Molecular Vision 2004; 10:738-749 <http://www.molvis.org/molvis/v10/a89/> Received 6 January 2004 | Accepted 7 September 2004 | Published 6 October 2004

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Characterization of 3',5' cyclic nucleotide phosphodiesterase activity in Y79 retinoblastoma cells: absence of functional PDE6

J. Brandon White,¹ W. Joseph Thompson,² Steven J. Pittler¹
 
 

¹Department of Physiological Optics, Vision Science Research Center, School of Optometry, University of Alabama at Birmingham, Birmingham, AL; ²OSI Pharmaceuticals, Farmingdale, NY

Correspondence to: Steven J. Pittler, University of Alabama at Birmingham, 924 18^th Street South, Birmingham, AL, 35294-4390; Phone: (205) 934-6744; FAX: (205) 934-5725; email: pittler@uab.edu
 
Dr. White is now at the Division of Regulatory Biology, The Salk Institute, La Jolla, CA.

Abstract

Purpose: Previous studies identified rod photoreceptor cyclic GMP phosphodiesterase (PDE6) transcripts in the human Y79 retinoblastoma cell line. To assess the potential to utilize this cell line for structure/function studies of PDE6, we analyzed 3',5' cyclic nucleotide phosphodiesterase activity focusing on expression of PDE6.

Methods: DEAE-chromatography was used to fractionate PDE activity from Y79 cell homogenates. PCR was performed on cDNA generated from Y79 cells and retina with PDE isoform specific primers. Western blots were performed with antibodies to PDE1, PDE4, or rod PDE6. DNA sequencing and protein truncation tests were performed with plasmids containing the entire coding region of Y79 rod PDE6 transcripts. Proteasome mediated degradation of PDE6 subunits was analyzed with a pathway specific inhibitor. Polysome isolation was performed by fractionation on sucrose gradients followed by RT-PCR for the PDE6 transcripts.

Results: Of three peaks of PDE activity, peaks 1 and 2 were activated by Ca²⁺/calmodulin, inhibited by dipyridamole and zaprinast, and were reactive with a PDE1 antibody. Peak 3 hydrolyzed only cAMP and was rolipram sensitive, indicative of PDE4. Transcripts for rod and cone PDE6 isoforms were detected in Y79 total RNA, however PDE6 antibodies recognized only a single 99 kDa polypeptide from immunoprecipitated ³⁵S labeled Y79 extracts. DNA sequencing of PDE6 α, β, γ, and PDE6 associated δ-subunit cDNA revealed some polymorphism, but no apparent mutations. Each of the PDE6 transcripts could be translated into protein of the correct length. The concentration of cGMP in the cells was greatly reduced in comparison to that reported in the photoreceptor cell. Addition of cyclic nucleotide analogues, zinc, or butyrate did not enhance the expression of PDE6. Transduction into Y79 cells of adenovirus expressing PDE6 subunits failed to produce functional enzyme

Conclusions: PDE1 and PDE4 enzyme activities predominate in Y79 cells. Despite the presence of PDE6 transcripts and the ability to translate each into protein in vitro, a functional PDE6 enzyme could not be detected. Attempts to enhance expression with cell culture or with introduction of virus expressing PDE6 were not successful. The results indicate that expression of a fully active stable PDE6 enzyme requires other post-transcriptional events that do not occur or are inhibited in Y79 cells.

Introduction

The phototransduction cascade within photoreceptors is mediated primarily through the action of three proteins: the receptor, rhodopsin, the G-protein, transducin, and 3',5' cyclic nucleotide phosphodiesterase type 6 (PDE6). PDE6 isoforms are the only PDE family enzymes [1,2] in the rod and cone photoreceptor outer segments. Rod photoreceptor PDE6 is composed of two catalytic subunits, α and β, and two identical partially inhibitory subunits, γ with calculated molecular masses of 99 kDa, 98 kDa, and 11 kDa, respectively [3]. Cone photoreceptor PDE6 is composed of two α' catalytic subunits, and a γ' inhibitory subunit with calculated molecular masses of 93 kDa and 13 kDa, respectively [4,5]. A 14 kDa protein (δ) originally called a fourth subunit of PDE6 was identified [6] that can solubilize PDE6 activity from the rod outer segment membrane and alter cGMP binding to noncatalytic binding sites [7,8] and reduce light stimulated hydrolysis of cGMP [9]. Other retinal and non-retinal proteins in other tissues have been shown to interact with the δ-subunit [10-12]. PDE6 is readily purified from isolated outer segments and can be expressed in a weakly active or inactive state; however, expression of a fully active stable enzyme that can be purified and remains active has not been demonstrated [13-18].

