Residues within the Polycationic Region of cGMP Phosphodiesterase gamma  Subunit Crucial for the Interaction with Transducin alpha  Subunit
IDENTIFICATION BY ENDOGENOUS ADP-RIBOSYLATION AND SITE-DIRECTED MUTAGENESIS*

(Received for publication, November 27, 1996, and in revised form, March 31, 1997)

Vladimir A. Bondarenko ab, Mit Desai ab, Salil Dua ab, Matsuyo Yamazaki ab, Rajesh Haresh Amin ab, Kirk K. Yousif ab, Tomoya Kinumi cd, Mamoru Ohashi c, Naoka Komori d, Hiroyuki Matsumoto d, Kenneth W. Jackson e, Fumio Hayashi f, Jiro Usukura g, Valery M. Lipkin h and Akio Yamazaki abij

From the a Kresge Eye Institute and the Departments of b Ophthalmology and i Pharmacology, Wayne State University, School of Medicine, Detroit, Michigan 48201, the c Department of Applied Physics and Chemistry, The University of Electro-Communications, Chofu, Tokyo, 182 Japan, the d Department of Biochemistry and Molecular Biology and the e William K. Warren Medical Research Institute and Department of Medicine, the University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, the f Department of Biology, Faculty of Science, Kobe University, Kobe, 657 Japan, the g Department of Anatomy, School of Medicine, Nagoya University, Nagoya, 466 Japan, and the h Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region, Russia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Interaction between the gamma  subunit (Pgamma ) of cGMP phosphodiesterase and the alpha  subunit (Talpha ) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an endogenous enzyme and site-directed mutagenesis of the Pgamma polycationic region to identify residues required for the interaction with Talpha . Pgamma , free or complexed with the alpha beta subunit (Palpha beta ) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32P]NAD. Identification of ADP-ribose in the radiolabeled Pgamma and radiolabeling of arginine-replaced mutant forms of Pgamma indicate that both arginine 33 and arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is modified at a time. Pgamma complexed with Talpha (both GTP- and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with Talpha , was ADP-ribosylated in the presence of Talpha , suggesting that a Pgamma domain containing these arginines is masked by Talpha . A Pgamma mutant (R33,36K), as well as wild type Pgamma , inhibited both GTP hydrolysis of Talpha and GTP binding to Talpha . Moreover, GTP-bound Talpha activated Palpha beta that had been inhibited by R33,36K. However, another Pgamma mutant (R33,36L) could not inhibit these Talpha functions. In addition, GTP-bound Talpha could not activate Palpha beta inhibited by R33,36L. These results indicate that a Pgamma domain containing these arginines is required for its interaction with Talpha , but not with Palpha beta , and that positive charges in these arginines are crucial for the interaction.


INTRODUCTION

Cyclic GMP phosphodiesterase (PDE),1 a key enzyme in phototransduction, is composed of Palpha beta and two Pgamma subunits (1-6). Palpha beta hydrolyzes cGMP (7, 8) and binds cGMP to its high affinity, noncatalytic sites (9-11). In amphibian ROS, Pgamma regulates these Palpha beta functions as an inhibitor of cGMP hydrolysis (12) and as a stimulator of cGMP binding to noncatalytic sites (13, 14). Different interactions between Palpha beta and Pgamma have been suggested to be required to express these two functions (15, 16). In bovine ROS, Pgamma inhibits cGMP hydrolysis by Palpha beta (17); however, the effect of Pgamma on the cGMP binding to noncatalytic sites has never been documented. In amphibian ROS, these Pgamma functions are interrupted by Pgamma release with GTP·Talpha from Palpha beta (12-14, 18). We have recently suggested that these functionally different Pgamma s are released in the different steps of phototransduction (15, 16). When [cGMP] is at the dark level, Pgamma responsible for the inhibition of cGMP hydrolysis is released. Consequently, cGMP is hydrolyzed by the activated PDE for photoexcitation. When [cGMP] becomes low, Pgamma responsible for the stimulation of cGMP binding is released, and the affinities of these noncatalytic sites to cGMP are drastically reduced. The resulting release of cGMP from these noncatalytic sites may facilitate the recovery of cytoplasmic [cGMP] to the dark level in ROS.

To understand physiological functions of protein-protein interaction, functional structures of the protein involved should be elucidated. Pgamma functional structure is especially interesting because such a small protein (87 amino acids) plays important roles in phototransduction by interacting with various proteins. At least four different interactions are considered: (a) Pgamma -Palpha beta interaction for the inhibition of cGMP hydrolysis by Palpha beta ; (b) Pgamma -Palpha beta interaction for the stimulation of cGMP binding to Palpha beta noncatalytic sites; (c) Pgamma -Talpha interaction for the release of Pgamma inhibitory strain from Palpha beta ; and (d) Pgamma -Talpha interaction for the release of Pgamma to reduce the affinity of Palpha beta noncatalytic sites to cGMP. Previous studies have focused on interactions (a) and (c). Peptides have been used to identify the polycationic region of Pgamma within residues 24-45 and the carboxyl-terminal region of Pgamma corresponding to residue 46-87 as the sites for the interaction (a) (19-22). Mutational analysis of Pgamma has also shown that the carboxyl-terminal residues and several positive charged residues in the polycationic region are also involved in the inhibition of cGMP hydrolysis (23, 24). Without impairing interaction with Palpha beta , a frameshift mutation of Pgamma has also revealed that the carboxyl-terminal residues are involved in the cGMP hydrolysis inhibition (15). In addition, the polycationic region and a site near the carboxyl terminus in Pgamma have been suggested as sites required for the interaction (c) (19, 23, 25-27). However, little is known about Pgamma domains involved in the interactions (b) and (d). The frameshift mutation of Pgamma has suggested that the amino-terminal residues are involved in the stimulation of cGMP binding to noncatalytic sites on Palpha beta (15). However, a Talpha interaction site on Pgamma , which is required for the Pgamma release to reduce the affinity of Palpha beta noncatalytic sites to cGMP, has not been identified.

In this study we have focused on identification of specific residues in the Pgamma polycationic region for following reasons. (i) The polycationic region has been suggested to be involved in the interaction with both Palpha beta and GTP·Talpha for the regulation of cGMP hydrolysis. Identification of amino acid residues in the polycationic region seems to be crucial to reveal the mechanism for the Pgamma release by GTP·Talpha . However, residues required for these interactions have not been identified. (ii) We found that the specific arginines in the Pgamma polycationic region were ADP-ribosylated by an endogenous enzyme. Thus, protein-protein interactions in which the polycationic region is involved may be monitored by tracing the Pgamma ADP-ribosylation under physiological conditions. (iii) We also found that the Pgamma ADP-ribosylation was regulated by the interaction between Pgamma and Talpha . Therefore, the ADP-ribosylation can be a useful tool to learn the interaction between Pgamma and Talpha . We describe that both arginine 33 and arginine 36 in the Pgamma , free or complexed with Palpha beta , are ADP-ribosylated by endogenous arginine-ADP-ribosyltransferase. The Pgamma ADP-ribosylation is inhibited when Pgamma is complexed with Talpha (both GTP- and GDP-bound forms), suggesting that the domain including these arginines is not exposed to ADP-ribosyltransferase when Pgamma is complexed with Talpha . Then, using forms of Pgamma mutated in these residues, we confirm that the domain is involved in the interaction with Talpha . Moreover, we find that positive charges in these arginine are important for the interaction with Talpha .


