From the Kresge Eye Institute and the Departments of
§ Ophthalmology and ** Pharmacology, Wayne State University,
School of Medicine, Detroit, Michigan 48201, the ¶ Unit of
Regulatory and Molecular Biology, Departments of Cell Biology and
Ophthalmology, University of Medicine and Dentistry of New Jersey,
Stratford, New Jersey 08084, and the
Department of Biology,
Faculty of Science, Kobe University, Kobe 657 Japan
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ABSTRACT |
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Retinal guanylyl cyclase-1 (retGC-1), a key
enzyme in phototransduction, is activated by guanylyl
cyclase-activating proteins (GCAPs) if [Ca2+] is
less than 300 nM. The activation is believed to be
essential for the recovery of photoreceptors to the dark state;
however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by
GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to
recombinant bovine GCAPs, constitutively active mutants of GCAPs that
activate retGC-1 in a [Ca2+]-independent manner and
bovine brain S100b that activates retGC-1 in the presence of ~10
µM [Ca2+] were used to investigate whether
these activations take place through a similar mechanism, and whether
[Ca2+] is directly involved in the dimerization. We found
that a monomeric form of retGC-1 (~110 kDa) was mainly observed
whenever GC activity was at basal or low levels. However, the 210-kDa
product was increased whenever the GC activity was stimulated by any
Ca2+-binding proteins used. We also found that
[Ca2+] did not directly regulate the formation of the
210-kDa product. The 210-kDa product was detected in a purified GC
preparation and did not contain GCAPs even when the formation of the
210-kDa product was stimulated by GCAPs. These data strongly suggest
that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and
that dimerization of retGC-1 may be an essential step for its
activation by active forms of GCAPs.
In outer segments of vertebrate retinal photoreceptors, rhodopsin
absorbs a photon which in turn triggers GTP-dependent
activation of cGMP phosphodiesterase. The activated phosphodiesterase
hydrolyzes cGMP. The resulting decrease in cytoplasmic [cGMP] leads
to reduction in the activity of cGMP-gated cation channels and
hyperpolarization of plasma membranes (1-4). Restoration of the dark
membrane potential requires recovery of the dark level of cytoplasmic
[cGMP]. Therefore, GC,1 the
enzyme that converts GTP to cGMP, has a crucial role in visual transduction. A retinal membrane GC has been purified from frog, toad,
and bovine photoreceptor outer segments (5, 6) and identified as a
~110 kDa protein. Isozymes of the GC have also been shown
biochemically (5, 7). Subsequently two forms of membrane GC (retGC-1,
ROS-GC1 or GC-E, and retGC-2, ROS-GC2 or GC-F) were cloned from human,
bovine, and rat retinal cDNA libraries (8-12). The structure of
these retGCs indicates that the enzyme is a member of the
peptide-regulated, membrane-bound GC family, although the retGC is not
activated by known peptides. Four functional domains in retGCs have
been predicted: a N-terminal extracellular domain, a transmembrane
domain, an intercellular protein-kinase-like domain, and a C-terminal
catalytic domain. Immunocytochemistry has shown that retGC-1 is
localized primarily in cone outer segments and to a lesser extent in
rod outer segments (13-15). RetGC-1 is also detected in the plexiform
layers of retina (13, 15), leading to speculation that the enzyme is
not unique in photoreceptor outer segments. RetGC-2 is in
photoreceptors (10); however, detailed localization of the enzyme in
the retina has not been demonstrated. RetGC-1 appears to be more
abundant than retGC-2 in the retina (10), and only retGC-1 may
contribute to the pool of cGMP essential to support phototransduction
in cone photoreceptors (16).
The reduction of cGMP-gated channel activity by lowering cytoplasmic
[cGMP] blocks Na2+ and Ca2+ influx, and
allows a Na2+/Ca2+, K+ exchanger to
decrease cytoplasmic [Ca2+] (4, 17). When
[Ca2+] is low, retGC is stimulated (18). In contrast to
other membrane-bound GCs that are regulated by binding of peptides to
their extracellular domain (19, 20), this Ca2+-sensitive
stimulation of retGC is mediated by at least two calmodulin-like Ca2+-binding proteins termed GCAPs, 1 and 2 (14, 21-23).
GCAPs interact with the intracellular domain of the enzyme (24, 25).
When free [Ca2+] is less than ~300 nM,
retGC-1 is activated by GCAPs. RetGC-2 has also been reported to be
stimulated by GCAP-2 under similar [Ca2+] (10, 12, 26).
In addition, GCAPs appear to inhibit the basal GC activity in the
presence of the higher [Ca2+] (more than ~500
nM) (27, 28). Thus, the regulatory mechanism of retGC is
completely different from that of peptide-regulated GCs. GCAP-1 is
mainly detected in cone outer segments, in particular, in disc membrane
regions (29-31). GCAP-1 is also observed in rod outer segments, but
the content is much lower than that in cone outer segments. Less GCAP-1
is also found in synaptic regions and inner segments of cones. GCAP-2
is predominantly observed in outer and inner segments of rods and cones
(23, 29-31). Synaptic regions are also labeled by a GCAP-2 antibody.
It has also been reported that Ca2+-binding proteins of the
S100 family, especially S100b, activate retGC-1 in the presence of high
[Ca2+] (32, 33). Half-maximal activation was observed at
about 40~50 µM [Ca2+] (33). Therefore, it
is believed that S100 proteins are not involved in phototransduction.
