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INTRODUCTION |
RetGC1 and RetGC2 are membrane guanylyl cyclases that catalyze the
conversion of GTP into cyclic GMP (cGMP) in vertebrate rods and cones.
RetGCs are implicated in restoring cGMP levels following activation of
cGMP phosphodiesterase by light (1-5). The ability of RetGC to
catalyze cGMP synthesis is sensitive to Ca2+ but only in
the presence of guanylyl cyclase activator proteins (GCAPs)1 (6-8).
GCAPs were identified and purified as Ca2+-binding proteins
that impart Ca2+ sensitivity to RetGCs in vitro
(9, 10). Two isoforms, GCAP-1 and GCAP-2, have been found. Both
stimulate RetGC activity in homogenates of rod outer segments (ROS) at
low free Ca2+ concentrations (below 200 nM) and
inhibit it at high free Ca2+ concentrations (11, 12).
GCAP-1 and GCAP-2 share the following primary structural features: (i)
four EF-hand motifs in the core of the protein, three of which bind
Ca2+, (ii) an acylated NH2 terminus, and (iii)
a molecular mass of roughly 24 kDa. However, there are significant
differences between GCAP-1 and GCAP-2. They display little sequence
conservation in their NH2 and COOH termini. Also, a
naturally occurring point mutation (13) affects the two proteins
differently (14, 15).
The ability of GCAPs to regulate RetGC in a Ca2+-sensitive
manner is well established (7, 10, 11, 16). However, specific structures within GCAPs that are responsible for regulating RetGCs have
not yet been clearly defined. Peptide competition experiments have
suggested that three structures in GCAP-1 are involved in activation of
RetGCs. The first is between residues Gly2 and
Glu28. The second one is contiguous with the first; it runs
from Glu28 to Glu57 (9), and the third one is
the EF-hand 3 motif (17).
In order to more precisely define sites in GCAP-1 that interact with
RetGC, we constructed deletion mutants of GCAP-1 and chimeras of GCAP-1
with recoverin, a closely related Ca2+-binding protein that
does not regulate RetGC. Chimeras were used in cases where deletions
were not desirable. For instance, deletions from the NH2
terminus are especially likely to complicate folding. Moreover, mere
deletions would change the length of the peptide, thus introducing
another variable into the experiments. Chimeras with recoverin, on the
other hand, allowed us to conserve the total length of the constructs
and improve the chances of proper folding.
These assumptions were borne out by the fact that nearly all constructs
displayed one or more kinds of assayable activity: (i) ability to
stimulate RetGC in low Ca2+, (ii) ability to inhibit it in
high Ca2+, (iii) ability to block activation of RetGC by wt
GCAP-1 in low Ca2+, or (iv) ability to block activation by
a Ca2+-insensitive GCAP-1 mutant in high
Ca2+.
In the study described here we have addressed the following specific
questions. Is regulation of RetGCs by GCAP-1 mediated by a single
contiguous stretch of GCAP-1 sequence? Or, if multiple regions of the
sequence contribute to the interaction and regulation, what are they
and what are their roles in regulating RetGC?
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EXPERIMENTAL PROCEDURES |
DNA Constructs--
All mutants were derived from bovine GCAP-1
and recoverin cDNA clones (18). All chimeras were generated by the
polymerase chain reaction-based "splicing by overlap extension"
method (19). Cloned Pfu polymerase (Stratagene) was used in all
polymerase chain reactions. GCAP-1 truncations were generated by
introducing a stop codon into the reverse polymerase chain reaction
primers used to amplify the cDNA. It was found that the wt sequence
of GCAP-1 does not provide for complete myristoylation of the protein in our expression system even at saturating myristate concentrations in
the media and with overexpression of yeast NMT. The wt GCAP-1 sequence
does not have a Ser in the sixth position. This residue is part of the
myristoylation consensus (20). The GCAP-1 sequence was mutated to
encode Ser in the sixth position. This substitution alone provided for
complete myristoylation of GCAP-1 as confirmed by mass spectrometry.
The properties of this D6S GCAP-1 in regard to activating and
inhibiting the cyclase were found to be indistinguishable from those of
fully myristoylated wt GCAP-1. For the sake of brevity the D6S GCAP-1
is referred to as wt in the rest of the paper. A
Ca2+-insensitive GCAP-1 mutant was produced by the
following substitutions in EF-hands 2, 3, and 4: E75Q, E111Q, D144N.