EXPERIMENTAL PROCEDURES

Materials

Mono Q (5 × 50 mm), Pep RPC HR5/5 (5 × 50 mm), TSK G2000SW (7.5 × 300 mm), DEAE-Sephacel, SP-Sepharose Fast Flow, and Blue Sepharose CL-6B were purchased from Pharmacia Biotech Inc. AG 1-X2 resin was obtained from Bio-Rad. Other materials were purchased from the following sources: [adenylate-32P]NAD, [3H]cGMP, [gamma 32P]GTP, and [35S]GTPgamma S from DuPont NEN; cGMP, GTP, GDP, GDPbeta S, Gpp(NH)p, and GTPgamma S from Boehringer Mannheim; novobiocin, snake venom phosphodiesterase, arginine methyl ester, cysteine methyl ester, PMSF, agmatine, and NAD-agarose from Sigma. Phosphatidylinositol-specific phospholipase C (from Bacillus thuringiensis) was obtained from ICN, and 1 unit was defined as the supplier described. Suppliers of materials for molecular biological experiments are described in these sections.

Buffer

Buffer A consisted of 100 mM Tris·HCl (pH 7.5), 5 mM DTT, and 0.1 mM PMSF. Buffer B consisted of 10 mM Tris·HCl (pH 7.5), 5 mM DTT, 5 mM MgCl2, 1 mM EGTA, and 0.1 mM PMSF. Buffer C consisted of 100 mM Tris·HCl (pH 7.5), 5 mM DTT, 5 mM MgCl2, 1 mM EGTA, and 0.1 mM PMSF. Buffer D consisted of 5 mM Tris·HCl (pH 7.5), 5 mM DTT, and 0.1 mM PMSF. Buffer E consisted of 50 mM Tris·HCl (pH 7.5), 2 mM EDTA, 1 mM DTT, 0.2 mM PMSF, and 50 mM NaCl. Buffer F consisted of 10 mM Tris·HCl (pH 7.5), 2 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 5 µM leupeptin, and 5 µM pepstatin. Buffer G consisted of 10 mM Tris·HCl (pH 7.5), 2 mM DTT, 5 mM MgCl2, and 1 mM EGTA. Buffer H consisted of 100 mM Tris·HCl (pH 7.5) and 10 mM MgCl2. Buffer I consisted of 100 mM Tris·HCl (pH 7.5), 1 mM DTT, and 5 mM MgCl2.

Preparation of ROS Membranes

Under dim red light, ROS were prepared from dark adapted bullfrogs (Rana catesbiana or Rana grylio) by 46% sucrose flotation in Buffer A. Although various amounts of ROS membranes were washed in this study, we used a similar method. Bleached ROS membranes from 20 frogs were suspended in 3 ml of Buffer B and passed through a no. 21 needle seven times. Membranes and soluble fractions were separated by centrifugation (200,000 × g, 4 °C, 15 min). ROS membranes were washed seven times in the same way, and these ROS membranes were termed prewashed ROS membranes. The prewashed ROS membranes were washed seven more times with 3 ml of Buffer C and seven times with 3 ml of Buffer C containing 400 µM GTP. Talpha (more than 90%) and Pgamma (about 50%) were released from these membranes. These membranes contain Pgamma -less (active) PDE and were termed as Pgamma -depleted ROS membranes. Residual Pgamma in these membranes is not sensitive to GTP·Talpha (12), suggesting that the membrane preparation contains a distinct subset of Pgamma which cannot be released by GTP·Talpha . This subset of Pgamma was termed GTP·Talpha -insensitive Pgamma . When Pgamma -depleted ROS membranes were used as a source for ADP-ribosyltransferase, these membranes were washed twice with 2 ml of Buffer C to remove residual GTP. When the Pgamma -depleted membranes were washed an additional seven times with 3 ml of Buffer D, Tbeta gamma (more than 90%) was released. These membranes are termed Pgamma - and transducin-depleted ROS membranes. It should be emphasized that ADP-ribosyltransferase activity was detected in Pgamma -depleted and Pgamma - and transducin-depleted membranes when frog or recombinant bovine Pgamma was used as a substrate. However, ADP-ribosylation of the residual Pgamma was not detected in these membranes. This suggests that GTP·Talpha -insensitive Pgamma cannot be ADP-ribosylated. Urea-treated ROS membranes were prepared as described (13).

Site-directed Mutagenesis of Pgamma

All DNA manipulations were carried out using standard procedures (28). Full-length bovine Pgamma cDNA (29), which was ligated into the EcoRI-HindIII-digested plasmid pALTER-1 (Promega), was used in the mutagenesis steps. All mutagenic oligonucleotide primers used (Table I) were purchased from DNAgency Inc. (Malvern, PA). Mutagenesis was carried out using Altered Site System Mutagenesis Kit (Promega). Mutant clones were identified by in situ hybridization with 32P-labeled mutagene oligonucleotides. The mutations were confirmed by double-stranded DNA sequencing using the fmol of DNA sequencing System (Promega). Two oligonucleotides: Up-5'-GCCAACCTGCATATGAACCTGGAGCC-3' and Down-5' GGGGTCGGATCCTAGATGATGCCATACTG-3' were used in a polymerase chain reaction to introduce NdeI and BamHI sites at the ends of Pgamma genes. The NdeI-BamHI fragment was cloned into the NdeI-BamHI-digested pET-IIA (Novogene). The vector was transferred to Escherichia coli BL21(DE3) (Novogene) for expression of Pgamma .

Table I. Oligonucleotides used in site-directed mutagenesis


Mutants Oligonucleotidesa

R11K A: 5'-GCCGAGATCAAATCGGCCACC-3'
R15K B: 5'-TCGGCCACAAAAGTGATGGGG-3'
R24E C: 5'-CCGTCACTCCCGAAAAAGGGCC-3'
R24K D: 5'-CCGTCACTCCCAACAAAGGGCCCCC-3'
R33K E: 5'-CGAAATTTAAGCAAAAACAAACCAGG-3'
R36K F: 5'-CGGCAAACAAAACAGTTCAAG-3'
R11,15K G: 5'-GCCGAGATCAAATCGGCCACCAAAGTGATGGGG-3'
R33,36L H: 5'-ATTTGAACTGCAGCGTCTGCAGCTGTTTGAA-3'
R33,36K I: 5'-TTTAAGCAGAAACAAACCAAGCAGTTCAAG-3'
R15,24K Oligo B + oligo D
R11,15,33,36K Oligo G + oligo I
R11,15,24,33,36K Oligo D + oligo G + oligo I

a Underlined letters indicate mutation sites.

