(Received for publication, May 9, 1995; and in revised form, July 19, 1995)
From the
Guanylyl cyclase activating protein (GCAP1) has been proposed to
act as a calcium-dependent regulator of retinal photoreceptor guanylyl
cyclase (GC) activity. Using immunocytochemical and biochemical
methods, we show here that GCAP1 is present in rod and cone
photoreceptor outer segments where phototransduction occurs.
Recombinant and native GCAP1 activate recombinant human retGC (outer
segment-specific GC) and endogenous GC(s) in rod outer segment (ROS)
membranes at low calcium. In addition, we isolate and clone a retinal
homolog, termed GCAP2, that shows 50% identity with GCAP1. Like
GCAP1, GCAP2 activates photoreceptor GC in a calcium-dependent manner.
Both GCAP1 and GCAP2 presumably act on GCs by a similar mechanism;
however, GCAP1 specifically localizes to photoreceptor outer segments,
while in these experiments GCAP2 was isolated from extracts of retina
but not ROS. These results demonstrate that GCAP1 is an activator of
ROS GC, while the finding of a second activator, GCAP2, suggests that a
similar mechanism of GC regulation may be present in outer segments,
other subcellular compartments of the photoreceptor, or other cell
types.
In vertebrate photoreceptor cells, the synthesis and hydrolysis
of cyclic GMP (cGMP) are critical steps in phototransduction. In
response to light, a cascade of reactions in the photoreceptor outer
segment leads to the hydrolysis of cGMP and the closure of cGMP-gated
cation channels in the outer segment plasma membrane. As a consequence,
there is a reduction in the amount of calcium entering the cell.
Calcium efflux owing to the Na:K
,
Ca
exchanger, however, is unaffected by light,
resulting in a net decrease in the concentration of internal free
calcium. This decrease in the calcium concentration leads to the
activation of guanylyl cyclase (GC), which in part restores the dark
conditions of the photoreceptor cell (reviewed by Lagnado and
Baylor(1992)).
Photoreceptor GC, a member of the particulate GC
family (Koch, 1991; Shyjan et al., 1992; Goraczniak et
al., 1994; Umbarger et al., 1992; Dizhoor et
al., 1994; Liu et al., 1994), responds to an activator
that senses changes in the calcium concentration (Lolley and Racz,
1982; Koch and Stryer, 1988). Recently, we proposed that a calcium
binding protein isolated from rod outer segments (ROS), ()guanylyl cyclase activating protein (GCAP, termed here
GCAP1), mediates this process (Gorczyca et al., 1994a), and
its molecular properties were described (Palczewski et al.,
1994). GCAP1 restores the calcium sensitivity of GC in a reconstituted
system, and it decreases the sensitivity, time-to-peak, and recovery
time of the light response following its introduction into intact ROS
(Gorczyca et al., 1994a). The molecular cloning of GCAP1 from
bovine, human, mouse, frog (Palczewski et al., 1994), and
chicken (
)retina and the genomic organization of mouse and
human GCAP1 (Subbaraya et al., 1994) demonstrate strong
sequence conservation between species, conservation of three putative
calcium binding loops, and relatedness to other neuronal
calcium-binding proteins of the calmodulin superfamily. Transcripts
encoding GCAP1 were localized to photoreceptor cells by in situ hybridization (Palczewski et al., 1994; Subbaraya et
al., 1994), but the precise localization of the protein is not
known. Independently, Dizhoor et al.(1994) proposed that
another protein, p24, was responsible for the calcium sensitivity of
photoreceptor GC.
In this paper, we describe the cellular localization of bovine GCAP1 by immunocytochemical and biochemical methods and provide further evidence that GCAP1 is a key element in the activation of photoreceptor GC. In addition, we show that the retina contains a second GC activator, GCAP2, that is identical with p24 and closely related to GCAP1.
Figure 5: cDNA sequence of bovine GCAP2 and amino acid sequence alignment of bovine GCAP2 and GCAP1. A, composite cDNA sequence derived from PCR clones G3 and G5. The degenerate sense primer W285 (I = inosine, Y = pyrimidines, P = purines) and the proteolytic GCAP2 peptide are highlighted. The three putative EF-hand calcium binding sites (EF2-4) are shaded. The amino acid and nucleotide numbering is shown on the right. B, amino acid sequence alignment of GCAP1 and GCAP2. The deduced GCAP2 sequence was aligned with bovine GCAP1, and, for optimal alignment, several gaps (hyphens) were introduced. Putative calcium binding loops are shaded in gray, while amino acids illustrated in white on black background represent identity or conservative replacement (L = I = V = M; Y = F; K = R; S = T = A). The bar above the GCAP2 sequence represents the sequence obtained by peptide microsequencing.
