(Received for publication, July 6, 1995; and in revised form, January 17, 1996)
From the
The membrane-bound guanylyl cyclase in vertebrate photoreceptor
cells is one of the key enzymes in visual transduction. It is highly
sensitive to the free calcium concentration
([Ca]). The activation process is
cooperative and mediated by a novel calcium-binding protein named GCAP
(guanylyl cyclase-activating protein). We isolated GCAP from bovine rod
outer segments, determined amino acid sequences of proteolytically
obtained peptides, and cloned its gene. The Ca
-bound
form of native GCAP has an apparent molecular mass of 20.5 kDa and the
Ca
-free form of 25 kDa as determined by
SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was
functionally expressed in Escherichia coli. Activation of
guanylyl cyclase in vertebrate photoreceptor cells by native acylated
GCAP was half-maximal at 100 nM free
[Ca
] with a Hill coefficient of 2.5.
Activation by recombinant nonacylated GCAP showed a lower degree of
cooperativity (n = 2.0), and half-maximal activation
was shifted to 261 nM free [Ca
].
Immunocytochemically we localized GCAP only in rod and cone cells of a
bovine retina.
Illumination of vertebrate photoreceptor cells triggers the
hydrolysis of guanosine 3`,5`-cyclic monophosphate (cGMP) by an
amplifying transduction cascade. Decrease of cytoplasmic cGMP leads to
the closure of cGMP-gated channels and results in the suppression of
the circulating dark current. This generates the electrical signal for
further processing in the retina. Restoration of the dark current
depends on effective shut-off mechanisms of all excitation steps in the
transduction cascade and on an efficient resynthesis of
cGMP(1, 2, 3, 4) . A calcium
feedback controls some of these events and is thought to be critical
for both recovery and light adaptation(5, 6) . Calcium
homeostasis in photoreceptor cells is maintained by the balance between
Ca influx through the cGMP-gated channel and
Ca
efflux via the Na:Ca,K exchanger. Closure of the
cGMP-gated channels prevents Ca
from entering the
cell. Because Ca
is continuously extruded by the
exchanger, a net decrease of the cytoplasmic calcium concentration
below the dark concentration of 400-500 nM accompanies
the light response (7, 8, 9, 10) .
One of the key events triggered by the decrease of cytoplasmic
[Ca] is a reinforced synthesis of cGMP
catalyzed by a 112-kDa guanylyl cyclase (GC) (
)that
represents a novel subtype among the membrane bound
GCs(11, 12, 13, 14, 15) . A
characteristic feature of this photoreceptor-specific GC (retGC) is its
strong dependence on the free [Ca
]. At low
[Ca
] the enzyme is activated by a soluble
calcium-binding protein(16) . Recent progress has been made in
the search for this protein. A calcium-binding protein named guanylyl
cyclase-activating protein (GCAP) with an apparent molecular mass of 20
kDa was shown to activate the retGC in a Ca
-dependent
manner(17) . Dialyzing GCAP into lizard rod outer segments
decreases the sensitivity, time to peak and recovery time of the light
response. Molecular cloning and sequence analysis showed the presence
of three Ca
-binding motifs (EF-hands) in
GCAP(18, 19) . Additionally, a 24-kDa calcium-binding
protein was isolated from retina extracts that was able to stimulate
retGC(20) . The relationship of these two activator proteins
and their cellular distribution in the retina is not clear at present.
An unexpected complexity has also emerged from the cloning of a second
photoreceptor-specific membrane guanylyl cyclase, retGC-2(21) .
(We simply refer to retGC activity, because we cannot distinguish
between retGC-1 and retGC-2 activities in our rod outer segment (ROS)
membrane preparations.) In order to gain further insight into the
control of retGC activity, we screened cytoplasmic extracts of bovine
ROS for guanylyl cyclase activating factors, isolated and cloned a
protein (GCAP), studied its distribution in the vertebrate retina, and
compared some of the molecular properties of two recombinant forms with
the native form.
A
154-base pair fragment that was found to harbor the sequences coding
for peptides 2, 4, and 5 was used to screen a bovine retina-specific
library in ZAPII (Stratagene). Labeled probes were prepared with a
DECAprime kit (Ambion) following the manufacturer's instructions.
Hybridization was in 5
SSC, 5
Denhardt's, 0.1 mg
ml
denatured herring testes DNA, 0.1% SDS at 64
°C for 14 h. Filters were washed in 1
SSC, 0.1% SDS at room
temperature, followed by two washes at 65 °C for 30 min. Filters
were exposed to x-ray films for
16 h at -80 °C. Both
strands of the longest isolated recombinant were sequenced according to
the dideoxy nucleotide chain termination technique (24) using
T7 DNA polymerase (Pharmacia). The sequence of the coding region and of
parts of nontranslated regions was confirmed by sequencing of three
independent recombinants.