Expression and Purification of Recombinant Pgamma

A fresh single colony was grown overnight at 37 °C in the presence of 100 µg/ml ampicillin. The overnight culture was diluted 1:100 into a medium (12 g of tryptone, 24 g of yeast extract, and 0.2 ml of 5 M NaOH/1,000 ml) containing 100 µg/ml ampicillin, and the culture was grown at 37 °C. At an A600 nm approx  0.6, protein expression was induced by the addition of 1 mM (final) isopropyl beta -D-thiogalactopyranoside, and cells were incubated for another 4 h at room temperature. Then, cells were spun down (1,700 × g, 20 min, 4 °C) and resuspended in 0.10 volume of Buffer E. After sonication, insoluble material was spun down (200,000 × g, 15 min, 4 °C), and the supernatant was loaded into an SP-Sepharose Fast Flow column (6 × 100 mm) that had been equilibrated with Buffer E. After washing with 10 bed volumes of Buffer E containing 100 mM NaCl, the protein was eluted using 10 ml of Buffer E with a gradient of 0.2-0.5 M NaCl. Following the measurement of PDE inhibitory activity, the active fractions were collected, heated at 80 °C for 5 min, and centrifuged (345,000 × g, 30 min, 4 °C). Pgamma and its mutants were further purified using Pep RPC HR5/5 column as described (12). The purity of Pgamma and its mutants was greater than 90%.

Purification of Proteins

Frog Pgamma was purified from the supernatant prepared by washing prewashed ROS membranes with Buffer C containing GTP (12). Siliconized tubes and pipette tips were used in all experiments using Pgamma except SDS-gel electrophoresis. Talpha (GTPgamma S- and GDP-bound forms) and Tbeta gamma were isolated as described (30). In some experiments partially purified ADP-ribosyltransferase was used. ADP-ribosyltransferase was solubilized in 10 ml of Buffer F containing phosphatidylinositol-specific phospholipase C (1 unit) from Pgamma -depleted membranes (120 mg of protein). Following incubation (37 °C, 30 min), the sample was centrifuged (200,000 × g, 20 min, 4 °C). The supernatant was applied to a Blue Sepharose CL-6B column (3 ml) that had been equilibrated with Buffer F and washed with 12 ml of Buffer F. Flow-through and washing fractions were collected and applied to NAD-agarose (2 ml) that had been equilibrated with Buffer F. The column was washed with 15 ml of Buffer F, and ADP-ribosyltransferase was eluted with 6 ml of Buffer F containing 100 µM NAD (its purity was about 80%). After dialysis against Buffer F, ADP-ribosyltransferase activity was measured.

ADP-ribosylation of Pgamma , Peptides, and Agmatine

ADP-ribosylation of Pgamma was carried out with various membrane preparations or enzyme preparations solubilized by detergents or phosphatidylinositol-specific phospholipase C. Frog Pgamma and recombinant bovine Pgamma were used. Both Pgamma s were ADP-ribosylated in a similar manner. The amounts of each components were slightly different in each experiment (see each figure legend); however, Pgamma ADP-ribosylation was performed in a similar way. The reaction mixture contained Pgamma (0.2-0.6 µg), NAD (10-50 µM; ~0.5 µCi) and proteins in the 50 µl of Buffer G. Pgamma ADP-ribosylation was initiated by the addition of [adenylate-32P]NAD and terminated by heating with SDS-sample buffer (5 min, 80 °C). These samples were analyzed by SDS-polyacrylamide gradient (8-16%) gel electrophoresis and autoradiography. The band corresponding to Pgamma (its apparent molecular weight is 13,000 in gels) was also excised from gels, and its radioactivity was measured. The radioactivity was proportional to the value obtained by densitometric scanning. As a control, the same procedure was performed without added Pgamma , and the radioactivity of the corresponding region was subtracted from the radioactivity of the Pgamma band. Without added Pgamma , no 13,000 band was detected, and the radioactivity was negligible. If one amino acid in Pgamma was radiolabeled, approximately 3-10% of Pgamma was apparently radiolabeled in the regular experiments, and the apparent maximum incorporation of radioactivity was 23% (see Fig. 2). Possible reasons for such a low incorporation of the apparent radioactivity will be discussed later. When pertussis toxin was used, pertussis toxin was activated as described (31). A peptide corresponding to Pgamma residues 30-39 (FKQRQTROFK) and its mutant forms was also ADP-ribosylated by Pgamma - and transducin-depleted membranes (76 µg of protein). After various amounts of these peptides (0.05-0.6 µg) were ADP-ribosylated (1 h, 33 °C) in 100 µl of Buffer G containing [adenylate-32P]NAD (10 µM; ~0.25 µCi), 40 µl of the reaction mixtures was spotted onto 2 × 2-cm pieces of Whatman P-81 phosphocellulose (32). Then, all of these pieces were washed five times with 400 ml of 1% trichloroacetic acid, rinsed with ethanol, and the radioactivity of each paper was measured. ADP-ribosylation of agmatine (20 mM) was carried out in a manner similar to that described above. ADP-ribosylated agmatine was isolated as described previously (33). Samples (100 µl) were applied to AG 1-X2 columns (1 ml) that had been equilibrated with 20 mM Tris·HCl (pH 7.5). [32P]ADP-ribosylated agmatine was eluted with 5 ml of water.


Fig. 2. Isolation of [adenylate-32P]NAD-radiolabeled Pgamma . Purified frog Pgamma (10 µg) was incubated with Pgamma - and transducin-depleted ROS membranes (276 µg of protein) in 160 µl of concentrated (×1.25) Buffer G for 30 min at 0 °C. Radiolabeling of Pgamma was performed by the addition of 20 µl of 500 µM NAD (~25 µCi) at 33 °C. After a 1-h incubation, an additional 20 µl of 500 µM NAD (~25 µCi) was applied to the reaction mixture, and the mixture was further incubated for 1 h. The reaction was terminated by the addition of 200 µl of 0.2 M formic acid and heating at 80 °C for 5 min. The supernatant was collected by centrifugation (345,000 × g, 30 min, 4 °C) and applied to a Pep RPC column. Pgamma was eluted with an acetonitrile gradient as described. Panel A, profile of absorbance at 280 nm. The arrow indicates the position of Pgamma eluted when purified frog Pgamma was applied to the column. Panel B, radioactivity (bullet ) and PDE inhibitory activity (open circle ) of each fraction. Each fraction (20 µl) was used to measure its 32P radioactivity. After drying each fraction (20 µl), its PDE inhibitory activity was measured using Pgamma -depleted ROS membranes. Panel C, purity of radiolabeled Pgamma . Lane A, Mr standards: a, 94,000; b, 68,000; c, 43,000; d, 30,000; e, 20,000; f, 14,000. Lane B, radiolabeled Pgamma (1 µg) visualized by Coomassie Blue. Lane C, autoradiography of radiolabeled Pgamma .
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Preparation of ADP-ribosylated Pgamma