Additional mechanisms and factors may also be involved in the
regulation of retGCs, including phosphorylation (34, 35), ATP binding
(36-38), actin binding (39), an inhibitor (guanylyl cyclase-inhibitory
protein) of GCAP-activated retGC in amphibian retina (40), and
inhibition of retGC-1 by RGS9 (41).
Characterizations of membrane-bound GC and adenylyl cyclase have
suggested that the mechanisms for the expression of these enzymatic
activities are closely related (42, 43), and that at least two cyclase
catalytic consensus domains may be required for the activity of these
cyclases. Membrane adenylyl cyclase contains two putative catalytic
domains that when separately expressed have no activity (44).
Peptide-regulated GCs have also been proposed to exist as dimeric or
oligomeric forms even in the absence of ligands (45-47). A recent
study has also suggested that retGCs form homodimers in photoreceptor
outer segments (48). However, it remains unknown whether dimerization
or oligomerization of retGCs is related to the regulation of retGC
activity. This question is important especially because the activity of
retGCs is regulated so differently from peptide-regulated GCs and the
regulatory mechanism of retGCs by GCAPs may not be identical to that of
peptide-regulated GCs. Thus, we investigated the relationship between
activation of retGC-1 by GCAPs and dimerization of the enzyme. In
addition to GCAPs, we used constitutively active mutants of GCAPs and
S100b to show that dimerization of retGC-1 is related to its
activation, not to Ca2+-binding proteins or
[Ca2+]. Dimerization of retGC-1 was monitored using a
cross-linker under the conditions similar to those used for the
measurement of the GC activity. Our results suggest that GCAPs activate
retGC-1 by enhancing its dimerization, and that transition of the
active form of retGC-1 to its inactive form is caused by either partial or complete dissociation of the dimeric form.
Materials--
Dark-adapted frozen bovine retinas were purchased
from Dr. Yee-Kin Ho (University of Illinois, Department of
Biochemistry, Chicago). Other materials were purchased from the
following sources: Sephacryl S-200 HR from Pharmacia Biotech Inc.;
[ Preparation of retGC and GCAPs--
Bovine ROS were prepared
from dark-adapted frozen retinas as described (49). Bleached ROS
membranes from 20 retinas were suspended in 3 ml of Buffer A (10 mM HEPES (pH 7.5), 5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µM
leupeptin, 5 µM pepstatin A, and 100 µM
CaCl2), homogenized by passing through a No. 21 needle 10 times and centrifuged (200,000 × g, 4 °C, 15 min)
(x 7). The membranes were further washed (3 times) with Buffer B (10 mM HEPES (pH 7.5), 5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluorine, 5 µM
leupeptin, and 5 µM pepstatin A), suspended in 3 ml of
Buffer B, frozen with liquid nitrogen, and stored at GC Activity Assay--
GC activity was measured as described
(5). Our preliminary studies indicated that 5 µg of protein of a ROS
homogenate had high GC activity with negligible hydrolysis of cGMP
under assay conditions. Thus, 5 µg of protein of ROS homogenate or
GCAPs-free ROS membranes were used for all studies. Briefly, ROS
membranes (5 µg of protein) were incubated with 200 µl of Buffer C
(50 mM HEPES (pH 7.5), 1 mM GTP, 1 mM cGMP, 2 mM 1-methyl-3-isobutylxanthine, 5 mM MgCl2, 15 mM phosphocreatine, 50 µg/ml creatine phosphokinase, ~5 µCi of
[ Cross-linking of retGC-1 by BS3--
In order to
obtain the exact relationship between dimerization of retGC-1 and its
activity, cross-linking reaction of retGC-1 was carried out under
conditions similar to that for the measurement of GC activity, although
the protein concentrations used were different because each experiment
was performed in its linear range. A bovine ROS homogenate, GCAPs-free
ROS membranes or COS-7 cell membranes containing recombinant bovine
retGC-1 were used. The preparation was suspended in 50 µl of Buffer D
(50 mM HEPES (pH 7.5), 1 mM GTP, 1 mM cGMP, and 5 mM MgCl2). Pilot
experiments indicated that the optimal conditions for the cross-linking
reaction by BS3 were the following: BS3
concentration, 50 µM (Fig. 1); temperature for the
cross-linking reaction, 0 °C; incubation period for the
cross-linking reaction, 30 min; and protein, 50 µg. We note that the
GTP regeneration system and proteinase inhibitors added in the GC assay
mixture did not affect the cross-linking reactions. We also note that the presence of 1 mM GTP in the cross-linking reaction
mixture slightly (less than 10%) inhibited the formation of the
210-kDa cross-linked product, but 1 mM cGMP did not. The
cross-linking reaction was terminated by addition of SDS-sample buffer
and boiling for 5 min. The cross-linked products were immediately
separated by SDS-PAGE (5-20% acrylamide gradient). After
electrophoresis, proteins were blotted to PVDF membranes and
cross-linked products of retGC-1 were detected with a retGC-1-specific
antibody (13) and chemiluminescent autoradiography using ULTRA Super
Signal substrate. The bands of cross-linked products were scanned by Paragon 1200A3 Pro Scanner and the relative density (mm2 × OD) was calculated by Molecular Analyst Software (Bio-Rad). It should
be emphasized that the Rf value of the 210-kDa cross-linked product was slightly different in each SDS-PAGE; however,
the molecular mass of the 210-kDa product was constantly monitored by
myosin (205 kDa) as a molecular standard. The molecular mass of the
>400-kDa cross-linked product(s) was estimated using high molecular
mass (97,400-584,400) standards.
