(Received for publication, November 27, 1996, and in revised form, March 31, 1997)
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
Interaction between the subunit
(P
) of cGMP phosphodiesterase and the
subunit (T
) 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 P
polycationic region to identify residues
required for the interaction with T
. P
, free or complexed with
the
subunit (P
) of cGMP phosphodiesterase, was
specifically radiolabeled by prewashed rod membranes in the presence of
[adenylate-32P]NAD. Identification of
ADP-ribose in the radiolabeled P
and radiolabeling of
arginine-replaced mutant forms of P
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.
P
complexed with T
(both GTP- and GDP-bound forms) was not
ADP-ribosylated; however, agmatine, which cannot interact with T
,
was ADP-ribosylated in the presence of T
, suggesting that a P
domain containing these arginines is masked by T
. A P
mutant
(R33,36K), as well as wild type P
, inhibited both GTP hydrolysis of
T
and GTP binding to T
. Moreover, GTP-bound T
activated
P
that had been inhibited by R33,36K. However, another P
mutant (R33,36L) could not inhibit these T
functions. In addition, GTP-bound T
could not activate P
inhibited by R33,36L. These results indicate that a P
domain containing these arginines is required for its interaction with T
, but not with P
, and that positive charges in these arginines are crucial for the
interaction.
Cyclic GMP phosphodiesterase (PDE),1 a
key enzyme in phototransduction, is composed of P and two P
subunits (1-6). P
hydrolyzes cGMP (7, 8) and binds cGMP to its
high affinity, noncatalytic sites (9-11). In amphibian ROS, P
regulates these P
functions as an inhibitor of cGMP hydrolysis
(12) and as a stimulator of cGMP binding to noncatalytic sites (13,
14). Different interactions between P
and P
have been
suggested to be required to express these two functions (15, 16). In bovine ROS, P
inhibits cGMP hydrolysis by P
(17); however, the
effect of P
on the cGMP binding to noncatalytic sites has never been
documented. In amphibian ROS, these P
functions are interrupted by
P
release with GTP·T
from P
(12-14, 18). We have
recently suggested that these functionally different P
s are released
in the different steps of phototransduction (15, 16). When [cGMP] is
at the dark level, P
responsible for the inhibition of cGMP
hydrolysis is released. Consequently, cGMP is hydrolyzed by the
activated PDE for photoexcitation. When [cGMP] becomes low, P
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.
P 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) P
-P
interaction for the inhibition of cGMP hydrolysis by P
;
(b) P
-P
interaction for the stimulation of cGMP
binding to P
noncatalytic sites; (c) P
-T
interaction for the release of P
inhibitory strain from P
; and
(d) P
-T
interaction for the release of P
to reduce
the affinity of P
noncatalytic sites to cGMP. Previous studies
have focused on interactions (a) and (c).
Peptides have been used to identify the polycationic region of P
within residues 24-45 and the carboxyl-terminal region of P
corresponding to residue 46-87 as the sites for the interaction (a) (19-22). Mutational analysis of P
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
P
, a frameshift mutation of P
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 P
have been suggested as sites required for
the interaction (c) (19, 23, 25-27). However, little is
known about P
domains involved in the interactions (b)
and (d). The frameshift mutation of P
has suggested that the amino-terminal residues are involved in the stimulation of cGMP
binding to noncatalytic sites on P
(15). However, a T
interaction site on P
, which is required for the P
release to reduce the affinity of P
noncatalytic sites to cGMP, has not been
identified.
In this study we have focused on identification of specific residues in
the P polycationic region for following reasons. (i) The
polycationic region has been suggested to be involved in the
interaction with both P
and GTP·T
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 P
release
by GTP·T
. However, residues required for these interactions have
not been identified. (ii) We found that the specific arginines in the
P
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 P
ADP-ribosylation under
physiological conditions. (iii) We also found that the P
ADP-ribosylation was regulated by the interaction between P
and
T
. Therefore, the ADP-ribosylation can be a useful tool to learn the
interaction between P
and T
. We describe that both arginine 33 and arginine 36 in the P
, free or complexed with P
, are
ADP-ribosylated by endogenous arginine-ADP-ribosyltransferase. The P
ADP-ribosylation is inhibited when P
is complexed with T
(both
GTP- and GDP-bound forms), suggesting that the domain including these
arginines is not exposed to ADP-ribosyltransferase when P
is
complexed with T
. Then, using forms of P
mutated in these
residues, we confirm that the domain is involved in the interaction
with T
. Moreover, we find that positive charges in these arginine
are important for the interaction with T
.
