INTRODUCTION

Phototransduction in the retina involves a cascade of regulated biochemical processes (for recent reviews, see Refs. 1 and 2). Key components in this light-activated biochemical cascade are the retinal specific phosphodiesterases (PDE6s).¹ These phosphodiesterases catalyze the conversion of cGMP to 5-GMP when activated by the G protein, transducin. The retinal phosphodiesterases are multimeric proteins composed of catalytic and inhibitory subunits. The large subunits of the rod and cone phosphodiesterases (the  and  subunits in rods and the subunit in cones) dimerize to form the catalytic core of the isozymes. The smaller 11- and 13-kDa subunits (rod  and cone ) serve as inhibitors of the rod and cone phosphodiesterases, respectively (3, 4, 5). Hurwitz et al. (6) identified a 17-kDa protein that immunoprecipitated with bovine retinal phosphodiesterases. Gillespie and Beavo (7) and Gillespie et al. (8) later demonstrated that a protein of this size co-purified with the isotonically soluble rod and cone phosphodiesterases, but not with the membrane-bound rod phosphodiesterase. A function for this 17-kDa protein subunit (also referred to as the  or 15-kDa subunit) has not been described.

The rod membrane-bound phosphodiesterase is loosely associated with the membrane at least in part due to C-terminal isoprenyl and carboxymethyl post-translational modifications. Several investigators (9, 10) have demonstrated that the  subunit is modified by a farnesyl (C-15) group and the  subunit is modified by a geranylgeranyl (C-20) group on their carboxyl termini. In addition both COOH termini are methylated as reported by Swanson and Applebury (11) and others (12). Mutation of the conserved cysteine residue to serine in the CAAX isoprenylation motif in the  and  subunits eliminated membrane binding of the expressed phosphodiesterase subunits (13).

Gillespie et al. (8) speculated that the 17-kDa subunit might confer solubility on the membrane-associated phosphodiesterases since it co-purifies with the isotonically soluble forms of the type 6 phosphodiesterases. Due to the insoluble nature of the isolated  subunit, it was not possible to test this hypothesis with  subunit purified from bovine retinas.

We report here the identification and expression of a clone encoding the rod-soluble  subunit protein. Recombinant  subunit solubilizes rod membrane-bound phosphodiesterase presumably by binding to one or both of the catalytic subunits' isoprenylated COOH termini. Localization studies confirm its presence in rod outer segments as well as its possible presence in nonretinal tissues.

EXPERIMENTAL PROCEDURES

Frozen dark-adapted bovine retinas were purchased from Hormel. Secondary fluorescent antibodies were purchased from Calbiochem. O.C.T. compound was purchased from Miles, Inc. Vectashield® was purchased from Vector Labs, Inc. Restriction enzymes were purchased from Life Technologies, Inc. High concentration ligase was purchased from U. S. Biochemical Corp. Tth DNA polymerase was obtained from Epicentre Technols. Inc. Hybond N⁺ membrane was purchased from Amersham Corp. Elutip-d columns were from Schleicher & Schuell. The vector pCR II was purchased from Invitrogen. The vector pVL1393 was the generous gift of Dr. D. Storm, University of Washington. The BakPak6 virus was obtained from Clontech. DE52 anion exchange resin was purchased from Whatmann. Gel filtration grade blue dextran, ovalbumin, chymotrypsinogen A, and RNase A standards were purchased from Pharmacia Biotech Inc. All other chemicals were obtained from Sigma or J. T. Baker Inc.

Purification of rod-soluble phosphodiesterase  subunit was accomplished as described by Gillespie et al. (8). For chymotryptic digestion, 200 pmol of rod-soluble 17-kDa subunit was incubated in 2% (w/w) -chymotrypsin in trifluoroacetic acid/acetonitrile, neutralized to a pH of 8.0 with ammonium bicarbonate. Proteolyzed samples were separated by reverse-phase high performance liquid chromatography (HPLC) as described below.

For cyanogen bromide digestion, rod-soluble 17-kDa subunit protein was dried under vacuum. The sample was reconstituted in ammonium bicarbonate buffer and incubated in 77 mM triethylammonium acetate, 0.077% tributylphosphine and 1.5% 4-vinylpyridine for 2 h at a pH of 9.5 at 37 °C under argon (14, 15, 16). The sample was dried and reconstituted in 88% formic acid, diluted to 70% formic acid, and incubated with cyanogen bromide under argon at room temperature for 24 h. The proteolyzed sample was lyophilized twice and resuspended in 70% formic acid. After incubating for 2 h, the sample was diluted to 9% formic acid and injected onto a C-8 reverse phase column.

High Performance Liquid Chromatography Separation of -Chymotryptic and Cyanogen Bromide Peptides

Chymotryptic fragments were separated by injecting the samples onto a 1.5 mm × 100-mm octyl (C-8) reverse phase column (Brownlee) on an HP 1090 HPLC. The HPLC buffers were 0.08% trifluoroacetic acid (HPLC buffer A) and 0.08% trifluoroacetic acid, 80% acetonitrile (HPLC buffer B). The fragments were separated using a 0-70% gradient of HPLC buffer B at a flow rate of 0.3 ml/min. Cyanogen bromide fragments were separated by the same procedure except the reverse-phase gradient was a 0-80% gradient of HPLC buffer B.

Edman degradation sequencing was performed on a Applied Biosystems model 470A gas-phase sequencer attached in line to a model 120A HPLC as described by Trong et al. (17).

Mass Spectral Analysis of the Expressed  Subunit

Approximately 40 µg of recombinant  subunit protein was subjected to reverse-phase HPLC as described above. The subunit was concentrated to approximately 20% of the original volume in a Speedvac concentrator at room temperature, then used for mass spectrum analysis. The sample was injected into a matrix-assisted laser desorption ionization mass spectrometer, model Voyager Elite BioSpectrometry Research Station (PerSeptive Biosystems, Framingham, MA). This mass spectrometer employs a nitrogen laser at a wavelength of 337 nm. An -cyano-4-hydroxycinnamic acid matrix was used. The spectrum was acquired in linear mode with an acceleration voltage of 30,000, a grid voltage set at 90.0%, and a guide wire voltage set at 0.300%. A positive ion spectra was collected. The data were externally calibrated to an accuracy of 0.1%. The mass reported is an average of two sample injections measured as doubly charged (M²⁺) and singly charged (M¹⁺) molecular ions. Each mass measurement is the result of 100 scans.