The retinoblastoma derived cell lines Y79 and WERI [19,20] have been shown to exhibit biochemical characteristics of the retinal photoreceptors as well as other retinal cell types [21,22]. Rod PDE6 α, β, and cone PDE6 α' transcripts were reported in Y79 cells at levels that could be detected by RNA blot analysis [23], however no protein analysis was performed. Early studies demonstrated that a Ca²⁺/calmodulin stimulated PDE activity was present in the cell line [24], but the full complement of Y79 PDE activities has not been characterized. To assess the potential usefulness for structure/function studies of rod PDE6, we have analyzed PDE activities in Y79 cells. We show that non-photoreceptor PDE activities predominate, and that despite the presence of intact transcripts encoding all of the known PDE6 subunits and associated proteins, no expression of functional enzyme activity is observed.

Methods

Cyclic-(8-³H)-AMP and cyclic-(8-³H)-GMP were from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). Snake venom, cAMP, cGMP, and Dowex 1X8-400 were from Sigma-Aldrich (St. Louis, MO). Triascryl M anion-exchange resin was from BioSepra (Ciphergen, Freemont, CA). 8-Methoxy-Methyl-IBMX, Sp-8-pCPT-cGMPs, and Rp-8-pCPT-cGMPs were from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). Y79 and WERI cell lines were obtained from ATCC (Rockville, MD), and grown in suspension culture as previously described [25].

Separation of PDE activity in Y79 cell extracts

Approximately 5x10⁸-10⁹ cells were homogenized on ice in 20 ml of buffer A (10 mM Pipes pH 7.0, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml antipain, 1 μg/ml pepstatin A, and 20 mM benzamidine) and 0.1% Triton X-100 using a Polytron homogenizer at medium speed for 10 s. The homogenate was diluted 10 fold in 10 mM Pipes (pH 7.0) and homogenized for two additional 10 s intervals. The homogenate was centrifuged for 1 h at 100,000 x g in a Ti-50 rotor at 4 °C. The supernatant was removed and the pellet resuspended in 20 ml of 10 mM Pipes (pH 7.0). DEAE-Trisacryl M was washed and equilibrated with 10 mM Pipes (pH 7.0) with a bed volume of 20 ml in a Pharmacia K16/20 column. The 100,000 x g supernatant was applied at a flow rate of 0.8 ml/min. Enzyme was eluted with 112 ml of a linear 0-0.8 M sodium acetate (NaOAc, pH 7.0) gradient at a flow rate of 0.8 ml/min. Fractions of 1.4 ml were collected at 4 °C, and the salt concentration in each fraction was determined by comparison to a standard curve using a conductivity meter.

PDE assays

PDE activity was assayed as previously described [26,27] with minor modification. Briefly, 10-20 μl of each fraction was added to assay buffer containing 0.25 μM [³H] cAMP or 0.5 μM [³H] cGMP (about 100,000 cpm/assay) for 10 min or 20 min (with inhibitors, Table 1) at 30 °C in a final volume of 0.4 ml. In this step of the assay, the PDEs present will hydrolyze the substrate, cGMP or cAMP at its cyclic bond to generate 5'-GMP or 5'-AMP, respectively. The reactions were boiled for 45 s, and 100 μl O. hannah snake venom (0.5 mg/ml), was added and incubated 10 min at 30 °C, followed by the addition of 1 ml methanol. Snake venom contains a phosphodiesterase 5'-nucleotidase activity that releases the phosphate from 5'-GMP or 5'-AMP to generate guanosine or adenosine, respectively. Samples were then applied to Dowex 1X8-400 columns (1 ml of 1:4 slurry), eluant collected, column washed with 1 ml methanol and collected, and total eluant counted in 6 ml of scintillation fluid (Ecoscint A, National Diagnostics, Atlanta, GA).

Western blot analysis

Approximately 40-80 μg of protein from column fractions corresponding to peaks of PDE activity were separated in 7.5% (C=2.6) SDS-PAGE gels. Immunoreactivity to PDE1 (Ab PDE1-A6, a gift from Dr. R.K. Sharma, Department of Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 1:2000) and PDE4 (GalK-hPDE4a, GST-PDE4 catalytic, 1:2000) was detected using BM Teton substrate (Roche Molecular Biochemicals, Indianapolis, IN). For PDE6 westerns, 80 μg of total protein was separated by 15.08% (C=0.5) low crosslinker SDS-PAGE [3] and transferred to PVDF membrane (BioRad, Hercules, CA). Immunoreactivity to PDE6 was detected by a commercially available antibody (CytoSignal Research, Irvine, CA) that recognizes the rod PDE6 catalytic subunits strongly, the γ-subunit more weakly and shows no cross-reactivity with cone PDE6. Proteins were visualized using an ECL detection kit (Amersham Pharmacia Biotech, Buckinghamshire, England).

