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J. Biol. Chem., Vol. 278, Issue 35, 33150-33160, August 29, 2003
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From the
Kresge Eye Institute and the Departments
of Ophthalmology and
¶Pharmacology, Wayne State University, School of
Medicine, Detroit, Michigan 48201 and the **Department
of Anatomy and Cell Biology, Nagoya University, School of Medicine, Nagoya
466, Japan
Received for publication, April 9, 2003 , and in revised form, May 27, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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500
nM to near 30 nM in photoreceptor OS
(4–6).
The decrease in free Ca2+ concentrations acts as a
trigger for cGMP synthesis (7,
8). Two membrane guanylyl
cyclases, retGC-1 and -2 (also referred to as ROS GC-1 and -2 or GC-E and
GC-F, respectively), are involved in the cGMP synthesis in photoreceptor OS
(9–14).
It has been shown that retGC-1 is localized in the photoreceptor layer and
that cone OS contains more retGC-1 than rod OS
(15,
16). In photoreceptor OS,
retGC-1 appears to be associated with the marginal region of disk membranes
and/or the plasma membranes
(15). retGC-2 is localized in
photoreceptors (12); however,
detailed localization of retGC-2 in retina has not been reported.
RetGC shares an overall molecular configuration similar to those of peptide-regulated GCs and contains an extracellular domain, a transmembrane domain, a kinase homology domain (KHD), and a catalytic domain (17, 18). However, it has been believed that regulation of retGC is different from those of peptide-regulated GCs. For example, GC-A, a typical peptide-regulated GC, is activated through binding of atrial natriuretic peptide to the extracellular domain (17, 18). In retGC, the extracellular domain appears not to be required for the stimulation, although its real function is unknown. Instead, the Ca2+-sensitive stimulation of retGC is mediated by binding of calmodulin-like Ca2+-binding proteins termed GCAPs (19, 20) to the intracellular domains (21, 22). Three GCAPs have been reported (19, 20, 23) and two GCAPs, 1 and 2, have been extensively studied. The mechanism of retGC activation by GCAPs is not clear; however, dimerization (or oligomerization) of retGC appears to be involved (24–27). It is also known that retGC is activated by S100 proteins, other Ca2+-binding proteins, at high Ca2+ concentrations (28, 29). This regulation is believed to be involved in retinal cells other than photoreceptor OS. It should be noted that the relationship between GCAP localization and their functions appears to be controversial. Immunocytochemical analysis (30, 31), with genetic analysis of cone degeneration (32, 33), suggests that GCAP1 is primarily expressed in cones, whereas GCAP2 is primarily detected in rods and, at a lower level, in cones. Very recent studies using double knockout mice (GCAP–/–) showed, as expected, that overexpression of bovine GCAP2 in GCAP–/– rescued the Ca2+ sensitivity of retGC and the time for the rod recovery from saturating flashes (34, 35). However, the GCAP2 overexpression could not restore the normal kinetics of response evoked by subsaturating flashes (34). On the contrary, Howes et al. (36) reported that GCAP1 expression in GCAP–/– restored the wild type properties of rod light response in the absence of GCAP2. They proposed that GCAP1 supports the generation of wild type flash responses in rods. In these studies using double knockout mice, it is not clear whether the expression of bovine GCAPs in mouse rods completely restores the function of the missing mouse GCAPs and whether the overexpression of GCAP2 disturbs normal functions. Moreover, it is not known whether all GCAPs expressed in GCAP–/– function properly and whether GCAP–/– can restore all normal properties if both GCAPs are expressed.
There is another crucial difference between peptide-regulated GCs and
retGC. ATP is obligatory in the stimulation of GC-A
(17,
18,
37). However, ATP has not been
reported to be essential for the GCAP stimulation of retGC. ATP has been
believed to only modify retGC activity; low ATP concentrations (less than
0.5 mM) slightly stimulate and high ATP concentrations (more
than 1 mM) significantly inhibit retGC activity in OS
membranes
(38–41).
Several studies also reported the synergetic effect of ATP and GCAPs on retGC
activity (21,
41–43),
although the ATP concentrations used in these studies (less than 0.5
mM) were lower than the physiological ATP concentrations in rods
(3–4 mM)
(44), and the activation was
not large in OS membranes. It should be emphasized that the significance of
the inhibition of retGC by physiological ATP concentrations has been
completely ignored in the previous studies of retGC regulation.
There is another fundamental question ignored in the previous retGC studies: whether the observed GCAP-stimulated retGC activity is sufficient to account for the recovery of cGMP level to the dark state. Needless to say, the activity of purified retGC is less than that of light/GTP-stimulated PDE (45–48). Sitaramayya et al. (39) estimated, based on published data obtained in vitro and their measurements of retGC activities in vitro, that the maximal rate of cGMP hydrolysis in light/GTP-activated PDE is 7–200 times greater than the potential retGC activity. In addition, the content of retGC in rods is not larger than that of PDE, although these estimations varied (from 1:1 to 1:10) (46–48). In addition, recent studies on GCAPs–/– mice showed that the mean single photon response amplitude was nearly five times larger than that of wild type rods (34, 35, 49). At all flash strengths examined, the light-evoked PDE activity measured as the rate of rise of the signal at early times was the same in GCAP–/– and control rods. These observations suggest that the level of GCAP-stimulated retGC activity in vivo may be similar or higher than that of light/GTP-activated PDE. Other electrophysiological studies also estimated the retGC activity in vivo as being higher than that measured in vitro (50, 51). These studies imply that there may be another mechanism for the retGC activation and/or that unknown components may be involved in the further stimulation of retGC by GCAPs in vivo.