Although the amounts of components in each reaction mixture were slightly different (see each figure legend), ADP-ribosylated Pgamma was prepared by incubation of purified Pgamma , Pgamma - and transducin-depleted ROS membranes, and [adenylate-32P]NAD. ADP-ribosylated Pgamma was isolated using a Pep RPC column (see Fig. 2) or SDS-gel electrophoresis (see Fig. 3). In the case of the Pep RPC column, the same volume of 0.2 M formic acid was added into the sample to terminate the reaction, and the mixture was heated for 5 min at 80 °C. Then, the sample was centrifuged (345,000 × g, 30 min, 4 °C). The process of the Pgamma extraction by formic acid was repeated two times. The pooled supernatant was applied to a Pep RPC HR5/5 column that had been equilibrated with 0.1% trifluoroacetic acid. Elution of Pgamma was carried out with an acetonitrile gradient (0-60%) containing 0.1% trifluoroacetic acid, as shown in Fig. 2. The flow rate was 1 ml/min, and the fraction volume was 0.5 ml. Following measurement of both radioactivity and Pgamma activity in each fraction, the active fractions were dried, suspended in water, and kept at -80 °C. In the case of SDS-gel electrophoresis, samples were heated with SDS-sample buffer to terminate the reaction. The gel was stained in 20% methanol containing 0.2% (w/v) Coomassie Blue and 0.5% (v/v) acetic acid (20 min) and destained with 30% (v/v) methanol (1 h). The visualized Pgamma was excised from the gel.


Fig. 3. Identification of ADP-ribose in Pgamma radiolabeled with [adenylate-32P]NAD. Purified frog Pgamma (4.0 µg) was incubated with [adenylate-32P]NAD (15 µM; ~4 µCi) and Pgamma - and transducin-depleted ROS membranes (85 µg of protein) in 50 µl of Buffer G for 60 min at 33 °C. The reaction was terminated by the addition of SDS-sample buffer (30 µl) and heating for 5 min at 80 °C. Following SDS-gel electrophoresis of the sample, the gel was rapidly stained and destained as described. After visualization, Pgamma was excised from the gel and cut into two pieces, and these two gel pieces were immersed overnight in 2 ml of water at 0 °C and then in 600 µl of acetonitrile for 20 min at room temperature (twice). These two gel pieces were dried under reduced pressure. One gel piece was incubated overnight in 50 mM glycine/NaOH (pH 10.0) at room temperature and spun (345,000 × g, 30 min, 4 °C). After the supernatant (275 µl) was adjusted to pH 7.0 with HCl, the volume of the sample was also adjusted to 1 ml with water and spun (345,000 × g, 30 min, 4 °C). The supernatant was analyzed by a Mono Q column as described. The other gel piece was swollen with 5 µl of Buffer H, and then snake venom phosphodiesterase (5 µg) suspended in 5 µl of Buffer H was added for the further swelling of the gel (10 min, room temperature). After the addition of Buffer H (590 µl) the gel piece was incubated at 37 °C for 3 h. Then, 400 µl of Buffer H was added to the mixture. The sample was spun (345,000 × g, 30 min, 4 °C), and the supernatant was analyzed by a Mono Q column. Panel A, chromatogram of mixture of NAD, AMP, and ADP-ribose. a, NAD; b, AMP; c, ADP-ribose. Panel B, Pgamma treated with pH 10.0 buffer. The supernatant of Pgamma treated with the pH 10.0 buffer (620 µl) was applied to the column with ADP-ribose (100 µM), and then the radioactivity of each fraction (200 µl) was measured. The yield of the radioactivity was 70%. Panel C, Pgamma treated with phosphodiesterase. The supernatant of Pgamma treated with phosphodiesterase (430 µl) was applied to the column with 100 µM AMP, and then the radioactivity of each fraction (200 µl) was measured. The yield of the radioactivity was 86%.
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Identification of ADP-ribose in the [Adenylate-32P]NAD-radiolabeled Pgamma

To identify ADP-ribose in the radiolabeled Pgamma , the radiolabeled frog Pgamma was incubated with glycine/NaOH buffer (pH 10.0) or treated with snake venom phosphodiesterase, as shown in Fig. 3. After centrifugation of these reaction mixtures (345,000 × g, 30 min, 4 °C), supernatants were applied with ADP-ribose (100 µM) or AMP (100 µM) to a Mono Q column that had been equilibrated with 10 mM sodium phosphate (pH 6.0). After washing the column, radioactive compounds were eluted. Chromatographic conditions were: A, 10 mM sodium phosphate (pH 6.0); and B, 10 mM sodium phosphate (pH 6.0) and 0.2 M NaCl; 0-100% B in 16 ml on a linear gradient. The flow rate was 1 ml/min, and the fraction volume was 0.5 ml.

Measurement of Molecular Ion Mass of ADP-ribosylated Pgamma

Fifty µl of ADP-ribosylated recombinant bovine Pgamma (0.2 mg/ml) was injected into a high performance liquid chromatography system that consisted of Ultrafast Microprotein Analyzer model 600 (Microm BioResources, Auburn, CA) equipped with a Reliasil C18 reverse phase column (5 µm particle size, 300 Å pore size, 1.0 × 150 mm) with a flow rate 40 µl/min. Chromatographic conditions were: A, 0.1% trifluoroacetic acid, 2% acetonitrile, H2O; and B, 0.07% trifluoroacetic acid, 90% acetonitrile, H2O, 0-56% B in 25 min on linear gradient. The flow was monitored at 215 nm, and the eluate was introduced directly into an API-III triple quadrupole mass spectrometer (Perkin-Elmer Sciex, Thornhill, Ontario, Canada) equipped with an electrospray atmospheric pressure ionization source. The tuning and calibration were done using polypropylene glycol. The mass spectrometer was set to scan in a positive ion mode at orifice potential of 90 V from m/z = 500-2,200 with a mass step of 0.4 Da. The mass data were analyzed by MacSpec 3.22 (Perkin-Elmer Sciex).

Analytical Methods

Activities of PDE and Pgamma were assayed as described (12). GTPase activity of Talpha and GTPgamma S binding to Talpha were measured as described (18). Immunological detection of Pgamma was carried out as described (34). SDS-polyacrylamide gel electrophoresis was performed as described (30). When [adenylate-32P]NAD was present in the electrophoresis, the gel was cut above the dye front to remove free radioactive NAD for the reduction of background in autoradiography. Therefore, we do not show the dye front in each picture of gel. Protein concentrations were assayed with bovine serum albumin as standard (35). The amount of Pgamma was assayed by densitometric scanning (12). To calculate the Pgamma concentration, 9,625 and 9,669 were used as molecular weights of frog (36) and recombinant bovine (see Fig. 4) Pgamma , respectively, although Pgamma was detected as a 13,000 band in SDS-gels. It should be emphasized that all experiments were carried out more than two times, and the results were similar. Data shown are representative of these experiments.