RT-PCR analysis and quantification

PCR amplification primer sets (Table 2) were designed to amplify portions of PDE1, PDE2, PDE4, PDE5, PDE6, and PDE7. Total RNA was used to generate first strand cDNA using Superscript Choice System (Invitrogen-Life Technologies, Carlsbad, CA). Two percent of the cDNA generated was used for optimized PCR amplification with primers specific for each of the PDE transcripts. For quantitative analysis twenty-five cycles were performed and 5 μl of product was analyzed by densitometry analysis using an EDAS 120 system and version 2 1-D software (Eastman Kodak, Rochester, NY). Each PDE6 sample was normalized to values obtained for GAPDH.

Immunoprecipitation of PDE6 subunits in Y79 protein extracts

Y79 cells were metabolically labeled with ³⁵S-methionine (Amersham Biosciences Corp, Piscataway, NJ). Briefly, after a starvation period of 30 min. in methionine/cysteine-free media, the cells were incubated for 3 h in the same media containing ³⁵S-Met. Cells were pelleted, washed briefly in 10 ml ice-cold PBS and re-pelleted. Total protein was extracted from the cells by brief homogenization with a teflon pestle in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 130 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 1 mg/ml BSA). Cell debris was pelleted and supernatant removed and stored at -80 °C. Protein A/G agarose beads (Calbiochem, San Diego, CA) were washed twice in immunoprecipitation buffer, pelleted, and resuspended 1:2. The beads (having a 20 μl bed volume) were coated with an antibody specific to PDE6 or to control IgG sera. The antibody was incubated 1 h with gentle agitation, pelleted, and washed two times with immunoprecipitation buffer. To remove material that nonspecifically adheres to the beads, labeled protein (1x10⁷ cpm) was applied to uncoated beads and incubated for 1 h at room temperature. The beads were pelleted, washed twice with one bed volume of immunoprecipitation buffer followed by centrifugation at low speed. The supernatants were combined and applied to the beads containing the nonspecific antibody and incubated 1 h. The beads were pelleted, supernatant removed, and washed twice with one bed volume of immunoprecipitation buffer. Beads were pelleted and the supernatants were combined. This combined supernatant lacked nonspecifically adhering materials and was applied to the beads containing the PDE6 antibody and incubated 1 h at room temperature. The beads were pelleted, washed four times with one bed volume of immunoprecipitation buffer and pelleted. Laemmli buffer (1X) [28] was used to elute bound proteins and the eluate was electrophoresed into a 15.08% (C=0.5) SDS-polyacrylamide gel. The gel was dried under vacuum and exposed to X-ray film for 18-24 h at -80 °C.

Cloning of Y79 PDE6 transcripts

Total RNA was isolated from the Y79 cell line followed by RT-PCR with the Superscript Choice System first strand cDNA kit (Invitrogen-Life Technologies, Rockville, MD). Each PDE6 primer set used contained a Kozak consensus sequence and a BamHI or BglII restriction site in the sense primer, and a stop codon and a BamHI or BglII restriction site in the antisense primer. Appropriate length PCR products were gel purified with a gel extraction kit (Qiagen, Valencia, CA) and cloned into the EcoRV site of pBS II KS vector. Qiagen mini-spin plasmid preps were used to isolate plasmid DNA, and the sequences were confirmed by dideoxy sequencing using a fmol cycle sequencing kit (Promega, Madison, WI).

Protein truncation tests (PTTs)

A coupled transcription-translation TnT reticulolysate System (Promega) was used with minor modification. Briefly, 2 μg of plasmid DNA containing the coding region for either the α, β, γ, or δ subunits of PDE6 was placed in a reaction containing ³⁵S-Met and other amino acids, incubated at 30 °C for 90 min and briefly centrifuged. Aliquots were removed and combined with equal amounts of 2X Laemmli buffer and electrophoresed into a 15.08% (C=0.5) SDS-polyacrylamide gel. The gel was dried under vacuum and exposed to X-ray film for 18-24 h at -80 °C.

Measurement of cGMP in Y79 cells

cGMP levels were measured in the Y79 cell line using an enzyme immunoassay kit from Biomol with minor modification. The kit contains a cGMP specific antibody that does not react with other cyclic nucleotides or related molecules. Briefly, 1-2x10⁷ cells were lysed in 100% ethanol, cell debris pelleted, and supernatant dried to completion, reconstituted in 100 μl of assay buffer and used directly or as a 1:2 dilution. Initial incubation was for 3 h instead of 2 h to increase sensitivity.