In this study, we have attempted to reconcile the difference in retGC activities observed in vivo and in vitro. We show that pretreatment of OS homogenates with adenine nucleotides (1–10 mM) enhances the level of retGC activity stimulated by GCAPs. Based on these observations, we propose a new mechanism for retGC activation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Preparation of retGC Samples—OS preparations used in this study were isolated from bovine retinas without separation of rod OS from cone OS. Thus, the OS preparations were mixtures of rod and cone OS, although ROS is expected to present mainly in the preparations. In addition, retGC-1 was not separated from retGC-2 in the preparations. Therefore, the retGC activity described here is the activity measured as the total activity of retGC-1 and –2, although the retGC-1 activity is expected to be dominant due to the low contents of retGC-2 (12). Bovine OS were prepared from dark-adapted retinas as described (52). GCAP-free membranes were prepared as described previously (25) with minor modifications. Briefly, OS isolated from 25 retinas were suspended in 5 ml of Buffer A (5 mM HEPES, pH 7.5, 1 mM DTT, 5 mM MgCl2, 100 µM CaCl2, 0.1 mM PMSF, 5 µM leupeptin, and 5 µM pepstatin A), homogenized by passing a needle with a 21-gauge diameter (seven times) and centrifuged (100,000 x g, 15 min, 4 °C). The process was repeated seven times. The membrane fraction was further washed in 5 ml of Buffer B (5 mM HEPES, pH 7.5, 1 mM DTT, 5 mM MgCl2, 2 mM EGTA, 0.1 mM PMSF, 5 µM leupeptin, and 5 µM pepstatin A). The process was repeated seven times. The membrane fraction (5 mg/ml) was suspended in Buffer C (10 mM HEPES, pH 7.5, 1 mM DTT, 2 mM MgCl2, 0.1 mM PMSF, 5 µM leupeptin, and 5 µM pepstatin A) and stored at –70 °C.
Preincubation of retGC with Adenine Nucleotides—An OS
preparation (150 µg) was homogenized in 300 µl of Buffer D (20
mM HEPES, pH 7.5, 5 mM MgCl2, 0.1
mM PMSF, 5 µM leupeptin, and 5 µM
pepstatin A) and incubated with 5 mM AMP-PNP. The preincubation was
carried out on ice for 30 min unless otherwise noted. After centrifugation
(100,000 x g, 10 min, 4 °C), the membrane fraction was
washed in 750 µl of buffer E (10 mM HEPES, pH 7.5, 1
mM DTT, 100 µM CaCl2, 0.1 mM
PMSF, 5 µM leupeptin, and 5 µM pepstatin A)
containing 5 mM AMP-PNP (x3) and then 750 µl of Buffer F (10
mM HEPES (pH 7.5), 1 mM DTT, 5 mM
MgCl2, 2mM EGTA, 0.1 mM PMSF, 5
µM leupeptin, and 5 µM pepstatin A) to exclude
AMP-PNP (two times). The membrane fraction was suspended in 270 µl of
Buffer F and used as retGC. Preincubation of OS homogenates with ATP (see
Fig. 1) was carried out under
slightly different conditions. Details of these conditions are described in
the figure legend. We estimate that the residual concentration of AMP-PNP in
the membrane fraction was less than 20 µM. This estimate is
based on experiments using the radioactive tracers [8H]cGMP and
[125I]anti-rabbit IgG whole antibody from goat. This estimation was
also confirmed by HPLC using TSKgel DEAE-2SW column as described below. We
also found that the AMP-PNP used was contaminated with 5% ADP. However,
preincubation of OS homogenates with 5 mM ADP only increased retGC
activity 15% as compared with OS membranes incubated without ADP,
suggesting that the ADP contamination in AMP-PNP was not involved in the
activation.
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Identification of Nucleotides—To estimate nucleotide concentrations in samples, the nucleotides were separated by HPLC, and their amounts were determined using the peak areas. The samples were prepared by centrifugation (100,000 x g, 30 min, 4 °C). After appropriate dilution, a portion of the supernatant was applied to a TSKgel DEAE-2SW column that had been equilibrated with Solution A (CH3CN in 0.1 M phosphate, pH 3.0, 20/80). The column was washed with 5 ml of Solution A, and the nucleotides were eluted by a 30-min linear gradient from Solution A to B (CH3CN in 0.5 M phosphate, pH 3.0, 20/80). The column chromatography was carried out with the flow rate 1.0 ml/min, and the nucleotides were detected by using a UV detector at 260 nm. The following are retention times of nucleotides measured under our conditions: cGMP, 7.6 min; ADP, 13.2 min; AMP-PNP, 18.8 min; GDP, 20.2 min; ATP, 21.6 min; and GTP, 28.9 min.