Fig. 4. Mass spectrum of bovine recombinant Pgamma and its ADP-ribosylated Pgamma . The ADP-ribosylated Pgamma was prepared with nonradioactive NAD and purified as shown in Fig. 3. Two major peaks with the m/z values of the multiple ion series are indicated in the figure. The ion series of the second major peak are underlined. The 100% relative intensity corresponds to 89,300 counts. Inset, deconvoluted mass spectrum.
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RESULTS

An Arginine Residue (Arg-33 or Arg-36) in Pgamma Is ADP-ribosylated by Endogenous Arginine-ADP-ribosyltransferase

In the presence of [adenylate-32P]NAD a 39-kDa protein in prewashed ROS membranes was radiolabeled by pertussis toxin (Fig. 1). However, the radiolabeling almost disappeared if ROS membranes were washed with a buffer containing GTP (data not shown). These data indicate that Talpha in prewashed ROS membranes is ADP-ribosylated by pertussis toxin, as described previously (31, 37, 38). In the absence of pertussis toxin, Talpha ADP-ribosylation was not detected, indicating that endogenous Talpha ADP-ribosylation (39, 40) is negligible under our conditions. Under the same conditions, a 13-kDa protein was also radiolabeled, and the radiolabeling was more clearly observed in the absence of pertussis toxin. The radiolabeling was increased by the addition of purified Pgamma . These data support the idea that the radiolabeled 13-kDa protein is Pgamma and that both endogenous and exogenous Pgamma are radiolabeled by an enzyme(s) in prewashed ROS membranes in the presence of [adenylate-32P]NAD. Following densitometric scanning of the protein band and measurement of its radioactivity, we estimate that approximately 50% of endogenous Pgamma was radiolabeled if one amino acid in Pgamma was radiolabeled.


Fig. 1. Pertussis toxin-independent radiolabeling of Pgamma by [adenylate-32P]NAD. After prewashed ROS membranes were divided into eight portions, each portion (145 µg of protein) was incubated (30 min, 0 °C) with or without frog Pgamma (0.6 µg) in 50 µl of Buffer G containing 16 µM GDP. Radiolabeling was initiated by the addition of NAD (final 10 µM; ~4 µCi) with or without pertussis toxin (11 µg). Radiolabeling was conducted for 20 min at 33 °C, and the reaction was terminated by the addition of 30 µl of SDS-sample buffer and heating at 80 °C for 5 min. Pgamma radiolabeling was analyzed by SDS-gel electrophoresis and autoradiography. PT, pertussis toxin. ROS Me, prewashed ROS membranes. Arrows A and B indicate molecular weights of 39,000 and 13,000, respectively.
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To confirm that the radiolabeled 13-kDa protein is Pgamma , the radiolabeling was conducted using purified Pgamma and Pgamma - and transducin-depleted membranes in the presence of [adenylate-32P]NAD. Then, the 13-kDa protein was isolated by using a reverse phase column (Fig. 2A). In the column chromatography, both radioactivity and PDE inhibitory activity were detected in the same fractions (Fig. 2B). Analysis of the radioactive fractions by SDS-gel electrophoresis and autoradiography of the gel indicate that the 13-kDa protein was isolated with a purity of more than 95% in these fractions and that the radioactivity was incorporated into the 13-kDa protein (Fig. 2C). Without Pgamma , the 13-kDa protein was not observed in the column chromatography (data not shown). Without Pgamma - and transducin-depleted membranes, the radiolabeling of the 13-kDa protein was not detected (data not shown). These data indicate that Pgamma is radiolabeled by a membrane-bound enzyme(s) in the presence of [adenylate-32P]NAD. If one amino acid in Pgamma was radiolabeled, approximately 23% of Pgamma was radiolabeled in the reconstituted system. Under these conditions Pgamma is roughly estimated as a mixture of free Pgamma (95%) and Pgamma complexed with Palpha beta (5%) if all of the Palpha beta in the membranes is occupied by exogenous Pgamma . Thus, these data indicate that free Pgamma is radiolabeled. We also note that Pgamma complexed with Palpha beta is radiolabeled. Under the conditions shown in Fig. 1, Pgamma appears to be complexed with Palpha beta for because (i) PDE activity in the ROS membranes was low, and (ii) addition of GTP or GTPgamma S stimulated PDE activity. We also radiolabeled bovine Palpha beta gamma 2 using partially purified frog ADP-ribosyltransferase after separation2 of Palpha beta gamma 2 from Palpha beta gamma and Palpha beta . We found that ~20% of Pgamma in the complex was radiolabeled (data not shown). Thus, we conclude that Pgamma , free or complexed with Palpha beta , is radiolabeled by a membrane-bound enzyme(s).

Radiolabeled Pgamma was treated under the following conditions: (i) incubation in a glycine/NaOH buffer (pH 10), and (ii) incubation with snake venom phosphodiesterase. These treatments have been used for the identification of ADP-ribose in the ADP-ribosylated alpha  subunit of G-protein (41-43). The radioactive products were then fractionated using a Mono Q column. As shown in Fig. 3, the radioactivity was detected in ADP-ribose fractions when the radioactive Pgamma was incubated in the glycine/NaOH buffer. In contrast, the radioactivity emerged in the AMP fractions when the Pgamma was incubated with phosphodiesterase. No other radioactive peak emerged in any fractions. It should be emphasized that nonenzymatic binding of NAD to Pgamma is not involved in the Pgamma radiolabeling, since the radioactivity was not detected in NAD fractions (fractions 4 and 5). We also note that nonenzymatic binding of ADP-ribose to Pgamma is excluded, since the radiolabeling of Pgamma by [adenylate-32P]NAD was not inhibited by preincubation of Pgamma with ADP-ribose (data not shown). These observations indicate that Pgamma is ADP-ribosylated.

We also analyzed the radiolabeled Pgamma by electrospray ionization mass spectrometry. Two different molecular ion masses were detected in the purified sample (Fig. 4). The estimated molecular ion mass of the major peak is 9,668.48 (standard deviation 0.97). The major peak is believed to be nonmodified Pgamma , since (i) without radiolabeling, Pgamma was detected as a single peak with the exact same molecular ion mass (data not shown); and (ii) the calculated molecular ion mass of the nonmodified Pgamma is 9,670.28. The estimated molecular ion mass of the second peak (approximately 20% of the major peak) was 10,209.90 (standard deviation 1.08). The difference in the observed masses of these two peaks is 541.52. This value is in fair agreement with gain in molecular ion mass of G-protein alpha  subunit by ADP-ribosylation (541.3). Taking into account the purity of radiolabeled Pgamma and the level of radiolabeling (Fig. 2), we conclude that the second peak is ADP-ribosylated Pgamma . These data indicate that a single ADP-ribosyl moiety is incorporated into Pgamma . These observations also confirm that nonenzymatic binding of NAD to Pgamma (663.4 increase in molecular ion mass) is excluded.

To identify an ADP-ribosylated amino acid, we treated the radiolabeled Pgamma under different conditions. Neither low pH (HCl, 0.1 M) nor HgCl2 (10 mM) reduced the radioactivity from the Pgamma (data not shown), suggesting that a cysteine in Pgamma is not ADP-ribosylated. This conclusion is also supported by the observation that L-cysteine methyl ester did not inhibit the radiolabeling of Pgamma (up to 20 mM) (data not shown). In contrast, as shown in Fig. 3, the radiolabeled Pgamma is sensitive to high pH (pH 10.0). The radioactivity was also decreased when the radioactive Pgamma was incubated with hydroxylamine (Fig. 5A). Moreover, the Pgamma radiolabeling was inhibited by novobiocin (Fig. 5Ba), an inhibitor of arginine-ADP-ribosyltransferase (42), and by L-arginine methyl ester (Fig. 5Bb). These observations indicate that an arginine in Pgamma is ADP-ribosylated.


