Regulatory modes of rod outer segment membrane guanylate cyclase
differ in catalytic efficiency and Ca
2+
-sensitivity
Ji-Young Hwang
1,
*, Christian Lange
1,
†, Andreas Helten
1
, Doris Ho¨ ppner-Heitmann
1
, Teresa Duda
2
,
Rameshwar K. Sharma
2
and Karl-Wilhelm Koch
1
1
Institut fu
¨
r Biologische Informationsverarbeitung 1, Forschungszentrum Ju
¨
lich, Ju
¨
lich, Germany;
2
The Unit of Regulatory and
Molecular Biology, Departments of Cell Biology and Ophthalmology, NJMS & SOM, UMDNJ, Stratford, NJ, USA
In rod phototransduction, cyclic GMP synthesis by mem-

free [Ca
2+
],
while that by GCAP-2 is at 100 n
M
. The findings show that
differences in catalytic efficiency and Ca
2+
-sensitivity of
ROS-GC1 are conferred by GCAP-1 and GCAP-2. The
results further indicate the concerted operation of two
ÔGCAP modesÕ that would extend the dynamic range of
cyclase regulation within the physiological range of free
cytoplasmic Ca
2+
in photoreceptor cells.
Keywords: phototransduction; guanylate cyclase; GCAP;
myristoylation; k
cat
/K
m
.
Photoexcitation of vertebrate photoreceptor cells leads to
the hydrolysis of cyclic GMP (cGMP) and subsequent
closure of the cyclic nucleotide-gated (CNG) channels in the
plasma membrane. Restoration of the dark state of the
photoreceptor cell requires the reopening of CNG-channels
(reviewed in [1–3]). A critical step in this recovery process is
synthesis of the second messenger, cGMP. Studies with
vertebrate photoreceptor cells, constituting mainly rods,

from retinal sources [19–23]. GCAP-1 and GCAP-2 are
both expressed in rod and cone cells of different species as
shown by immunocytochemistry [21,22,24,25]. Expression
of GCAP-3 is more restricted; it is present in human cones,
fish rods and cones, but not in mice photoreceptor cells [26].
Thus, GCAP-3 does not appear to be a general sensor of
Ca
2+
-pulses linked with phototransduction.
GCAP-1 and GCAP-2 contain one nonfunctional and
three functional EF-hands. Through functional hands they
detect changes in the intracellular Ca
2+
-concentration
[Ca
2+
] and modulate ROS-GC1. Dark adapted vertebrate
photoreceptor cells have a cytoplasmic free [Ca
2+
] of 500–
650 n
M
. This falls below 100 n
M
upon illumination [27–30].
GCAPs detect the fall and in their Ca
2+
-free form, activate
ROS-GC1 [4,5,19–23]. The generated cyclic GMP replen-
ishes the depleted pool and restores the channels in their

Eur. J. Biochem. 270, 3814–3821 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03770.x
that these Ca
2+
-binding-proteins are powerful activators of
the ROS-GCs [4,5,19–23], there is no unanimity on their
specific expression in rods or cones and to which ROS-GC
they are paired with. Some immunocytochemical studies
show that GCAP-1 is the predominant form in cones and
GCAP-2 in rods [31,32]; and recently the physiological role
of GCAP-2 has been questioned, because the expression of
GCAP-2 in transgenic GCAPs null mice did not fully
restore the normal flash response of the wild-type [33]; and
Howes et al. [34] in a subsequent study on transgenic mice
showed, that the wild-type phenotype flash response could
be rescued by the expression of GCAP-1 in the absence of
GCAP-2.
To better define the Ca
2+
-modulated ROS-GC trans-
duction component in rod cells we addressed the following
questions in the present work: how much of GCAP-1 and
GCAP-2 is present in rod outer segments? Are there
differences between GCAP-1 and GCAP-2 in their regula-
tory properties and if so, what is the physiological signifi-
cance of such a difference?
The study demonstrates that GCAP1 and GCAP2
modulate the Ca
2+
signaling of ROS-GC1 in different ways.
Experimental procedures

