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INTRODUCTION |
Rod and cone photoreceptor cells respond to light by closure of
cGMP-gated ion channels in the plasma membrane of the outer segment.
Light triggers an amplifying cascade of enzymatic reactions leading to
the hydrolysis of the intracellular messenger cGMP (1, 2). A further
consequence of illumination is the decrease in the cytoplasmic
[Ca2+]. Changes in [Ca2+] are sensed by
Ca2+-binding proteins that control the activity of several
proteins in the enzyme cascade (3, 4). Recovery from the light response requires the termination of all activating steps in the cascade and the
resynthesis of cGMP. Synthesis of cGMP is catalyzed by a membrane-bound
retina-specific guanylyl cyclase in rod outer segments
(ROS-GC).1 Two isoforms of
this enzyme are expressed in photoreceptor cells (5-11). They are
members of the family of receptor guanylyl cyclases (12, 13). Their
mechanism of activation is different from other family members:
Ca2+-binding proteins act on an intracellular domain of
ROS-GC (14-16, 17), whereas other membrane guanylyl cyclases are
activated by extracellular peptide hormones (12, 13). The activity of the ROS-GC forms is regulated by a photoreceptor-specific
Ca2+-binding protein named guanylyl cyclase-activating
protein (GCAP) that is also expressed in two isoforms (GCAP-1 and
GCAP-2) (18-22). These proteins activate ROS-GC to restore the
depleted cGMP pool after illumination. In the dark, when the
[Ca2+ ] is high (500 nM), ROS-GC activity is
low, and it is stimulated at least 10-fold during the light response,
when the [Ca2+] drops to
100 nM. Both GCAP
forms are heterogeneously acylated at the N terminus by C14:0, C14:1,
C14:2, and C12:0 fatty acids. Although the N-terminal fatty acyl group
may be important for regulation of ROS-GC, it is probably not essential
for membrane binding (18, 20, 21, 23, 24). Several experiments have indicated that GCAP-1 is associated with rod outer segment membranes or
ROS-GC independent of [Ca2+] (16, 24).
Ca2+-loaded GCAP-1 competes with a constitutively active
GCAP-1 mutant for binding to ROS-GC (25), which also led to the
conclusion that GCAP-1 forms a stable complex with ROS-GC independent
of [Ca2+]. In contrast, GCAP-2 does show a
Ca2+ sensitivity of its membrane association; it binds more
efficiently to membranes at [Ca2+] < 200 nM
(23).
Knowledge about the precise interaction domains in GCAP-1 and GCAP-2 is
incomplete or nonexistent. So far, peptide competition studies have
indicated that the N-terminal 20 amino acids of GCAP-1 are involved in
regulation of ROS-GC (18, 24). In addition, two peptides
(Glu57-Lys86 and
Asp37-Asp163 in GCAP-1) including the first
and third functional Ca2+-binding motifs (EF-hands)
inhibited stimulation of ROS-GC (24). A 25-amino acid truncation at the
N terminus of GCAP-1 abolished the ability to increase ROS-GC activity
almost completely (24).
It was the aim of our project to identify the domain(s) in GCAP-1 that
is directly involved in the interaction with ROS-GC. We synthesized an
oligopeptide library of overlapping peptides spanning the entire amino
acid sequence of GCAP-1. Peptides were used in competitive screening
assays and column affinity binding studies. By this approach, we found
that ROS-GC and tubulin bind as a complex to a 15-amino acid peptide of
GCAP-1. Coimmunoprecipitation studies demonstrated the presence of an
interacting complex of ROS-GC, GCAP-1, and tubulin independent of
[Ca2+].
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MATERIALS AND METHODS |
Synthesis of an Oligopeptide Library--
Peptides spanning the
whole amino acid sequence of GCAP-1 were synthesized using a Multipin
peptide synthesis kit (Chiron Mimotopes) according to the
manufacturer's recommendations. Each peptide was 12 amino acids long
and overlapped with the preceding one by 10 amino acids. All peptides
were obtained in their amide form at the carboxyl terminus.