Lactacystin studies of PDE6 in Y79 cells

To assess the contribution of the proteasome degradation pathway on PDE6 in Y79 cells, 1x10⁶ cells were plated in 35 mm tissue culture dishes that were previously treated with poly-d-lysine and fibronectin. The cells were incubated in the presence of 10 μM lactacystin (provided by Dr. M. Obin, Tufts University, Boston, MA) or DMSO (as carrier) for 8 h under standard growth conditions. Media was removed, cells were lysed in 0.2% Triton X-100 in 100 mM potassium phosphate buffer (pH 7.8) and collected. Samples were boiled in 1X Laemmli buffer and loaded onto 7.5% (C=2.6) SDS polyacrylamide gels. Gels were transferred to PVDF membrane, and western analysis was performed with the PDE6 antibody as previously described or with an antibody that recognizes ubiquinated proteins (provided by M. Obin, Tufts University) using ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ).

Polysome profile analysis

Polysomes were isolated from Y79 cells as previously described [29]. For experiments in which EDTA was used to release the RNA from polysomes, the cells were lysed as described with the inclusion of 30 mM EDTA in all buffers (free of MgCl₂) prior to loading on the gradient.

Expression of PDE6 in Y79 cells

Adenovirus expressing PDE6β was a gift of Dr. Jean Bennett, University of Pennsylvania, Philadelphia, PA. An adenovirus expressing PDE6α was generated by Quantum Biotechnologies, Inc. (Quebec, Canada). Viruses were grown and titered at the UAB Cystic Fibrosis Gene Therapy Core Facility and were stored at -80 °C until use. HEK293 cells were transduced with a multiplicity of infection (MOI) of 20 and 50. Y79 cells were transduced with virus at an MOI of 200, 500, and 1000. Cells were lysed 72 h post-transduction in 0.1% Triton X-100, 1 mM PMSF, 10 mM Tris-Cl (pH 8.0), combined with an equal volume of 2X Laemmli buffer and boiled for 10 min. The extract was loaded onto 7.5% (C = 2.6) SDS polyacrylamide gels. Proteins were transferred to PVDF membrane, and western analysis was performed with a PDE6 antibody as described.

Results

Characterization of the PDE activity in Y79 cells

To characterize the major cyclic nucleotide phosphodiesterase activities in Y79 cell extracts, PDE hydrolysis was first determined in crude cytosolic and membrane fractions. Ninety-eight percent of the PDE activity was found in the cytosol of Y79 cells, whereas only 2% was membrane bound (not shown). Further fractionation of the cytosol through DEAE-Trisacryl revealed three distinct peaks of activity that accounted for approximately 95% of the total soluble PDE activity (Figure 1A). Peak 2 sometimes was split into major and minor peaks as shown, and in other runs was not resolved into two peaks. There was some variability in apparent salt elution profiles and fraction distribution from run to run (n=4). The first peak of enzyme activity from the Y79 column eluted between 55 and 100 mM sodium acetate. This enzyme was stimulated approximately 7 fold by Ca²⁺/calmodulin (Figure 1B) and could hydrolyze cGMP and cAMP equally well. The K_m value in the absence of Ca²⁺/calmodulin for cGMP was 2.37 μM and for cAMP was 0.64 μM (Table 3). This first peak of enzyme activity was inhibited by zaprinast with IC₅₀ values of 2.37 μM with cGMP as substrate and 1.98 μM with cAMP as substrate. The enzyme activity present in peak 2 eluted from the column between 167 and 339 mM sodium acetate. It was stimulated to a lesser extent than peak 1 by Ca²⁺/calmodulin (about 3 fold) and could hydrolyze both cGMP and cAMP equally well. The K_m value in the absence of Ca²⁺/calmodulin for cGMP was 0.70 μM and for cAMP was 0.77 μM. Peak 2 was also inhibited by zaprinast with IC₅₀ values of 1.69 μM with cGMP as substrate and 1.77 μM with cAMP as substrate. Other drug inhibitors (Table 1) used showed no drug inhibition (>50 μM for Rolipram, E4021, and Trequinsin). The third peak of enzyme activity eluted between 450 and 585 mM sodium acetate. The enzyme contained in this peak hydrolyzed only cAMP as substrate and was inhibited by rolipram with an IC₅₀ value of 2.81 μM with cAMP as substrate. Kinetic analysis with cAMP as substrate yielded a K_m value of 1.25 μM for cAMP; no detectable cGMP hydrolysis was observed.