Gel Filtration Column Chromatography--
The freshly
solubilized retGC was prepared by incubation of ROS membranes with 1 ml
of Buffer E (20 mM HEPES (pH 7.5), 1 mM dithiothreitol, 5 mM MgCl2, 0.1 mM
phenylmethylsulfonyl fluoride, 5 µM leupeptin, 5 µM pepstatin A, and 150 mM NaCl) containing 5% n-dodecyl- Analytical Methods--
SDS-PAGE was performed as described
(51). Protein concentrations were assayed with bovine serum albumin as
standard (52). Protein concentration in samples containing a detergent
was monitored as described (5). Ca-EGTA buffers were calculated (26)
and prepared (53). Free [Ca2+] was verified by
Ca2+-electrode and Rod-2 titration. 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.
Detection of a retGC-1 Dimer with Molecular Mass ~210
kDa--
Dimerization or oligomerization of peptide-regulated GCs have
been monitored using several methods including cross-linking (45),
measurement of molecular weight (46), and the yeast two-hybrid system
(47). In this study, to obtain the exact relationship between
dimerization of retGC-1 and its activity, we attempted to fix dimeric
(or oligomeric) forms of retGC-1 by cross-linking under a condition
similar to these used for measurement of GC activity. We found that two
oligomeric forms of the retGC-1, ~210 and >400 kDa, were fixed by
less than 60 µM BS3 in a ROS homogenate (Fig.
1, upper inset). The amounts
of both cross-linked products were increased in a
[BS3]-dependent manner; however, the 210-kDa
product was detected by a much lower [BS3] than the
>400-kDa product. These cross-linked products in ROS homogenates were
also formed with different cross-linkers, such as
3,3'-dithiobis(sulfosuccinimidyl propionate) and disuccinimidyl suberate.
These two cross-linked products of retGC-1 were also observed in a
purified retGC preparation (Fig. 1, lower inset), indicating that these oligomers are made from retGC. Because the molecular mass
(~210 kDa) of the cross-linking product is similar to the calculated
molecular mass of the retGC-1 dimer (~220 kDa), we conclude that the
210-kDa product is a dimer of retGC-1. We note that the 210-kDa
cross-linked product was detected in ROS homogenates only when
[Ca2+] was low (data not shown), and that the amount of
210-kDa product in the purified retGC-1 preparation was much less than
that in ROS homogenates.
RetGC-1 has been shown to be regulated by proteins, such as GCAPs (14,
21-23) and RGS9 (41), in photoreceptor outer segments. It is possible
that the 210-kDa cross-linked product of retGC-1 in the ROS homogenate
(Fig. 1) is a complex of retGC-1 with these retGC regulators. As shown
in Fig. 2, the ROS homogenate contained GCAPs; however, GCAPs were not detected in the 210-kDa cross-linked product although under these conditions the formation of the 210-kDa cross-linked product was stimulated by GCAPs, as described below. In
addition, the ROS homogenate also contained RGS9; however, RGS9 was not
present in the 210-kDa product. These observations support our
conclusion that the 210-kDa cross-linked product is a homodimer of
retGC-1, but not a monomeric form of retGC-1 cross-linked with other
proteins, such as GCAPs and RGS9.
Effects of Ca2+ and GCAPs on the GC Activity and the
Formation of the 210-kDa Cross-linked Product of retGC-1--
The GC
activity in a ROS homogenate was high in the presence of low
[Ca2+], but inhibited to its basal activity when
[Ca2+] was higher than 400 nM (Fig.
3A). This behavior of retGCs
is consistent with the interpretation that GCAPs in the ROS homogenate function as activators of retGCs in the presence of low
[Ca2+]. Under these conditions, the 210-kDa cross-linked
product of retGC-1 was clearly formed; however, the product was
drastically decreased when [Ca2+] was higher than 500 nM (Fig. 3, B and C). In other words,
if the GC activity was basal, the 110-kDa retGC-1 was distinctly observed; however, the 210-kDa product was increased when the GC
activity was increased. These data strongly indicate a relationship between the formation of the 210-kDa cross-linked product of retGC-1 and the retGC activity. In this experiment, the amounts of the retGC-1
cross-linked product(s) with molecular mass >400-kDa appear to be
parallel to the retGC activity. The possible mechanism for the
formation of this high molecular weight complex and meanings of this
observation will be discussed later.
In order to strengthen our conclusion that the 210-kDa cross-linked
product of retGC-1 was formed when the activity of retGC-1 was
increased by GCAPs, the effect of recombinant GCAP-1 on the GC activity
and the formation of the 210-kDa cross-linked product of retGC-1 in
GCAPs-free ROS membranes were investigated (Fig. 4). A constitutively active mutant of
GCAP-1, Y99C, was also used not only to strengthen our argument but
also to indicate that [Ca2+] does not directly regulate
the formation of the 210-kDa product. The GC activity of these
membranes was low even when [Ca2+] was low (Fig.