A bovine retinal cDNA library (18) was screened with the following redundant oligonucleotides: TGNGCNGGCATCATYTGN(G/C)RYTC (17NO1) and C(G/T)RAAYTTYTCCATYTGYTCNGC (17NO-2B) (19, 20).

Most nucleic acid sequencing was performed as described elsewhere (21, 23, 24) with slight modifications. The procedure of Andersen et al. (21) was modified by annealing template DNA and sequencing oligonucleotide on ice for 5-10 min in place of the annealing step at 37 °C.² One region of the full-length 17-kDa clones consistently resulted in band compressions using our standard sequencing procedure, therefore several other sequencing methods were employed (22, 23, 24, 25, 26) to sequence this ``GC''-rich region.

Messenger RNA was isolated using Invitrogen's FastTrack mRNA isolation kit (version 3.1) and quantitated (20). Total RNA was isolated using the methods of Chomczynski and Sacchi (27). Messenger RNA was electrophoresed in a denaturing formaldehyde agarose gel (20). Nucleic acids were transferred to Hybond-N⁺ membrane overnight. Immediately after cross-linking, the membrane was stained (28), then incubated with random prime labeled cDNA probes (29, 30). Blots were hybridized and then washed under increasingly stringent conditions (19). The most stringent wash conditions were at 55-60 °C in 0.25 × SSC, 0.1% SDS for at least 20 min.

Approximately 0.7 µg of poly(A)-selected mRNA isolated as described above was reverse transcribed with Thermus thermophilus (Tth) DNA polymerase (31). The oligonucleotide used for the reverse transcription reaction was 17K-AS6: 5-CCTTGCCAAAGTATCTTCCC-3. The oligonucleotides used for the first set of PCR reactions were 17K-S5 (5-AGCGGGAGCTGAGGGGAG-3) and 17K-AS6. Southern analysis was performed (20) using a 350-bp XhoI DNA fragment from clone 17K-11 random prime labeled with [-³²P]dATP (29, 30). Control reactions contained no template DNA or mRNA.

To further select for a 17-kDa-specific DNA, a nested amplification reaction was performed on the RT-PCR products using the oligonucleotides, 17K-S5, 17K-AS8 (5-CTCAGCATCCCGAAGGTTC-3), and Thermus aquaticus (Taq) DNA polymerase. Control reactions contained either no DNA template, 17K-10 DNA (identical to clone 17K-11) in place of RT-PCR product, or substituted the oligonucleotide T7-24 (5-CGACGGCCAGTGAATTGTAATACG-3) for the oligonucleotide 17K-S5. A 220-bp DNA product was isolated and subcloned into the vector, pCR II, using standard techniques.

The following peptides: EKFRLEQKVYFKGQV (17K-I) and RDAETGKILWQGTEDLSVP (17K-II), were synthesized for producing 17-kDa subunitspecific anti-peptide antibodies. The sequence, RSQILMWSANKVFEELTDVE, was synthesized for producing a type 6 phosphodiesterase catalytic subunit-specific anti-peptide antibody (18, 32). The peptides were conjugated to keyhole limpet hemocyanin in the presence of 20 mM glutaraldehyde in 100 mM sodium phosphate buffer, pH 7.5. After addition of diluted RIBI adjuvant, approximately 1.6 ml of the final conjugated peptide sample was injected into each rabbit. The rabbits were boosted 26 days after the first injection with freshly conjugated peptide. Serums were tested using an enzyme-linked immunosorbent assay plate (33). For Western analysis, proteins were electrophoresed in SDS-PAGE gels, transferred to nitrocellulose, and probed with antibodies (33, 34).

Polyclonal anti-peptide antibody 17K-I was affinity-purified against the  subunit from 60 µg of rod-soluble phosphodiesterase bound to nitrocellulose (33). The -bound antibody was eluted from the nitrocellulose with 500 µl of 100 mM glycine, pH 2.5, and neutralized by addition of 50 µl of 1 M Tris-Cl, pH 8.0. Monoclonal antibody ROS1 was purified over a protein A column under high salt conditions at pH 9.0 (33). The concentration of the purified ROS1 antibody was approximately 6 mg/ml.

Bovine eyes were obtained at a local slaughterhouse and stored on ice until dissection (approximately 1-2 h). Dissection was performed under normal room illumination. Tissue was fixed in 4% paraformaldehyde dissolved in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄·7H₂O, 1.4 mM KH₂PO₄, pH 7.4) for approximately 2 h on ice (20, 35). After rinsing in PBS, pH 7.4, the eye cups were floated in 30% sucrose, 130 mM sodium phosphate, pH 7.3, for 2-18 h, then embedded in O.C.T. compound on a dry ice block. Twenty-micrometer sections were cut on a Tissue-Tek II Cryostat set at 18 to 20 °C (20).

Sections were incubated in blocking solution (1 mg/ml bovine serum albumin, 0.05% (v/v) Triton X-100, 2% (v/v) goat serum, 2% (v/v) bovine serum diluted into PBS, pH 7.4) for 1 h at room temperature. Protein A-purified monoclonal antibody ROS1 and affinity-purified 17K-I were diluted 1:500 and 1:10, respectively, in antibody diluent (1 mg/ml bovine serum albumin, 0.05% (v/v) Triton X-100, 1% (v/v) goat serum, 1% (v/v) bovine serum in PBS, pH 7.4) and incubated for longer than 1 h at room temperature. After three 5-min washes in rinse solution (PBS containing 0.05% (v/v) Tween 20, pH 7.3), sections were incubated in a 1:50 dilution of the appropriate secondary antibody (goat anti-rabbit antibody conjugated to fluorescein isothiocyanate or goat anti-mouse antibody conjugated to tetramethylrhodamine isothiocyanate). After rinsing three times for 5 min each, sections were mounted in Vectashield®. Signals were visualized and recorded on a Leitz Dialux 20 microscope equipped with a 100-watt high pressure mercury lamp and a Leitz vario-orthomat camera. Alternatively, data were collected on a Bio-Rad MRC-600 confocal laser scanning microscope utilizing a krypton/argon laser with emissions at 488 and 568 nm.