RT-PCR identification of PDE Isozymes in Y79 cells

RT-PCR analysis using primer pairs specific for transcripts encoding PDE1A, PDE1B, PDE2, PDE4, rod and cone PDE6 subunits, and PDE7 amplified appropriately sized products from cDNA generated from Y79 and WERI total RNA (Figure 2A). Transcripts to PDE2 were also detected, but based on cGMP stimulation studies, did not correlate with enzyme activity. However, a PDE3 isoform activity was detected from these same studies based on the inhibition of enzyme when assayed with 5 mM cGMP (Figure 1B). However, this activity was found within the PDE1 peaks (peaks 1 and 2, Figure 1A) and represented only about 2% of the total PDE activity. PDE7 was also detected by RT-PCR analysis, but a complete analysis could not be performed because of lack of specific inhibitors and antibodies to distinguish it from PDE4. The cAMP specific PDE identified (peak 3, Figure 1A) was sensitive to rolipram, a known PDE4 inhibitor [30] that does not inhibit PDE7 [31].

Quantitation of PDE6 transcripts in Y79, WERI and human retina

Transcripts encoding all of the PDE6 subunits (cone and rod transcripts) were detectable in Y79 and WERI cells, further extending previously reported results on a subset of PDE6 transcripts [23]. To compare the mRNA steady state levels for all of the PDE6 transcripts in Y79, WERI and human retina, the linear phase of amplification of each cDNA was empirically determined so that gene expression could be quantitatively compared, when normalized to G3PDH. Based on densitometric analysis, the transcripts encoding rod PDE6 subunits were reduced about 3 fold relative to retinal RNA (Figure 2E) however, PDE6 associated δ was at near equivalent levels (Figure 2A). The PDE6α transcript was 5 fold more abundant in Y79 than WERI cells. PDE6β and γ transcripts were at apparent equal levels in both Y79 and WERI RNA. Cone PDE6α' transcript was barely detectable in Y79 RNA and cone PDE6γ' was about 7 fold less than the level in WERI cells.

Western analysis of Y79 PDE1 and PDE6

To correlate RNA levels with protein expression, western blot analysis was performed with pooled fractions representative of each peak of enzyme activity using antibodies specific to PDE1, PDE4, and PDE6. PDE1 was detected in peaks 1 and 2 (Figure 2D) and PDE4 was detected in peak 3 (not shown). PDE6 could not be detected in any of the peaks from the column as compared to a positive control of total retinal proteins indicating PDE6. Furthermore, trypsin activated PDE6 activity [32] was not detectable in any of the column fractions. The failure to detect any PDE6 protein or enzyme activity indicates the absence of this enzyme; however individual inactive subunits may be present. To increase the sensitivity of detection metabolically labeled protein was used in an immunoprecipitation reaction with an antibody that recognizes the α, β, and γ subunits of PDE6. Figure 3 shows that a single protein of 99 kDa was eluted from the antibody column. It was not possible to distinguish between the two catalytic subunits because the antibody used cannot differentiate the α and β subunits.

Analysis of Y79 PDE6 transcripts

As a first step in analyzing the integrity of the coding region sequences, the protein truncation translation (PTT) test was performed for each of the coding regions of the PDE6 subunits (α, β, γ), and for δ. The PTT test provides a rapid means of assessing coding regions for premature stop codon mutations. Previous studies on animal models have demonstrated a requirement for all three subunits of rod PDE6 to form a functional enzyme [33-36]. Additionally, mutations in the α and β-subunits in patients with retinitis pigmentosa establish the general requirement for both of these gene products for enzyme activity [36,37]. Each of the four cloned transcripts generated a polypeptide of the predicted size in a rabbit reticulocyte lysate system, thus ruling out the possibility of a premature stop codon in one of the coding regions (Figure 4). To verify the integrity of the entire coding region for each rod PDE6 gene and the δ subunit, the more arduous task of DNA sequencing was carried out. The sequence obtained from the cloned Y79 PDE6 mRNAs and δ were identical to the previously published sequences [38-42], with the exception of three silent polymorphisms in the PDE6A coding region [C->T at +6 (A of ATG is +1), C->A at 1042, and C->T at 1084]. These results are consistent with intact coding regions for all of the rod PDE6 subunits and δ capable of producing full-length functional subunits.