4A), indicating that functional amounts of GCAPs had been
washed out from membranes. Addition of GCAP-1 increased GC activity of
membranes if [Ca2+] was low; however, the GC activation
was drastically diminished when [Ca2+] was increased
(Fig. 4A). On the other hand, even without Ca2+,
the 210-kDa product in GCAPs-free membranes was reduced to ~35% of
that found in the ROS homogenate (Fig. 4, B and
C). Addition of GCAP-1 recovered the 210-kDa product to
~85% of that found in the ROS homogenate if [Ca2+] was
low; however, the recovery was not detected in the presence of more
than 500 nM [Ca2+] (Fig. 4, B and
C). When the constitutively active mutant of GCAP-1 was
added to these membranes, the GC activity in the membranes was
constantly high, although the GC activity was slightly reduced if
[Ca2+] was increased (Fig. 4A). Under these
conditions, the amount of the 210-kDa product was also consistently
high although the 210-kDa product decreased slightly if
[Ca2+] was increased (Fig. 4, B and
C). These observations indicate that the 210-kDa
cross-linked product of retGC-1 is less when the retGC activity is
basal, and that the formation of the 210-kDa product is stimulated when
the GC activity was stimulated by GCAP-1 or its mutant. Moreover,
[Ca2+] is not directly involved in the regulation of the
210-kDa product formation. We note that, when [Ca2+] was
more than 500 nM, GCAP-1 did not inhibit the basal activity of retGC in membranes (Fig. 4A), although amounts of the
210-kDa product were reduced to ~20% of the basal level (Fig. 4,
B and C). We speculate that the ability of GCAP-1
to inhibit retGC basal activity is weak. Alternatively, the final
concentration of GCAP-1 in the cross-linking mixture may be higher than
that in the mixture for the assay of enzymatic activity because larger
amounts of membranes were used for the cross-linking reaction; that is,
the residual amounts of GCAP-1 in membranes may be much higher than that for the enzyme assay. We also note that the cross-linked product(s) of retGC-1 with >400-kDa molecular mass was not detected under these conditions.
GCAP-2 and its constitutively active mutant, E80Q/ E160Q/D158N,
showed similar effects on the GC activity and the formation of the
210-kDa cross-linked product of retGC-1 in the GCAPs-free ROS membranes
(Fig. 5). In addition, both the GC
activity and the formation of the 210-kDa product were reduced to less
than these basal levels if [Ca2+] was higher than 1 µM. These observations clearly indicate the relationship
between the GC activation and the formation of the 210-kDa cross-linked
product of retGC-1 in ROS membranes.
Effects of GCAPs on the GC Activity of Recombinant retGC-1 and the
Formation of the 210-kDa Cross-linked Product of
retGC-1--
Recombinant retGC-1 expressed in COS-7 cells was also
used to check the relationship between the activation of retGC-1 and the formation of the 210-kDa cross-linked product (Fig.
6). The GC activity in these membranes
was low and the activity was not changed with or without
Ca2+, indicating that GCAPs were not expressed (Fig.
6B). Addition of GCAPs to these membranes activated retGC-1
in the absence of Ca2+; however, in the presence of 1.5 µM Ca2+, the GC activation was drastically
reduced. Addition of constitutively active mutants of GCAPs, Y99C, and
E80Q/E160Q/D158N, to these membranes enhanced the retGC-1 activity and
this enhancement was virtually unaffected by the high
[Ca2+]. These results indicate that active forms of GCAPs
affect the retGC-1 activity in these membranes the same as in
photoreceptor membranes. Under these conditions, when GCAPs were added
to membranes and Ca2+ was not present, the formation of the
210-kDa product was clearly detected; however, the 210-kDa product was
not observed in the presence of 1.5 µM Ca2+
(Fig. 6A). When constitutively active mutants of GCAPs were
added to membranes, the formation of the 210-kDa product was detected and the formation was not sensitive to Ca2+. These results
indicate the clear relationship between the activation of retGC-1 by
GCAPs and the formation of the 210-kDa cross-linked product of retGC-1.
Moreover, these observations demonstrate that Ca2+ is not
directly involved in the formation of the 210-kDa product. We note that
the retGC-1 complex(s) with molecular mass >400 kDa was constantly
detected in these membranes even without BS3, and that
formation of the complex was not related to the GC activity.
The Effect of S100b on the GC Activity and the Formation of the
210-kDa Cross-linked Product of retGC-1--
The GC activity in ROS
membranes is activated by the S100 family proteins in the presence of
very high [Ca2+] (32, 33). The relationship between the
activation of retGC and the formation of 210-kDa product was also
investigated using GCAPs-free membranes and S100b (Fig.
7). The GC activity in GCAPs-free membranes used was ~27% of the GC activity in the original ROS homogenate (Fig. 7A), indicating that GCAPs have been washed
out from these membranes. Under these conditions, the GC activity in
membranes was increased by the addition of 2 µM S100b in
a [Ca2+]-dependent manner. The half-maximal
activation was observed to be ~40 µM
[Ca2+]. Under the same conditions, the amounts of the
210-kDa product were increased when [Ca2+] was increased
(Fig. 7, B and C). We note that without S100b such high [Ca2+] stimulated neither the GC activity nor
the formation of the 210-kDa product (data not shown). These results
indicate that the clear relationship exists between the activation of
retGC by S100b and the formation of the 210-kDa product of retGC-1. Since GCAPs also stimulate the formation of the 210-kDa product in the
presence of low [Ca2+] (Figs. 4-6), these observations
imply that the 210-kDa product is formed whenever retGC is activated by
any activator, and that Ca2+ is only involved in the
regulation of activators.
Molecular Mass of Various GC Preparations from Bovine ROS Measured
by Gel Filtration--
The purified retGC has been shown to behave as
a very large molecular mass complex or an aggregated form (5). We
confirmed that a large portion of retGC purified from ROS membranes
appeared to be aggregated (Fig.