Fmoc-protected amino acids were attached via carbodiimide chemistry.
After 12 cycles of coupling, the N terminus of each peptide was
acetylated by incubating with 3% acetanhydride and 0.5%
N-ethyldiisopropylamine in dimethylformamide for 100 min.
For synthesis of the myristoylated form of peptide 1, the acetylation
was omitted. Instead, myristic acid was coupled to the peptide's free
N terminus using the same chemistry as for coupling Fmoc-protected
amino acids. Deprotection, cleavage, precipitation, and washing of
peptides were done as recommended by the manufacturer. Lyophilized
peptides were analyzed by high performance liquid chromatography.
Peptide 37/38 and the GCAP-2 and the recoverin peptides were
synthesized in larger amounts as described (26).
Peptide Affinity Chromatography--
Peptides were coupled to
activated thiol-Sepharose 4B (Amersham Pharmacia Biotech) via a thiol
group according to the manufacturer's description. The
peptide-Sepharose was equilibrated with 1 mM dodecyl
maltoside, 50 mM KCl, 20 mM Hepes-KOH (pH 7.4)
and incubated with an extract of membrane and/or cytoskeletal proteins
from rod outer segments (ROS). ROS and the extract were obtained as described (5, 20). Briefly, ROS (9-13 mg/ml rhodopsin) were diluted
30-fold with a low salt buffer (10 mM Hepes-KOH (pH 7.4), 0.1 mM EGTA, 0.1 mM phenylmethylsulfonyl
fluoride, and 1 mM DTT) and centrifuged at 30,000 × g for 30 min. Washed ROS membranes were homogenized and
solubilized in 5% Triton X-100, 4 mM MgCl2, 10 mM Hepes-KOH (pH 7.4), and 1 mM DTT. Subsequent
centrifugation removed the majority of membrane proteins, including
rhodopsin and the cGMP-gated channel (present in the Triton X-100
supernatant). The pellet was solubilized with 20 mM dodecyl
maltoside, 20 mM Hepes (pH 7.4), 1 M KCl, and 1 mM DTT and centrifuged. This extract (dodecyl maltoside
extract) was enriched in ROS-GC and contained tubulin, actin, and a few
other unidentified proteins in minor quantities. Before application to
the column, the extract was adjusted to 1 mM dodecyl
maltoside, 50 mM KCl, and 20 mM Hepes (pH 7.4).
The extract was incubated with the Sepharose at 4 °C for at least
2 h. The unbound fraction was collected, and the Sepharose was
washed with 1 mM dodecyl maltoside, 20 mM
Hepes-KOH (pH 7.4), and 0.5 M NaCl (10-fold the volume of
the column) and subsequently with the same buffer containing 1 M NaCl. Salt was removed by washing with 20 mM
Hepes (pH 7.4). Bound proteins were eluted with 0.5% SDS and 20 mM Hepes-KOH (pH 7.4). The Triton X-100 extract (5) was
also used to test binding of tubulin to peptide columns in the absence
of ROS-GC.
Guanylyl Cyclase Assay--
The activity of ROS-GC was
determined as described previously (5, 20). In all experiments, the
incubation volume was 50 µl. Added peptides were dissolved in 0.5%
Me2SO and 0.05% Tween 20, which caused little interference
with the guanylyl cyclase activity. Controls were incubated under
identical buffer conditions. Free Ca2+ was adjusted by
Ca2+/EGTA buffers as described (20).
Expression and Purification of GCAP-1--
The coding sequence
of bovine GCAP-1 was amplified from a full-length cDNA clone (20)
via polymerase chain reaction using the following primers:
5'-sense primer, CAGGGATCCACCATGGGGAACATTATGGAC; and 3'-antisense
primer, AGGGAATTCTATCAGCCGTCGGCCTCCGCG. The product obtained
after 15 cycles of polymerase chain reaction was subcloned into
pBluescript SK vector via the introduced BamHI and
EcoRI sites. One of the resulting clones was sequenced to
exclude that errors had been introduced by the polymerase chain reaction.