cGMP metabolism in Y79 cells

Because each of the catalytic subunits contains two noncatalytic binding sites for cGMP [43] we speculated that cGMP may be required for expression and proper protein folding. The levels of cGMP were assessed directly in cell extracts by immunoassay. Basal levels were 0.0519 pmols cGMP/mg of soluble protein, which is approximately 100,000 fold lower than that found in ROS (Table 4). In contrast, cAMP levels were 80 fold higher than cGMP in Y79 cells and were comparable to levels previously reported [44]. Western blots with antibodies to retinal specific guanylate cyclases 1 and 2 failed detection of a protein of the appropriate MW in Y79 cell extracts, but the antibodies did successfully recognize the cyclases in protein extracted from bovine ROS (data not shown). To test the effects of elevating cGMP levels in the Y79 cells, we incubated the cells in the presence of a specific PDE1 inhibitor (8-Methoxymethyl-IBMX) with either of two cGMP analogues (Sp-8-pCPT-cGMP or Rp-8-pCPT-cGMP). Increasing intracellular cyclic nucleotide concentrations had no effect on PDE6 expression assessed by western and PDE6 activity assay (not shown).

Proteasome inhibitor studies in Y79 cells

We next postulated that the lack of PDE6 could be due to enhanced proteasome mediated degradation [45]. To analyze proteasome mediated degradation an antibody that detects ubiquinated proteins was used in the presence or absence of the proteasome inhibitor, lactacystin. Lactacystin (10 μM) placed on the cells for 8 h caused an increase in conjugated lactacystin as compared to untreated cells (Figure 5, cf. mock vs lactacystin treated). A corresponding increase in ubiquitinated PDE6 subunits was not observed (not shown). Therefore, either the protein is degraded through an alternate pathway or the ribosomes bind PDE6 mRNA but it can not be translated.

Polysome analysis of PDE6 transcripts from Y79 cells

To examine the proportion of PDE6 and δ transcripts binding to ribosomes, polysomes were isolated from a linear sucrose gradient and RT-PCR performed to determine the location of each of the transcripts. Nested PCR results indicated that the PDE6 transcripts (α, β, γ) and δ could bind ribosomes in Y79 cells (Figure 6). Upon addition of EDTA (which causes ribosomes to dissociate from mRNA) the profile was shifted to the lower sucrose gradient fractions. This was true for all PDE6 transcripts, but not for δ. However, an antibody that recognizes δ was positive for detection of an appropriate molecular weight protein in Y79 cells and an identically sized protein in a human retinal extract (data not shown). Spurious bands were detected in the sucrose gradient fractions but these bands were also seen in the non-sucrose positive control from human retinal RNA indicating false annealing of the primers.

Expression of PDE6 in Y79 cells

We next attempted to saturate the apparent inhibition of translation by transduction of adenovirus expressing α and β into HEK293, COS7, or Y79 cells. Both viruses were positive for expression in control HEK293 and COS7 cell lines (Figure 7). In the Y79 cell line, only PDE6β, but not α was expressed at a detectable level. Moreover, PDE6β expression in Y79 cells was greatly reduced compared to expression in HEK293 and COS7 cell lines. The increase in levels of PDE6β in Y79 cells was insufficient to produce PDE6 enzyme activity (data not shown).

Discussion

Rod PDE6 is the most complex PDE family member, requiring at least three distinct subunits to produce a functional enzyme: catalytic α and β subunits, and two γ inhibitory subunits. However, once PDE6 is made and shuttled to the outer segments, the enzyme can be purified to homogeneity, and exhibits remarkable stability over time. There have been several attempts to express rod and cone PDE6 in heterologous systems [13-18], but none have yielded stable active enzyme in sufficient quantities for structure/function analysis. The most successful attempt used the baculovirus system which yielded only up to 100 μg/L of functional enzyme [16] (compare to expression of PDE5 (5 mg/L) personal communication, N. Artemyev), and it was not established that the expressed enzyme could be purified to homogeneity and retain activity. The finding of relatively high levels of PDE6 transcripts in Y79 cells [23] suggested that these cells may be ideal for structure/function studies of the enzyme. Unlike photoreceptor cell outer segments, Y79 cells were likely to contain more than one PDE activity. Consistent with an earlier preliminary characterization of the cells [24] we found a predominant PDE1 activity in the cells (Figure 1, Table 3). Surprisingly, the activity identified as PDE1 based on several criterion including column elution profile, CaM activation and substrate specificity, was not inhibited by the PDE1 inhibitor vinpocetine. This could be due to a block of the inhibition by factors present in the crude homogenate. We cannot rule out the possibility, however that the Y79 cells contain a novel vinpocetine insensitive PDE1. Another indication of this possibility is that the low Km for both cGMP and cAMP (especially in peak 2) is indicative of a PDE1C isoform, which has a molecular weight of 80 kDa not the 60 kDa that we observed with a pan-PDE1 antibody (Figure 2D) [46]. PDE1A and PDE1B show greater preference for cGMP as substrate and generally have K_ms for cAMP in the range of 3-100 μM. Yet RT-PCR indicates that PDE1A and PDE1B related isozymes are present and not PDE1C. Thus, we conclude that a complete characterization of the PDE1 activity in Y79 cells, while beyond the scope of this study, may uncover novel isozymes in this PDE family. A PDE activity profile of the WERI cell line produced very similar results with three peaks of enzyme activity (data not shown). The relatively low level of PDE6α transcript in WERI cells and the high levels of PDE6α' and γ' (Figure 2B) suggested that the WERI cells are more cone-like and Y79 more rod-like, thus Y79 cells were used for all subsequent analyses.