Sections were preincubated in a solution of 10% normal goat serum, 0.5% Triton X-100 in PB for 1 h. The primary antibody was diluted 1:600 in 3% normal goat serum, 0.5% Triton X-100, 0.02% sodium azide in PB, and sections were incubated at room temperature overnight. After several rinses in PB, sections were incubated in goat anti-rabbit carboxymethylindocyanin (cy3) conjugate (Dianova) diluted 1:1000 in 1% bovine serum albumin, 0.5% Triton X-100 in PB for 2 h. After washing in PB, sections were coverslipped in Mowiol and examined under epifluorescence.
Figure 1:
A,
purification of GCAP from bovine ROS. Upper part, a
cytoplasmic ROS extract was concentrated to 1.4 ml by ultrafiltration
(centricon 10) and applied to a gel filtration column (Superdex 75
16/60). The chromatography was performed at a flow rate of 1 ml
min. Fractions containing retGC-stimulating activity (shaded area in the elution profile between 26 and 36 ml) were
pooled and applied to an anion exchange chromatography on a MonoQ
HR5/5 column (lower part). Bound proteins were eluted by a
continuous NaCl gradient (0-500 mM). B,
SDS-PAGE analysis of fractions obtained from purification of GCAP on a
12.5% polyacrylamide gel. LMW lane, low molecular mass
standard; light extract lane, cytoplasmic extract of ROS
proteins (18 µg); gel filtration lane, fraction of gel
filtration step with retGC-stimulating activity (1.6 µg); MonoQ
lane, purified GCAP after anion exchange chromatography (0.2
µg). A sample from the shaded area of the lower part of A was electrophoresed. The gel was stained with
Coomassie Blue. C, property of native GCAP to activate retGC
in washed ROS membrane preparations (30 ng of GCAP and 40 µg of
rhodopsin). Incubations were performed at high (8 µM [Ca
], open columns) and low
[Ca
] (1 nM, hatched
columns). Washed ROS membranes only exhibited the basal retGC
activity. The addition of purified native GCAP stimulated retGC
activity about 3-fold. Low concentrations of ATP (100 µM)
enhanced retGC activity independent of the free
[Ca
] about 2-fold. The addition of ATP and
GCAP resulted in a 5-fold activation. The data are the means of four
different measurements.
Activity
of retGC at high [Ca] was 2-3 nmol
cGMP min
mg
rhod (Fig. 1C). When free [Ca
]
was lowered to less than 100 nM native GCAP activated retGC in
washed ROS membranes to 5-7 nmol cGMP min
mg
rhod (Fig. 1C). The
addition of 100 µM ATP enhanced retGC activity independent
of the free [Ca
] about 2-fold (Fig. 1C, control + ATP). Maximal
activity of retGC was obtained at low free
[Ca
] with native GCAP and 100 µM ATP resulting in 12-14 nmol cGMP min
mg
rhod (Fig. 1C and Fig. 4B). Low concentrations of ATP (0.1-0.2
mM) were reported to enhance retGC activity in whole ROS
preparations (26, 27, 28) and in a
reconstituted system with purified GCAP(27) . Contrary,
Sitaramayya et al. reported of an inhibitory effect of ATP on
retGC activity(29) . We only observed inhibition of retGC at
higher ATP concentrations (1-2 mM). The inhibition by
ATP probably reflects competition with the GTP binding site as also
suggested by Gorczyca et al.(27) . Occasionally we
observed retGC activities at low free [Ca
]
as high as 23 nmol cGMP min
mg
rhod in whole ROS and also with purified native GCAP.
Figure 4:
Characterization of native and recombinant
GCAP. A, electrophoretic mobility of the native (lane
a) and the two recombinant GCAP forms (lane b, GCAP
corresponding to subclone pG-GCAPT; lane c, GCAP corresponding
to subclone pG-GCAPX) in the presence of 2 mM Ca or 2 mM EGTA. SDS-PAGE was performed on a 12.5%
polyacrylamide gel, and the gel was stained with Coomassie Blue. B, activation of retGC by native and recombinant GCAPT in
dependence of [Ca
]. GCAP was reconstituted
with washed ROS membranes containing retGC. Ratio of native GCAP to
rhodopsin was 1:800 (open circles). Molar ratios of
recombinant GCAPT to rhodopsin was 1:20 (open squares). All
incubations were done in the presence of 0.1 mM ATP. No GCAP
was added in the control (filled diamonds). The data represent
triplicates.