Sf9 cells were grown on plates or in spinner flasks in TNM-FH medium supplemented with 100 units/ml penicillin G, 100 µg/ml of streptomycin sulfate and 10% fetal calf serum (20, 36, 37). To infect cells for protein production, spinner flasks were infected at cell densities of 1-3 × 10⁶ cells/ml; plates were infected at cell densities that were 60-80% confluent (36, 37). Titers were conducted using the end point dilution procedure of O'Reilly et al. (37) and Reed and Muench (38). Most infections were done at a multiplicity of infection of 10 or greater.

The EcoRV-BamHI fragment of the 17-kDa cDNA clone, 17K-11, was subcloned into the SmaI and BglII sites of the baculovirus vector, pVL1393 (19, 20). Five µg of the resulting transfer plasmid, pVL17-17, and 5 µl of Bsu36I-digested BacPAK6 DNA (Clontech) were combined with 500 µl of Sf9 cells at a cell density of 2 × 10⁶ cells/ml (39). After electroporation, 1.5 ml of complete TNM-FH medium was added, and cells were plated in a 35-mm dish. Following incubation for 4 days at 27 °C, extracellular virus particles were collected and immediately subjected to agarose overlays (36). Viral plaques were screened (40) and the identity of the expressed protein was confirmed by Western analysis. Viral stocks were amplified according to the procedures of Summers and Smith (36) and O'Reilly et al. (37).

Purification of the Expressed  Subunit

The  subunit was overexpressed in Sf9 cells (36, 37). After 3 days, the cells were collected by centrifugation at 200 × g and 22 °C for 5 min. The cell pellet was washed with PBS, pH 7.4, containing 14 mM benzamidine, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, and 1 mM dithiothreitol. After repelleting, the cells were homogenized in low salt buffer (20 mM Tris-Cl, 1 mM Na₂EDTA, pH 7.4) supplemented with the same protease inhibitors and dithiothreitol. The homogenate was centrifuged at 16,000 × g for 30 min at 4 °C to separate the supernatant fraction from the pellet fraction. The supernatant was added to DEAE anion exchange resin (DE52) preequilibrated with low salt buffer, pH 7.4, at a concentration of 3-10 mg of protein/ml of resin. After incubating for 1-3 h at 4 °C with constant rotation, the resin was loaded into a 1.5 cm × 13.5-cm column (bed height was approximately 6 cm). The column was washed with 2-3 volumes of low salt buffer. A linear gradient was run from 20 mM Tris-Cl, 1 mM Na₂EDTA, pH 7.4, to 300 mM NaCl, 20 mM Tris-Cl, 1 mM Na₂EDTA, pH 7.4, at a flow rate of approximately 14 ml/h at 4 °C. The total gradient volume was approximately six times the bed volume. Approximately 400-µl fractions were collected and assayed for  subunit using Coomassie-stained SDS-PAGE and Western analysis. The fractions containing  subunit were pooled. For a larger scale procedure, the method was essentially the same except that a 2.5 cm × 22-cm column was used, and approximately 1.1-ml fractions were collected.

The pooled DEAE fractions were sequentially filtered through Amicon Centricon 100 and 50 concentrators prerinsed with ROS buffer (60 mM KCl, 30 mM NaCl, 20 mM MOPS, 2 mM MgCl₂, pH 7.2), as follows. The Centricon 100 concentrators were centrifuged for 30-60 min at 1000 × g. The filtrates containing approximately 99% of the partially purified  subunit were collected and loaded onto Amicon Centricon 50 concentrators. The Centricon 50 concentrators were centrifuged for 30 min at 5000 × g. The filtrates were collected and concentrated on Amicon Centricon 10 concentrators for 60-120 min at 5000 × g. To increase recovery of the 17-kDa subunit protein, the Centricon 50 retentates were diluted with 2 ml of ROS buffer, pH 7.2, and centrifuged again. Purified expressed  subunit was stored at 4 °C. For larger scale purification, Amicon Centriprep concentrators were used.

For assay of  subunit activity, purified rod outer segments were isolated from 50 frozen dark-adapted bovine retinas as described by Papermaster and Dreyer (41) with modification by Uhl et al. (42). This procedure releases nearly all of the endogenous -bound rod and cone phosphodiesterase, but not the rod membrane-bound phosphodiesterase. The rod outer segment membranes were resuspended in ROS buffer, aliquoted, frozen in liquid nitrogen or on dry ice, and stored at 70 °C. Protein concentration was determined either with the Bradford (43) assay or by measuring its absorbance at 280 nm. The concentration of the visual pigment, rhodopsin, was determined spectrophotometrically (44). To calculate phosphodiesterase concentration in rod outer segments, we assumed that 67% of the total protein was rhodopsin and that phosphodiesterase was present at 1/100 the rhodopsin concentration (45). The value obtained using this calculation is within 2-fold the value obtained by back calculating from phosphodiesterase activity assuming a cGMP hydrolytic rate of 2000 µmol of cGMP hydrolyzed/min/mg of protein (data not shown).

Subunit Activity Assay

Expressed  subunit was combined with rod outer segment membranes diluted in ROS buffer, pH 7.2, and incubated for the times and temperatures indicated. The samples were centrifuged at 4 °C for 30 min at 16,000 × g to separate rod outer segment membranes and supernatants. The membranes were resuspended in ROS buffer, pH 7.2, or hypotonic buffer (5 mM Tris-Cl, pH 7.5) as indicated. Each fraction was assayed for phosphodiesterase activity using either the phosphate release assay (7, 8) or the [³H]cGMP assay (46). Phosphodiesterase was activated by preincubating samples with 0.02 mg/ml TPCK-trypsin for 8-10 min on ice followed by addition of 0.125 mg/ml soybean trypsin inhibitor on ice.