transfected with ROS-GC1 in pcDNA3.1 expression vector
by the calcium phosphate method [16]. The following
modifications were applied: 22 h post-transfection cells were
washed sequentially with NaCl/P
i
,NaCl/P
i
plus EDTA and
NaCl/P
i
and incubated in medium for 20–22 h. Cells were
harvested by a short centrifugation step (200 g;5min;
4 °C), resuspended in NaCl/P
i
and centrifuged again. The
pellet was resuspended in lysis buffer (10 m
M
Hepes/KOH
pH 7.5, 1 m
M
dithiothreitol), sonicated and centrifuged to
remove cellular debris (400 g;5min;4 °C). The supernatant
was then centrifuged at 125 000 g for 15 min at 4 °Cto
pellet the membranes. The membranes were resuspended in
10 m
M
Hepes/KOH pH 7.5, 250 m
M
KCl, 10 m
M

calibration curve. The predominant membrane guanylate
cyclase in bovine ROS is ROS-GC1, the other isoform
(ROS-GC2) that is expressed in photoreceptor cells consti-
tutes less than 10% of the amount of ROS-GC1
(A. Helten and K W. Koch, unpublished observation).
Amounts of GCAPs were obtained by a similar proce-
dure but instead of a Luminograph we used a Kodak Image
Station. Exact amounts of purified GCAP-1 or GCAP-2 for
SDS/PAGE and Western blotting were determined from
GCAP specific protein standard curves. These curves were
created as follows: purified preparations of GCAP-1 and
GCAP-2 were used to determine their molar extinction
coefficients as described in [40]. The values were
e
280
¼ 28378
M
)1
Æcm
)1
for GCAP-1 and e
280
¼ 37512
M
)1
Æcm
)1
for GCAP-2. Exact stock solutions of GCAP-1
and GCAP-2 were prepared using the molar absorbance
coefficients and a calibration curve was made using the

for each GCAP.
Analysis of enzyme kinetics
ROS-GC1 in washed ROS membranes was reconstituted
with 2 l
M
of purified myr- or nonmyr-forms of GCAP-1
and GCAP-2. Guanylate cyclase activity was assayed at
2m
M
EGTA (£ 10 n
M
free [Ca
2+
]) as a function of the
substrate GTP. We used Mg
2+
as cofactor and kept the
ratio of Mg
2+
to GTP at 5 : 1. Analysis of data was
performed with
ORIGIN
6.1 and
SIGMA PLOT
4.2 software. A
direct plot of activity vs. [GTP] gave in all cases a sigmoidal
curve indicating cooperative substrate binding. In order to
analyze data of a sigmoidal dependence in a linear
Lineweaver–Burk plot [41], we determined a Hill coefficient
n from a fit of the direct plot. Values of V

to 10
)1
M
free [Ca
2+
].
Results
The Ca
2+
-sensor proteins GCAP-1 and GCAP-2
are almost equally expressed in native bovine ROS
membranes
Molar concentrations of GCAPs have not been reported so
far, although they are indispensable for a full quantitative
description of phototransduction. We determined these
values by the following procedure: 0.5–9 ng of purified
GCAP-1 and 2–10 ng of GCAP-2 were loaded onto PAGE.
ROS containing 5–25 lg of rhodopsin were loaded on the
same gel. After electrophoresis, proteins were electrotrans-
ferred to a blot membrane and probed with antibodies
against GCAP-1 or GCAP-2. Bands were visualized by
chemiluminescence. Chemiluminescence intensity was line-
arly dependent on the amount of antigen and was used to
quantify the amounts of GCAPs. An example is shown in
Fig. 1A,B, where the amount of GCAP standards showed a
linear increase in chemiluminescence intensity. The analysis
of several Western blots revealed a ratio of 1 : 1200 ± 360
(N ¼ 12) of GCAP-1 to rhodopsin and 1 : 1100 ± 560
(N ¼ 5) of GCAP-2 to rhodopsin. About 25% of GCAP-1
is lost during the purification of ROS on a sucrose gradient,

ratio of 1 : 1340 GCAP-2 to rhodopsin.
3816 J Y. Hwang et al. (Eur. J. Biochem. 270) Ó FEBS 2003
contained 28 ng of ROS-GC1 (not shown). This relates to a
molar ratio of 1 : 200 ROS-GC1 to rhodopsin. Analyzing
four different blots revealed a mean ratio of 1 : 260 ± 60,
which is consistent with previous estimates [6,9]. The
functional unit of ROS-GC1 is a dimer [16,42,43] that is
correspondingly present in a ratio to rhodopsin of 1 : 520.
The cellular concentration of the ROS-GC1 dimer therefore
is 5.8 l
M
(cellular concentration of rhodopsin ¼ 3m
M
). We
used this value to calculate the turnover number of ROS-
GC1: V
max
in washed ROS membranes was 3.0 nmol
cGMPÆmin
)1
per mg Rh, which is synthesized by
4.8 · 10
)2
nmol ROS-GC1 dimer (1 mg of Rh ¼ 25 nmol
Rh; ratio of ROS-GC1 to Rh is 1 : 520). Thus, the resulting
turnover number or k
cat
of ROS-GC1 in native membranes is
1.0 s
)1