Unlike the rod photoreceptors where PDE6 is the only predominant PDE activity, Y79 cells were known to contain at least one other predominant PDE activity with properties of what is now classified as PDE1 [24]. In addition to PDE1, we found other PDE transcripts and activities, but the cells did not contain any PDE6 activity (as assessed by trypsin activation of the individual column fractions), and only very low levels of PDE6 subunits were detectable in metabolically labeled cells (Figure 3). We first speculated that one or more of the PDE6 subunits may be mutated, however, a scan for nonsense mutations (Figure 4) and complete DNA sequencing of each of the PDE6 subunits in Y79 cells failed to uncover any potentially pathogenic changes. Only for the β-subunit was there any sign of some translational failure indicated by truncated translation products (Figure 4). The apparent lower translational efficiency for PDE6β is consistent with a recent report demonstrating differential expression of PDE6α and β at both the transcriptional and translational levels [47].

We next considered supplementing the media with molecules previously shown to enhance expression of other proteins. The finding of exceedingly low cGMP levels in the Y79 cells (Table 4) led us to try supplementation with cGMP analogues. It was hypothesized that cGMP binding at non-catalytic sites may be required for enzyme folding and stabilization. It was also possible that altered activity of cGMP dependent kinases or phosphatases may prevent formation of the enzyme by direct or indirect alteration of post-translational modification. Elevating cGMP, however had no effect on PDE6 expression. Additionally, media supplementation with zinc, a PDE6 cofactor [48], or butyrate, a known inducer of gene expression in retinoblastoma (Rb) and many other cell types [49] also had no effect on PDE6 expression.

Another possible mechanism to explain the paucity of PDE6 in Y79 cells was rapid degradation of newly synthesized subunits. Regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T) can signal protein degradation through the proteasome pathway [50]. Two regions in PDE6α with highly probable PEST degradation sequences (HSPSSMEESEI, a.a. 50-60, PEST score=14.51; ANVPNTEEDEH, a.a. 151-161, PEST score=8.99) were found. This could at least partly explain the reduced levels of PDE6α protein expressed in the three cell lines used in this study (Figure 7). An examination of the other subunits of PDE6 did not reveal additional strong PEST candidate regions. Inhibition of the common proteasome degradation pathway with lactacystin increased the steady state amount of ubiquitinated proteins (Figure 5); however, an antibody recognizing all three subunits of PDE6 failed to detect an increase in ubiquinated protein, suggesting the possibility that PDE6 is being degraded by other, non-proteasome pathways.

The in vitro translation results established the presence of full-length open reading frames for all of the rod PDE6 transcripts, but did not address the ability of each endogenous PDE6 mRNA in the Y79 cells to be translated into protein. To examine the translation of the mRNA we first looked at the ability of each mRNA to form a polysome complex. The increased density of polysomes allows for gradient fractionation of polysome complexes and free mRNA, which can be followed by RT-PCR of the RNA in each fraction. In the absence of cations polysomes dissociate, so chelators such as EDTA should shift the amplified RNA pool to reduced sucrose fractions. For PDE6α and β there is a clear shift, however the shift observed in the PDE6γ gradients is apparent, but less pronounced possibly due to some γ mRNA remaining unassociated with ribosomes or still associated in a protein complex. The distribution of δ across the gradient in both the presence and absence of EDTA indicates that δ is not bound to ribosomes. However, antibodies against δ readily recognize the protein in Y79 cell extracts. This may be due to the apparent high abundance of δ mRNA, or alternatively it could be that the mRNA is present in two pools. In the presence of EDTA there is a pool of mRNA that remains in a non-dissociable complex, followed by a fraction where no mRNA was detected (Figure 6, δ panel, from left lanes 1-3). The absence of detectable δ mRNA indicates that there was a shift similar to that seen for PDE6γ.