Construction of Three-dimensional Models of KHD of
retGC-1—The amino acid sequence of retGC-1 (bovine guanylyl cyclase
D; Swiss-Protein code P55203
[GenBank]
) was aligned using Clustal X
(53) with the catalytic
domains of four protein kinases for which crystal structures have been
determined. The KHD sequence of retGC-1 (residues 536–830 using the
Swiss Protein sequence numbers) as predicted by the multiple sequence
alignment was then modeled using the Swiss PdbViewer 3.7b2 in conjunction with
the Swiss Model program (54).
For modeling of the unstimulated conformation of retGC1, the coordinates of
the nonactivated insulin receptor kinase domain (Protein Data Bank code 1IRK
[PDB]
)
were used as the template; fitting of the model to the template gave a root
mean square deviation of 0.40 Å for 1080 carbon-
atoms. For the
AMP-PNP-stimulated conformation, the coordinates for the activated insulin
receptor kinase domain with bound AMP-PNP (Protein Data Bank code 1IR3
[PDB]
) were
used; fitting gave a root mean square value of 0.56 Å for 1030
carbon- atoms.
Measurement of retGC Activity—The activity of retGC was
measured as described (25,
46,
55). Under the assay
conditions, the hydrolysis of cGMP formed was negligible, and a linear
relationship exists between the retGC activity measured and the protein
amounts used. The linear relationship was established even in the highly
activated retGC (see Fig.
2B). All of the results about retGC activities were
analyzed using the computer program Prism (GraphPad). We note that the
concentration of protein used for the enzyme assay is expressed as total
protein rather than the rhodopsin concentration frequently used for retGC
activity. We also note that the protein concentration measured before the
adenine nucleotide preincubation was used to calculate the enzyme activities.
This calculation underestimates the actual specific activity of membrane-bound
retGC because it includes soluble proteins that are subsequently removed by
membrane washes. Measurements of protein concentrations indicated that
82% of the total proteins in OS homogenates were membrane-bound, implying
that the actual enzyme activity may be 20% higher than the activity
shown.
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Other Analytical Methods—Dimerization of retGC was monitored using a cross-linker, bis(sulfosuccinimidyl) suberate, and Western blotting with a retGC-1-specific antibody (25). SDS-PAGE was performed as described (56). Protein concentration was measured with bovine serum albumin as standard (57). Ca2+-EGTA buffers were prepared as described (25). It should be emphasized that individual points obtained in all experiments represent the average values of duplicate assays and that all experiments were carried out more than two times, and the results were similar. The data shown are representative of these experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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10 times larger than the GCAP-stimulated retGC
activities in GCAP-free membranes (47.6–56.2 as compared with 5.44 nmol
cGMP/min/mg formed in the presence of 500 nM GCAP2). Such large
activation of retGC was totally unexpected. We also found that the
preincubation in the presence of high Ca2+
concentrations was more effective than in its low concentration
(56.2–47.6 nmol cGMP/min/mg formed in the presence of 500 nM
GCAP2). It should be emphasized that the activity is expressed as specific
activity rather than the activation level compared with that obtained without
ATP and that GCAP2-stimulated retGC activities in GCAP-free membranes were
measured without the preincubation. Thus, the large activation by ATP is not
due to the reduction of retGC control activity by instability of
retGC/membranes incubated without ATP
(58). Indeed, the GCAP2
sensitivity of retGC in OS homogenates preincubated without ATP was less than
that in GCAP-free membranes, suggesting the instability of retGC/membranes
incubated without ATP. These observations strongly suggest that ATP is
positively involved in the stimulation of retGC by GCAP2.
However, mechanisms underlying the ATP stimulation are difficult to
elucidate because under these conditions ATP may affect retGC activity in
various ways. For example, ATP may express its functions by binding to retGC.
ATP may also be a phosphate donor in the phosphorylation of proteins involved
in the retGC regulation. We note that phosphatase inhibitors were added to the
ATP preincubation. We also found that OS washed membranes contained high ATP
hydrolytic activity under retGC assay conditions (33 °C, 10 min,
5–10 µg of protein/200 µl). For example, 60% of 5
mM ATP was hydrolyzed to ADP under our assay conditions, even with
an ATP-regenerating system present. This high ATP hydrolytic activity makes
data interpretation difficult. For example, 0.1–0.5 mM ATP
was added to the assay mixture in some previous studies; however, the final
ATP concentrations may be substantially lower. In addition, it is possible
that ATP metabolites may affect retGC activity.
To focus on the ATP binding to retGC, as a first step in elucidating the
ATP stimulation, and to exclude other possibilities and difficulty in data
interpretation, we preincubated the OS homogenate (on ice, 30 min) with
AMP-PNP (Fig. 2). AMP-PNP, a
hydrolysis-resistant ATP analog, has been considered to bind to retGC but not
to serve as a phosphate donor in protein phosphorylation
(58). The use of ice
temperature prevented the inactivation of retGC previously shown in incubation
at 30–37 °C (58). We
found that retGC activity, measured without GCAPs, was increased slightly but
consistently by the AMP-PNP preincubation (from 1.56–2.10 to
2.99–3.15 nmol of cGMP/min/mg formed). Without AMP-PNP, GCAP2 at 500
nM stimulated three to four times the control activity of retGC
(from 1.56–2.10 to 6.28–6.94 nmol cGMP/min/mg formed). However,
with 5 mM AMP-PNP, 500 nM GCAP2 stimulated the control
activity about 10–13 times (from 1.56–2.10 to 20.6–21.4 nmol
cGMP/min/mg formed). In the GCAP2-stimulated activity, a linear relationship
existed between retGC activity and membranes added
(Fig. 2B). These
results show that the AMP-PNP preincubation also enhances GCAP-stimulated
retGC activity, although the activation level was less than that by the ATP
preincubation (Fig. 1). These
observations strongly suggest that binding of adenine nucleotide may be
involved in the large activation of retGC by GCAP2. It should be emphasized
that the preincubation of OS homogenates with GTP slightly enhanced the
GCAP2-stimulated retGC activity; however, the level of the enhancement was
10% of those obtained by the AMP-PNP preincubation (data not shown). This
indicates that adenine nucleotides are required for this activation. We also
note that GCAP2 (500 nM) activated retGC in the GCAP-free membranes
approximately four times under our assay conditions (from 0.877 to 3.83 nmol
cGMP/min/mg formed) (Fig.