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,
[32P]GTP, and [35S]GTP
S from DuPont
NEN; cGMP, GTP, GDP, GDP
S, Gpp(NH)p, and GTP
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 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 MembranesUnder 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. T (more than 90%) and P
(about 50%) were released from these membranes. These membranes contain P
-less (active) PDE and were termed as P
-depleted ROS membranes. Residual P
in these membranes is not sensitive to GTP·T
(12), suggesting that the membrane preparation contains a
distinct subset of P
which cannot be released by GTP·T
. This subset of P
was termed GTP·T
-insensitive P
. When
P
-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 P
-depleted membranes were
washed an additional seven times with 3 ml of Buffer D, T
(more
than 90%) was released. These membranes are termed P
- and
transducin-depleted ROS membranes. It should be emphasized that
ADP-ribosyltransferase activity was detected in P
-depleted and P
-
and transducin-depleted membranes when frog or recombinant bovine P
was used as a substrate. However, ADP-ribosylation of the residual P
was not detected in these membranes. This suggests that
GTP·T
-insensitive P
cannot be ADP-ribosylated. Urea-treated ROS
membranes were prepared as described (13).
All DNA manipulations
were carried out using standard procedures (28). Full-length bovine
P 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 P
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 P
.
|
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 0.6, protein expression was induced by the addition of 1 mM (final) isopropyl
-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). P
and its mutants were further purified using Pep RPC HR5/5 column as described (12). The purity of P
and its mutants was greater than
90%.
Frog P 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 P
except SDS-gel electrophoresis. T
(GTP
S- and GDP-bound forms) and T
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
P
-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 P was carried out with various
membrane preparations or enzyme preparations solubilized by detergents or phosphatidylinositol-specific phospholipase C. Frog P
and recombinant bovine P
were used. Both P
s were ADP-ribosylated in a
similar manner. The amounts of each components were slightly different
in each experiment (see each figure legend); however, P
ADP-ribosylation was performed in a similar way. The reaction mixture
contained P
(0.2-0.6 µg), NAD (10-50 µM; ~0.5
µCi) and proteins in the 50 µl of Buffer G. P
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 P
(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 P
, and the radioactivity of
the corresponding region was subtracted from the radioactivity of the
P
band. Without added P
, no 13,000 band was detected, and the
radioactivity was negligible. If one amino acid in P
was
radiolabeled, approximately 3-10% of P
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 P
residues 30-39
(FKQRQTROFK) and its mutant forms was also ADP-ribosylated by P
- 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.
Preparation of ADP-ribosylated P
Although the amounts of
components in each reaction mixture were slightly different (see each
figure legend), ADP-ribosylated P was prepared by incubation of
purified P
, P
- and transducin-depleted ROS membranes, and
[adenylate-32P]NAD. ADP-ribosylated P
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 P
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
P
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 P
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 P
was excised from the gel.
Identification of ADP-ribose in the [Adenylate-32P]NAD-radiolabeled P
To identify
ADP-ribose in the radiolabeled P, the radiolabeled frog P
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.
Fifty µl of ADP-ribosylated recombinant bovine P (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).
Activities of PDE and P were assayed
as described (12). GTPase activity of T
and GTP
S binding to T
were measured as described (18). Immunological detection of P
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 P
was assayed by densitometric scanning (12). To
calculate the P
concentration, 9,625 and 9,669 were used as
molecular weights of frog (36) and recombinant bovine (see Fig. 4)
P
, respectively, although P
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.
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 T in prewashed ROS membranes is
ADP-ribosylated by pertussis toxin, as described previously (31, 37,
38). In the absence of pertussis toxin, T
ADP-ribosylation was not detected, indicating that endogenous T
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 P
. These data support the idea
that the radiolabeled 13-kDa protein is P
and that both endogenous
and exogenous P
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 P
was radiolabeled if
one amino acid in P
was radiolabeled.
To confirm that the radiolabeled 13-kDa protein is P, the
radiolabeling was conducted using purified P
and P
- 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 P
, the 13-kDa protein
was not observed in the column chromatography (data not shown). Without
P
- and transducin-depleted membranes, the radiolabeling of the
13-kDa protein was not detected (data not shown). These data indicate
that P
is radiolabeled by a membrane-bound enzyme(s) in the presence
of [adenylate-32P]NAD. If one amino acid in
P
was radiolabeled, approximately 23% of P
was radiolabeled in
the reconstituted system. Under these conditions P
is roughly
estimated as a mixture of free P
(95%) and P
complexed with
P
(5%) if all of the P
in the membranes is occupied by
exogenous P
. Thus, these data indicate that free P
is
radiolabeled. We also note that P
complexed with P
is
radiolabeled. Under the conditions shown in Fig. 1, P
appears to be
complexed with P
for because (i) PDE activity in the ROS
membranes was low, and (ii) addition of GTP or GTP
S stimulated PDE
activity. We also radiolabeled bovine P
2 using partially
purified frog ADP-ribosyltransferase after
separation2 of P
2 from P
and P
. We found that ~20% of P
in the complex was
radiolabeled (data not shown). Thus, we conclude that P
, free or
complexed with P
, is radiolabeled by a membrane-bound enzyme(s).