Another possibility to explain the lack of PDE6 expression is that the messenger RNA levels are not at adult levels and, as a result, the protein is not expressed. The mRNA levels are above leaky transcription, but clearly below the levels detected in the retina. It has been suggested that RNA levels must increase to a certain level in the retina before protein is detected [51-55]. Developmental studies of opsin gene expression delineates three temporal categories of mRNA transcription in cattle retina: basal levels of transcription occurring before 6.5 months of gestation, which are <10% of the adult level; enhanced levels of fetal transcription occurring after 7.5 months of gestation, which are 75-80% of the adult level by birth (9 months of gestation); and adult levels of transcription occurring at birth [56]. It is unknown whether the transcripts must reach a certain level of abundance before translation will occur, or an inhibitory factor is bound that is released upon the temporally regulated expression of an additional factor. Rb tumors are classified as primitive neuroectodermal tumors that likely originate from cells in the process of commitment to becoming photoreceptors. Therefore the cells may be locked in a developmental state where transcript accumulation is occurring, but translation is blocked. It is well known that there is a lag phase of transcript accumulation of photoreceptor specific mRNAs prior to the onset of translation many days later. This idea appears inconsistent with one study of PDE6 activities in retinoblastoma cells where measurable levels of histone activatable PDE6 was identified [21]. The primary difference in this study and ours was that our study used Y79 cells in continuous culture over many years and the prior study only analyzed primary Rb tumors. Therefore, the discrepancy in results may be due to a dedifferentiation process which is common with continuous cell cultures derived from primary tumors and has been reported for Rb derived lines [57-59]. It is intriguing to speculate that the dedifferentiation process triggers expression of a protein factor to be turned on or off thereby preventing PDE6 assembly. The use of microarrays or a subtractive approach may be a good way to prove this hypothesis. Because transcript levels could account for lack of expression we reasoned that introduction of expression constructs encoding the PDE6 subunits could raise the mRNA levels and increase expression levels. Because of the low transfection efficiency of Y79 and WERI cells [25], we used adenovirus to deliver the PDE6 expression cassettes which in our hands has yield >70% transduction efficiencies. PDE6β was expressed in control COS and HEK cells at reasonable levels and was barely detectable in Y79 cells (Figure 7). PDE6α expressed at very low levels in the control cells, but was not detectable in Y79 extracts. While it could be argued that modification of the PDE6α expression construct to increase yield may lead to production of functional PDE6, in a recent report stable expression of PDE6α' in Y79 cells also failed to produce detectable protein [18]. Another possibility is translational regulation through the 5'-, 3'-untranslated or coding regions of one or all of the PDE6 subunits. Indeed, it has been reported that the coding regions of both PDEα and β transcripts contribute to overall translational efficiency of the encoded proteins [18]. While it was established in this study [18] that PDE6α and β mRNA exhibit a five fold difference in translation efficiency, it is less clear that the basis for the difference found in cell free reticulocyte lysates will be valid in the photoreceptor. Nonetheless, translational regulation may be involved in equalizing expression levels of the catalytic subunits.

In summary, we have analyzed PDE enzyme activity in Y79 RB cells, and have identified PDE1 and PDE4 isoforms that appear to account for the majority of activity in these cells. PDE2, PDE6, PDE7 transcripts were also found, however corresponding enzyme activity if existent was at levels below detection. Despite relative abundance of PDE6 transcripts no functional PDE6 was observed. All attempts to induce expression of functional PDE6 were unsuccessful. We speculate that a specific pathway may be turned on or off in continuous Rb cell lines that mimics the lag phase that occurs during photoreceptor maturation. Further studies will be required to uncover the molecular basis for this translational repression. Two recently published papers indicate that AIPL1 may be the missing protein we suggested was required for PDE6 assembly. Absence of this protein specifically reduces levels of PDE6 in murine retinas [60,61].

Acknowledgements

The authors thank Gina Capley and Druhan Lowry (University of South Alabama) for expert technical assistance, the Cystic Fibrosis Gene Therapy Core Facility at UAB (NIH NIDDK T30 DK54781) for amplifying the adenoviruses used in this study, and Dr. John Wang for critical comments on the manuscript. The authors also acknowledge with gratitude Drs. Martin Obin (Tufts University), and Raj Sharma (Department of Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada) for providing antibodies and inhibitors. This research was supported in part by National Institutes of Health grant R01 EY09924. Some of these data were presented in abstract form [62] at the 2001 meeting of the Association for Research in Vision and Ophthalmology.

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