2A). Because the GCAP stimulation has been measured using
GCAP-free membranes without preincubation with adenine nucleotides, our
results indicate that most, if not all, activities of retGC/membranes
described in previous studies represent only a small portion of the potential
retGC activity in photoreceptors. We note that the control activity of retGC
preincubated without AMP-PNP is used to express the level of retGC activation
by the AMP-PNP preincubation. This comparison may be suitable to compare the
activation of retGC preincubated with AMP-PNP with that preincubated without
AMP-PNP and to compare the activity of retGC preincubated with AMP-PNP with
the activities reported previously that were measured without preincubation
with adenine nucleotides.
In AMP-PNP-pretreated OS homogenates, the retGC activity was enhanced in a
GCAP2 concentration-dependent manner (Fig.
2C). The GCAP2 concentration required for 50% enhancement
appears to be similar at all AMP-PNP concentrations used (mean for five
concentrations = 47.9 ± 2.7 nM). These results indicate that
the AMP-PNP preincubation increases the Vmax of
GCAP2-stimulated retGC activity but does not alter the affinity of retGC for
GCAP2. We note that the real EC50 may be slightly higher than the
average concentration because the membranes already contain some endogenous
GCAP2, as described below. Generally the EC50 obtained here was
similar to those reported previously
(5,
59). The AMP-PNP concentration
required for the 50% enhancement is 0.65 ± 0.20 mM
(Fig. 2D). The maximum
stimulation was achieved by 5 mM AMP-PNP, and the stimulation
was not changed even after the preincubation with 10 mM
AMP-PNP.
The effect of Ca2+ on the AMP-PNP preincubation was also investigated (Fig. 2A). Without AMP-PNP, the activity of retGC preincubated under high Ca2+ concentrations was consistently lower than that preincubated under low Ca2+ concentrations (2 mM EGTA) (1.56–2.10 nmol cGMP/min/mg formed). With AMP-PNP, but without exogenous GCAP2, OS homogenates preincubated in low Ca2+ concentrations also show slightly higher retGC activity than that in high Ca2+ concentrations (3.15–2.99 nmol cGMP/min/mg formed). However, with GCAP2, the maximum activity of retGC preincubated in high Ca2+ concentrations was slightly higher than that in low Ca2+ concentrations (21.4–20.6 nmol cGMP/min/mg formed in the presence of 500 nM GCAP2). These Ca2+ effects were measured five times, and the observations were consistent. The higher retGC activity with the preincubation in the presence of high Ca2+ concentrations was more clearly observed in the ATP preincubation (Fig. 1). Although the physiological significance of the slight increase in retGC activity by the incubation in the presence of high Ca2+ concentrations is not clear now, these results indicate that the reduction of Ca2+ concentrations, essential for the retGC activation by GCAPs, is not required during the AMP-PNP preincubation.
We also checked the effect of Mg2+ concentrations in
the preincubation on the subsequent activation of retGC by GCAP2. The
preincubation was also carried out with 5 mM AMP-PNP. We found that
the preincubation with 10 mM MgCl2 most effectively
enhanced the retGC activity. However, the differences with varying
Mg2+ concentrations were small. For example, the
activity of retGC preincubated in 10 mM MgCl2 was
110 and 105% of those preincubated in 5 and 15 mM
MgCl2, respectively. Thus, throughout this study we used 5
mM MgCl2 as a source of Mg2+ ion
in the preincubation.
RetGC Stimulation by GCAP2 after Preincubation of OS Homogenates with AMP-PNP Is Sensitive to High Ca2+ Concentrations—The activation of retGC by GCAP2 in OS homogenates preincubated with AMP-PNP was abolished by high Ca2+ concentrations in the assay, and this Ca2+ inhibition was consistently observed regardless of GCAP2 concentrations (Fig. 3A). The sensitivity to high Ca2+ concentrations was the same as that observed in membranes preincubated without AMP-PNP (Fig. 3B). In the preincubation without AMP-PNP, the IC50 for calcium inhibition was 180 nM, and the Hill coefficient was 1.42; with AMP-PNP, the IC50 was 178 nM, and the Hill coefficient was 1.36. The sensitivity to high Ca2+ concentrations was also the same as that observed in GCAP2-stimulated retGC in GCAP-free membranes (data not shown). These results indicate that the Ca2+ sensitivity of GCAP-stimulated retGC is preserved after OS homogenates were preincubated with AMP-PNP and that the highly activated retGC can be regulated by change in physiological Ca2+ concentrations in OS.