The GCAP-1/pBluescript plasmid was linearized by digestion with
NcoI, and the ends were filled in with Klenow polymerase. The GCAP-1 coding sequence was then released by digestion with EcoRI and inserted between the NdeI and
EcoRI restriction sites of the pET21a(+) expression vector (Novagene).
GCAP-1 protein was expressed from GCAP-1/pET21a(+) in an
Escherichia coli BL21(DE3) strain carrying the plasmid
pBB131 (a kind gift of Dr. J. Gordon) encoding for yeast
N-myristoyltransferase. Cells were grown at 37 °C to an
A600 of ~0.5 in LB medium containing 100 µg/ml ampicillin and 30 µg/ml kanamycin and then supplemented with
50 µg/ml myristic acid. Thirty minutes after addition of myristic
acid, the expression of GCAP-1 was induced by addition of
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 0.5 mM. Expression was allowed to proceed
for 4 h. Cells from 1 liter of expression culture were collected
by centrifugation, resuspended in 250 ml of 50 mM Tris-HCl
(pH 8), and stored frozen at
20 °C until further use. The thawed
cell suspension was incubated at 30 °C for 30 min in the presence of
0.1% Tween 20, 100 µg/ml lysozyme (SERVA), and 10 units/ml DNase I
(TaKaRa). Lysis was terminated by addition of 1 mM DTT and
0.1 mM phenylmethylsulfonyl fluoride. The insoluble
fraction was collected by centrifugation at 370,000 × g for 20 min and solubilized by stirring in 250 ml of buffer
containing 6 M guanidin chloride, 0.1 mM
phenylmethylsulfonyl fluoride, and 2.5 mM DTT for 1 h
at room temperature. The resulting suspension was filtered and dialyzed
at 4 °C against three changes of 10 mM sodium phosphate
buffer (pH 7) containing 1 mM EDTA and 1 mM
DTT, followed by two changes of 10 mM sodium phosphate
buffer containing 200 µM CaCl2 and 1 mM DTT. Precipitated material was removed by centrifugation
at 370,000 × g for 20 min. The supernatant was
concentrated using Centriplus devices (Amicon, Inc.) with a molecular
mass cutoff of 10 kDa. The concentrate was applied to a Superdex 75 XK16/60 gel filtration column (Amersham Pharmacia Biotech) in 10 mM sodium phosphate (pH 7), 100 mM NaCl, and
200 µM CaCl2. Fractions containing
recombinant GCAP-1 were pooled and bound to ceramic hydroxylapatite
type I (Bio-Rad) in an HR5/5 FPLC column (Amersham Pharmacia Biotech).
Elution was performed by a 0-100% gradient of 300 mM
sodium phosphate (pH 7) in 10 mM sodium phosphate (pH 7)
and 200 µM CaCl2. Recombinant GCAP-1 was obtained in >90% purity and stored at
80 °C.
Immunoprecipitation--
Protein A-Sepharose was equilibrated
with 100 mM Tris (pH 8.0). Polyclonal anti-GCAP-1 serum
from immunized rabbit (20) was adjusted to 100 mM Tris (pH
8.0) and incubated with protein A-Sepharose for 1 h at room
temperature. Afterward, the Sepharose was washed with 40 ml of 100 mM Tris (pH 8.0) and with 40 ml of 10 mM Tris
(pH 8.0) and equilibrated with 0.2 M sodium borate buffer
(pH 9.0). The Sepharose beads were resuspended in 40 ml of sodium
borate, and 1.1 g of dimethyl pimelimidate was added to the
suspension. After incubation for 30 min at room temperature, the
reaction was stopped with 0.2 M ethanolamine.