The dose-response curves were analyzed using the program, SigmaPlot. They were fit with a rectangular hyperbole.

Purified recombinant  subunit protein was incubated with rod outer segment membranes as described above or with hypotonically extracted rod outer segment protein (47), or trypsin-released rod outer segment protein (5). Fifty µM Pefabloc® was included in all experiments except those requiring trypsin treatment. Each sample was loaded onto a Sephadex G100 gel filtration column (1 cm × 26 cm), then eluted with 200 mM NaCl buffer (200 mM NaCl, 20 mM Tris-Cl, 1 mM Na₂EDTA, pH 7.4) at a flow rate of 8-9 ml/h at 8 °C. Fractions were pooled and concentrated on Amicon Centricon 10 concentrators, and equal volumes were loaded onto an SDS-PAGE gel. The gels were stained with Coomassie (48) or silver (7), or were analyzed by Western analysis (33).

For trypsin-released rod outer segment protein, rod outer segment membranes were incubated in 0.02 mg/ml TPCK-trypsin for 1 min at 22 °C, and the reaction was stopped with the addition of 0.125 mg/ml soybean trypsin inhibitor. Reactions were centrifuged for 30 min at 16,000 × g and 4 °C to separate bound proteins from soluble proteins. The trypsin conditions were optimized to allow for maximal release of PDE activity from the membrane without significant degradation of the inhibitory subunits (5).

RESULTS

To obtain amino acid sequence for screening the retinal cDNA library, the rod-soluble  subunit was purified by reverse-phase HPLC and cleaved with cyanogen bromide and -chymotrypsin. After isolation on C-8 columns, the peptide fragments were analyzed using Edman degradation. The five peptide sequences identified for each of the protease treatments are listed in Table I, as well as the number of times that each sequence was observed. Most of the peptides were observed more than once, and two of the cyanogen bromide and chymotryptic peptide sequences overlapped by several amino acids.

Table I. Peptide sequences isolated from the rod-soluble phosphodiesterase  subunit Peptide sequences are listed in the order that they appear in the deduced amino acid sequence in Fig. 1B. Cyanogen bromidea  -Chymotrypsina Peptide sequenceb,c 2 2 NLRDAETGKILwQGTEDLSVP-  VE-hAsV-D 2 NFSSAEQMEKF 1 1 EKFRLEQKVYFKGQVLe 3 QSLIEAAPESQMmPAh 1 KLETAKAElmsw a  The numbers indicate the number of times each peptide was observed during Edman degradation for each protease treatment listed. b  Capital letters indicate a strong signal during Edman degradation. Small letters indicate a weaker signal during Edman degradation. c  Dash indicates no specific amino acid could be assigned to this cycle during Edman degradation.

Based on the sequences listed in Table I, redundant oligonucleotides were synthesized and used to screen the bovine retinal cDNA library of Li et al. (18). Two lifts were taken of each plate and probed with each of two redundant antisense oligonucleotides encoding the amino acid sequences, ESQMMPAH and AEQMEKF. Only viral plaques that were positive with both oligonucleotides were selected for further screening.

From the final screen, two groups of clones were identified. The first clone, 17K-14, contained approximately 90% of the open reading frame encoding the 17-kDa subunit and 35 bp of the 3-noncoding sequence. However, this clone also contained a library cloning ``artifact'' or perhaps was derived from an incompletely processed RNA transcript. It contained an in-frame stop codon 9 amino acids NH₂-terminal to the  subunit peptide sequence, LRDAE. There was no methionine between this in-frame stop codon and the  subunit peptide sequence. The second group of clones contained two identical 1123-bp full-length clones (referred to as clone 17K-11 henceforth). A map of the sequencing strategies and the final cDNA sequence is shown in Fig. 1, A and B, respectively.

cDNA sequence and predicted amino acid sequence of the rod soluble phosphodiesterase  subunit.

The NH₂-terminal-most peptide sequence recognized from the Edman degradation analysis is ``NLRDAE'' (see underlined amino acid sequence, Fig. 1B). This left us with two possible starting methionines, the methionine immediately preceding this sequence (predicted molecular mass, 15,087 Da) and the methionine 20 amino acids NH<sub>2</sub>-terminal to this sequence (predicted molecular mass, 17,390 Da). It seems likely that the starting amino acid sequence must be the latter at the ``MSA ...'' sequence for several reasons. 1) Neither ATG start site contains an ideal Kozak sequence at positions 3 and +4; however, the ATG site at nucleotide 166 is clearly the preferred codon of the two (49). 2) The sequence immediately upstream from the ATG sequence at nucleotide 166 is highly ``GC''-enriched, often a signature of regions immediately preceding start ``ATGs'' (49). 3) There are no in-frame stop codons between ATG¹⁶⁶ and ATG²²³.

We knew that the expressed subunit aligned with the native rod soluble subunit in SDS-PAGE chromatography and Western analysis (Fig. 8 and data not shown). However, we could not determine whether its molecular mass was 15 or 17 kDa in SDS-PAGE chromatography. In addition, the size of this subunit was reported as both 15 kDa (8) and 17 kDa (6). To determine its molecular mass, we performed mass spectral analysis on the recombinant  subunit. The analysis indicated that the mass of the recombinant protein was 17,395 ± 4 Da (data not shown) indicating that ATG¹⁶⁶ is the preferred start site for translation of the expressed subunit.

Expressed  subunit shifts its mobility in G100 size exclusion chromatography when preincubated with ROS membranes.