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GCAP1, but Not S100B, Also Enhances retGC Activity after Preincubation of OS Homogenates with AMP-PNP—After preincubation of OS homogenates with or without AMP-PNP, GCAP1-stimulated retGC activity was measured (Fig. 4A). Without AMP-PNP, the retGC activity was stimulated by GCAP1 (1.5 µM) approximately six times (from 1.69 to 9.57 nmol cGMP/min/mg formed), whereas with AMP-PNP (5 mM), the GCAP1-stimulated retGC activity was about 15 times of the control activity (from 1.69 to 25.9 nmol cGMP/min/mg formed). The retGC control activity was also increased by the AMP-PNP preincubation (from 1.69 to 2.62 nmol cGMP/min/mg formed). As was the case with GCAP2, the concentration for half-maximal stimulation by GCAP1 was similar in the absence (EC50 = 298 nM) or presence (EC50 = 308 nM) of AMP-PNP in the preincubation. The EC50 was similar to that reported previously (43). However, when retGC in these membranes was activated by S100B, the AMP-PNP preincubation only negligibly increased the retGC activity (Fig. 4B). In the experiments, the AMP-PNP preincubation was carried out with high or low Ca2+ concentrations (100 µM CaCl2 or2mM EGTA), indicating that the high activation does not occur regardless of Ca2+ concentrations in the preincubation. Together, observations shown here suggest that the large stimulation of retGC in OS homogenates preincubated with AMP-PNP occurs only when retGC is stimulated by GCAPs.
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Endogenous GCAPs Remain Bound to retGC/Membranes during Washing after the AMP-PNP Preincubation—As described above, the AMP-PNP preincubation is critical for the subsequent large activation of retGC by GCAPs. Thus, we have closely studied retGC activities with various preincubation conditions. We found that the retGC activity in OS homogenates preincubated with adenine nucleotides (1 mM) was approximately two times those preincubated without adenine nucleotides (Fig. 5A). The high activities were detected regardless of Ca2+ concentrations in the preincubation, although preincubation with a low Ca2+ concentration (1 mM EGTA) showed slightly higher retGC activities than those with a high Ca2+ concentration. We also found that these activities were sensitive to high Ca2+ concentrations (data not shown). These results indicate that endogenous GCAPs remain bound to retGC/membranes during washing and function as retGC activators in the adenine nucleotide preincubation. We note that these results are similar to those observed in the preincubation with 5 mM AMP-PNP (Fig. 2), suggesting that similar protein-protein interactions occur in the preincubation. Experiments using a GCAP2-specific antibody (Fig. 5B) support this notion. We detected GCAP2 in membranes after washing only when OS homogenates were preincubated with adenine nucleotides. The result apparently indicates that much of endogenous GCAP2 was associated with retGC/membranes. The reduction of retGC activity by high Ca2+ concentrations (Fig. 5A) may be due to the weaker binding of GCAP2 to membranes (and/or to retGC) in the presence of high Ca2+ (60).
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To further test this notion, experiments were carried out using GCAP-free membranes reconstituted with exogenous GCAP2 in the presence or absence of adenine nucleotides (Fig. 5C). We found that only membranes pretreated with adenine nucleotides in combination with GCAP2 had higher retGC activities. A previous study showing that ATP (0.4 mM) increased the efficacy of GCAP1 to stimulate retGC-1 (43) suggests that GCAP1 also shows the similar interaction with retGC in the presence of adenine nucleotides. The simplest explanation of these phenomena is that GCAP-binding sites directly connected with retGC activation in membranes were increased by the incubation with adenine nucleotide and more endogenous GCAPs bound to these sites. In the preincubation, washing conditions used were not enough to exclude all GCAPs bound. Another explanation may be that after the incubation with adenine nucleotides, endogenous GCAPs became tightly associated with retGC/membranes, remained bound to the membranes during washing, and functioned as a retGC activator. However, the possibility of this explanation may not be high because the retGC affinity to GCAP2 was not changed by the preincubation with or without adenine nucleotides (Fig. 2C).
RetGC Is Present as a Monomer during the Preincubation with Adenine
Nucleotides—In our previous study, we suggested using a
cross-linker that dimerization of retGC is essential for the activation of
retGC by GCAPs (25). Here,
using the same cross-linking procedure, we investigated whether the retGC
dimers are formed by endogenous GCAPs during the preincubation. Because the
preincubation, with high or low Ca2+ concentrations,
enhances the GCAP-activation of retGC (Figs.
1 and
2A), we examine
conditions with high or low Ca2+ concentrations. In the
presence of low Ca2+ concentrations (2 mM
EGTA), relatively high activity of retGC was detected because of the
stimulation of retGC by endogenous GCAPs
(Fig. 6A). However,
the activity was inhibited by AMP-PNP in a concentration-dependent manner, and
5 mM AMP-PNP, the AMP-PNP concentration used to preincubate OS
homogenates, suppressed 85% of the activity. The inhibition was similar
in the presence of low (5 mM) or high (20 mM)
MgCl2, indicating that the inhibition is not due to the shortage of
Mg2+. We also found that high concentrations of adenine
nucleotides inhibited the formation of a 210-kDa retGC complex
(Fig. 6B). The retGC
complex has been shown to be a homodimer of retGC
(25). The inhibition of the
retGC complex formation appears to be parallel to the reduction of retGC
activity, strongly suggesting that the inhibition of retGC dimerization is
involved in the retGC inhibition by adenine nucleotides, although we do not
have any data to exclude the possibility that the adenine nucleotide
inhibition is due to binding of adenine nucleotides at the catalytic site. In
the presence of high Ca2+ concentrations, retGC in OS
homogenates preincubated with AMP-PNP was inactive
(Fig. 3A), and retGC
exists as a monomeric form even in the absence of AMP-PNP
(25). Together, these
observations indicate that retGC exists as a monomer during preincubation of
OS homogenates with adenine nucleotides regardless of
Ca2+ concentrations.