Ten-µm cryosections were blocked with 1% (w/v) bovine serum
albumin, 1% (v/v) normal goat serum, 0.2% Triton X-100 in 0.1 M phosphate buffer, pH 7.4, for 1 h at room temperature. Sections
were incubated with anti-GCAP antibodies at 0.8 mg/ml total IgG either
for 2 h or overnight. After several washes, sections were incubated for
1 h with a biotinyl-goat anti-rabbit IgG at a concentration of 3
µg/ml. Antibody binding was detected using streptavidin-Texas red
at 1 µg/ml. (Antibodies and streptavidin-Texas red were diluted
with blocking solution.) Sections were rinsed, dehydrated with ethanol,
cleared with xylene, coverslipped, and viewed with a Zeiss Universal
fluorescence microscope using a 25 neofluar objective.
Figure 1:
Reverse phase
chromatography of truncated GST-GCAP1 fusion protein (GST-GCAP180). The
expressed GST-GCAP180 was isolated as inclusion bodies and solubilized
in 5 M urea (Palczewski et al., 1994). The protein
was dialyzed extensively against 10 mM BTP buffer, pH 7.5, and
treated with thrombin (Qin and Baehr, 1993). Acetonitrile was then
added to yield a final concentration of 15%, and the sample was loaded
on a C-4 column (W-Porex 5 C4, 4.6 150 mm, Phenomenex)
equilibrated with 30% acetonitrile in 5 mM BTP, pH 7.5
(Gorczyca et al., 1994a). GCAP180 was eluted with a linear
gradient of acetonitrile (30-60% during 20 min) in 5 mM BTP, pH 7.5, at a flow rate of 1.5 ml/min, and 0.75-ml fractions
were collected. GCAP180 was eluted at
40% acetonitrile. SDS-PAGE
analysis is presented on the combined fraction eluted at
10
min.
Figure 2: Anti-GCAP1 antibody staining of bovine retina fixed in paraformaldehyde and cryoprotected with sucrose (see ``Materials and Methods''). Bovine rod and cone outer segments were stained with IgG purified antibodies to GCAP1 (A). Arrow shows a cone outer segment. GCAP1 antibody binding was significantly reduced by a 1-h preincubation of the antibody with native GCAP1 (B). In some experiments, cone inner segments and synaptic processes were stained in addition to rod and cone outer segments (C). Arrow indicates labeling of a cone process.
We further investigated the distribution of GCAP1 in purified bovine ROS and retinas using monoclonal antibody (mAb) G-2 coupled to CNBr-activated Sepharose. When a ROS extract was applied to the mAb G-2 column, all GC-stimulating activity was absorbed. At low pH, the activity was almost quantitatively eluted as a single peak (Fig. 3A, top panel). The GC-stimulating activity correlated with the elution of GCAP1, as identified by immunoblotting (inset in Fig. 3A, top panel) and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (Fig. 3B, top panel).
Figure 3:
Immunoaffinity chromatography of ROS and
retinal extracts. GCAP1 was extracted from 25 retinas or from ROS
prepared from 50 retinas, as described under ``Materials and
Methods.'' The extracts were loaded onto an immunoaffinity column
containing mAb G-2 coupled to CNBr-Sepharose. The fractions which
passed through the column during loading were collected (fractions
1-6), the column was washed with 10 mM BTP buffer, pH
7.5, containing 200 mM NaCl, and then with 10 mM BTP,
pH 7.5 (fractions 7-19), and bound proteins were eluted with 0.1 M glycine, pH 2.5 (fractions 20-27). Eluted fractions
were immediately neutralized with 1 M Tris/HCl, pH 8.4. A, GC stimulating activity in ROS and retinal extracts.
5-µl aliquots of the fractions were tested to determine their
effect on GC activity using washed ROS membranes in the presence of
either 45 nM Ca (open circles) or 1
µM Ca
(closed circles).