Clone 17K-11 contains 165 bp of a 5-noncoding sequence and 505 bp of a 3-noncoding sequence. The open reading frame consists of 453 bp with a deduced amino acid sequence of 150 amino acids, predicting a molecular mass of 17,390 Da. In addition, a polyadenylation signal is present near the end of the 3-noncoding sequence in this clone. The predicted amino acid sequence is not related to any other known protein (GenBank^TM release no. 92). Since clone 17K-14 and clone 17K-11 overlapped in only 88% of the open reading frame (see Fig. 1A), we did a RT-PCR experiment to confirm that the 5 end of clone 17K-11 did not contain any ``artifacts,'' as reported by Li et al. (18) for other cDNAs isolated from this library and as identified in our clone 17K-14. Oligonucleotides flanking the region of interest were used to reverse transcribe then amplify bovine retinal mRNA unrelated to the bovine retinal cDNA library. Two clones were identified whose sequence matched that of clone 17K-11 in the NH₂-terminal portion of the open reading frame. The region of overlap between the RT-PCR product, 17K-15, and clone 17K-11 is indicated in Fig. 1A.

A Partial Human Deduced Amino Acid Sequence Is Nearly Identical to the Bovine Rod Phosphodiesterase  Subunit

Recently, two sequences derived from a human placental cDNA library were submitted to GenBank^TM as part of the Merck-Washington University EST Project (accession nos. N41734[GenBank] and R81870[GenBank]). The cDNA, accession no. R81870[GenBank], has a 78% nucleotide sequence similarity to part of the 3-noncoding region of clone 17K-11 (data not shown). The cDNA, accession no. N41734[GenBank], has a 90% nucleotide sequence identity, and a 96% amino acid sequence identity to the bovine  sequence amino acids Met¹ to Val⁸⁰, and contains no in-frame stop codon (Fig. 1B and data not shown).

To initiate studies on the tissue distribution of the 17-kDa subunit and to confirm that our cDNA represented a message enriched in retina, we isolated messenger RNA from eight bovine tissues and analyzed them by Northern analysis. The Northern blot was probed with a cDNA probe that included the entire open reading frame of clone 17K-11. The results of this Northern analysis are shown in Fig. 2. The predicted size of the mRNA is approximately 1300 bases.

Northern analysis of soluble rod  subunit message in different bovine tissues.

As expected, the probe produced an intense band in the retinal mRNA lane. The adrenal and brain mRNA lanes also contained a detectable signal even after a high stringency wash (Fig. 2, A and B). The bands in the adrenal gland and brain are approximately the same size as that in the retina (Fig. 2A). Because of this unexpected result, several different Northern blots were probed using two different preparations of mRNA to confirm the presence of a signal in the bovine brain and adrenal preparations (Fig. 2, A and B). Analysis of the radiolabeled signals on the Northern blot using a PhosphorImager indicate that the adrenal and brain messages are present at approximately 25-30% of retinal message (Fig. 2A, bottom). We conclude that the  subunit message, while highly enriched in the bovine retina, may also be present to a lesser extent in nonretinal tissues. Relatively little is known about the localization of other PDE6 subunits in nonretinal tissues (see ``Discussion'').

In addition to cloning a cDNA encoding this subunit, we used the sequence data to synthesize two polyclonal anti-peptide antibodies. Although both antibodies recognized proteins in immunocytochemistry experiments (see below) and immunoabsorbent assays (data not shown), only the anti-peptide antibody, 17K-II, identified a 17-kDa band in Western analysis (Fig. 3, middle panel). As can be seen, the antibody identifies specifically a 17-kDa band in purified cone phosphodiesterase and rod-soluble phosphodiesterase. It does not recognize the 11-kDa subunit, 13-kDa subunit, or the larger 84-, 88-, and 92-kDa subunits of the rod and cone phosphodiesterases (compare silver-stained proteins to Western immunoreactivity, Fig. 3, left and middle panels).

In addition to the 17-kDa subunit anti-peptide antibodies, three anti-peptide antibodies were produced against peptides encoding portions of bovine cone subunit. One of the antibodies, PDE6 Cat pAb, identifies the large catalytic subunit of the rod and cone phosphodiesterases (Fig. 3, right panel) specifically. We used this antibody to identify the phosphodiesterase subunits in the gel filtration experiments described below.

Immunocytochemical Localization of  Subunit to Rod Outer Segments

Gillespie and Beavo (7) and Gillespie et al. (8) isolated cone and rod phosphodiesterase isozymes that contained a 17-kDa subunit. To confirm that the  subunit was localized to cone and rod outer segments and to confirm that our peptide sequence coded for a photoreceptor cell-specific subunit, we performed immunocytochemistry experiments on bovine retinal sections using the anti-peptide antibodies. The anti-peptide antibody, 17K-I, appeared to identify only rod outer segments (Fig. 4A). The signal was absent when 17K-I antibody was preincubated with rod-soluble phosphodiesterase (Fig. 4B). Preimmune sera or the absence of primary antibody from the histochemical reactions yielded similar results to the competition experiment (Fig. 4, C and D). Labeling by the other anti-peptide antibody, 17K-II, yielded similar results (data not shown).

The  anti-peptide antibody, 17K-I, labels bovine retinal outer segments in immunocytochemical analysis.

To further examine these results, double-labeling experiments were performed using a mouse monoclonal antibody, ROS1, and affinity-purified 17K-I with visualization on a confocal microscope. The results of the 17K-I and ROS1 labeling are shown in Fig. 5, A and B, respectively. Hurwitz et al. (50) previously demonstrated that ROS1 labels both cone outer segments and rod outer segments in several species including bovine. Fig. 5A demonstrates the labeling of bovine retina sections with the 17K-I antibody in green. In Fig. 5B, the labeling of rod and cone outer segments by the ROS1 antibody is shown in red. In Fig. 5C, the images from Fig. 5, A and B, were merged. The yellow color indicates areas of signal overlap, demonstrating that 17K-I signal and ROS1 signals overlap in rod outer segments but not in cone inner or outer segments (Fig. 5C, arrows). The apparant discrepancy between the immunocytochemistry results (Fig. 5) and the Western analysis (Fig. 3) probably results from an exchange of the rod  subunit into the cone phosphodiesterase preparation during purification (see ``Discussion'').