The mutagenesis strategy is described in Ref. 11. The mutant protein
activated RetGC in high Ca2+ in our assay system. Some
constructs were confirmed by DNA sequencing. The masses of all of the
expressed proteins were confirmed to be correct by electrospray mass
spectrometry. All constructs were at least 90% myristoylated.
Expression of GCAP-1 Mutants--
The cDNA constructs were
ligated into pET11d or pET11a vectors (Novagen) using the
NcoI or NdeI sites respectively at the 5' and
BamHI site at the 3' end. The expression plasmids were transformed into Escherichia coli (BL21 DE3pLysE) that
harbored p88131 encoding yeast N-myristoyl transferase (NMT)
and kanamycin resistance. Expression was carried out essentially as
described in Ref. 11. 30 min prior to induction of expression with 1 mM isopropyl-1-thio-
-D-galactopyranoside
bacterial media were supplemented with free myristic acid. After
expression (2-5 h) cells were collected and sonicated on ice in lysis
buffer: 40 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 mM
-mercapthoethanol, 20 µg/ml leupeptin, and 100 µM phenylmethylsulfonyl fluoride. The resulting lysate
was centrifuged at 30,000 × g. Most of the expressed
protein was recovered in the pellet. The pellet was washed twice with
lysis buffer by resuspension and centrifugation at 30,000 × g. It was then dissolved in 6 M urea and
dialyzed 3 times against 1000 volumes of lysis buffer without protease
inhibitors. The renatured fraction typically contained 50% or more of
the expressed protein. Construction of the
LQ truncation mutant
required an introduction of non-endogenous TAA stop codon. The use of
the endogenous stop codon in this truncation produced a read-through
peptide of a higher than predicted molecular weight.
Expression of RetGC1 and RetGC2--
Bovine GC1 and GC2 were
expressed in the HEK293 cell line. The expression vector pCDNA3
(Invitrogen) containing corresponding cDNAs was a gift from Dr. R. Sharma (24). Cells at 80% confluency were transfected using calcium
phosphate. 15 µg of vector DNA was used per 100-mm dish. Cells were
harvested 48 h after transfection and lysed by passaging three
times through a 26-gauge needle in hypotonic buffer. A 500 × g supernatant was collected and centrifuged at 400,000 × g for 10 min. The resulting pellet was resuspended in the
buffer containing 10 mM Tris (pH 7.5) and 10 mM
-mercapthoethanol to the concentration of 4 µg/µl of total
protein as measured by the Bradford assay.
Circular Dichroism--
All experiments were performed on
circular dichroism spectrometer 62A DS from AVIVTM,
Lakewood, NJ, in a 1-mm optical path cell. We used purified proteins at
20-30 µM in 10 mM phosphate buffer (pH 7.0)
and 50 µM EDTA. Denaturation curves were obtained by
monitoring ellipticity at 222 nm. Ellipticities were normalized
according to the formula:
MRW =
oMr/lc, where
o is observed ellipticity in degrees,
Mr is the average molecular weight of an amino
acid in the protein, l is the optical path length in mm, and
c is protein concentration in grams/liter.
GC Assays--
The expressed proteins were assayed for their
ability to regulate RetGCs in parallel with wtGCAP-1 and nonspecific
protein (BSA or recoverin). The assays were carried out as described
previously (10). In brief, rod outer segments were washed to remove
endogenous GCAPs and were then assayed for GC activity under infrared
illumination. Ca2+ concentrations were controlled by 1 mM EGTA or EGTA/Ca2+ buffers. The substrate was
5 mM cold GTP and 0.1 µCi of [
-32P]GTP
(Amersham). The reactions were carried out at 30 °C for 20 min, and
the products were analyzed by TLC and scintillation counting.
Typically, synthesized cGMP was labeled to 1,000-10,000 cpm. The
background was typically 50 cpm. The amount of cGMP hydrolysis was
controlled in every reaction by adding 25 mM cold cGMP and 20,000 dpm of 3H-labeled cGMP. For recombinant cyclases
each assay point contained membranes with 10 µg of total membrane protein.
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RESULTS |
Recoverin Does Not Regulate RetGC in Our Experimental
System--
In order to perform an interpretable analysis of chimeras
we first established that recoverin is not a regulator of RetGC in our
system. Bovine recoverin and GCAP-1 share roughly 30% amino acid
sequence identity (Fig. 1A).