8A). This observation is also
supported by the presence of substantial amounts of the oligomeric
form(s) of retGC-1 with molecular mass >400-kDa in the purified GC
even without a cross-linker (Fig. 1). Since the activity of purified retGC is basal (5) and the amounts of the large molecular mass complex(es) was not proportional to the retGC activity (data not shown), the large molecular mass complex should not be related to the
activation of retGC. In order to estimate the formation mechanism of
the large molecular mass complex, we measured the molecular mass of the
retGC solubilized freshly from photoreceptor membranes. As shown in
Fig. 8B, the enzyme solubilized freshly was eluted in a peak
corresponding to a molecule with a Stoke's radius of 48.9 Å and an
estimated molecular mass of ~150 kDa. The size of the retGC is
consistent with that of a monomeric form of retGC with detergent bound
and/or that of elongated retGC. However, when the same retGC
preparation was stored overnight and applied to the column, the retGC
was eluted in fractions similar to that of the purified retGC (Fig.
8B). Aparicio and Applebury (54) also reported similar data.
These results suggest that retGC can be present as a monomeric form in
a fresh preparation, and that the storage of retGC preparations makes
the enzyme aggregated. These observations imply that the aggregated
form of the purified retGC may be formed during its purification.
In retinal photoreceptor cells, retGC is known to be activated by
GCAPs when [Ca2+] is less than 300 nM. The
activation is believed to be essential for the recovery of
photoreceptors to the dark state, although the molecular mechanism of
the activation is unknown. In this study, we have shown that retGC-1
exists in a monomeric form (~110 kDa) whenever GC activity is at
basal or low level, and the 210-kDa cross-linked product is increased
whenever the GC activity is stimulated. We have also indicated that the
210-kDa cross-linked product is a homodimer of retGC-1, and the
[Ca2+] directly regulates neither the GC activity nor
formation of the 210-kDa cross-linked product. Thus, we conclude that
dimerization of retGC-1 is involved in its activation by active forms
of GCAPs, as summarized in Fig. 9. To
reach these conclusions, we monitored the GC activity and formation of
the 210-kDa cross-linked product of retGC-1 under similar conditions.
Moreover, we used constitutively active mutants of GCAPs and S100b, not
only to strengthen our argument but also to indicate that
[Ca2+] does not directly regulate the formation of the
210-kDa product. In addition to retGC-1 expressed in COS cells, ROS
homogenates and GCAPs-free ROS membranes were used. Then, the total GC
activity in these membranes was compared with the amounts of
cross-linked products detected by a retGC-1-specific antibody. Although
these ROS membranes contain two retGCs (retGC-1 and -2) (8-12, 48), our results show a clear relationship between retGC activity and the
210-kDa cross-linked product of retGC-1. It is possible that retGC-2 is
also dimerized when activated by GCAPs because retGC-2 is similar to
retGC-1 in its activation by GCAPs (8-12). We could not use retGC-2
expressed in COS cells to investigate this possibility because of low
activity. Alternatively, the GC activity measured may be mainly
contributed by retGC-1 because retGC-2 may be less abundant (10).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]GTP and [3H]cGMP from NEN Life
Science Products Inc.; cGMP and GTP from Roche Molecular Biochemicals;
AG 1-X2 resin from Bio-Rad; alumina N-Super I from ICN; creatine
phosphokinase, phosphocreatine, phenylmethylsulfonyl fluoride,
leupeptin, pepstatin A, 1-methyl-3-isobutylxanthine, n-dodecyl-
-D-maltoside, S100b, GTP-agarose
resin, and high molecular weight makers for SDS-PAGE from Sigma; Ultra
Super Signal substrate, PVDF membranes, BS3,
3,3'-dithiobis(sulfosuccinimidyl propionate), and disuccinimidyl suberate from Pierce. Antibodies against retGC-1 (13), GCAPs (23, 31),
and RGS9 (41) were prepared as described.
80 °C. The
washed membranes are termed GCAPs-free ROS membranes. Purified retGC
from bovine ROS was prepared using a GTP-agarose column (5). The purity
of the preparation was greater than 95%. The purified retGC was stored
in liquid nitrogen until used (up to months). Preparation of
recombinant bovine retGC-1 expressed in COS-7 cells (24), bovine GCAPs
in Escherichia coli (28, 50), and constitutively active
mutants of GCAP-1 (Y99C) (28) and GCAP-2 (E80Q/E160Q/D158N) (27) has
been described.
-32P]GTP, and ~0.5 µCi of
[3H]cGMP). The reaction was initiated by addition of GTP
and cGMP. Following incubation (37 °C for 10 min), the reaction was
terminated by adding 40 µl of 1 N HCl and boiling for 2 min. [32P]cGMP derived from [
-32P]GTP
was isolated by alumina and AG 1-X2 columns (5), and both
3H and 32P radioactivities in samples were counted.
-D-maltoside for 2 h at
0 °C (5). After centrifugation (100,000 × g,
4 °C, 30 min), a portion of the supernatant (1.5 mg of protein, 200 µl) was immediately applied to a Sephacryl S-200 HR column (9 × 600 mm) which had been equilibrated with Buffer E containing 0.1%
n-dodecyl-
-D-maltoside. Another portion of
the supernatant was stored on ice overnight, and applied to the column
under the same conditions. Purified retGC (8 µg) was also applied to
the column under the same conditions. Column chromatography conditions
were as follows: flow rate, 1.5 ml/10 min; and fraction volume, 1.0 ml.