Double labeling bovine retinal sections with ROS1 (a rod + cone cell antibody) and the  anti-peptide antibody, 17K-I, demonstrates that 17K-I labels rod outer segments only.

Expression of  Subunit in Baculovirus

The cDNA containing the entire open reading frame and most of the noncoding regions of the rod-soluble  subunit was subcloned into a baculovirus expression vector, pVL1393, and a recombinant baculovirus, BCV11, was isolated. Fig. 6A demonstrates the presence of a 17-kDa protein expressed specifically in cells infected with the recombinant virus and identified by the anti-peptide antibody, 17K-II, in Western analysis.

Recombinant  subunit Western analysis, purification using anion exchange chromatography and size filtration and its activity in ROS membranes.

Purification of Expressed  Subunit from Baculovirus Extracts

Since the purification of the  subunit using the methods of Gillespie and co-workers (7, 8) results in an insoluble  subunit, we devised a new method of purification for the expressed subunit protein. The expressed protein binds to anion exchange (DE52) resin under low salt conditions (20 mM Tris-Cl, 1 mM Na₂EDTA, pH 7.4) and elutes early in a 0 to 300 mM sodium chloride gradient (see Fig. 6B, the heavy line below the graph and the inset) with the midpoint of the elution at approximately 60 mM NaCl. The expressed protein in the pooled fractions represents approximately 10% of the total protein present in the pool as determined by densitometry scans of Coomassie-stained gels (see Fig. 6C, first lane). In the pooled fractions, most of the Sf9 and viral proteins were much larger than the expressed 17-kDa protein. We therefore reasoned that we might be able to use a sizing step to further purify the protein.

The results of filtering the protein through a series of Amicon filters are shown in Fig. 6C. Approximately 99% of the  subunit filtered through an Amicon 100 concentrator resulting in a 3-fold purification of the anion exchange pool and elimination of many high molecular mass contaminants (Fig. 6C, compare lanes 100R, 100F, and 50R). Nearly all of the remaining contaminants were eliminated from the preparation by filtering the  subunit through an Amicon 50 concentrator. Since a fraction of the  subunit was retained by this filter, the retentate was diluted and refiltered to increase the yield of  protein (Fig. 6C, compare lanes 50R and 50-2R). The purified subunit was concentrated using an Amicon 10 concentrator (Fig. 6C, lane 10R). Analysis of Coomassie-stained SDS-PAGE gels using densitometry scanning indicates that the recombinant  subunit protein was approximately 95% of the total protein (Fig. 6C, lane 10R). In general, we can purify 1-2 mg of recombinant  per liter of Sf9 culture (1.5 × 10⁹ cells).

Purified Recombinant  Solubilize Membrane-bound Rod Phosphodiesterase Activity

Gillespie et al. (8) suggested that the 17-kDa subunit may solubilize rod membrane phosphodiesterase since it associated with the isotonically soluble isozymes, but they were unable to test this hypothesis because the buffers required to keep the 17-kDa preparation available in solution were highly denaturing. Therefore we developed an assay to determine if the recombinant subunit protein is capable of removing membrane-bound rod outer segment phosphodiesterase. Purified recombinant  was incubated with rod outer segments under isotonic salt conditions. The membrane fraction and soluble fraction were isolated by centrifugation, and each fraction was analyzed for phosphodiesterase activity. Fig. 6D demonstrates the ability of the recombinant protein to release phosphodiesterase activity into the supernatant fraction, whereas isotonic buffer alone released little membrane-bound phosphodiesterase activity.

Solubilization of the Rod Membrane Phosphodiesterase Depends on the Concentration of Purified  Subunit

Fig. 7A demonstrates a dose-dependent solubilization of phosphodiesterase activity by increasing concentrations of purified expressed  subunit protein. The apparent EC₅₀ for  activity at 5 nM phosphodiesterase was 14 ± 2 nM (assuming that active  is a 17-kDa monomer and that 100% of the expressed  subunit is active). Since equilibrium could not be reached at concentrations of phosphodiesterase lower than 5 nM (data not shown), the dose-response curve might represent a titration event and not a measure of affinity.

Dose-dependent solubilization of ROS phosphodiesterase activity by  and trypsin-activated ROS membrane phosphodiesterase activity in the presence and absence of  subunit.

The Expressed  Subunit Does Not Alter the Activity of ROS Phosphodiesterase

Gillespie et al. (8) determined that the purified rod-soluble and rod membrane-bound phosphodiesterase had similar activation and kinetic properties. However, Gillespie et al. (8) worked with purified enzymes and did not have the ability to test for activity in the presence of added  subunit protein. It seemed possible that the  subunit protein, in addition to solubilizing membrane bound phosphodiesterase activity, could directly alter its activity. Therefore we assayed rod membrane phosphodiesterase activity in the presence and absence of expressed  subunit (Fig. 7B). The ROS membranes were preincubated with  subunit prior to dilution and trypsin activation of the phosphodiesterase. Fig. 7B demonstrates that addition of purified expressed  subunit protein does not appear to alter the K_m or V_max activities of trypsin-activated ROS phosphodiesterase activity in the presence of membranes.

Gel Filtration Chromatography Indicates That the  Subunit Protein Binds to PDE Catalytic Subunits

To further demonstrate that the  subunit was interacting with the membrane associated rod phosphodiesterase, we used gel filtration in combination with Western analysis and phosphodiesterase activity assays to confirm that the  subunit protein interacts with phosphodiesterase. Purified  subunit was incubated with rod outer segments for 30 min at 30 °C, and the supernatant and pellet fractions were separated by centrifugation. The supernatant fraction was then fractionated on a G100 gel filtration column. Purified recombinant  eluted near the included volume of the column (Fig. 8A). When recombinant  subunit was first preincubated with rod outer segment membranes, a fraction of recombinant  co-migrated in the void volume with phosphodiesterase activity and catalytic subunit immunoreactivity (Figs. 8B and 9A). In addition, the PDE6-specific ROS1 antibody co-immunoprecipitated recombinant  and catalytic subunit immunoreactivity and phosphodiesterase activity (data not shown). The -solubilization activity assay, the size exclusion chromatography, and the immunoprecipitation results strongly suggest that the  subunit binds to the membrane-bound phosphodiesterase.