Previous studies have shown that pure recoverin does not stimulate
photoreceptor guanylyl cyclase (RetGC) (21). To confirm this result in
our system and to determine whether or not recoverin inhibits RetGC we
assayed GC activity in washed ROS membranes titrated with recombinant myristoylated recoverin (Fig. 1B). Recoverin did not
stimulate RetGC even at concentrations up to 30 µM
whereas GCAP-1 stimulated it 4-fold at 10 µM
concentration. Similarly, recoverin did not inhibit RetGC in >10
µM Ca2+ while GCAP-1 did (Fig.
1C).

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Fig. 1.
Effects of GCAP-1 and recoverin on RetGC
activity and lack of PDE activation. A, an alignment of
bovine GCAP-1, recoverin, and GCAP-2. Identical residues are
boxed. EF-hands are underlined in dashed
lines. B, purified recombinant myristoylated GCAP-1 and
recoverin were added to washed ROS membranes, and RetGC activity was
assayed in 1 mM EGTA. denotes GCAP-1, denotes
recoverin. Solid lines represent guanylate cyclase activity,
broken and dashed lines represent cGMP levels.
The left y axis is plotted in percent of maximal GC
activity, the right y axis in percent unhydrolyzed cGMP
recovered from the assay. C, GC activity was assayed in the
presence of >10 µM Ca2+. The data shown are
the average of duplicate data points in one experiment. They are
representative of two or more independent experiments.
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These results confirmed that recoverin indeed does not regulate RetGC
in our assay. None of the structural elements in our chimeras derived
from recoverin are in themselves sufficient to regulate RetGC. This
suggested that we could indeed use chimeric proteins to identify
GCAP-1-specific structural elements that are responsible for activating
and inhibiting RetGC.
Lack of Phosphodiesterase Activation in the Assay System--
It
is a formal possibility that the changing levels of cGMP in our assay
system result from variations in PDE activity present in ROS
preparations. We monitored hydrolysis of cGMP in all our assays as
described under "Experimental Procedures." As evident from the
dashed lines on Fig. 1, B and C, the
level of cGMP hydrolysis did not depend on increasing concentrations of
GCAP-1 and recoverin in low as well as high Ca2+. Similarly
we observed no effect on cGMP hydrolysis by any of the mutants we
produced (data not shown). We were able to conclude that the varying
amounts of cGMP in our assay system result solely from varied guanylyl
cyclase activity.
The Role of the COOH Terminus--
For the purpose of this work we
consider the COOH terminus of GCAP-1 as the residues from
Phe156 at the end of EF-hand 4 to the very COOH-terminal
Gly205. To study the role of the COOH terminus in
regulating RetGC we constructed several truncation mutants and chimeras
with the COOH terminus of recoverin (Fig.
2, A and B). The
truncation mutant that ended after Gly159 (
VQ) had only
7% of the stimulatory activity of wt GCAP-1 (low Ca2+
conditions) when its concentration in the assay was 25 µM
(Fig. 3A). The stimulatory
effect of GCAP-1 saturated below 10 µM (Fig. 1B).

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Fig. 2.
COOH-terminal constructs. A,
an alignment of the COOH-terminal sequences of GCAP-1, GCAP-2, and
recoverin. B, a series of truncation mutants:
VQ, SL, RI, LQ is represented here. EF4 is a chimera.
Closed area denotes GCAP-1, and open area denotes
recoverin. A minimum 2-fold difference from the negative control (BSA)
was considered as a positive effect (+). Less than 2-fold
difference was considered as no effect ( ). LQ is the shortest
truncation mutant that can activate GC. The difference between LQ
and the next shortest truncation RI is the sequence RIVRR.
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Fig. 3.
A, effect of COOH-terminal mutants on
RetGC in low Ca2+. The mutant proteins shown in Fig. 2 were
tested in a GC assay using washed ROS. All assays were performed in a
25-µl volume in the presence of 1 mM EGTA. The following
concentrations were used: VQ at 43 µM, SL at 87 µM, RI at 56 µM, LQ at 52 µM, EF4 at 65 µM, GCAP-1 at 1 µM. The data shown are the average of duplicate data
points in one experiment. They are representative of two or more
independent experiments. Basal GC activity in the presence of a
nonspecific protein (BSA) was taken to be 0 and % maximal activation
was calculated using the formula: %(X) = ((activity
X basal activity)/(maximal stimulated activity basal activity))*100%. A saturating amount of GCAP-1 was taken as
100% activation (3-5-fold stimulation depending on the ROS
preparation). Suppression of the activity below the basal level is
represented as a negative value. B, inhibition of GC in high
Ca2+ by COOH-terminal mutants. denotes VQ; ,
SL; , EF4; , GCAP-1. The mutant proteins were tested in a GC
assay using washed ROS membranes. All assays were performed in a
25-µl volume in the presence of >10 µM free
Ca2+. The data shown are the average of duplicate data
points in one experiment. They are representative of two or more
independent experiments. Basal GC activity in the presence of a
nonspecific protein (BSA) was taken as 100%. Saturation with wt GCAP-1
is reached at 2.5 µM.
