region (NCBI, the human gene map). This region of chromosome 6 has been shown to have closely linked markers for the genes responsible for two hereditary retinal dystrophies: CORD7, an autosomal dominant cone-rod dystrophy [20], and STGD3, an autosomal dominant Stargardt-like macular dystrophy [44]. Thus, PRL-1 is a good candidate gene for these dystrophies since the cone-rod dystrophies initially cause degeneration of the cone photoreceptors, and STGD3 is characterized, in part, by reduced visual acuity implicating cones in its pathogenesis. Screening of affected CORD7 and STGD3 individuals for PRL-1 gene mutations is currently underway.

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Acknowledgements The authors wish to thank J. Nathans for the cone opsin antibodies. This work was supported by National Institutes of Health (NIH) grants EY11286 (CBR), EY11389 (DWR) EY06463 and EY11515 (GSH) and by an unrestricted grant from Research to Prevent Blindness, Inc. Support for the University of Iowa DNA facility is partially derived from the Diabetes Endocrinology Research Center with NIH grant DK25295 and the College of Medicine.

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