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Low AMP-PNP concentrations slightly enhanced the retGC activity in OS homogenates (Fig. 6A). This is consistent with data reported previously (38–41). Is the enhancement due to the stimulation of retGC dimerization by low adenine nucleotide concentrations? The result (Fig. 6B) suggests that the level of retGC dimerization appears not to be affected by low adenine nucleotide concentrations. We did the same experiment five times; however, the results were not consistent. The lack of reproducibility suggests that clear evidence for the effect of low adenine nucleotide concentrations on retGC dimerization may be difficult to obtain, in part because the effect is small. In addition, concentrations of adenine nucleotides used were less than the physiological concentration of ATP (3–4 mM). Moreover, it is doubtful that the ATP concentration in ROS becomes so low under normal conditions, because the average ATP concentration is not changed by light, as reported previously (61). Thus, the significance of the activation of retGC by low concentrations of adenine nucleotides is unclear.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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13-fold over the control activity,
and the level of stimulated retGC activity is similar or higher than that of
light-activated PDE (34,
35,
49). Our observations are in
good agreement with those in vivo studies. We feel that the large
stimulation of retGC reported here represents a major portion of the mechanism
involved in the retGC activation detected in vivo. In the previous model, the retGC activation by GCAPs is a one-step mechanism: activation by GCAPs when the Ca2+ concentration in OS is reduced. Low concentrations (less than 0.5 mM) of adenine nucleotides in the assay mixture were shown to stimulate retGC activity; however, the activation was generally small in OS membranes (less than 1.5 times) (Refs. 21 and 38–43 and Fig. 6A). Thus, adenine nucleotides were believed to modify the activity but not to be essential. Most aspects, if not all, of retGC including its physiological roles, its regulations, and its function-structure relationships appear to be interpreted based on the one-step mechanism. Here, based on the results described, we propose a two-step mechanism for the retGC activation: retGC first interacts with ATP to produce a conformational change and is then highly activated by GCAPs when the Ca2+ concentration is reduced. As described above, we estimated that less than 20 µM AMP-PNP were carried over from the preincubation mixture to the retGC preparation. This indicates that less than 1 µM AMP-PNP presented in the assay mixture because 10 µl of the retGC preparation was added to assay mixture (200 µl). This AMP-PNP concentration was too small to activate retGC in the assay mixture (Fig. 6A). Under the conditions, GCAPs stimulated retGC (Fig. 2). Thus, in this mechanism, the step for the ATP interaction and the step for the GCAP activation are separate and distinct, and both steps are essential for the large stimulation. This adenine nucleotide interaction requires concentrations in the millimolar range (EC50 = 0.65 mM for AMP-PNP) along with other factor(s) (discussed below) and is indifferent to the calcium concentration. On the other hand, the adenine nucleotide stimulation in the assay mixtures requires adenine nucleotide concentrations below the millimolar range because higher concentrations inhibit retGC activity either directly or through inhibition of dimerization. In addition, the stimulation in the assay mixtures requires low calcium concentrations for the activation by GCAPs.
Is the mechanism for retGC activation by the adenine nucleotide preincubation different from the activation by low concentrations of adenine nucleotides in the assay mixture? The retGC activity in AMP-PNP-preincubated OS membranes was further activated by the low concentrations of AMP-PNP in the assay mixture.2 This implies that the AMP-PNP in the assay mixture further activates retGC already stimulated by the AMP-PNP preincubation and that the AMP-PNP activation in the assay mixture does not require the unknown factor(s) (discussed below) required for the activation by AMP-PNP preincubation. These implications are suggestive of the mechanistic difference but not conclusive. On the other hand, in some studies retGC activities similar to those stimulated by AMP-PNP preincubation were reported without the adenine nucleotide preincubation (58, 60). These activities were measured with low concentrations (<0.5 mM) of ATP in the assay mixture. Although these high retGC activities were not constantly observed even in the same research groups and the ATP contribution to the high activities is unknown, these activities may suggest that under certain conditions a mechanism similar to that for the activation by adenine nucleotide preincubation may function in the assay mixture.