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The longer truncation mutants
SL (ends at Arg172) and
RI (ends at Thr176) also do not stimulate RetGC. In fact
they suppress RetGC in low Ca2+ below the basal level (Fig.
3A).
The truncation mutant
LQ ends after Arg182. In contrast
to the shorter deletion mutants it stimulated RetGC to 93% of the wt GCAP-1 level when assayed at 50 µM. Essentially, only
LQ of all truncation mutants described here is capable of activating
RetGC to significant levels.
The truncation mutants that failed to activate RetGC do inhibit RetGC
in high Ca2+ (>10 µM) as shown in Fig.
3B. They also block activation by wt GCAP-1 in low
Ca2+ in a competition experiment with a half-maximal effect
reached at a molar excess of 35-100 (data not shown). Even the most
extensive COOH-terminal truncation mutant,
VQ, and the EF4 chimera
inhibited the cyclase in high Ca2+. The EF4 chimera
contains GCAP-1 sequence from the NH2 terminus down to
Phe156 following EF-hand 4 (Fig. 2). The rest of the
chimera consists of Ile172 to Leu202 of
recoverin. The length of this chimera exceeds the lengths of
VQ,
SL, and
RI.
Since the EF4 chimera does not activate RetGC (Fig. 3A) a
specific sequence in the COOH-terminal region is required for
activation, not simply any sequence of a suitable length. More
precisely the presence of the sequence RIVRR flanked by
Arg177 and Arg182 appears to be crucial for
activation but not for inhibition. The residues COOH-terminal of
Arg182 are not essential for stimulating RetGC. The actual
structural requirements provided by the RIVRR structure are not yet
clear. Results of a preliminary alanine scanning mutagenesis study
suggest that none of the specific residues within the RIVRR sequence
are essential for RetGC regulation (data not shown).
A chimera, EF3-4
, has the region between EF-hands 3 and
4 substituted with the corresponding recoverin sequence (see Fig. 7 and
discussion on core sequences below). It can stimulate RetGC as shown in
Fig. 8A. Based on the EF3-4
chimera and the
LQ truncation mutant we conclude that all elements essential for
RetGC activation that lie in the COOH terminus are localized within
residues Glu155 and Arg182.
The Role of the NH2 Terminus--
We consider the
NH2 terminus of GCAP-1 as residues from Gly2 to
Thr27. It is 31% identical to the corresponding region of
recoverin. Since recoverin does not regulate RetGC, we constructed and
analyzed chimeras that have increasing portions of the GCAP-1
NH2 terminus replaced by recoverin (Fig.
4). The "VEEL" chimera with sequence from the NH2 terminus to Val10 replaced by
recoverin stimulates RetGC in low Ca2+ (data not shown).
The chimera referred to as "WYK" has recoverin sequence from the
NH2 terminus to Trp21. This chimera also
stimulated RetGC as shown in Fig.
5A. However, replacing only 6 more residues of the native GCAP-1 sequence produced a chimera,
"TEC," that was completely inactive (Fig. 5A). This can
be because TEC lacks sequence elements necessary to activate RetGC.
Alternatively, misfolding could cause TEC to be inactive.

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Fig. 4.
NH2-terminal constructs.
A, an alignment of the NH2-terminal sequences of
GCAP-1, GCAP-2, and recoverin. B, black denotes
recoverin sequences, white denotes GCAP-1. A minimum 2-fold
difference from the negative control with BSA was considered as a
positive effect (+). Less than 2-fold difference was
considered as no effect ( ). The sequence WYKKFMT appears to be
critical for the ability to activate GC.
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Fig. 5.
A, effects of NH2-terminal
chimeras on RetGC activity in low Ca2+. denotes GCAP-1,
WYK, TEC. The constructs were tested in a GC assay using washed
ROS membranes. WYK and TEC were purified by affinity chromatography and
GCAP-1 was purified on a gel filtration column. All assays were
performed in a 25-µl volume in the presence of 1 mM EGTA.