Coimmunoprecipitation of GCAP-1 and ROS-GC was done as follows. Soluble
ROS proteins were separated from the membranes by centrifugation of a
5-fold diluted suspension of ROS in 10 mM Tris (pH 7.5)
(45,000 rpm for 15 min; Beckman TLA-45). The supernatant was
concentrated with a Centricon device (cutoff = 10 kDa), adjusted
to 150 mM KCl, and incubated with the anti-GCAP
antibody-Sepharose equilibrated in the same buffer (200 µl of
supernatant and 100 µl of Sepharose). The unbound fraction was
separated from the beads by a quick spin using 0.45 micropure
separators (Amicon, Inc.). Pelleted ROS membranes (see above) were
resuspended in 10 mM Tris (pH 7.5); solubilized in 500 µl
of buffer containing 2% Triton X-100, 0.5 M KCl, and 20 mM Tris (pH 7.4); and centrifuged for 15 min at 45,000 rpm. The supernatant was incubated with the anti-GCAP antibody-Sepharose beads. Samples were run in parallel with either 1 mM
CaCl2 or 1 mM EGTA present, including all
subsequent washing steps. Samples were incubated for 10 min at room
temperature, for 30 min at 4 °C, and for 10 min at room temperature
and then spun. The filtrates were collected (unbound fractions), and
the beads were washed with 0.2% Triton X-100, 0.15 M KCl,
and 20 mM Tris (pH 7.5). These washing cycles were done
five times and were followed by a washing step with 10 mM
Tris (pH 7.5). Bound proteins were eluted first with 100 mM
glycine (pH 2.5) and second with 2% SDS, 5 mM EDTA, and 20 mM Tris (pH 7.5).
SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
SDS-polyacrylamide gel electrophoresis and Western
blotting were performed as described (20). The polyclonal anti-ROS-GC antibody (27) was diluted 1:5000; the polyclonal anti-GCAP-1 antibody
(20) was diluted 1:10,000; and the monoclonal anti-
-tubulin antibody
(Sigma) was diluted 1:50,000. The secondary horseradish peroxidase-coupled antibodies were diluted 1:5000. Immunoreactive bands
were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).
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RESULTS |
Identification of an Interaction Domain in GCAP-1--
Ninety-six
peptides encompassing the entire amino acid sequence of GCAP-1 were
tested at a concentration of 0.1 mM for their efficiency to
inhibit the ROS-GC activity in the presence of GCAP and
10
9 M Ca2+. Most peptides caused
a weak inhibition from 0 to 10% (Fig.
1). A few peptides even showed a small
stimulatory effect (see, for example, peptides 8-10). The most
effective peptides were peptides 37 and 38 (74MEYVAALSLVLKGK87); they caused 30-40%
inhibition (Fig. 1). Peptides of intermediate efficiency (20-30%)
were peptides 1 (amino acids 2-15), 15 and 18 (amino acids 30-47),
and 44 and 45 (amino acids 88-102). Peptides that displayed inhibition
20% were further examined at increasing concentrations (0.1-0.3
mM). The results for peptides 1, 38, and 44 are shown in
Fig. 2A (peptide 36 was chosen
as a control). The normalized ROS-GC activity was lowered to 30% with
0.3 mM peptide 38. Less strong, but still significant were
the inhibitory effects of peptides 1 and 44 (60-70% normalized ROS-GC
activity) and peptide 37 (60-70% normalized ROS-GC activity) (data
not shown). Peptides 15, 18, and 45 were of similar efficiency as
peptide 44 (data not shown). A small effect was seen with peptide 36 (Fig. 2A). Because native GCAP-1 contains a myristoyl group
at the N terminus, we synthesized a myristoylated and a
nonmyristoylated form of the N-terminal peptide. The nonmyristoylated
peptide did not change the ROS-GC activity (data not shown), whereas
the myristoylated peptide displayed the modest inhibitory effect shown
in Fig. 1 (peptide 1). Basal ROS-GC activity in washed ROS membranes
depleted of GCAP was not influenced by most peptides, with the
exception of peptides 45-47, which showed a weak inhibition of 20%
(data not shown). In conclusion, peptides 37 and 38 were the most
effective in interfering with ROS-GC activity. In further studies, we
used a peptide that encompassed the sequences of both peptides 37 and 38 (peptide 37/38) (Fig.