For column calibration, proteins with known Stokes radii were
chromatographed under identical conditions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Formation of the 210-kDa retGC-1 cross-linked
product in a bovine ROS homogenate. With various
[BS3], bovine ROS homogenate (50 µg) was incubated
(0 °C, 30 min) in 50 µl of Buffer D containing 1 mM
EGTA. The cross-linking reaction was quenched by the addition of SDS
sample buffer and boiling for 5 min. The cross-linked protein products
were immediately separated by SDS-PAGE, transferred to a PVDF membrane,
and detected by Western immunoblotting analysis with a chemiluminescent
substrate using a retGC-1-specific antibody. The upper inset
shows the profile of cross-linked products of retGC-1 in ROS
homogenates. The 210-kDa product was scanned and its relative density
was calculated as described under "Experimental Procedures." One
hundred % indicates the amounts of the 210-kDa product formed in the
presence of 60 µM BS3. Purified retGC (~0.5
µg) was also incubated with or without 50 µM
BS3 under the same conditions. The lower inset
shows the profile of cross-linked products of retGC-1 in the purified
preparation. A ~110-kDa retGC-1 is a monomeric form.
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Fig. 2.
Cross-linked products of retGC-1 and its
regulators in a bovine ROS homogenate. Bovine ROS homogenate (50 µg of protein) was incubated in 50 µl of Buffer D containing 1 mM EGTA in the presence or absence of 50 µM
BS3 (0 °C and 30 min). The cross-linking reaction was
quenched by the addition of SDS sample buffer and boiling for 5 min.
The cross-linked products were separated by SDS-PAGE and transferred to
PVDF membranes. The cross-linked products of retGC-1, RGS9, and GCAPs
were detected by Western immunoblotting analysis using rabbit
antibodies specific to these proteins and a chemiluminescent substrate.
To avoid a larger figure, parts of gels are shown. There is no
chemiluminescent band between 55 and 110 kDa.
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Fig. 3.
Effects of Ca2+ on GC activity
and formation of the 210-kDa retGC-1 cross-linked product in a bovine
ROS homogenate. A, GC activity. The GC activity of
bovine ROS homogenate (5 µg) was measured in 200 µl of Buffer C
containing various [Ca2+]. Results represent mean values
from three separate experiments performed in duplicate. B
and C, all cross-linked products (B) and relative
amounts of the 210-kDa product (C). ROS homogenate (50 µg)
was incubated in 50 µl of Buffer D containing various
[Ca2+]. The cross-linking reaction (30 min, 0 °C) was
carried out using 50 µM BS3 and quenched by
the addition of SDS sample buffer and boiling for 5 min. The
cross-linked products were immediately isolated by SDS-PAGE,
transferred to PVDF membrane, and detected by Western immunoblotting
analysis with a chemiluminescent substrate using a retGC-1-specific
antibody. The 210-kDa cross-linked product in B was scanned
and the relative density was shown in C. One hundred % indicates the amounts of the 210-kDa product formed without
Ca2+. * indicates the 210-kDa cross-linked product formed
without BS3.
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Fig. 4.
Effects of GCAP-1 or its constitutively
active mutant on GC activity and formation of the 210-kDa retGC-1
cross-linked product in GCAPs-free ROS membranes. A, GC
activity. With or without 2 µM GCAP-1 or its
constitutively active mutant, Y99C (GCAP-1m), the GC
activity in GCAPs-free ROS membranes (equal to 5 µg of ROS
homogenate) was measured in 200 µl of Buffer C containing various
[Ca2+]. Results represent mean values from three separate
experiments performed in duplicate. , GCAPs-free ROS membranes only;
, +GCAP-1;
, +GCAP-1m. B and C, all
cross-linked products (B) and relative amounts of the
210-kDa product (C). With or without 2 µM
GCAP-1 or its constitutively active mutant (GCAP-1m),
GCAPs-free ROS membranes (equal to 50 µg of ROS homogenate) were
incubated in 50 µl of Buffer D containing various
[Ca2+]. The cross-linking reaction (30 min, 0 °C) was
carried out with 50 µM BS3 and quenched by
the addition of SDS sample buffer and boiling for 5 min. As controls,
cross-linking reaction of ROS homogenate (50 µg) was also performed
with or without BS3. The cross-linked products were
immediately isolated by SDS-PAGE, transferred to PVDF membrane, and
detected by Western immunoblotting analysis with a chemiluminescent
substrate using a retGC-1-specific antibody. The 210-kDa cross-linked
product in B was scanned and the relative density was shown
in C. One hundred % indicates the amounts of the 210-kDa
product formed without Ca2+ in the ROS homogenate.
ROS, ROS homogenate; Ca-W-Me, GCAPs-free
membranes; R*, ROS homogenate without BS3;
R, ROS homogenate with BS3; C,
GCAPs-free ROS membranes with BS3; +GCAP-1,
GCAPs-free ROS membranes and GCAP-1 in the presence of BS3;
+GCAP-1m, GCAPs-free ROS membranes and GCAP-1m in the
presence of BS3.
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Fig. 5.