The data shown are the average of duplicate data points in one
experiment. They are representative of two or more independent
experiments. B, inhibition of RetGC in high Ca2+
by NH2-terminal chimeras. The constructs were tested in a
GC assay using ROS washed membranes. All assays were performed in a
25-µl volume in the presence of >10 µM free
Ca2+. The data shown are the average of duplicate data
points in one experiment. They are representative of two or more
independent experiments. The following concentrations were used: GCAP-1
at 4 µM, WYK at 15 µM, TEC at 6 µM, ECP at 11 µM.
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In order to evaluate the folding state of the TEC chimera we used
circular dichroism (Fig. 6). GCAP-1
displayed a spectrum with an ellipticity 1.5 times higher than that of
TEC. However, the shape of the two spectra are virtually
indistinguishable and both are characteristic of a folded protein (Fig.
6A). The ellipticity at 222 nm decreased as a function of
temperature in a similar fashion for TEC and wt GCAP-1 (Fig.
6B). Ellipticity at this wavelength is indicative of the
helical content of a protein. It is routinely used to monitor
temperature denaturation of proteins.

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Fig. 6.
Circular dichroism of TEC and GCAP-1.
A, ellipticity was monitored at 25 °C. denotes
GCAP-1, TEC. B, ellipticity at 222 nm was monitored as a
function of temperature. The data were normalized according to the
formula described under "Experimental Procedures."
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These data suggest the
-helical content of TEC is similar to that of
GCAP-1 at room temperature. The smaller ellipticity of TEC may be
explained by the tendency of the recoverin NH2 terminus to
stay unfolded (22, 23). However, the CD spectra indicate that most, if
not all, of TEC is indeed folded. Since WYK activates RetGC and TEC
does not, we conclude that the GCAP-1 sequence from Trp21
to Thr27, WYKKFMT, is required for activation. Even though
this sequence is essential, other residues in the core also contribute
to activation. This is apparent from the properties of core
substitution mutants we describe in the following section. A summary of
the NH2-terminal chimeras and their properties is presented
in Fig. 4B.
An essential inhibitory structure also resides within the GCAP-1
NH2 terminus. None of the recoverin/GCAP-1 chimeras VEEL, TEC, and WYK inhibit RetGC in high Ca2+ (Fig.
5B). Despite its ability to stimulate, WYK did not block activation of Ret GC by a Ca2+-insensitive GCAP-1 mutant in
high Ca2+ at up to 30-fold molar access (data not shown).
The NH2 terminus is not in itself sufficient for
inhibition, however. A chimera, "ECP," consisting of the complete
NH2 terminus from GCAP-1 up to EF-hand 1 and the rest of
the sequence from recoverin fails to inhibit RetGC (Fig.
5B).
The Role of the Core Sequences--
We consider the sequence
between Glu28 and Met157 as the core of GCAP-1;
it includes EF-hands 1 through 4 (Fig.
7A). We constructed chimeras that
replaced native GCAP-1 core sequences with the corresponding sequences
of recoverin. The "EF4" chimera was spliced at Phe156
at the end of EF-hand 4 giving it the least recoverin and most GCAP-1
sequence. We also produced chimeras spliced after Glu111 at
the end of EF-hand 3 ("EF3") and at Val77 at the end of
EF-hand 2 ("EF2").

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Fig. 7.
The core chimeras. A shows an
alignment of the core sequences of GCAP-1, GCAP-2 and recoverin.
Highlighting represents the region necessary for activation.
B, EF2, EF3, and EF4 chimeras consist of an
NH2-terminal GCAP-1 stretch (shown in black) and
a COOH-terminal recoverin stretch (shown in white). For
stimulation and inhibition at least a 2-fold effect on the basal
activity of GC was considered as positive (+). Less than
2-fold difference was considered to be no effect ( ).
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The chimera EF1-2
has the region between EF-hands 1 and
2 replaced by recoverin. Similarly, EF2-3
and
EF3-4
have recoverin sequences between the
corresponding EF-hands.
EF4, EF3, and EF2 inhibit RetGC in high Ca2+ although the
concentrations required for inhibition are higher than for wtGCAP-1 (Fig. 8B). The EF2 chimera,
which has the least GCAP-1 sequence, also blocked activation of RetGC
in high Ca2+ by a Ca2+-insensitive GCAP-1
mutant (data not shown). Out of EF1-2
,
EF2-3
, and EF3-4
, only
EF3-4
failed to inhibit RetGC (data not shown). This may
suggest that the region between EF-hands 3 and 4 is involved in
inhibiting RetGC. Alternatively, the recoverin sequence introduced into
this chimera may interfere with the correct conformation required for inhibition.