Fig. 5. Effect of chemicals on Pgamma ADP-ribosylation. Panel A, sensitivity of ADP-ribosylated Pgamma to hydroxylamine. Following radiolabeling of bovine recombinant Pgamma (1.2 µg) with [adenylate-32P]NAD (50 µM; ~0.5 µCi) in the presence of Pgamma - and transducin-depleted ROS membranes, the Pgamma radiolabeling was terminated by heating (2.5 min, 80 °C). Hydroxylamine (2 M, pH 7.0) or water (as a control) was added to the reaction mixture and incubated for various periods (33 °C). Following dilution of the samples (50 µl) with water to 500 µl and dialysis of the reaction mixture against water and SDS (0.1%), the reaction mixture was analyzed by SDS-gel electrophoresis and autoradiography. Panel B, effects of inhibitors of ADP-ribosyltransferase on Pgamma ADP-ribosylation. In the presence of the various concentrations of inhibitors, purified frog Pgamma (0.26 µg) was incubated (30 min, 33 °C) with Pgamma -depleted ROS membranes (80 µg of protein) in 50 µl of Buffer G. Pgamma radiolabeling was initiated by the addition of [adenylate-32P]NAD (10 µM; ~0.6 µCi). To terminate the reaction, 30 µl of SDS-sample buffer was added, and the reaction mixture was heated at 80 °C for 5 min. Radiolabeled Pgamma was isolated by SDS-gel electrophoresis and autoradiography. a, novobiocin; b, L-arginine methyl ester.
[View Larger Version of this Image (28K GIF file)]

Bovine Pgamma contains five arginine residues: Arg-11, Arg-15, Arg-24, Arg-33, and Arg-36 (29). We attempted to isolate a peptide(s) containing an ADP-ribosyl arginine after proteolytic digestion of the radiolabeled Pgamma , but we failed. This is probably because the ADP-ribosyl moiety was released from the radiolabeled Pgamma during proteolytic digestion. The radioactive Pgamma may be sensitive to longer incubation (room temperature, 18~24 h) in these buffers (pH 8.0-8.5). In fact, after incubation of the radiolabeled Pgamma under the same conditions (except without proteinase), a large portion of radioactivity was detected in the flow-through fractions in the reverse phase column chromatography. Therefore, to identify an arginine for ADP-ribosylation, we created mutant forms of Pgamma in which an arginine was replaced by a lysine. PDE inhibitory activities of these Pgamma mutants, summarized in Table II, show that all mutants have inhibitory activities similar to that of wild type Pgamma . These observations suggest that the mutation does not cause drastic change in the Pgamma conformation required for the inhibition of cGMP hydrolysis. As shown in Fig. 6, these Pgamma mutants were radiolabeled if each arginine was singly replaced by lysine (R11K, R15K, R24K, R33K, and R36K). However, the Pgamma radiolabeling was abolished if both Arg-33 and Arg-36 are replaced by lysines (R11,15,24,33,36K; R11,15,33,36K, and R33,36K). In contrast, the Pgamma radiolabeling was detected even if two other arginines are replaced by lysines (R11,15K and R15,24K). We also confirmed these data using a peptide corresponding to residues 30-39 of Pgamma (FKQRQTROFK) and its mutant forms. The wild type peptide and mutant forms of the peptide (R33K and R36K) were similarly ADP-ribosylated by Pgamma - and transducin-depleted membranes. However, a mutant form of the peptide (R33,36K) was not modified under the same conditions (data not shown). Together with data that one ADP-ribose is incorporated in the radiolabeled Pgamma (Fig. 4), these results indicate that Arg-33 and Arg-36 in Pgamma can be ADP-ribosylated; however, only one ADP-ribose is incorporated at a time into one of these two arginines. The time course of radiolabeling on both R33K and R36K mutants appeared similar (Fig. 6), suggesting that the possibility for the radiolabeling of Arg-33 and Arg-36 is similar under these conditions.

Table II. PDE inhibitory activity of Pgamma and its mutants


Pgamma and its mutant   Amino acid sequence of Pgamma (10-37) IC50a

nM
 11  15       24       33 36
Wild type  -IRSATRVMGGPVTPRKGPPKFKQRQTRQ- 3.2  ± 1.0
R11K   K   R        R        R  R 2.6  ± 0.8
R15K   R   K        R        R  R 3.7  ± 1.0
R24E   R   R        E        R  R 3.2  ± 0.6
R24K   R   R        K        R  R 3.3  ± 0.9
R33K   R   R        R        K  R 4.4  ± 1.2
R36K   R   R        R        R  K 4.3  ± 1.2
R11,15K   K   K        R        R  R 3.3  ± 0.9
R15,24K   R   K        K        R  R 3.5  ± 1.1
R33,36L   R   R        R        L  L 3.1  ± 0.8
R33,36K   R   R        R        K  K 3.4  ± 0.7
R11,15,33,36K   K   K        R        K  K 2.0  ± 0.8
R11,15,24,33,36K   K   K        K        K  K 3.4  ± 1.0

a Concentration of Pgamma and its mutants for 50% inhibition of Pgamma -depleted PDE activity. Inhibitory effects of bovine recombinant Pgamma and its mutants were investigated using Pgamma -depleted ROS membranes and various amounts of Pgamma . The specific inhibitory activity values (inhibition/mg of protein) and maximal inhibitory effect on Palpha beta were similar for wild type and mutant Pgamma s (±5%).


Fig. 6. ADP-ribosylation of arginine-mutant Pgamma s. In the presence of Pgamma - and transducin-depleted ROS membranes (320 µg), mutant Pgamma s (0.8 µg) were incubated at 33 °C in 250 µl of Buffer G. Pgamma radiolabeling was initiated by the addition of [adenylate-32P]NAD (50 µM; ~2 µCi). Following incubation for various periods, an aliquot (50 µl) was mixed with 20 µl of SDS-sample buffer and heated at 80 °C for 5 min. After SDS-gel electrophoresis and autoradiography, the Pgamma band was excised from gel, and its radioactivity was measured. Based on the radioactivity of wild type Pgamma after a 60-min incubation, the radioactivity of each mutant was calculated. a, wild type Pgamma ; b, R11K; c, R15K; d, R11,15K; e, R33K; f, R36K; g, R33,36K; h, R24K; i, R11,15,33,36K; and j, R11,15,24,33,36K. A mutant R15,24K shows the same radiolabeling as R11,15K; however, the data are not shown because of the limited space.
[View Larger Version of this Image (29K GIF file)]

Effect of Talpha on Pgamma ADP-ribosylation

ADP-ribosylation of Pgamma by partially purified ADP-ribosyltransferase was inhibited by both GTPgamma S- and GDP-bound forms of Talpha (Fig. 7). ADP-ribosylation of Pgamma by the enzyme solubilized from ROS membranes by n-dodecyl-beta -D-maltoside was also inhibited by Talpha (data not shown). We note that Pgamma forms a complex with both GTPgamma S·Talpha and GDP·Talpha (12, 34). These observations suggest the following two possibilities: (i) after complex formation with Talpha (both GTP- and GDP-bound forms), Pgamma is not a substrate for ADP-ribosyltransferase; and/or (ii) ADP-ribosyltransferase is inhibited directly by Talpha .