Effects of GCAP-2 on GC activity and
formation of the 210-kDa cross-linked product in GCAPs-free ROS
membranes. A, GC activity. With or without 2 µM GCAP-2 or its constitutively active mutant,
E80Q/E160Q/D158N (GCAP-2m), the GC activity in GCAPs-free
ROS membranes (equal to 5 µg of ROS homogenate) was measured in 200 µl of Buffer C containing various [Ca2+]. Results
represent mean values from three separate experiments performed in
duplicate. , GCAPs-free ROS membranes only;
, +GCAP-2;
,
+GCAP-2m. B and C, all cross-linked products
(B) and relative amounts of the 210-kDa product
(C). With or without 2 µM GCAP-2 or its
constitutively active mutant (GCAP-2m), GCAPs-free ROS membranes (equal
to 50 µg of ROS homogenate) were incubated in 50 µl of Buffer D
containing various [Ca2+]. The cross-linking reaction (30 min, 0 °C) was carried out with 50 µM BS3
and quenched by the addition of SDS sample buffer and boiling for 5 min. As controls, cross-linking reaction of ROS homogenate (50 µg)
was also performed with or without BS3. The cross-linked
products were immediately isolated by SDS-PAGE, transferred to PVDF
membrane, and detected by Western immunoblotting analysis with a
chemiluminescent substrate using a retGC-1-specific antibody. The
210-kDa cross-linked product in B was scanned and the
relative density was shown in C. One hundred % indicates
the amounts of the 210-kDa product formed without Ca2+ in
the ROS homogenate. ROS, ROS homogenate; Ca-W-Me,
GCAPs-free ROS membranes; R*, ROS homogenate without
BS3; R, ROS homogenate with BS3;
C, GCAPs-free ROS membranes with BS3;
+GCAP-2, GCAPs-free ROS membranes and GCAP-2 in the presence
of BS3; +GCAP-2m, GCAPs-free ROS membranes and
GCAP-2m in the presence of BS3.
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Fig. 6.
Effects of GCAPs on GC activity and formation
of the 210-kDa cross-linked product of retGC-1 in COS-7 cell
membranes. A, cross-linked products. With or without 2 µM GCAPs (GCAP-1 and GCAP-2) or their constitutively
active mutants, Y99C (GCAP-1m) and E80Q/E160Q/D158N (GCAP-2m), retGC-1
expressed in COS-7 cells (50 µg) was incubated in 50 µl of Buffer D
in the presence or absence of 1.5 µM Ca2+.
The cross-linked reaction (30 min, 0 °C) was carried out with or
without 50 µM BS3 and quenched by the
addition of SDS sample buffer and boiling for 5 min. As controls, the
cross-linking reaction of ROS homogenate (50 µg) was also carried out
with or without BS3. The cross-linked products were
immediately isolated by SDS-PAGE, transferred to a PVDF membrane. The
retGC-1 complexes were detected by Western immunoblotting analysis
using an anti-retGC-1 antibody and a chemiluminescent substrate.
ROS, ROS homogenate; Re-GC1, retGC-1 expressed in
COS cell membranes. B, GC activity. With or without 2 µM GCAPs (GACP-1 and GCAP-2) or their constitutively
active mutants (GCAP-1m and GCAP-2m), GC activity in COS cell membranes
(20 µg) was measured in the presence or absence of 1.5 µM Ca2+. Results are shown as fold
stimulation to the basal activity of membranes (Contr., 30 pmol/min/mg). Results represent mean values from three separate
experiments performed in duplicate.
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Fig. 7.
Effect of S100b on GC activity and formation
of the 210-kDa cross-linked product in GCAPs-free ROS membranes.
A, GC activity. With 2 µM S100b, the GC
activity in GCAPs-free ROS membranes (equal to 5 µg of ROS
homogenate) was measured in 200 µl of Buffer C containing various
[Ca2+]. Results represent mean values from three separate
experiments performed in duplicate. As a control, GC activity of ROS
homogenate (5 µg) was also measured without Ca2+. ,
GCAPs-free ROS membranes;
, ROS homogenate. B and
C, all cross-linked products (B) and relative
amounts of the 210-kDa product (C). With or without 5 µM S100b, GCAPs-free ROS membranes (equal to 50 µg of
ROS homogenate) were incubated in 50 µl of Buffer D containing
various [Ca2+]. The cross-linking reaction (30 min,
0 °C) was carried out with 50 µM BS3 and
quenched by the addition of SDS sample buffer and boiling for 5 min. As
a control, cross-linking reaction of ROS homogenate (50 µg) was also
performed in the absence of Ca2+. The cross-linked products
were immediately isolated by SDS-PAGE, transferred to PVDF membrane,
and detected by Western immunoblotting analysis with a chemiluminescent
substrate using a retGC-1-specific antibody. The 210-kDa cross-linked
product in B was scanned and the relative density was shown
in C. One hundred % indicates the amounts of the 210-kDa
product formed without Ca2+ in the ROS homogenate.
ROS, ROS homogenate; Ca-W-Me, GCAPs-free ROS
membranes.
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Fig. 8.
Activity profiles of various retGC
preparations eluted from a Sephacryl S-200 HR column. After
Sephacryl S-200 HR column chromatography of various retGC samples, GC
activity of each fraction was measured. As molecular mass standards,
aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa),
and chymotrypsinogen A (25 kDa) were used. A, purified retGC
(8 µg). B, solubilized retGCs. After freshly solubilized
by 5% of n-dodecyl- -D-maltoside from ROS
membranes, one portion of the supernatant (1.5 mg) was applied
immediately to the Sephacryl S-200 HR column (
). The other portion
(1.5 mg) was incubated overnight on ice and then applied to the column
(
). The conditions for the chromatography are described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Relationship between monomer, dimer, and
oligomer of retGC-1 in the molecular mechanism of retGC-1 activation by
GCAPs.