Radiolabeled P 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
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
P
was incubated in the glycine/NaOH buffer. In contrast, the
radioactivity emerged in the AMP fractions when the P
was incubated
with phosphodiesterase. No other radioactive peak emerged in any
fractions. It should be emphasized that nonenzymatic binding of NAD to
P
is not involved in the P
radiolabeling, since the radioactivity
was not detected in NAD fractions (fractions 4 and 5). We also note
that nonenzymatic binding of ADP-ribose to P
is excluded, since the
radiolabeling of P
by [adenylate-32P]NAD
was not inhibited by preincubation of P
with ADP-ribose (data not
shown). These observations indicate that P
is ADP-ribosylated.
We also analyzed the radiolabeled P 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 P
, since (i) without radiolabeling, P
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 P
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
subunit by
ADP-ribosylation (541.3). Taking into account the purity of
radiolabeled P
and the level of radiolabeling (Fig. 2), we conclude
that the second peak is ADP-ribosylated P
. These data indicate that
a single ADP-ribosyl moiety is incorporated into P
. These
observations also confirm that nonenzymatic binding of NAD to P
(663.4 increase in molecular ion mass) is excluded.
To identify an ADP-ribosylated amino acid, we treated the radiolabeled
P under different conditions. Neither low pH (HCl, 0.1 M) nor HgCl2 (10 mM) reduced the
radioactivity from the P
(data not shown), suggesting that a
cysteine in P
is not ADP-ribosylated. This conclusion is also
supported by the observation that L-cysteine methyl ester
did not inhibit the radiolabeling of P
(up to 20 mM)
(data not shown). In contrast, as shown in Fig. 3, the radiolabeled P
is sensitive to high pH (pH 10.0). The radioactivity was also decreased when the radioactive P
was incubated with hydroxylamine (Fig. 5A). Moreover, the P
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 P
is ADP-ribosylated.
Bovine P 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 P
, but we failed. This is probably because the
ADP-ribosyl moiety was released from the radiolabeled P
during proteolytic digestion. The radioactive P
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 P
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 P
in which an arginine
was replaced by a lysine. PDE inhibitory activities of these P
mutants, summarized in Table II, show that all mutants
have inhibitory activities similar to that of wild type P
. These
observations suggest that the mutation does not cause drastic change in
the P
conformation required for the inhibition of cGMP hydrolysis.
As shown in Fig. 6, these P
mutants were radiolabeled
if each arginine was singly replaced by lysine (R11K, R15K, R24K, R33K,
and R36K). However, the P
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 P
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 P
(FKQRQTROFK) and its mutant forms. The wild type peptide
and mutant forms of the peptide (R33K and R36K) were similarly
ADP-ribosylated by P
- 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 P
(Fig. 4), these results indicate
that Arg-33 and Arg-36 in P
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.
|
Effect of T
ADP-ribosylation of
P by partially purified ADP-ribosyltransferase was inhibited by both
GTP
S- and GDP-bound forms of T
(Fig. 7).
ADP-ribosylation of P
by the enzyme solubilized from ROS membranes
by n-dodecyl-
-D-maltoside was also inhibited
by T
(data not shown). We note that P
forms a complex with both GTP
S·T
and GDP·T
(12, 34). These observations suggest the following two possibilities: (i) after complex formation with T
(both GTP- and GDP-bound forms), P
is not a substrate for ADP-ribosyltransferase; and/or (ii) ADP-ribosyltransferase is inhibited
directly by T
.
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 T (GTP
S- or GDP-bound forms). As
shown in Fig. 8, agmatine was ADP-ribosylated; however, the ADP-ribosylation was not affected by T
. Using a TSK-250 column (34), we confirmed that agmatine does not form a complex with T
(data not shown). These observations indicate that
ADP-ribosyltransferase is not inhibited by T
. Therefore, we conclude
that P
is no longer a substrate for ADP-ribosyltransferase after
complex formation with T
. The simplest explanation for these
phenomena is that both Arg-33 and Arg-36 in P
are masked by T
.
Thus, a domain including these arginines is involved directly in the
P
interaction with T
. In contrast, P
complexed with P
is
a substrate for ADP-ribosyltransferase, as described above. Thus, these
arginines seem to be exposed to the enzyme when P
is complexed with
P
, suggesting that these arginines are not directly involved in
the P
interaction with P
.