catalytic efficiency and Ca
2+
-sensitivity.
First, how does the interaction with GCAP-1 and GCAP-
2 influence k
cat
/K
m
of ROS-GC1? ROS-GC1 in washed
ROS membranes was reconstituted with a constant amount
of myr- and nonmyr-forms of GCAP-1 or GCAP-2. As the
half-maximal activation (EC
50
)ofROS-GC1byGCAP-1
and GCAP-2 occurs well below 1 l
M
[37],wechosea
saturating concentration of 2 l
M
for all experiments. The
ROS-GC1 activity was measured as a function of [Mg-
GTP] at a constant free [Ca
2+
]of5n
M
. The catalytic
parameters were then determined from a Lineweaver–Burk
plot (Fig. 2A–D). The results are listed in Table 1. The
values of k
cat

value was 2.6 · 10
3
M
)1
Æs
)1
. When the
effect of GCAP-2 was assayed in the same manner the
results were different. Both GCAP-2 forms, myristoylated
and nonmyristoylated, increased V
max
and decreased K
m
to
a similar degree, i.e., for GCAP-2, the influence of the
Table 1. Catalytic parameters of GCAP-dependent activation of ROS-GC1 in washed ROS membranes. Assays were performed at low [Ca
2+
](2 m
M
EGTA).
V
max
(nmolÆmin
)1
per mg Rh) K
m
(m
M
) k
cat

)1
.Plotwas
linearized by using the apparent Hill
coefficient n ¼ 1.68 (Table 1) to yield 1/[S]
n
.
Reciprocal activity 1/V is expressed as
nmol
)1
Æmin per mg Rh. Kinetic analysis was
performed in the same manner with 2 l
M
nonmyryistoylated GCAP-1 (B), 2 l
M
myris-
toylated GCAP-2 (C) and 2 l
M
nonmyris-
toylated GCAP-2 (D). Data points represent
mean of triplicates ± SD. Details are given in
(A) and in Table 1.
Ó FEBS 2003 Differential Ca
2+
-modulation of guanylate cyclase (Eur. J. Biochem. 270) 3817
myristoyl group was not very pronounced. Myr-GCAP-2
increased the catalytic efficiency k
cat
/K
m
by 13-fold, while

6
S-GCAP-1.
The results were reproducible with different preparations of
bovine ROS and GCAPs: the dose–response curve was
alwaysshiftedtolower[Ca
2+
]withGCAP-2.TheIC
50
value for Ca
2+
determined from two to three independent
titration curves for GCAP-2 was 100 ± 32 n
M
and that for
D
6
S-GCAP-1, was 707 ± 122 n
M
. The dose–response
curves were, in both cases, cooperative with Hill coefficients
of n ¼ 1.46 ± 0.11 and n ¼ 2.4 ± 0.0 for GCAP-1 and
GCAP-2, respectively. These different Ca
2+
-sensitivities of
D
6
S-GCAP-1 and GCAP-2 were independent of GCAP
concentrations, as similar activation profiles were obtained
1 l
M

physiological concentrations can reproduce the activation
profile of ROS-GC1 in native ROS. We conclude from
these results that GCAP-1 and GCAP-2 confer different
Ca
2+
-sensitivities to ROS-GC1 in bovine rods.
We then proceeded to study systems of heterologously
expressed ROS-GC1 and purified GCAPs to answer the
question whether the reconstitution of the native activation
profile requires components specific to native ROS mem-
brane preparations or not. Thus, ROS-GC1 was hetero-
logously expressed in HEK293 tsA cells, HEK293 cells and
COS cells. Cell membranes were reconstituted with D
6
S-
GCAP-1 and GCAP-2 and the EC
50
and IC
50
values were
determined. EC
50
values were similar as described previ-
ously [25,44] and maximal activity at saturating GCAP
concentration was similar (data not shown). When we
analyzed the activation profile as a function of free [Ca
2+
],
we obtained for ROS-GC1 that was heterologously
expressed in HEK293 or HEK tsA cells, similar results as