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Fig. 8.
Effects of core chimeras on RetGC activity in
low Ca2+. A, the chimeras were tested in a
GC assay using washed ROS membranes in the presence of 1 mM
EGTA. Basal GC activity in the presence of a nonspecific protein (BSA)
was taken to be 0 and % maximal activation was calculated using the
formula: %(X) = ((activity X basal
activity)/(maximal stimulated activity basal activity))*100%.
The following concentrations were used: GCAP-1 at 1 µM,
EF1-2 at 100 µM, EF2-3 at
50 µM, EF3-4 at 58 µM, EF2
21 µM, EF3 at 26 µM. Chimeras EF3,
EF2-3 , and EF2 suppressed RetGC below the basal level.
EF3-4 displayed the ability to stimulate RetGC 2-fold
above the basal level. The inset shows a titration of ROS
with EF3-4 protein in low Ca2+. denotes
EF3-4 , denotes basal activity. The data
shown are the average of duplicate data points in a one experiment.
They are representative of two or more independent experiments.
B, inhibition in high Ca2+ by core chimeras. denotes EF2, EF3, EF4, + denotes HINGE, GCAP-1. The
chimeras were tested in a GC assay using washed ROS membranes. All
assays were performed in the presence of >10 µM free
Ca2+. The data shown are the average of duplicate data
points in one experiment. They are representative of two or more
independent experiments. Basal GC activity in the presence of BSA was
taken to be 100%.
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None of the chimeras with the C terminus replaced by recoverin (EF4,
EF3, and EF2) activate RetGC (Fig. 8A). This agrees with our
finding described above that the COOH-terminal RIVRR structure is
needed for activation. This sequence is not present in recoverin.
The chimera EF3-4
activates RetGC in low
Ca2+ by 2-fold above the basal level. This constitutes 28%
of the wt GCAP-1 level of activation in this experiment (Fig. 8A,
inset). In this chimera the GCAP-1 sequence between
Glu111 and Phe156 is replaced by recoverin.
Since the conservation between GCAP-1 and recoverin is quite low here,
we suggest that this region is not essential for RetGC activation.
The regions between EF-hands 1 and 2 and between 2 and 3 could not be
replaced without complete loss of the ability to activate RetGC. It
appears unlikely that this whole 71-amino acid stretch interacts with
the cyclase. Rather, it may provide for the proper configuration of the
activating elements that we identified in the NH2 and COOH
termini. Since both EF1-2
and EF2-3
can
inhibit RetGC in high Ca2+ it appears that they can bind to
the cyclase but fail to activate it.
Effects of Key Mutants on Recombinant RetGC1 and RetGC2--
To
study the effects our mutants may have on the known retinal guanylyl
cyclases we tested several mutant proteins on bovine recombinant RetGC1
and GC2 referred to as OS GC1 and OS GC2. Fig. 9 shows the effects of key mutants in low
Ca2+. The key COOH-terminal truncations,
LQ and
RI,
exhibited regulatory properties toward recombinant RetGC1 and GC2 that
are similar to those of the total ROS guanylyl cyclase activity. The
longer mutant
LQ stimulated RetGC1 by 10-fold, while the shorter
mutant
RI which lacks the critical structure represented by
"RIVRR" sequence had a less than 2-fold effect on GC1. For the less
active recombinant RetGC2 the effects were: 2.6-fold for
LQ and
under 2-fold for
RI. Similarly the key NH2-terminal
chimeras WYK and TEC affected the recombinant cyclases much like the
RetGC activity in ROS preparations. Both for expressed RetGC1 and GC2
WYK stimulated the activity and TEC did not have a significant effect
(Fig. 9). We conclude that the essential GCAP-1 stimulatory sequences
we identified in the native system are also essential for activation of
recombinant RetGC1 and GC2. We were not able to assess the inhibitory
effects of our mutants on recombinant RetGC1 and GC2 due to the very
low basal activity of the bovine recombinant cyclases.

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Fig. 9.
Effect of mutants on recombinant GC1 and
GC2. GCAP-1 and critical mutants were added to the membrane
fraction of HEK293 cells expressing (A) RetGC1 and
(B) RetGC2 and cyclase activity was assayed. All assays were
performed in the presence of 1 mM EGTA. The following
protein concentrations were used: GCAP-1 at 18 µM, WYK at
57 and 16 µM, TEC at 16 µM, LQ at 78 µM, RI at 76 µM. The data shown are
representative of three independent experiments. Each value is the
average of two measurements. The error bars are negligible
where not visible.