3A).

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Fig. 1.
Inhibition of GCAP-1-dependent
activation of ROS-GC at low [Ca2+] by peptides shown as
percentage of inhibition. Peptides at 0.1 mM were
added to suspensions of bovine ROS, and ROS-GC activity was measured at
low [Ca2+] (~1 nM). Data represent the
mean ± S.D. of two to four measurements.
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Fig. 2.
A, normalized ROS-GC activity at
different peptide concentrations. Activity was measured at 1 nM Ca2+ in whole ROS. ROS-GC activity without
peptide was set to 1. B, comparison of the inhibitory effect
of peptides 37/38 ( ) and 38 ( ) at increasing peptide
concentrations. The IC50 values were 0.07 and 0.14 mM for peptides 37/38 and 38, respectively. Curves were
fitted to a modified Hill equation. Data represent mean ± S.D.
(triplicates).
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Fig. 3.
A, domain structure of GCAP-1 in the
myristoylated (myr) form and localization of peptides 37/38
and 38. The nonfunctional EF-hand motif (EF1) is shown as
shaded box; the three functional EF-hand motifs
(EF2-EF4) are shown as black boxes.
B, sequence alignment of the identified interaction domain
in GCAP-1 (Phe73-Lys87) and the corresponding
regions in GCAP-2 (Phe78-Thr92) (22),
recoverin (Rec; Phe83-Lys97) (29),
and calmodulin (CaM; Phe65-Thr79
(31). Identical amino acids are shaded. C,
percentage inhibition of ROS-GC activity at low [Ca2+] by
peptides from GCAP-1, GCAP-2, and recoverin (sequences shown in
B) at 0.1 mM.
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Inhibition of ROS-GC was saturable at a concentration of 0.3 mM with peptide 37/38
(73FMEYVAALSLVLKGK87), but the maximal
inhibition did not exceed 80% (Fig. 2B). A shorter section
of this putative interaction domain represented by peptide 38 inhibited
ROS-GC to the same maximal extent. The IC50 value of 0.14 mM, however, was larger than the one observed for peptide
37/38 (0.07 mM). We conclude that the three additional amino acids (Phe73-Glu75) in peptide 37/38
determine the affinity of ROS-GC for GCAP-1. It is interesting that
flanking regions of this domain (represented by peptides 36 (Fig.
2A) and 39 (data not shown)) showed only a marginal effect.
Comparison of the GCAP-1 Peptide with Related Peptides from GCAP-2
and Recoverin--
A peptide from recoverin related to peptide 37/38
caused only 15% inhibition of ROS-GC at low [Ca2+],
whereas the related peptide from GCAP-2 showed 50% inhibition (GCAP-1
peptide 37/38 caused 75% inhibition (Fig. 3, B and
C); all peptides at 0.1 mM). These results
indicated that the GCAP-1 peptide (and the almost identical GCAP-2
peptide) acts specifically on ROS-GC and that the amino acid
differences between recoverin and GCAP peptides are critical for the
interaction with ROS-GC.
Inhibition by Peptides Is Reversible--
Recombinant GCAP-1 was
added to a suspension of ROS in which ROS-GC was inhibited by either
peptide 37/38 or 38. GCAP-1 at 12 µM could completely
relieve the inhibition by both peptides. Addition of GCAP-1 to a ROS
suspension without peptides did not change the maximal rate of ROS-GC
activity (Fig. 4). This experiment clearly showed that the observed inhibition is caused by a direct interference of the peptides with the GCAP-1/ROS-GC interaction, which
can be overcome by an additional amount of GCAP-1.

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Fig. 4.