control determinations without added GCAPs. These data were
averaged from at least three experiments. The activity unit is nmol
cGMPÆmin
)1
per mg rhodopsin. The data were fitted by the modified
Hill equation; V/V
max
¼ –Z[Ca
2+
]
n
/([Ca
2+
]
n
+ K
n
m
)+1; V is the
activity of ROS-GC1, V
max
is the maximal activity of ROS-GC1, n is
the Hill cooperativity, K
m
corresponds to IC
50
of Ca
2+
-dependent
ROS-GC1 activity, and Z is a constant taking into account that

dependency on the membrane source for ROS-GC1 could
also indicate that cyclase lacks some modification when it is
expressed in COS cells for instance and this modification
could be necessary to exert different actions of GCAP-1 or
GCAP-2, but we have no experimental proof for this
speculation.
From the results obtained with native membranes, the
native reconstituted membranes and with the reconstituted
heterologous expression systems of HEK tsa and HEK 293
cells, we conclude that the differences in the Ca
2+
-sensitive
regulation of ROS-GC1 by GCAP-1 and GCAP-2 are an
intrinsic property of the ROS-GC1/GCAP complex. The
effect does not particularly depend on the presence of ROS
membranes and therefore does not require a specific
component found exclusively in ROS.
Discussion
A key finding of this study is that the cellular concentration
of GCAP-1 and GCAP-2 approximately sums to the
cellular concentration of a functional ROS-GC1 dimer
(about 6 l
M
). As the apparent affinities of GCAPs for
native ROS-GC1 are very similar [37], one molecule of
GCAP-1 or GCAP-2 could assemble with one ROS-GC1
dimer and form a functional unit. This would lead to two
different ROS-GC1 complexes, i.e., ROS-GC1/GCAP-1
and ROS-GC1/GCAP-2. Alternatively, one ROS-GC1
dimer could assemble with one GCAP-1 and one GCAP-

sensitive to different levels of free [Ca
2+
]. The detection
level of GCAP-2 is one order of magnitude lower than
that of GCAP-1. However, at intermediate levels, both
GCAPs can detect the changes in Ca
2+
intensity.
Although the Ca
2+
IC
50
values of ROS-GC1 regulation
reported in the literature vary over a large range [4], these
differences have not been addressed as specific distinct
properties of GCAP-1 and GCAP-2. On the contrary, it
has been shown that the Ca
2+
-dependent activation
profiles of GCAP-1 and GCAP-2 coincide (Fig. 8C of
[21]). Only occasionally have differences between GCAP-1
and GCAP-2 activation profiles been noted but not
explicitly discussed [36,46]. Therefore, we investigated this
problem in a systematic manner by using different
membrane systems as a source for ROS-GC1, i.e., native
ROS membranes, HEK293tsA, HEK293 and COS cells.
In addition we varied the concentration of GCAPs, when
testing the activation profiles. Different Ca
2+
-sensitivities

6
S-GCAP-1 (d)orGCAP-2(s). IC
50
values were 609 n
M
(D
6
S-GCAP-1) and 47 n
M
(GCAP-2). (B) Activity of ROS-GC1
expresssedinCOScellsasafunctionoffree[Ca
2+
] at a constant
amount of either 4 l
M
D
6
S-GCAP-1 (d)or15l
M
GCAP-2 (s). IC
50
values were 109 n
M
(D
6
S-GCAP-1) and 111 n
M
(GCAP-2).
Ó FEBS 2003 Differential Ca
2+

begun to fall. The observed undershoot indicates a delayed
activation of cyclase by GCAP-2. This is also consistent
with our result, that GCAP-2 is activated at lower free
[Ca
2+
] than GCAP-1. The operating range of free [Ca
2+
]
for GCAP-2 will be reached later after flash illumination.
On the other hand, expression of GCAP-1 can fully reverse
the observed effect of the lack of GCAP-1 and GCAP-2.
This observation can be explained by our results, that
GCAP-1 activates ROS-GC1 over a larger range of free
[Ca
2+
], at higher free [Ca
2+
] and therefore with an onset far
earlier in the timeframe of a single-flash response than
GCAP-2 does. GCAP-1 can therefore compensate the lack
of GCAP-2.
In summary, we have found that both GCAPs are
necessary to restore the native [Ca
2+
]-dependent activity
profile of ROS-GC1. Their combined presence in a complex
with the catalytic ROS-GC1 dimer may serve to extend the
working range of the Ca
2+
-feedback on ROS-GC1 and

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