Normalized GC activity was calculated by assuming that the total
stimulatory activity present in each fraction was recovered in the
volume of 1 ml. Insets: 1-µl aliquots from the indicated
fractions were separated by SDS-PAGE, transferred to Immobilon P, and
immunostained with pAb UW-14 (lane L, loaded extract). B, SDS-PAGE analysis. 20 µl of the indicated fractions
from ROS (upper panel) or a retinal extract (middle
panel) were separated using 12% acrylamide gels and stained with
Coomassie Brilliant Blue R-250. Lanes L, extracts before
chromatography; lanes S, standard proteins of known molecular
weights (from the top: 92-, 67-, 43-, 30-, 21-, and 14-kDa
standards; Pharmacia Biotech Inc.). Open arrow indicates
position of GCAP2. In control experiments, a mouse monoclonal antibody
raised against purified peripherin (concentration of antibody and
purification identical with mAb G-2 and the size of the column
comparable to the mAb G-2 column) was coupled to CNBr-activated
Sepharose, and ROS and retinal extracts were passed through the column.
Only proteins that interact with immunoglobulins and Sepharose bound
(like transducin from ROS extract) and were subsequently eluted, while
GCAP1 and GCAP2 passed through. Note the high density of mAb G-2 (6
mg/ml) that resulted in a complete adsorption of GCAP1 or GCAP2;
however, it also yielded nonspecific adsorption of other contaminating
proteins. C, reactivity of purified ROS and retinal extracts
with anti-GCAP1 antibodies. 1-µl aliquots from fraction 21 after
immunoaffinity chromatography of ROS (a) or a retinal extract (b) were separated by SDS-PAGE, transferred to Immobilon, and
immunostained with antibodies: I, mAb G-2 (monoclonal against
expressed GCAP1; II, pAb UW-14 (polyclonal against expressed
GCAP1); III, pAb GS-31 (polyclonal against C-terminal peptide
from GCAP1); and IV, pAb GS-35 (polyclonal against N-terminal
peptide from GCAP1). Open arrow indicates GCAP2 staining with
mAb G-2 only.
Thus, biochemical analyses revealed that GCAP1 is associated with photoreceptor outer segments, consistent with immunolocalization studies, while in these experiments GCAP2 was detected in retinal but not ROS preparations.
Figure 4: Separation of GCAP1 and GCAP2 by immunoaffinity chromatography of the retinal extract on pAb GS-31 and mAb G-2. The retinal extract was loaded on pAb GS-31 as described in the legend to Fig. 2. GCAP1 was eluted with a 1 mM concentration of the C-terminal peptide, and the GC-stimulating activity was determined (solid line, top). The column was washed with 0.1 M glycine (pH 2.5) and re-equilibrated with 10 mM BTP, pH 7.5. The procedure was repeated 4 times. The flow-through from the pAb GS-31 column (containing GCAP2) was loaded on mAb G-2 column, and GCAP2 was eluted at low pH as described in Fig. 2. The activity profile is shown as the solid lines, and the bar represents 2 nmol/min/mg GC activity. Inset, SDS-PAGE of the eluted fractions from pAb GS-31 (lane 1) and mAb G-2 (lane 2). S denotes molecular weight standards, p18 indicates the electrophoretic mobility of GCAP2.
To clone GCAP2,
peptides were generated by in situ proteolysis with Lys-C
endoproteinase and purified by reverse phase HPLC. One GCAP2 peptide
was blocked and not suitable for Edman degradation, while a second
peptide yielded amino acid sequence (VPDNEEATQYVEAMFRAFDTNGDNTIDFLEY)
homologous to GCAP1 (Fig. 5A). Importantly, the
sequence obtained for the fragment of GCAP2 is identical with the
corresponding portion of p24, a protein purified by Dizhoor et
al.(1994). A degenerate oligonucleotide primer reversely
translated to the portion of the peptide that was most different from
GCAP1 (underlined) was used to clone GCAP2 after PCR amplification. The
results show that the molecular mass, based on the predicted amino acid
composition (Fig. 5A) (204 residues), is 23,759
daltons, and that the amino acid sequence is homologous to GCAP1
(53%) (Fig. 5B). The amino acid sequence of GCAP2
showed that it is also related to other Ca
-binding
proteins of the EF-hand superfamily, including calmodulin and troponin
C. Members of this family contain 2-4 canonical calcium binding
motifs composed of ``loops'' of 12 contiguous residues with
oxygen atoms involved in calcium coordination and two flanking
-helices that stabilize this complex (Kretsinger, 1980; Strynadka
and James, 1989). GCAPs are most closely related to recoverin (Fig. 6); however, they differ in the number of functional
calcium binding loops (three versus two) and they are more
acidic (pK
= 4.1-4.4 versus 5.1). GCAP2 also has Gly
that is a putative
site of heterogeneous tissue-specific acylation (Johnson et
al., 1994), as well as a consensus sequence for protein kinase A
phosphorylation at residue Ser
.