The expressed  subunit co-migrates with soluble phosphodiesterase immunoreactivity and activity but not trypsin-released phosphodiesterase immunoreactivity and activity.

To determine if the 17-kDa subunit protein could bind to soluble phosphodiesterase, the purified  subunit protein was incubated with hypotonically extracted rod outer segment protein in the presence of a serine protease inhibitor prior to size exclusion chromatography. As shown in Fig. 9B, the 17-kDa subunit protein migrated in the void volume when incubated with hypotonically extracted phosphodiesterase.

To determine if the phosphodiesterase catalytic subunit COOH termini containing the isoprenyl and carboxymethyl modifications might be involved in the solubilization of phos-phodiesterase by the 17-kDa subunit protein, we repeated the size exclusion experiments in the presence of trypsin-released rod outer segment proteins. To determine optimum conditions for release of phosphodiesterase activity from the membrane, the trypsin release experiments described by Wensel and Stryer (5) were repeated. The purified expressed  subunit protein was incubated with trypsin-released phosphodiesterase and applied to the G100 column. The results of this experiment, shown in Fig. 9C, demonstrate that the recombinant  no longer migrates in the void volume. The phosphodiesterase activity profiles for each of these experiments is shown below the Western analysis indicating the location of the phosphodiesterase activity in the column profile.

In Fig. 9D, an equal amount of phosphodiesterase activity present in the void volumes of the experiments shown in Fig. 9, A-C, were analyzed with the 17K-II anti-peptide antibody and catalytic subunit-specific anti-peptide antibody in Western analysis. This experiment demonstrates the inability of the  subunit to co-migrate with the trypsin-released phosphodiesterase, suggesting that the catalytic subunits' COOH termini may contain a  subunit binding site.

DISCUSSION

This is the first report demonstrating a function for the rod  subunit of retinal phosphodiesterase. It appears to be one of only a few proteins thus far characterized that are capable of solubilizing a membrane-bound enzyme. It is, to our knowledge, the only protein described to date that solubilizes a protein modified by both a farnesyl and geranylgeranyl group. In addition, we demonstrate that it may interact directly with one or both isoprenylated COOH termini of the phosphodiesterase catalytic subunits. The localization of the  subunit's message in other tissues suggests that it also may interact with phosphodiesterases or other proteins in nonretinal tissues.

To initiate studies examining the biochemical function of the  subunit of the soluble rod phosphodiesterase, we obtained a full-length cDNA clone encoding the soluble rod phosphodiesterase  subunit. Confirmation that the clone encodes the rod phosphodiesterase  subunit is based on the following criteria. 1) The deduced amino acid sequence contains 4 of the 5 peptides isolated and sequenced from the soluble rod phosphodiesterase  subunit and codes for a 17-kDa protein. 2) A probe made from the  subunit cDNA clone binds to bovine retina mRNA in Northern analysis. 3) The expressed  subunit co-migrates with native  subunit in SDS-PAGE analysis (Fig. 8). 4) A  subunit-specific anti-peptide antibody identifies a 17-kDa protein expressed by our recombinant baculovirus. 5) A  subunit-specific anti-peptide antibody labels bovine rod outer segments specifically.

The predicted molecular mass of the 17-kDa subunit protein is 17,390 Da, which corresponds closely to the observed mobility of the expressed protein in SDS-PAGE electrophoresis and is the same mass obtained in mass spectral analysis. Reverse-transcription and polymerase chain reaction analysis demonstrated that the 5 end of the open reading frame of clone 17K-11 does not contain a cloning artifact. From the Northern analysis, we conclude that clone 17K-11 contains approximately 88% of the full-length transcript and all of the coding sequence.

We found evidence in the Northern analysis to indicate that the 17-kDa message may be present in brain and adrenal tissue as well as in the retina. This is the first report of evidence indicating that a subunit of retinal phosphodiesterase may be present in adrenal tissue. Recently, Collins et al. (51), reported that human PDE6  subunit message is present in brain. Carcamo et al. (52) recently demonstrated the presence of a cone-type phosphodiesterase in mammalian pineal gland. The rat pineal extract contained a 15-kDa protein that may or may not be the same as our  subunit. The identification of a highly conserved ``'' or ``''-like cDNA sequence from a human placental library supports the conclusion that 's message is present in nonretinal tissues. The distribution of this polypeptide's message in nonretinal tissues may indicate that it interacts with other phosphodiesterases or proteins. The unusually high degree of amino acid similarity between the human placental  and rod bovine -deduced amino acid sequences indicates that at least a portion of the cDNA is highly conserved within mammals and suggests a conserved function for the retinal PDE6  subunit.

The immunocytochemistry results shown here indicate that the  subunit protein in the retina is localized only to the rod outer segments in bovine retinal sections, data apparently in conflict with previous results (7, 8) and the data presented in Fig. 3. There are several possible explanations for these results. 1) It is possible that free or excess  subunit exists in the rods and exchanges with cone phosphodiesterase during purification of cone phosphodiesterase. Hamilton and Hurley (4) indicated that the rod phosphodiesterase  subunit may bind to the cone enzyme during purification. 2) The rod and cone  subunits may share partial, but not complete, sequence homology; therefore, the polyclonal antibodies may identify different epitopes in Western analysis and immunocytochemical analysis. 3) It is also possible that, in the immunocytochemistry experiments, the  subunit protein was not accessible to the antibody in cone cells, although capable of labeling rod outer segments. Whether the 17-kDa subunit protein is present in a complex with cone phosphodiesterase in situ remains uncertain.