Using AMP-PNP, we suggest that binding of ATP to retGC produces the large activation by facilitating subsequent interaction with GCAPs. However, it should be emphasized that binding of ATP to retGC is speculative based on the observation that preincubation of OS homogenates with AMP-PNP was required for high activation of retGC. However, this speculation is also supported by previous studies as follows. (a) RetGC has a molecular configuration similar to those of peptide-regulated GCs (17, 18, 37), implying that the regulatory mechanism of retGC may be similar to those of peptide-regulated GCs. In peptide-regulated GCs, ATP is an important factor for their regulation (17, 18, 37). It has been hypothesized in these GCs that ATP binds to the KHD, serves as an intracellular allosteric regulator, and releases an inhibitory constraint of the KHD for the activation. (b) A previous study already suggested that AMP-PNP binds to retGC (58). Although the study did not directly show the AMP-PNP binding to retGC, the AMP-PNP binding appears to be a reasonable explanation. c) As discussed above, activation of retGC by low concentrations of ATP and its analogs has been reported in previous studies (38–41), although the concentration of adenine nucleotides is different from those in this study. This stimulation has been speculated to be due to the binding of ATP to the KHD in retGC.
To test the possibility that adenine nucleotides function as allosteric regulators by binding to the KHD, we used three-dimensional modeling. The availability of crystal structures for the inactive and active conformations of the insulin receptor kinase domain (62) allowed construction of models for the KHD of retGC with and without bound AMP-PNP. For this purpose, an alignment of bovine retGC-1 with the ATP binding sites of the insulin receptor and other kinases was carried out to determine the boundaries of the KHD within the retGC-1 sequence. Fig. 7 displays the alignment that indicates sequence similarity allowing three-dimensional modeling. Also indicated is the lack of the GXGXXG motif (the boxed region 552–557) necessary for kinases in the retGC-1 sequence, a feature previously noted (37, 63).
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In the absence of AMP-PNP binding, the KHD was modeled in a conformation corresponding to the inactive insulin receptor kinase domain. Fig. 8A shows the model generated for the AMP-PNP-free form of retGC-1 with a loop shown in orange that corresponds to the activation loop (the boxed region 690–710 of Fig. 7) of the insulin receptor. In this conformation, the putative activation loop occupies the potential adenine nucleotide-binding site. We propose that in this conformational state, GCAPs may not effectively interact with retGC to stimulate activity. Upon binding of AMP-PNP, the KHD of retGC can be modeled in a conformation corresponding to the activated insulin receptor kinase (Fig. 8B). The putative activation loop of retGC (shown here in cyan) is now exposed along with a groove corresponding to the peptide-binding site of the insulin receptor kinase, allowing new intra- and inter-protein interactions such as with GCAPs. Fig. 8C shows a stereo view of the superposition of the two conformations. Modeling of inactive and active conformations of retGC-2 produced similar results (data not shown).
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However, it should be emphasized that this study does not exclude the
possibility that protein phosphorylation also plays a role in the large
activation of retGC. The preincubation of OS homogenates with adenine
nucleotides was initially carried out under the conditions for protein
phosphorylation. Comparison of specific activities of retGC shown in Figs.
1 and
2 shows that ATP is
approximately twice as effective as AMP-PNP for the retGC stimulation under
our conditions. Moreover, phosphatase inhibitors (10 nM okadaic
acid and 10 mM NaF) further increased the retGC
activity.2 These results suggest that phosphorylation of retGC
and/or proteins involved in the retGC regulation may further enhance the
GCAP-dependent retGC activity that has already been stimulated by binding of
adenine nucleotides. For retGC, we have already detected incorporation of the
-phosphate of ATP into the protein under the conditions used for
preincubation.3
Previous studies also reported that retGC might be regulated by
phosphorylation
(64–66).
Moreover, it has been reported that phosphorylation, in addition to ATP
binding, is important for the activation of GC-A
(18,
67). For proteins involved in
the retGC regulation, GCAPs are the established candidates, although
phosphorylation of an unknown protein regulator(s) (discussed below) may also
be possible. It has been reported that GCAP2 is phosphorylated by cyclic
nucleotide-dependent protein kinases, but the phosphorylation had little
effect on retGC activation by GCAP2
(68). The study appears to
have been conducted under conditions for the single-step mechanism. It may not
be surprising to detect that GCAP phosphorylation is involved in the retGC
regulation under the two-step mechanism reported here. We also note that the
GCAP2-stimulated activity of retGC preincubated in the presence of high
Ca2+ concentrations was slightly but consistently higher
than that in the presence of low Ca2+ concentrations
when AMP-PNP was added to the preincubation mixture
(Fig. 2A). It is
possible that the difference found in the AMP-PNP preincubation may be due to
protein dephosphorylation because phosphatase inhibitors were not in the
preincubation mixture. This Ca2+ effect was detected
more clearly when ATP was used (37 °C)
(Fig. 1). These observations
imply that if protein phosphorylation is involved in the large activation, the
phosphorylation level may be Ca2+-regulated.
Because this study is the first report for the two-step mechanism, new questions have arisen that will require answers. We found that addition of GCAP2 to prewashed membranes followed by the incubation with AMP-PNP was not enough to obtain the large activation.2 These observations suggest that the simple binding of AMP-PNP to retGC with GCAP2 was not enough for the high activation. The possibility that AMP-PNP cannot bind to retGC in washed membranes may be excluded by a previous study suggesting that AMP-PNP appears to change the retGC structure in washed membranes (58). The simplest explanation to our observations is that a factor(s) in OS homogenates may be required in the AMP-PNP preincubation.