Figure 6:
Dendogram of calcium-binding proteins.
Computated dendogram (PC-Gene, Intelligenetics, Inc.) for protein
sequence similarities between GCAPs and recoverin. The sequences were
taken from the following references: bovine, frog, human, and mouse
GCAP1 (Palczewski et al., 1994; Subbaraya et al.,
1994); chicken GCAP1; human GCAP2 (W. Baehr, unpublished
data); bovine recoverin (Dizhoor et al., 1991); mouse
recoverin (McGinnis et al., 1992); human recoverin (Murakami et al., 1992); and frog recoverin (W. Baehr and I. Subbaraya,
unpublished data).
The discrepancy in the mobility of GCAP2 noted in the literature is likely a result of addition of EGTA to the sample buffer during SDS-PAGE (Dizhoor et al., 1994) and absence of the chelator in our samples as shown in Fig. 7.
Figure 7:
SDS-PAGE mobility of GCAP1 and GCAP2 at
low and high calcium. SDS-PAGE of the mixture of GCAP1 and GCAP2 in the
presence (+) and absence(-) of 1 mM
CaCl. The sample without CaCl
contained 1
mM EGTA.
Figure 8:
Stimulation of GC in ROS membranes by
GCAP1 and GCAP2. A, the saturable effects of GCAP1 and GCAP2
on GC activity. Washed ROS (30 µM rhodopsin in 64 µl)
in 50 mM Hepes, pH 7.8, containing 60 mM KCl, 20
mM NaCl, 10 mM MgCl at 45 nM free calcium were incubated with increasing amounts of GCAP1 or
GCAP2. The GC activity was determined as described under
``Materials and Methods.'' B, lack of the additive
stimulation of GC in ROS membranes by GCAP1 and GCAP2. Washed ROS (30
µM rhodopsin in 64 µl) in 50 mM Hepes, pH
7.8, containing 60 mM KCl, 20 mM NaCl, 10 mM MgCl
at 45 nM free calcium were incubated
with either 1.2 µg of GCAP1, 1.0 µg of GCAP2, or both. C, calcium titration of GC activity. The calcium sensitivity
of GC was assayed in washed ROS membranes with either 0.3 µg of
GCAP1 (closed circles) or with 0.25 µg of GCAP2 (closed squares). The free calcium concentrations were
adjusted by EGTA-Ca buffer as described under ``Materials and
Methods.'' Experimental conditions were identical with those
described by Gorczyca et al. (1994a); however, note that in
experiments in A, GC was maximally stimulated by GCAP1 or
GCAP2 before addition of GCAP2 or GCAP1, respectively. The GC activity
in C is a calcium titration in the presence of GCAPs at
half-saturating concentrations.
Washed ROS were incubated with various amounts of purified GCAP1, the binding of GCAP1 then was determined by immunoblotting, and the GC activity was assessed in aliquots from the same samples. GCAP1 bound to membranes in the presence of moderate ionic strength buffer, and the binding of GCAP1 correlated with increased GC activity in ROS membranes (Fig. 9). The binding was independent of the calcium concentration. These results suggest that at physiological conditions, GCAP1 remains in a complex with GC/membranes despite changes in the free calcium concentration.
Figure 9:
Binding of GCAP1 to ROS membranes. Washed
ROS (30 µM rhodopsin in 64 µl) in 50 mM Hepes, pH 7.8, containing 60 mM KCl, 20 mM NaCl,
10 mM MgCl at 45 nM free calcium were
incubated with increasing amounts of purified GCAP. The membranes were
collected by centrifugation, washed with 100 µl of the same buffer,
and finally suspended in 50 µl of 10 mM Hepes, pH 7.5. A
portion of each sample was assayed for GC activity, while GCAP was
identified by Western blot analysis using mAb G-2 (inset).
Figure 10:
Stimulation of GC in ROS membranes or
recombinant retGC with expressed GCAP1. GCAP1 (0.25 µg) was assayed
for GC-stimulating activity in low (-; 30 nM) and high
(+; 1 µM) calcium using GC in washed ROS membranes (open bars, 80 µg of rhodopsin) or expressed retGC (shaded bars, 120 µg of total protein). retGC was
expressed in human embryonic kidney 293 cells that were produced as
described by Shyjan et al.(1992). Expression of GCAP1 in
High-Five insect cells was described under ``Materials and
Methods.'' Note that the GC activity is measured with GTP for
retGC and GTPS for GC in ROS membranes, and the figure represents
qualitatively similar activation of both GCs at low free calcium
concentration.