Inhibition of ROS-GC activity by GCAP-1
peptides is reversible. ROS preparations were incubated at low
[Ca2+]. The sample contained endogenous GCAP-1;
additional GCAP-1 did not further increase maximal ROS-GC activity
(black and white control columns).
Peptides 38 and 37/38 at 0.1 mM suppressed the stimulation
of ROS-GC activity. Addition of GCAP-1 compensated the inhibitory
effects. The specific activity of ROS-GC is calculated in relation to
the amount of rhodopsin present in the incubation mixture. Data show
mean ± S.D.
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Influence of Peptides on the Ca2+ Regulation of
ROS-GC--
We next examined whether inhibitory peptides could change
the [Ca2+] at which the activation of ROS-GC was
half-maximal. The inhibitory effect of peptides 37/38 and 38 (0.1 mM peptide) was studied within the physiologically
important range of [Ca2+] (Fig.
5). Peptide 37/38 was more efficient than
peptide 38, consistent with the results in Fig. 2B. Peptides
at 0.1 mM did not significantly change the Ca2+
regulation of ROS-GC activity (Fig. 5, inset). The
activation of ROS-GC was half-maximal at 60 nM
Ca2+ and cooperative (nH = 1.6)
independent of peptide addition. The EC50 of 60 nM is at the lower range of reported values in the literature (50-280 nM) (12). ROS-GC activity at high
[Ca2+] was not influenced by these peptides. Basal ROS-GC
activity in ROS membranes depleted of GCAP was also not influenced at
high or low [Ca2+]. Therefore, peptides 37/38 and 38 interfere with the GCAP-1/ROS-GC interaction and not with basal ROS-GC
activity.

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Fig. 5.
Ca2+-dependent
regulation of ROS-GC in the presence of peptides 37/38 ( ) and 38 ( ) at 0.1 mM and without peptides
(control; ). The EC50 values are
70, 54, and 60 nM for peptides 38 and 37/38 and the
control, respectively. The specific activity of ROS-GC is calculated in
relation to the amount of rhodopsin present in the incubation mixture.
The inset shows the normalized ROS-GC activity for the
incubation with and without peptides. Maximal ROS-GC activity at low
[Ca2+] was set to 1. Data show mean ± S.D.
(triplicates).
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Binding of ROS-GC and Tubulin to a Peptide Affinity Column--
We
tested direct binding of ROS-GC to the most effective GCAP peptide
(peptide 37/38). Peptide 37/38 was covalently coupled to an activated
thiol-Sepharose matrix via an additional N-terminal cysteine and a
glycine spacer (37/38-Sepharose). The 37/38-Sepharose was incubated
with an extract containing ROS-GC and other ROS membrane and
cytoskeletal proteins. Two controls were used, Sepharose columns to
which either cysteine (Cys-Sepharose) or peptide 53 (53-Sepharose) was
coupled. GCAP-1 peptide 53 (102CIDRDELLTIIR113)
was chosen as a control because it did not show a significant effect in
our screening assay (Fig. 1) and could be coupled to the column matrix
via its terminal cysteine. No protein was retained by these columns
(Fig. 6A, bound,
C and 53 lanes), and the unbound fractions of the
Cys-Sepharose and 53-Sepharose were identical in their protein
composition when compared with the starting material (C and
53 lanes). Occasionally, some proteins bound nonspecifically to these columns (data not shown). Two major polypeptides of 112 and 55 kDa, respectively, were bound to the 37/38-Sepharose. They were
completely removed from the ROS extract (Fig. 6A,
nonbound, 37/38 lane) and could be eluted from the Sepharose
by 0.5% SDS (bound, 37/38 lane). Western blotting of the
unbound and bound fractions revealed that the band at 112 kDa is ROS-GC
(Fig. 6B), whereas the protein at 55 kDa was identified as
tubulin (Fig. 6D, DE, B lane). A few other
proteins appeared in both the bound and unbound fractions of the
37/38-Sepharose. Therefore, binding of these proteins to the
37/38-Sepharose was not specific, whereas it was for ROS-GC and
tubulin. Tubulin in a control extract without ROS-GC (Fig.