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DISCUSSION |
In this study we evaluated the ability of deletion mutants and
GCAP-1/recoverin chimeras to regulate RetGC. The ability of each
protein to stimulate RetGC was studied at low free Ca2+
levels buffered by EGTA. The ability to inhibit was assayed at >10
µM free Ca2+. Here we correlate the effects
of the mutants on RetGC activity with the presence of specific GCAP-1
sequences. We use this correlation to map GCAP-1 sequences critical for
RetGC regulation. We do not distinguish here between sequences that
directly interact with RetGC and those required for any other reason,
e.g. for proper scaffolding of non-contiguous interacting
side chains.
Experimental Strategy--
In order to simplify the analysis of
the mutants, we broke down the sequence into three major stretches: the
NH2 terminus (Gly2 to Thr27), the
core (Glu28 to Phe156), and COOH terminus
(Met157 to Gly205). In this discussion we take
a qualitative approach to describing the ability of each protein to
activate and inhibit RetGC. When a mutant was able to regulate RetGC we
frequently found that its apparent affinity for RetGC was altered
(Table I). This suggests that the
chimeras may not reproduce all features of the wild type GCAP-1
conformation correctly. Nonetheless, the stimulation and inhibition of
RetGC by these mutant proteins were reproducible. We only considered
2-fold or greater effects as significant. This cut-off clearly
differentiated between a specific effect and background variation that
we routinely observe with nonspecific proteins (e.g. BSA,
recoverin) in our assay. The effect of these proteins on GC activity
typically does not exceed 10% of the basal level.
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Table I
IC50 and fold maximal inhibition for COOH-terminal core
constructs
The values were calculated from fitted curves within the range of
collected data points. IC50 concentrations were calculated for
each construct individually, i.e. based on its maximal fold
inhibition.
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The presence of two distinct guanylyl cyclases, RetGC1 and RetGC2, is
established in ROS of humans and other species (1-5). These cyclases
are referred to as ROS GC1 and ROS GC2 in the literature. At present it
is not clear if they account for all cyclase activity in ROS or if
other cyclases are also present. In this study we have focused on the
regions of GCAP-1 which are essential for the interaction with
cyclases. We therefore used ROS preparations to make all cyclases that
are regulated by GCAP-1 in vivo available to the GCAP-1
mutants in our assays. However, we have also confirmed that the key
COOH-terminal deletion mutants and NH2-terminal chimeras affect recombinant ROS GC1 and ROS GC2 the same way they affect GC
activity in ROS preparations (Fig. 9).
The Role of the COOH Terminus--
In the COOH terminus of GCAP-1
a structure represented by the sequence RIVRR appears crucial for
activation. A mutant truncated immediately after this sequence
activates RetGC whereas a truncation that stops immediately before it
does not. Paradoxically, none of the residues in the RIVRR sequence
seems essential for RetGC activation based on the results of a
preliminary point mutagenesis study. Our results localize all elements
essential for activation in the COOH terminus to residues from
Glu155 to Arg182.
None of the structures in the COOH terminus of GCAP-1 are required to
inhibit RetGC. All the truncation mutants as well as chimeras with the
COOH terminus of recoverin inhibit the cyclase.
The Role of the NH2 Terminus and the Core--
As
evident from the TEC chimera the NH2 terminus is critical
for activating RetGC. TEC displays CD spectra resembling those of wt
GCAP-1 (Fig. 6, A and B) arguing that it is a
folded protein. It does not, however, stimulate RetGC in low
Ca2+ (Fig. 5A) nor does it block stimulation by
wt GCAP-1 (data not shown). Another chimera, WYK, that included only 7 more residues of GCAP-1 than TEC activates RetGC by over 2-fold. We
conclude that these 7 residues, WYKKFMT, are essential for activation.
Replacing the NH2 terminus of GCAP-1 with recoverin
sequence to Ser9 (as in the VEEL chimera, Fig.
5B) abolishes inhibition but not activation. This shows that
a structure within GCAP-1 between Gly2 and Ser9
is specifically required for inhibition. It has been shown in a
different study that an NH2-terminal peptide derived from
GCAP-1 blocks activation of RetGC by GCAP-1 (IC50 of 10 µM) (4, 8). The role of the NH2 terminus is
summarized in Fig. 4B.