Another puzzling point is that the large activation was detected even after washing out of soluble components from OS homogenates. This indicates that the effect of AMP-PNP is retained on retGC after the washing, although EC50 of AMP-PNP for the large activation is relatively high (0.64 mM). The mechanism to retain the effect of AMP-PNP on retGC is unclear. We speculate that after interaction with AMP-PNP, retGC changes its conformation, and the conformational change prevents AMP-PNP from being released from retGC. Alternatively, AMP-PNP may be released from retGC during the washing, but the conformational change in retGC is retained by a factor(s) in OS homogenates. Although there may be other explanations, it is crucial to elucidate all of the components in OS homogenates involved for understanding of the large activation of retGC.
We have shown that endogenous GCAP2 is associated with retGC/membranes after preincubation of OS homogenates with adenine nucleotides (Fig. 5) and that the large activation of the retGC is accomplished by addition of exogenous GCAP2 (Fig. 2). How many GCAP-binding sites are on the adenine nucleotide-treated retGC? As a model for one binding site, we presume two retGC conformations in equilibrium: an inactive form without enzyme activity and possessing no ability to be stimulated by GCAP2 and an active form with enzyme activity that is dependent on GCAP2. Thus, the observed activity is dependent upon the proportion of the active form and the content of GCAP2. Without adenine nucleotide treatment, only a small fraction of retGC exists as the active form. The small increase of the retGC activity by exogenous GCAP2 would be due to that portion of the active form whose GCAP2 sites are not already filled by endogenous GCAP2, i.e. exogenous GCAP2 binds to the residual GCAP-free active form to express retGC activity. However, because the fraction of total retGC present as active form is small, the observed retGC activity is not large (Fig. 2). The requirement for exogenous GCAP2 may be due to low content of GCAP2 compared with retGC in the preparations used and/or the loss by washing. This explanation can also be applied to the basal activity and GCAP-dependent activation of retGC in previous studies. Treatment with adenine nucleotides shifts the equilibrium from the inactive form toward the active form. The large activation by adenine nucleotide preincubation (Fig. 2) is completed by binding of exogenous GCAP2 to the resulting larger fraction of retGC in the active form. This model is supported by the observations that the affinity of retGC for GCAP2 is not changed by preincubation with or without AMP-PNP (Fig. 2C) and that the level of retGC activity observed with saturating amounts of GCAP2 is determined by the concentration of AMP-PNP in the preincubation (Fig. 2D).
Other explanations including multiple GCAP2-binding sites are also possible. The GCAP2 added exogenously may bind to a retGC domain(s) different from the domain with which endogenous GCAP2 is associated. It is possible that the new conformation of retGC, a product of adenine nucleotide binding to retGC, opens a new binding site(s) for GCAP2. We believe that GCAP1 also associates with retGC under the same conditions, as described above. If so, which GCAP combinations can more effectively activate retGC? This question may lead not only to an explanation for the presence of two GCAPs, 1 and 2, in ROS (30, 31) but also to a new mechanism of retGC regulation.
It should be emphasized that a new preincubation of OS homogenates with adenine nucleotides is required for the large activation of retGC, although the adenine nucleotide effect is retained after washing of membranes. This implies that the adenine nucleotide effect is initiated and then terminated under certain conditions, i.e. there are mechanisms to turn on and turn off the ATP effect in OS. In particular, the mechanism to turn off the highly activated retGC may be as important as turning off light/GTP-activated PDE in the overall control of cGMP metabolism in OS during normal function and adaptation. Several mechanisms for the inhibition of retGC have been reported (69, 70), although these inhibitions cannot be easily incorporated in the current model of retGC regulation: the single-step mechanism. Under the new mechanism of retGC regulation reported here, these retGC inhibitory mechanisms may be incorporated more easily. We especially emphasize that retGC inhibition by RGS9 (70, 71) is attractive because under the large activation of retGC, the mutual regulation between the retGC system and the PDE system is more important, and RGS9 has been proposed to function in both the PDE and retGC systems. Obviously, further studies are needed to establish the mechanism suggested here and to answer puzzling points. Moreover, it is important to reveal mechanisms to overcome the retGC inhibition by physiological ATP concentrations in the system proposed here. It is clear, however, that this study opens a new field of retGC regulation in photoreceptor OS.
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FOOTNOTES |
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|| To whom correspondence should be addressed. Tel.: 313-577-2009; Fax: 313-577-0238; E-mail: ayamazak{at}med.wayne.edu.
1 The abbreviations used are: PDE, cGMP phosphodiesterase; retGC, retinal
guanylyl cyclase; GC, membrane-bound guanylyl cyclase; KHD, kinase homology
domain in GCs; OS, outer segments of retinal photoreceptors; ROS, rod outer
segments; GCAPs, retGC-activating proteins; AMP-PNP, adenylyl
imidodiphosphate; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol;
HPLC, high pressure liquid chromatography.
2 A. Yamazaki, unpublished observations.
3 H. Yu and A. Yamazaki, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) or without (
) 1 mM ATP (37 °C, 30 min).
The preincubation was also carried out in the presence of high (•) or low
(
). GCAP-free membranes
were not preincubated. The enzyme activities shown are the averages of two
experiments.
, 1 mM; 
, 2 mM; and 
,
5mM; and •, 10mM. D, AMP-PNP
concentrations in the preincubation of OS homogenates. The data shown in
C were replotted to determine the GCAP2 concentration required for
the large activation of retGC. The following are the GCAP2 concentrations
used: 
strand; cylindrical symbols
indicate 