Fig. 7. Inhibitory effect of Talpha on Pgamma ADP-ribosylation. After partial purification of ADP-ribosyltransferase, ADP-ribosylation of Pgamma was measured with recombinant bovine Pgamma (0.5 µg) and the enzyme (2.0 µg) in the presence of GTPgamma S·Talpha (12.5 µg) or GDP·Talpha (12.5 µg) in 300 µl of Buffer G. Pgamma ADP-ribosylation was initiated by the addition of NAD (50 µM; ~2 µCi). Following incubation for various periods (33 °C), the Pgamma ADP-ribosylation in 50 µl of the reaction mixture was terminated by the addition of 20 µl of SDS-sample buffer. Samples were analyzed by SDS-gel electrophoresis and autoradiography. a, control; b, GTPgamma S·Talpha ; c, GDP·Talpha .
[View Larger Version of this Image (29K GIF file)]

Agmatine is a simple arginine derivative often used as an artificial substrate for arginine-ADP-ribosyltransferase (33). ADP-ribosylation of agmatine by partially purified ADP-ribosyltransferase was carried out in the presence or absence of Talpha (GTPgamma S- or GDP-bound forms). As shown in Fig. 8, agmatine was ADP-ribosylated; however, the ADP-ribosylation was not affected by Talpha . Using a TSK-250 column (34), we confirmed that agmatine does not form a complex with Talpha (data not shown). These observations indicate that ADP-ribosyltransferase is not inhibited by Talpha . Therefore, we conclude that Pgamma is no longer a substrate for ADP-ribosyltransferase after complex formation with Talpha . The simplest explanation for these phenomena is that both Arg-33 and Arg-36 in Pgamma are masked by Talpha . Thus, a domain including these arginines is involved directly in the Pgamma interaction with Talpha . In contrast, Pgamma complexed with Palpha beta is a substrate for ADP-ribosyltransferase, as described above. Thus, these arginines seem to be exposed to the enzyme when Pgamma is complexed with Palpha beta , suggesting that these arginines are not directly involved in the Pgamma interaction with Palpha beta .


Fig. 8. Effect of Talpha on ADP-ribosylation of agmatine. ADP-ribosylation of agmatine (20 mM) was carried out with partially purified ADP-ribosyltransferase (2.0 µg) in the presence or absence of GTPgamma S·Talpha (25 µg) or GDP·Talpha (25 µg) in 1,100 µl of Buffer G. ADP-ribosylation was initiated by the addition of [adenylate-32P]NAD (50 µM; ~2 µCi). Following incubation for various periods (33 °C), samples (100 µl) were applied to an AG 1-X2 column (1 ml). [32P]ADP-ribosylagmatine was eluted with 5 ml of H2O. bullet , control; open circle , GTPgamma S·Talpha ; and black-triangle, GDP·Talpha .
[View Larger Version of this Image (15K GIF file)]

Effect of Site-directed mutagenesis of Arg-33 and Arg-36 in Pgamma on the interaction between Pgamma and Talpha

To confirm the role of Arg-33 and Arg-36 in Pgamma in the interaction with Talpha , both Arg-33 and Arg-36 were replaced by lysine or leucine. These mutants inhibited PDE activity similarly to wild type Pgamma (Table II). These data support our conclusion that these arginines are not crucial for the interaction with Palpha beta to inhibit PDE activity. Then, GTPase activity of Talpha and GTPgamma S binding to Talpha was measured in the presence of various amounts of these Pgamma mutants. We have already shown that wild type Pgamma inhibits both GTPase activity of Talpha and GTPgamma S binding to Talpha under our conditions and that these phenomena are used as evidence for the interaction between Talpha and Pgamma (34, 44). As shown in Fig. 9A, the Pgamma mutant R33,36K inhibited GTPase activity; however, the Pgamma mutant R33,36L did not inhibit GTPase activity. Moreover, the R33,36K mutant inhibited GTPgamma S binding to Talpha , but the R33,36L mutant did not inhibit GTPgamma S binding (Fig. 9B). Furthermore, GTPgamma S·Talpha activated PDE that had been inhibited by the R33,36K mutant, but not PDE that had been inhibited by the R33,36L mutant (Fig. 10). These data indicate that arginines 33 and 36 are involved in the Pgamma interaction with Talpha and that positive charges of these arginines are important for the interaction between Talpha and Pgamma . We note that another arginine in the polycationic region, Arg-24, is not involved in the interaction with Talpha . A mutant form of Pgamma , R24E, inhibited GTPase activity in the same manner as wild type Pgamma (data not shown). This indicates that arginines 33 and 36 in the Pgamma polycationic region have a special function for the interaction with Talpha .


Fig. 9. Effect of Pgamma mutants on the GTPase activity of Talpha and GTPgamma S binding to Talpha . Bovine recombinant Pgamma and its mutants were used. open circle , wild type Pgamma ; black-square, R33,36K; black-triangle, R33,36L. Panel A, GTPase activity. GTPase activity was measured in 100 µl of Buffer G containing Talpha (1.0 µg), Tbeta gamma (0.5 µg), urea-treated frog ROS membranes (2.0 µg), and various amounts of Pgamma . GTP hydrolysis was initiated by the addition of [gamma -32P]GTP (1 µM; ~0.1 µCi). Following incubation (30 min, 33 °C), the reaction was terminated by the addition of 500 µl of stop solution (6% charcoal, 10% trichloroacetic acid, and 5 mM NaH2PO4). After spinning (1,000 × g, 10 min), radioactivity of the supernatant (100 µl) was measured. Panel B, GTPgamma S binding. GTPgamma S binding to Talpha was measured in 100 µl of Buffer G containing Talpha (0.8 µg), Tbeta gamma (0.33 µg), urea-treated ROS membranes (2.0 µg), and various amounts of Pgamma . Reactions were initiated by the addition of [35S]GTPgamma S (1 µM; 0.06 µCi). Following incubation (30 min, 0 °C), 80 µl of the reaction mixture was applied to a membrane filter (Millipore, HA, pore size 0.45 µm) and washed with 4 ml of Buffer G (four times). Radioactivity bound was measured.
[View Larger Version of this Image (13K GIF file)]


Fig. 10. GTPgamma S·Talpha -dependent activation of PDE inhibited by Pgamma mutants. Bovine recombinant Pgamma and its mutants were used. open circle , wild type Pgamma ; black-square, R33,36K; black-triangle, R33,36L. Inhibited PDE was prepared with Pgamma -depleted membranes (130 µg) and Pgamma (10 µg) in 100 µl of Buffer I. After incubation (10 min, 25 °C), the mixture was spun down (200,000 × g, 10 min, 4 °C), and membranes were washed with 200 µl of Buffer I (twice) and suspended in 200 µl of Buffer I. Hydrolysis of cGMP (1 mM) by the inhibited PDE (8.0 µg of protein) was measured with various amounts of GTPgamma S·Talpha in 100 µl of Buffer G for 10 min at 33 °C.
[View Larger Version of this Image (20K GIF file)]