To further confirm the identity of the cDNA sequence, to initiate studies examining the biochemical mechanism of  subunit protein activity and to initiate physiological studies determining the role of the soluble phosphodiesterase in phototransduction, the cDNA encoding the  subunit protein was expressed in a baculovirus expression system. The recombinant baculovirus expressed a protein of the expected molecular mass that is recognized by the anti-peptide antibody, 17K-II. The protein appears to be expressed at relatively high levels for a phosphodiesterase subunit (approximately 1-4% of total protein in cell extracts).³ Purification of this recombinant subunit proved to be relatively simple due to the lack of low molecular mass proteins in baculovirus infected extracts. Purification was obtained by a 2-step procedure, anion exchange chromatography and size filtration. Although the fold purification varies somewhat depending on expression levels, with most preparations >85% purity is reached after approximately 50-fold purification. The fact that most of the expressed protein filtered through Amicon 100 and 50 concentrators indicates that the recombinant protein is not highly aggregated.

A functional assay was developed to test the hypothesis that the  subunit protein is capable of solubilizing the membrane-bound rod phosphodiesterase under physiological (e.g. isotonic) salt concentrations. The results of the functional assay indicate that a majority of the phosphodiesterase can be released into the supernatant due to the presence of the 17-kDa subunit protein. However, in all the experiments conducted thus far we have yet to see more than 60% of the phosphodiesterase solubilized. It is unclear why 40% of the phosphodiesterase activity in rod outer segment cannot be solubilized by the  subunit. It is possible that  is capable of binding to the phosphodiesterase only when it is in a certain membrane compartment (i.e. disc membranes versus plasma membranes), when it is in a specific tertiary or quaternary structure, when the active dimer is composed of certain subunits (i.e. / versus / versus /), when the phosphodiesterase is in association with other molecules, or when the phosphodiesterase is present in a specific lipid environment (e.g. positively charged lipid head groups). It is also possible that only a portion of the phosphodiesterase is accessible in our assay, that is, the disc membranes may be inside-out or otherwise not accessible or the membranes may not be completely permeable to small protein molecules.

The release of phosphodiesterase activity into the supernatant fraction is dependent on the concentration of  subunit. The apparent EC₅₀ for the solubilization is approximately 14 nM (assuming a monomer molecular mass and 100% activity of the protein). Since we could not perform the assay under more dilute phosphodiesterase concentrations in our experimental system, we could not determine whether the EC₅₀ is a measure of titratable phosphodiesterase activity or whether it approximates the affinity of  subunit for membrane-bound phosphodiesterase. The EC₅₀ value obtained may therefore represent a lower limit of the affinity of  subunit for membrane-bound phosphodiesterase. A high affinity of  for phosphodiesterase is indicated by the tight association of  in the purified rod-soluble phosphodiesterase (8), the nM EC₅₀ obtained here, and the lack of dissociation of  during size exclusion chromatography or immunoprecipitation. Consistent with the experiments of Gillespie et al. (8), who compared the catalytic activities of the rod soluble and rod membrane phosphodiesterases, recombinant  subunit had no apparent effect on trypsin-activated ROS phosphodiesterase activity.

Since the results of the solubilization assay implied that the expressed  subunit protein was binding directly to the phosphodiesterase catalytic subunits and solubilizing them, we chose to examine this event using size-exclusion chromatography. Rod outer segment proteins extracted under isotonic conditions by the  subunit caused a shift in the  subunit protein mobility, indicating that the  subunit protein had combined with one or more proteins to form a larger complex. Western analysis as well as phosphodiesterase activity assays demonstrated the co-elution of the  subunit protein and phosphodiesterase catalytic subunits in the void volume of the column. The observation that rod membrane phosphodiesterase activity is solubilized in the presence but not absence of expressed  subunit, the shift in molecular mass of the  subunit in the presence but not absence of rod outer segment membranes, and the immunoprecipitation of -bound rod membrane phosphodiesterase strongly implies that the  subunit is binding the phosphodiesterase directly to solubilize its activity.

After the isolation and purification of a rod-soluble phosphodiesterase, there was some question as to the relationship of the soluble rod and membrane-bound rod phosphodiesterase catalytic subunits. The results presented here imply that the critical difference between the membrane-bound rod and soluble rod phosphodiesterases is the presence of the  subunit and that the catalytic subunits are not likely to be different gene products.

Several groups have recently reported the presence of isoprenyl groups on the carboxyl termini of the  and  subunits of the rod membrane phosphodiesterase (9, 10). It is generally concluded that one or both of the isoprenyl groups serve to anchor the phosphodiesterase subunits on the membrane. We hypothesized that the  protein could cause solubilization of the phosphodiesterase by binding to the isoprenylated catalytic subunits at their carboxyl termini. When phosphodiesterase activity was released from the membranes under conditions that proteolyzed the catalytic subunits' COOH termini but kept  intact, the  subunit protein no longer migrated in the void volume of the G100 size exclusion column. These experiments suggest that the  subunit protein is interacting with the COOH-terminal region of either or both of the large catalytic subunits and that either the COOH-terminal sequence, the isoprenyl groups, or both are necessary for binding.

Only a few other proteins have been characterized that interact with lipid groups on post-translationally modified proteins. The protein, guanine-dissociation inhibitor, has been shown to solubilize geranylgeranylated membrane-bound proteins (53, 54, 55, 56). In addition, a family of proteins known as the fatty acid-binding proteins are capable of binding to fatty acid moieties. The chick retina fatty acid protein displays little expression in post-embryonic chick retinas (57, 58, 59). Neither of these proteins share any sequence identity to the rod soluble  subunit cDNA reported here.

The function of the soluble phosphodiesterase in visual transduction is unclear. Solubilization of the enzyme by the  subunit protein may serve to uncouple the phosphodiesterase from its activator molecule, transducin. The  subunit solubilization activity may be responsive to one or several mechanisms of light adaptation in vertebrate photoreceptors. The availability of an active, purified expressed  subunit protein should allow us to elucidate the physiological role of the soluble phosphodiesterase in the process of phototransduction.