Several independent lines of evidence suggest that GCAPs are involved in the calcium-sensitive regulation of GC. (i) A combination of chromatographic procedures, independent of the calcium binding properties of the proteins, led to the purification of GCAP1 and GCAP2, which are homologous proteins. Chromatographic profiles of protein purification and GC stimulating activity are consistent with GCAP1 and GCAP2 activating GC in reconstitution experiments using washed ROS membranes (Palczewski et al., 1994; Gorczyca et al., 1994a, 1994b; Dizhoor et al., 1994) and expressed retinal GC (Fig. 10). Furthermore, expressed GCAP1 activates GC in a calcium- and dose-dependent manner, similar to the native protein (Fig. 10). (ii) mAb G-2, raised against bacterially expressed GCAP1, inhibits the calcium regulation of GC (Palczewski et al., 1994), and selectively purifies GCAPs from retinal extracts. (iii) GCAP1 is present in rod and cone outer segments, sites of calcium-sensitive regulation of GC. The occasional staining of cone processes may correlate with the presence of cGMP-regulated calcium channels (Cook et al., 1989; Rieke and Schwartz, 1994). Alternatively, the variability in cone labeling may reflect the adaptational state of the tissues. The bovine, human, and monkey retinas used in the immunohistochemical experiments are at least partially light-adapted, and placement of these autopsy tissues in the dark before fixation may not reproducibly reset the mechanisms governing the distribution of GCAP1 in cone cells. pAb UW-14 used for immunohistochemical staining did not react with mouse retinas, which would normally be used to explore differences between light- and dark-adapted conditions.
The major GC activity in ROS membranes can be attributed to retGC (Shyjan et al., 1992; Goraczniak et al., 1994; Umbarger et al., 1992; Koch, 1991; Dizhoor et al., 1994; Liu et al., 1994). A second retina-specific GC (GC F; retGC-2) has been identified recently (Yang et al., 1995; Lowe et al., 1995); however, the subcellular localization of this cyclase remains to be established. Considering the present data, one intriguing hypothesis would propose that both cyclases, retGC-1 (retGC) and retGC-2, are regulated in a calcium-dependent manner by their respective calcium sensors, GCAP1 and GCAP2. Indeed, retGC-2 is activated by purified GCAP2 (Lowe et al., 1995) in the same calcium-dependent manner as shown in this report (Fig. 8). The availability of recombinant GC(s) and GCAPs will assist in further deciphering their interactions, their subcellular localization, kinetics, and calcium binding properties.
The localization of GCAP2 is not yet known; however, it was not detected in our ROS extracts (Fig. 3). The amount of GCAP2 in ROS compared to retinal extracts suggests that this protein may be localized in the photoreceptor inner segments or in other parts of the retina. If correct, these observations suggest that regulation of particulate GCs by a small, acidic, calcium-binding protein might be a general phenomenon occurring in other retinal cells and possibly other sensory transduction systems. Alternatively, GCAP2, in our experiments, may be selectively lost during the purification of ROS and, like GCAP1, therefore would be involved in the regulation of retGC.
While more than 200 calcium-binding proteins of the EF-hand superfamily have been identified, the function is known for relatively few. Owing to the elucidation of the biochemical steps involved in vertebrate phototransduction, the function of several calcium-binding proteins has been assigned. Calmodulin regulates the sensitivity of the cyclic GMP-gated channel in response to light-induced changes in the levels of ligand (Hsu and Molday, 1993). Recoverin (Dizhoor et al., 1991; Polans et al., 1991; Kawamura and Murakami, 1991; Lambrecht and Koch, 1991) alters the kinetics of photoresponse recovery (Gray-Keller et al., 1993), perhaps by inhibiting the phosphorylation of rhodopsin (Kawamura, 1993) through direct interaction with rhodopsin kinase (Gorodovikova and Philippov, 1993; Gorodovikova et al., 1994a, 1994b). In this and previous reports, we demonstrate that GCAPs regulate the activity of photoreceptor GC(s). Unlike other calcium-binding proteins, GCAPs activate their target at low calcium concentrations. Of general interest, calcium and cyclic nucleotides act as second messengers to mediate a variety of physiological responses; GCAPs provide an intersection between these two small molecules and may offer the cell additional stages of regulation. It will be interesting to see whether molecules similar to GCAPs are associated with other transduction pathways involving cyclic nucleotides and calcium.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L43001[GenBank].