Effect of Site-directed mutagenesis of Arg-33 and Arg-36 in P
To confirm the role of
Arg-33 and Arg-36 in P in the interaction with T
, both Arg-33 and
Arg-36 were replaced by lysine or leucine. These mutants inhibited PDE
activity similarly to wild type P
(Table II). These data support our
conclusion that these arginines are not crucial for the interaction
with P
to inhibit PDE activity. Then, GTPase activity of T
and
GTP
S binding to T
was measured in the presence of various amounts
of these P
mutants. We have already shown that wild type P
inhibits both GTPase activity of T
and GTP
S binding to T
under
our conditions and that these phenomena are used as evidence for the
interaction between T
and P
(34, 44). As shown in Fig.
9A, the P
mutant R33,36K inhibited GTPase
activity; however, the P
mutant R33,36L did not inhibit GTPase
activity. Moreover, the R33,36K mutant inhibited GTP
S binding to
T
, but the R33,36L mutant did not inhibit GTP
S binding (Fig.
9B). Furthermore, GTP
S·T
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 P
interaction with T
and
that positive charges of these arginines are important for the
interaction between T
and P
. We note that another arginine in the
polycationic region, Arg-24, is not involved in the interaction with
T
. A mutant form of P
, R24E, inhibited GTPase activity in the
same manner as wild type P
(data not shown). This indicates that
arginines 33 and 36 in the P
polycationic region have a special
function for the interaction with T
.
PDE, a key protein to regulate the level of cGMP in retinal
photoreceptors, is composed of P and two P
subunits. P
has two roles in P
regulation: inhibiting cGMP hydrolysis by P
(12, 17) and stimulating cGMP binding to high affinity, noncatalytic sites on P
(13, 14). We have recently indicated that an identical
P
expresses these different functions by binding to different sites
on P
(16) and that different regions in P
are involved in
these functions (15). Since these P
functions are expressed by
interaction with P
and interrupted by P
release with
GTP·T
, the functional structure of P
required for these interactions should be clarified to understand the regulation of these
P
functions. In this study we have shown that two arginines, 33 and
36, in the P
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 P
is complexed with P
. However, the
ADP-ribosylation was inhibited when P
is complexed with T
(GTP-
and GDP-bound forms). These data imply that these arginines are masked
when P
is complexed with T
. Then, site-directed mutagenesis was
applied to replace these arginines with lysines or leucines, and the
effects of these P
mutants on T
functions were measured. These
experiments confirm that these arginines are crucial for the
interaction with T
. We have also shown that arginine 24, another
arginine in the P
polycationic region, is not involved in the P
interaction with T
. Thus, it is concluded that the polycationic
region in P
may be divided into at least two subdomains, and a
subdomain containing arginines 33 and 36 appears to be involved in the
interaction with T
, but not in the interaction with P
.
As summarized in the Introduction, various methods have been applied to
identify specific domains in P. Proteolytic digestion of P
has
also been applied to identify a specific domain in P
(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 P
reduces
not only its inhibitory activity of cGMP hydrolysis but also its
ability to interact with P
(15). Therefore, we seek a method to
identify specific residues in the P
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 P and P
- and
transducin-depleted ROS membranes, the maximum level of P
ADP-ribosylation is estimated about 20% by the measurement of P
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 P
complexed with P
may be a better substrate for
ADP-ribosyltransferase, especially in membranes, because high P
ADP-ribosylation (about 50%) was detected in native membranes (Fig.
1). We note that all P
appeared to be complexed with P
under
conditions described in Fig. 1; however, ~95% of added P
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 P
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 P
. 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 P complexed with GTP·T
is
phosphorylated by a kinase; however, P
complexed with P
is not a substrate for the kinase (36, 44). The phosphorylation of P
inverts the relative affinities of P
to GTP·T
and to P
, and the change in the relative affinities may function in the turnoff
mechanism of GTP·T
-activated PDE without GTP hydrolysis. In this
study we have shown that P
complexed with P
, but not with
T
, is ADP-ribosylated by arginine-ADP-ribosyltransferase in ROS
membranes. We have utilized the P
ADP-ribosylation as a tool to
identify arginines in the polycationic region which are involved in the
interaction with T
. However, the physiological significance of the
P
ADP-ribosylation in phototransduction remains unsolved. We
anticipate that P
ADP-ribosylation may control phototransduction through regulation of P
interaction with specific proteins involved in phototransduction. The information obtained in this study will also
be useful to reveal the physiological significance of the P
ADP-ribosylation.
We thank Drs. W. H. Miller, R. B. Needleman, D. R. Pepperberg, and R. K. Yamazaki for critical reading of the manuscript.