6C, TE) did not bind to the 37/38-Sepharose (Fig. 6D, TE, NB lane). Sometimes tubulin
was lost, probably due to aggregation when ROS-GC was not present.

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Fig. 6.
Peptide affinity column chromatography.
A dodecyl maltoside extract (DE) containing ROS-GC was
incubated with three kinds of Sepharose: Cys-Sepharose (C
lanes), 53-Sepharose (53 lanes), and 37/38-Sepharose
(37/38 lanes). Unbound and bound fractions were
electrophoresed, and the gel was stained with Coomassie Blue
(A) or blotted afterward for immunostaining (B)
using a polyclonal anti-ROS-GC antibody. C shows the
distribution of tubulin and ROS-GC in Triton X-100-solubilized ROS
membranes (TS lanes) and in Triton X-100 extracts (TE
lanes) tested by immunoblotting. D shows the results
from affinity chromatography of tubulin-containing extracts on the
37/38-Sepharose. Tubulin was found in the unbound (nonbound (NB
lanes)) fraction when no ROS-GC was present, and it was found in
the bound (B lanes) fraction when ROS-GC was present
(DE).
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Coimmunoprecipitation of GCAP-1, ROS-GC, and Tubulin--
The
results of the peptide affinity chromatography suggested that ROS-GC
and tubulin bind as a complex to GCAP-1. We tested this by
coimmunoprecipitation using a polyclonal GCAP-1 antibody (20). This
antibody, covalently coupled to protein A-Sepharose, specifically
immunoprecipitated GCAP-1. GCAP-1 was partially eluted from the beads
by glycine; the remaining GCAP-1 was eluted by SDS (data not shown).
Both ROS-GC and tubulin coimmunoprecipitated with GCAP-1 (Fig.
7, A and B,
SDS elution). Tubulin could be partially eluted by glycine,
whereas ROS-GC needed an SDS buffer for elution. The presence of either
1 mM CaCl2 or 1 mM EGTA did not
change the binding of the ROS-GC·tubulin complex to GCAP-1. Thus, the interaction of the ROS-GC·tubulin complex with GCAP-1 seemed to be
independent of Ca2+. A small amount of ROS-GC and tubulin
was not immunoprecipitated (Fig. 7, A and B,
nonbound); however, GCAP-1, ROS-GC, and tubulin were not
bound to protein A-Sepharose without immobilized GCAP-1 antibody
(c lanes). These results confirmed that ROS-GC and tubulin form a complex with GCAP-1.

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Fig. 7.
Coimmunoprecipitation experiments. A
cytoplasmic extract of ROS proteins was incubated with anti-GCAP
antibody beads to allow binding of GCAP-1 to the antibody. Solubilized
ROS membranes (RM lanes) were incubated with the beads
either with 1 mM CaCl2 (Ca lanes) or
with 1 mM EGTA (EGTA lanes). Bound fractions
were collected by elution with glycine or SDS. Unbound and bound
samples were analyzed by Western blotting. Blots were probed with
antibodies against ROS-GC (A) and tubulin (B).
Control incubations (c lanes) were performed with protein
A-Sepharose beads without attached antibodies.
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 |
DISCUSSION |
We have identified a 15-amino acid region located C-terminally
from the second EF-hand (Phe73-Lys87) in
GCAP-1 as the main interaction domain (Fig. 3) for ROS-GC. The peptide
interfered with the GCAP-1-dependent activation of ROS-GC
at low [Ca2+], which indicates that it critically
disturbs the Ca2+-dependent activation of
ROS-GC by GCAP-1. We cannot exclude that the peptides also compete with
the action of GCAP-2. However, based on other reports (21, 28) and our
previous observations (20), GCAP-1 is the major component of bovine ROS
homogenates. We compared the region with regions adjacent to the second
EF-hand in GCAP-2, recoverin, and calmodulin, three other
Ca2+-binding proteins involved in regulation of
phototransduction (21, 22, 29, 30). GCAP-1 and GCAP-2 are almost
identical in this region and, not surprisingly, showed the same
inhibition of ROS-GC activity (Fig. 3, B and C).