Peptide-regulated GCs have been proposed to be present as oligomeric forms even without activation by peptides (42-44). In this study, we have also shown that retGC-1 complexes with molecular mass >400 kDa was observed in several GC preparations. As summarized in Fig. 9, we believe that the high molecular mass complex(es) of retGC-1 is formed artificially from monomeric and/or dimeric forms of retGC-1 for the following reasons. (a) The detection of the high molecular mass complex was not constant in ROS membranes. For example, the complex was detected in ROS homogenates shown in Figs. 3, 5, and 7, but not in Figs. 2 and 4. In addition, these complexes were detected in the GCAP-free membranes shown in Fig. 5, but not in Fig. 4. Moreover, the presence or absence of these complexes appears not to be related to the GC activity. Thus, the high molecular mass complex may be formed during preparation of membranes. (b) We have shown that a retGC preparation becomes aggregated if stored, and that a purified retGC preparation behaves in a gel filtration column as the retGC preparation stored overnight (Fig. 8). Moreover, the purified preparation contains substantial amounts of the retGC complex with molecular mass >400 kDa (Fig. 1). These observations imply that the high molecular mass complex in the purified sample is self-aggregated by storage or purification procedures. (c) In recombinant retGC-1 expressed in COS-7 cells, the high molecular mass complex was detected without cross-linker (Fig. 6). The high molecular mass complex was also observed even when the GC activity was basal and the content of the complex was not changed even when the activity of retGC-1 was changed. The high molecular mass complex was also detected in retGC-2 expressed in COS cells (data not shown). These results suggest that the high molecular mass complex is not related to the GC activity. It is possible that the complex is a self-aggregated form and/or a form of misfolding retGC-1 during its expression.
Although it is difficult to rule out completely the possibility that the 210-kDa cross-linked product is a retGC-1 complexed with proteins other than retGC-1, we anticipate that the 210-kDa product is a dimer form of retGC-1 for the following reasons. (a) The molecular mass of the cross-linked product (~210 kDa) is similar to the calculated molecular mass (~220 kDa) of the retGC-1 dimer. (b) The 210-kDa product was detected in a preparation of purified retGC-1 (Fig. 1). This retGC preparation did not contain GCAPs and RGS9 (data not shown). Formation of the 210-kDa product in the purified retGC preparation was much less than that in ROS homogenates because the purified preparation did not contain any activator and its GC activity is low. Even if GCAPs are contaminated in the purified preparation, its GC activity should be basal because GCAPs do not function as retGC activators in the presence of a detergent (6). (c) The 210-kDa product was also detected in a preparation of recombinant retGC-1 expressed in COS-7 cells (Fig. 6). (d) The 210-kDa product is increased whenever the GC activity is stimulated (Figs. 1 and 3-7). These observations exclude the possibility that inhibitory regulators, such as guanylyl cyclase-inhibitory protein (40) and RGS9 (41), are complexed with retGC-1 to form the 210-kDa product. Using a RGS9-specific antibody (41) we have shown that RGS9 is not involved in the 210-kDa product (Fig. 2). (e) The 210-kDa product does not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. Western blotting of the 210-kDa product using GCAP-specific antibodies has indicated that the 210-kDa product does not contain GCAPs under our conditions (Fig. 2). (f) The 210-kDa product in ROS homogenates was also observed by different cross-linkers, such as 3,3'-dithiobis(sulfosuccinimidyl propionate) and disuccinimidyl suberate, when the GC activity in the preparation was stimulated by lowering [Ca2+] (data not shown).
GCAPs were not found in the ~210-kDa cross-linked product of retGC-1
when the dimerization of retGC-1 was stimulated by GCAPs (Fig. 2).
However, it should be emphasized that this study does not exclude the
possibility that the real retGC dimer contains Ca2+
activators if the formation of the retGC dimer is enhanced by Ca2+ activators. As depicted in Fig. 9, we rather believe
that Ca2+ activators were contained in the retGC dimer
before cross-linking reaction if the dimer formation was stimulated by
Ca2+ activators. We believe that our conditions for the
cross-linking reaction fix the interaction between retGCs, but not
between retGC and GCAPs. A previous study has already shown that a high
molecular mass cross-linked product contains retGC-1 and GCAP-1 (24). Previous studies have suggested that a different region of retGC-1 appears to be required for its interaction with each Ca2+
activator (24, 25, 55). Thus, it is possible that each Ca2+
activator forms a retGC-1 dimer in a different way, and that each
retGC-1 dimer may be complexed with a different Ca2+
activator. A slight difference in the conformation of the retGC-1 dimers may be important for the fine regulation of free [cGMP] in
photoreceptor outer segments.
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ACKNOWLEDGEMENT |
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We thank Dr. R. B. Needleman for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants EY07546 and EY09631 (to A. Y), EY11522 (to A. M. D), EY10828 (to R. K. S), and HL58151 (to T. D), a Career Development Award from Research to Prevent Blindness (to A. M. D.), and by affiliated supports from Research to Prevent Blindness.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.
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:
akio_yamazaki{at}wayne.edu.
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ABBREVIATIONS |
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The abbreviations used are: GC, guanylyl cyclase; retGC, retinal guanylyl cyclase; GCAP, guanylyl cyclase-activating protein; ROS, rod outer segments; GCAP-1m, a constitutively active mutant of GCAP-1, Y99C; GCAP-2m, a constitutively active mutant of GCAP-2, E80Q/E160Q/D158N; BS3, bis(sulfosuccinimidyl) suberate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.
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REFERENCES |
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