DISCUSSION

PDE, a key protein to regulate the level of cGMP in retinal photoreceptors, is composed of Palpha beta and two Pgamma subunits. Pgamma has two roles in Palpha beta regulation: inhibiting cGMP hydrolysis by Palpha beta (12, 17) and stimulating cGMP binding to high affinity, noncatalytic sites on Palpha beta (13, 14). We have recently indicated that an identical Pgamma expresses these different functions by binding to different sites on Palpha beta (16) and that different regions in Pgamma are involved in these functions (15). Since these Pgamma functions are expressed by interaction with Palpha beta and interrupted by Pgamma release with GTP·Talpha , the functional structure of Pgamma required for these interactions should be clarified to understand the regulation of these Pgamma functions. In this study we have shown that two arginines, 33 and 36, in the Pgamma polycationic region are equally ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is ADP-ribosylated at a time. We speculate that steric hindrance may contribute to ADP-ribosylation of one arginine. The ADP-ribosylation was detected when Pgamma is complexed with Palpha beta . However, the ADP-ribosylation was inhibited when Pgamma is complexed with Talpha (GTP- and GDP-bound forms). These data imply that these arginines are masked when Pgamma is complexed with Talpha . Then, site-directed mutagenesis was applied to replace these arginines with lysines or leucines, and the effects of these Pgamma mutants on Talpha functions were measured. These experiments confirm that these arginines are crucial for the interaction with Talpha . We have also shown that arginine 24, another arginine in the Pgamma polycationic region, is not involved in the Pgamma interaction with Talpha . Thus, it is concluded that the polycationic region in Pgamma may be divided into at least two subdomains, and a subdomain containing arginines 33 and 36 appears to be involved in the interaction with Talpha , but not in the interaction with Palpha beta .

As summarized in the Introduction, various methods have been applied to identify specific domains in Pgamma . Proteolytic digestion of Pgamma has also been applied to identify a specific domain in Pgamma (25). Although these methods are potentially useful in identifying specific domains in proteins, they have also serious problems. For example, one cannot be sure that conformation of the peptide corresponding to the specific region is the same or similar to that of the region in the protein. Site-directed mutagenesis of the specific residues does not necessarily change only the conformation of the region in which these residues are involved. Moreover, deletion of the specific residues does not necessarily delete functions of the specific residues. We have already shown that deletion of the carboxyl-terminal residues of Pgamma reduces not only its inhibitory activity of cGMP hydrolysis but also its ability to interact with Palpha beta (15). Therefore, we seek a method to identify specific residues in the Pgamma polycationic region under more physiological conditions. In this study we have used ADP-ribosylation to identify two arginines in the polycationic region. The ADP-ribosylation was carried out by endogenous ADP-ribosyltransferase under physiological conditions. Then, we used peptides and site-directed mutagenesis to confirm data obtained by the ADP-ribosylation.

In a system reconstituted by exogenous Pgamma and Pgamma - and transducin-depleted ROS membranes, the maximum level of Pgamma ADP-ribosylation is estimated about 20% by the measurement of Pgamma ADP-ribosylation after SDS-gel electrophoresis (Fig. 2). Although we do not think that the conclusions in this study are affected by the low level of ADP-ribosylation, we try to specify the reasons for such a low level of ADP-ribosylation. One possible interpretation is that Pgamma complexed with Palpha beta may be a better substrate for ADP-ribosyltransferase, especially in membranes, because high Pgamma ADP-ribosylation (about 50%) was detected in native membranes (Fig. 1). We note that all Pgamma appeared to be complexed with Palpha beta under conditions described in Fig. 1; however, ~95% of added Pgamma was estimated to be free under the conditions described in Fig. 2. Moreover, we anticipate that an activator of ADP-ribosyltransferase may be present in these membranes because ADP-ribosyltransferase might be regulated by several proteins.3 Another possible interpretation is that the apparent incorporation of ADP-ribose to Pgamma measured after SDS-gel electrophoresis may be underestimated. In this study, the pH values of the separating gel buffer and the running buffer are 8.8 and 8.4, respectively. SDS-gel electrophoresis was carried out without a cooling system. It is possible that these conditions accelerate the release of ADP-ribose from Pgamma . This speculation is supported by the observation that ADP-ribose-arginine linkage was sensitive to in buffers (pH 8.0-8.5) as described above. Zolkiewska and Moss (43) also described possible breakdown of ADP-ribose-arginine linkage during electrophoresis.

We have shown previously that Pgamma complexed with GTP·Talpha is phosphorylated by a kinase; however, Pgamma complexed with Palpha beta is not a substrate for the kinase (36, 44). The phosphorylation of Pgamma inverts the relative affinities of Pgamma to GTP·Talpha and to Palpha beta , and the change in the relative affinities may function in the turnoff mechanism of GTP·Talpha -activated PDE without GTP hydrolysis. In this study we have shown that Pgamma complexed with Palpha beta , but not with Talpha , is ADP-ribosylated by arginine-ADP-ribosyltransferase in ROS membranes. We have utilized the Pgamma ADP-ribosylation as a tool to identify arginines in the polycationic region which are involved in the interaction with Talpha . However, the physiological significance of the Pgamma ADP-ribosylation in phototransduction remains unsolved. We anticipate that Pgamma ADP-ribosylation may control phototransduction through regulation of Pgamma interaction with specific proteins involved in phototransduction. The information obtained in this study will also be useful to reveal the physiological significance of the Pgamma ADP-ribosylation.


FOOTNOTES

*   This work was supported in part by National Institute of Health Grants EY07546 and EY09631 (to A. Y.) and EY06595 (to H. M.), an unrestricted grant from Research to Prevent Blindness, and grant-in-aids from the Ministry of Education, Science, and Culture of Japan (to M. O., F. H., and J. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
j    Supported by a Jules and Doris Stein Professorship from Research to Prevent Blindness. To whom all correspondence should be addressed: Kresge Eye Institute, Wayne State University, School of Medicine, 4717 St. Antoine St., Detroit, MI 48201. Tel.: 313-577-2009; Fax: 313-577-0238; E-mail: ayamazak{at}med.wayne.edu.
1   The abbreviations used are: PDE, cGMP phosphodiesterase; Palpha beta , catalytic subunits of PDE; Pgamma , gamma  subunit of PDE; ROS, rod outer segments; Talpha and Tbeta gamma , subunits of transducin, retinal G-protein; GDPbeta S, guanyl-5'-yl thiophosphate; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); Gpp(NH)p, guanyl-5'-yl beta ,gamma -imidotriphosphate; PMSF, phenylmethylsulfoxyl fluoride; DTT, dithiothreitol.
2   A. Yamazaki, unpublished method.
3   V. A. Bondarenko and A. Yamazaki, unpublished observation.

ACKNOWLEDGEMENTS

We thank Drs. W. H. Miller, R. B. Needleman, D. R. Pepperberg, and R. K. Yamazaki for critical reading of the manuscript.


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