We identified no GCAP-1-specific sequences within the core of the
protein (Glu28 to Phe156) that are required for
inhibition. The chimera EF2 with all GCAP-1 sequence from EF-hand 2 to
the COOH terminus replaced by recoverin sequence inhibits RetGC (Fig.
8B). EF1-2
with GCAP-1 sequence between
EF-hand 1 and EF-hand 2 replaced by the corresponding recoverin
sequence also inhibits RetGC (data not shown). Based on the ability of
the EF1-2
chimera to inhibit we conclude that the region
of GCAP-1 from Gln33 to Val77 is not
specifically required for inhibition. To summarize, the first 9 amino
acids of GCAP-1 are specifically required for inhibition of RetGC in
high Ca2+. Other residues in the NH2 terminus
and the core, however, are likely to contribute to inhibition in a
nonspecific way, e.g. by providing scaffolding for
inhibitory structures. For example, chimera ECP that contains the whole
NH2 terminus of GCAP-1 down to Thr27, with the
rest of it derived from recoverin, fails to inhibit RetGC in high
Ca2+.
Chimeras EF1-2
and EF2-3
do not stimulate
RetGC. That shows that the GCAP-1 region between EF-hands 1 and 3 is
necessary for RetGC activation. Since this is a long stretch, it
appears unlikely that all of it is involved in a direct contact with
RetGC. This region of GCAP-1 may provide for the correct scaffolding of
activating sequences, while the corresponding region of recoverin does
not fulfill this role. The role of the core sequences is summarized in
Fig. 7B.
Activation Versus Inhibition--
A summary of our findings is
shown in Fig. 10. The inhibitory and
stimulatory effects of GCAP-1 on RetGC appear to require different
GCAP-1 structures. Stimulation requires both the COOH-terminal RIVRR
and the NH2-terminal WYKKFMT sequences, whereas inhibition appears to require the first 9 amino acids which are distinct from
either of the stimulatory determinants. Moreover, structures between
EF-hands 1 and 3 are required for activation but not for inhibition.

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Fig. 10.
Summary of GCAP-1 sequences necessary for
activation and inhibition of RetGC. Regions necessary for
inhibition are labeled with ; regions needed for activation are
underscored with +. × represents regions whose role in inhibition or
activation is tentative. Bold script highlights essential
activation or inhibition sequences. The functional EF-hands are
boxed with a solid line, the non-functional
EF-hand is boxed with a dotted line.
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Comparison with GCAP-2--
Both GCAP-1 and GCAP-2 inhibit and
stimulate RetGC, yet there are substantial differences in their
sequences. In particular the NH2 and the COOH termini of
the two proteins have few common primary sequence features. A parallel
study using chimeras of GCAP-2 with neurocalcin (see accompanying
article, Ref. 25) showed that a sequence near the COOH terminus of
GCAP-2 is specifically required for activation. This correlates with
our finding that a specific sequence in the GCAP-1 COOH terminus is
essential for activation but not inhibition of RetGC.
The GCAP-2 study also identified a sequence flanking EF1 of GCAP-2 as
important for activation and inhibition. According to our results a
sequence, WYKKFMT, which flanks EF1 in GCAP-1 is necessary for
activation of RetGC. In fact part of this stretch, WYKKF, is conserved
between the two proteins.
There are also significant differences between the findings in the
GCAP-1 and GCAP-2 studies. GCAP-1 but not GCAP-2 appears to require the
9 NH2-terminal residues for inhibition and the region
between EF-hands 1 and 3 for activation of RetGC. We have produced a
GCAP-1 chimera whose region between Ser53 and
Ile122 is replaced by the corresponding recoverin sequence.
A similar GCAP-2/neurocalcin chimera displayed reversed
Ca2+ sensitivity in the GCAP-2 study. GCAP-1/recoverin
chimera we have constructed, however, does not exhibit a reversed
Ca2+ sensitivity. It inhibits RetGC in high
Ca2+ and does not stimulate it in low Ca2+
(data not shown). These differences between GCAP-1 and GCAP-2 may
reflect the divergence of the primary sequences of the two proteins.
For instance, a mutation in GCAP-1 and GCAP-2 has been shown to affect
their activity differently (13-15). Alternatively, the differences may
arise from the experimental systems used to make chimeras in the two
studies since recoverin has less homology to GCAP-1 than neurocalcin to
GCAP-2. Further structural analyses will be required to clarify the
precise functions of the regions identified in these studies for
binding, activation, and inhibition of RetGCs.