Microsequencing of proteolytically obtained peptides of the 20.5-kDa
protein revealed six partially overlapping peptides (Fig. 2).
N-terminal sequencing failed due to N-terminal modification (covalent
acylation; see ``Discussion''). Based on the sequences of
peptides 2 and 5 degenerate oligonucleotide primers were designed for
PCR on bovine retinal cDNA. PCR products were subcloned and sequenced.
A fragment that was found to harbor the sequence coding for peptides 2
and 5 as well as for peptide 4 was used to screen a bovine
retina-specific library. The longest isolated recombinant consisted of
945 base pairs. One long open reading frame was identified. The
translation initiation site was assigned to the first ATG codon
(nucleotides 238-240) that appears downstream of a nonsense
sequence for eukaryotic initiation sites (CC(A/G)CCATGG; (30) ). An in-frame TGA translation termination codon is found
at positions 616-618. The deduced polypeptide sequence consisted
of 205 amino acids with a calculated molecular mass of 23,510 daltons.
All microsequenced peptides of the native protein were found in the
full-length clone. While this work was in progress, the cDNA derived
amino acid sequence of GCAP from several vertebrate species was
published representing a novel subfamily of
Ca-binding proteins(18, 19) . Our
sequence is identical to the bovine homologue. Three canonical
Ca
-binding motifs (EF-hands) were present in the
sequence (see shaded parts in Fig. 2).
Figure 2:
cDNA and deduced amino acid sequence
(single letter code). Sequences of proteolytically derived peptides
(P1-P6) of native GCAP are marked by black lines. Three
putative canonical EF-hands for Ca binding are shaded. Primers 1 and 2 were used to amplify a fragment via
PCR.
Figure 3: Purification of recombinant GCAP. SDS-PAGE summary of the different purification steps: low molecular mass (LMW) standard; 2 µg each of E. coli lysate, unbound fraction of the glutathione S-transferase-agarose chromatography, eluate from glutathione S-transferase-agarose after thrombin cleavage, and MonoQ fraction containing GCAP activity.
The
calcium-dependent activation of retGC by the native and the two
recombinant GCAP forms was tested at different free
[Ca] from 1 nM to 150
µM. Native GCAP stimulated GC activity 5-10-fold
with high cooperativity in the physiologically significant range from
50 to 400 nM (EC
= 100 nM; Hill
coefficient n = 2.5). The ratio of GCAP to rhodopsin in Fig. 4B was 1:800. Activation of retGC by recombinant
GCAP was also Ca
-dependent and maximal retGC activity
at low Ca
increased with the amount of GCAP (data not
shown). However, several differences to the native GCAP were observed.
The degree of cooperativity was lower (Hill coefficient n = 2.0) and the EC
value has shifted to a
higher value of 261 nM (Fig. 4B). A lower
degree of cooperativity and a shift in the EC
value was
observed at different concentrations of GCAP tested. Sometimes we
observed a slight decrease of retGC activity at free
[Ca
] below 50 nM where an excess
of EGTA is used (see for example Fig. 4B). When we
evaluated our data in Fig. 4B by applying the curve
fitting program only to the values in the range from 150 µM to 50 nM free [Ca
], we
obtained for native GCAP an EC
of 97 nM and a
Hill coefficient of 2.3 and for recombinant GCAPT an EC
of
210 nM and a Hill coefficient of 1.5. GCAP harboring six
additional amino acids at the N terminus (GCAPX) activated retGC in a
similar fashion.
The effect of recombinant GCAP was most efficient
at rather high concentrations of GCAP. In order to exclude any
nonspecific protein-protein interactions, samples were incubated in the
presence of 0.03 mg of bovine serum albumin. Incubation with bovine
serum albumin alone did only result in basal nonstimulated retGC activity. The addition of anti-GCAP antibody inhibited the
Ca-sensitive regulation of retGC.
Figure 5: Immunodetection of GCAP. A, ROS proteins separated by SDS-PAGE and stained with Coomassie Blue (CB). GCAP in ROS was detected by the anti-GCAP antibody (1:5000) after Western blotting (WB). B, Nomarski micrograph showing the retinal layers. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. C, GCAP-like immunoreactivity as revealed by indirect immunofluorescence. Staining was found throughout the photoreceptor layer but was most prominent in the outer segments of rods and cones. Arrows indicate some cone outer segments. The arrowhead indicates a cone soma. Cone axons project to the outer plexiform layer where processes from their pedicles form a dense network. Somata and processes of rods are less intensely stained. Scale bar indicates 50 µm.