The related peptide from recoverin was much less effective. Sequence
identity between GCAP-1 and recoverin is only found at 8 out of 15 positions. Significant differences in recoverin compared with GCAP-1
are Lys84 (corresponding to Met74 in GCAP-1)
and the stretch of five amino acids at
His91-Ala95
(Ser81-Lys85 in GCAP-1), which is more
hydrophobic in GCAP-1. We conclude that the differences in amino acid
sequences in the compared regions are essential for different target
recognition of recoverin and GCAP-1.
Other regions that were less effective in the competition studies might
also participate in controlling ROS-GC activity. For example, a
myristoylated N-terminal peptide significantly decreased ROS-GC
activity, whereas a nonmyristoylated form failed to show an effect.
This result is in agreement with two previous studies. One study showed
that a myristoylated peptide from the N terminus decreased
GCAP-1-stimulated ROS-GC activity (18); in the other study, it was
reported that an N-terminally truncated mutant of GCAP-1 was unable to
stimulate ROS-GC (24). The myristoylated N terminus of GCAP-1 might be
involved in locating GCAP-1 close to the membrane in the right
orientation to interact with ROS-GC. Otto-Bruc et al. (24)
identified three larger regions in GCAP-1 as putative interaction
domains. These regions correspond to peptides 1-7, 27-38, and 68-75.
Although our screening assay narrowed down two of their regions to a
stretch of 15 amino acids (peptides 1 and 37/38), we did not observe
any significant inhibitory effect with peptides from the C-terminal
part (peptide 68-75 in Fig. 1). Incubation with a peptide mixture that
covered this entire region also showed no significant inhibitory effect
(data not shown). One explanation for these two different results is
that the interaction domain represented by this C-terminal region needs a correct folding, which might be present in the larger peptide of
Otto-Bruc et al. (24).
We also demonstrated direct binding of ROS-GC to peptide 37/38 by
affinity chromatography. ROS-GC bound to this peptide in a complex with
tubulin. The same complex formation was observed when ROS-GC was
coimmunoprecipitated with GCAP-1. These two independent experiments
strongly suggest that there exists a tight association of ROS-GC with
the cytoskeletal protein tubulin.
Association of ROS-GC with the outer segment cytoskeleton has been
noted by different investigators based on fractionation, solubilization, and overlay studies (5, 32, 33). So far, only actin has
been identified as one of the interacting components (33). Our finding
that tubulin and ROS-GC form a complex with GCAP-1 indicates that
Ca2+/cGMP signaling in photoreceptor cells occurs in close
proximity to cytoskeletal elements, but it is unclear whether
cytoskeletal proteins are directly involved in enzyme regulation.
The immunoprecipitation studies also indicate that GCAP-1 and ROS-GC
form a stable complex independent of the free [Ca2+].
This is consistent with previous cross-linking experiments in which
GCAP-1 was found to be in close proximity to ROS-GC at high and low
[Ca2+] (16). Competition experiments of
Ca2+-loaded GCAP-1 with a constitutively active GCAP-1
mutant also showed that Ca2+-bound GCAP binds to ROS-GC
(25). All these findings support a model in which a change in cytosolic
[Ca2+] would be detected by GCAP-1 while it is bound to
ROS-GC. Activation occurs by a conformational switch in GCAP-1 that is
transmitted to the cyclase. Such a regulatory mechanism would be
different from classical calmodulin-regulated systems, in which
calmodulin controls the activity of its targets by
Ca2+-dependent binding and dissociation (31),
but the mechanism would resemble the regulation of calmodulin
targets containing IQ motifs (34).