The anti-GCAP antibody was used to localize GCAP in vertical sections of the bovine retina. GCAP-like immunoreactivity was found in the photoreceptor layer. Staining was prominent in outer segments of both rods and cones but was also present in other compartments of the photoreceptor like soma, axon, and axon terminal. In addition to rods, cones were heavily labeled. Cone outer segments, somata, axons, and pedicles could be clearly identified (Fig. 5). In the inner retina, only occasionally small somata in the inner nuclear layer and the ganglion cell layer were stained (not shown). Except for this extremely rare amacrine cell population, no other cell types were found to be immunoreactive. No staining was observed without the primary antibody and after preadsorption with recombinant GCAP (not shown).
The decrease of cytoplasmic [Ca]
in a vertebrate photoreceptor cell is considered to be a principal
control step that modulates phototransduction and is critical for light
adaptation. One of the key events for recovery from a light pulse and
for light adaptation is the increase of retGC activity at low
[Ca
]. We have demonstrated that a
calcium-binding protein with a calculated molecular mass of 23.51 kDa
serves as a cytoplasmic activator of retGC. Following a different
strategy in purification and cloning we identified the same activator
protein that Palczewski et al. have described as
GCAP(18) . Our retGC activities were comparable with values in
the literature for whole ROS (16, 22, 25, 28) . Activation of
retGC in ROS membranes by purified native GCAP was identical to the
activation measured in whole ROS regarding Ca
sensitivity, cooperativity, and maximal stimulation. Using the
physiological substrate GTP we measured higher activities than Gorczyca et al. obtained with a phosphorothioate analogue of
GTP(17, 27) .
Functional expression of recombinant
GCAP has not been reported so far and allowed us a functional
comparison with the native protein. Furthermore we could address
specific questions concerning the molar ratios (i.e. cellular
concentrations) of retGC and GCAP and the role of fatty acid
modification on GCAP. Native and recombinant GCAP exhibited different
properties with respect to activation of retGC. The maximal activation
at low [Ca] with native GCAP was exactly
the same as it is observed in whole ROS preparations. It was
half-maximal at 100 nM and showed a high cooperativity (Fig. 4B). Efficient activation with recombinant GCAP
was achieved at higher concentrations of GCAP (Fig. 4B). We observed a change in the EC
value and a lower degree of cooperativity when recombinant GCAP
was assayed. Several reasons for the observed differences are
conceivable: 1) Because heterologous expression in E. coli yields recombinant proteins without post-translational
modification, a lack of N-terminal acylation in recombinant GCAP could
cause the change in the EC
value and in cooperativity. The
amino acid sequence of GCAP contains the consensus sequence for
N-terminal myristoylation at Gly
. Heterogeneous N-terminal
acylation of native GCAP was demonstrated by mass
spectometry(18) . In addition, the N-terminal part of GCAP
seems to be critical for the activation process as investigated by
peptide competition experiments(18) . (2) Our
recombinant GCAP preparation could have an impaired Ca
binding or could be partially denatured or misfolded due to
sonification during the lysis step (see ``Experimental
Procedures''). However, the Ca
-induced
conformational change monitored by the mobility shift assay (Fig. 4A) works to the same extent in native and
recombinant GCAP forms.
Preliminary cross-linking experiments (data not shown) indicated that GCAP interacts with retGC and that retGC exists in native ROS membranes as an oligomer. An oligomeric form of retGC was also suggested from previous gel filtration studies that showed elution of GC activity in association with a high molecular weight complex(11, 31, 32) . We speculate that GCAP could act on an oligomeric form of retGC.
We cannot
exclude at the moment that additional factors play a significant role
in retGC activation. Dizhoor et al. reported that human retGC
could be activated at low [Ca] by an
extract partially purified from whole retinae(20) . Enrichment
of a 24-kDa protein correlated with retGC stimulating activity.
Starting from fresh bovine ROS preparations, retGC stimulating activity
correlated in our chromatographic fractions only with GCAP. As far as
we can judge from Coomassie Blue- or silver-stained gels, no other
protein of similar molecular mass (16-26 kDa) was detectable.
Low concentrations of ATP (100 µM, below the
concentration that causes a competitive interference with the
substrate) enhanced retGC activity independent of the free
[Ca]. We used 100 µM ATP in
all retGC assays to maximize the response. Wolbring and Schnetkamp
recently reported that retGC activity can be increased by
ATP(28) . This increase was inhibited by specific protein
kinase C inhibitors, suggesting that a phosphorylation step plays an
additional modulatory function.
The polyclonal anti-GCAP antibody was an effective tool in immunocytochemical and Western blotting studies. Strong labeling of the photoreceptor layer in the bovine retina is in agreement with a specialized function of GCAP in controlling retGC activity. Labeling of cone cells also indicates that the same or a very similar form of GCAP exists in both rods and cones.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X95352[GenBank].