(Received for publication, January 24, 1996; and in revised form, March 5, 1996)
From the
Retinal guanylyl cyclase-1 (RetGC-1) is a membrane guanylyl
cyclase found in photoreceptor outer segments. It consists of an
apparent extracellular domain (ECD) linked by a single transmembrane
segment to an intracellular domain (ICD). Guanylyl cyclase activating
protein-2 (GCAP-2) is a Ca-binding protein that
activates RetGC-1 in a Ca
-sensitive manner. To
establish whether GCAP-2 stimulates RetGC-1 through the ECD or ICD, we
made deletion mutants lacking either the ECD or both the ECD and
transmembrane domains (TMD) of RetGC-1. Recombinant wild type RetGC-1
and both deletion mutants were expressed in HEK 293 cells, and their
sensitivities to GCAP-2, Ca
, and ATP were compared.
Our data demonstrate that both deletion mutants are regulated similarly
to wild type RetGC-1 with indistinguishable EC
values for
Ca
and similar K
values for
activation by GCAP-2. This shows that GCAP-2 functions through the ICD
of RetGC-1 and that removal of the ECD and TMD do not significantly
alter regulation by these factors. Our data also show that ATP
potentiates stimulation of guanylyl cyclase activity by GCAP-2 and that
neither the ECD nor the TMD of RetGC-1 participate in its regulation by
ATP.
Photoexcitation of retinal rod cells stimulates hydrolysis of
intracellular cGMP. This reduces the activity of cGMP gated cation
channels in the rod outer segment plasma membrane, blocks Na and Ca
influx, and allows a
Na
/Ca
,K
exchanger
to decrease the concentration of free cytoplasmic Ca
(reviewed in Lagnado and Baylor(1992)). At low concentrations of
free Ca
, a soluble factor stimulates guanylyl cyclase
(GC) (
)activity in photoreceptor membranes (Koch and Stryer,
1988). It has now been shown that this stimulatory activity is
represented by at least two Ca
-binding proteins,
GCAP-1 and GCAP-2 (Dizhoor et al., 1995; Gorczyca et
al., 1995). It has been proposed that the light-induced decrease
in free cytoplasmic Ca
concentration stimulates GC
activity in vivo. Such a feedback mechanism would stimulate
resynthesis of cGMP and enhance photoreceptor recovery and/or light
adaptation following photoexcitation (reviewed in Lagnado and
Baylor(1992) and McNaughton(1990)).
RetGC-1 and RetGC-2 are two
photoreceptor GCs that were cloned from a human retinal cDNA library
(Shyjan et al., 1992; Lowe et al., 1995). ()Homologues of RetGC-1 have also been cloned from bovine
and rat eye cDNA libraries, and a homologue of RetGC-2 was isolated
from a rat eye cDNA library (Goraczniak et al., 1994; Yang et al., 1995). RetGC-1 and RetGC-2 are expressed in
photoreceptors and associate with the membrane fraction of
photoreceptor outer segments (OS) (Shyjan et al., 1992,
Dizhoor et al., 1994; Lowe et al., 1995).
Immunofluorescence studies suggest that RetGC-1 is localized primarily
in cone OS and to a lesser extent in rod OS (Dizhoor et al.,
1994; Liu et al., 1994). Electron microscopy studies further
indicate that RetGC-1 is associated with the membrane-rich regions of
OS (Liu et al., 1994). The GC activities of OS membranes and
recombinant RetGC-1 are activated by the photoreceptor
Ca
-binding proteins GCAP-1 and GCAP-2, and
recombinant RetGC-2 is activated by GCAP-2 (Dizhoor et al.,
1995; Gorczyca et al., 1995; Lowe et al., 1995).
Activation by either GCAP is inhibited by Ca
with an
EC
for Ca
near 200 nM (Dizhoor et al., 1994; Gorczyca et al., 1994a, 1995; Dizhoor et al., 1995). This value agrees well with the range of bulk
free Ca
(50-550 nM) recently measured
in intact OS in darkness and following a flash of light (Gray-Keller
and Detwiler, 1994). The localization of RetGC-1 and RetGC-2 and their
sensitivity to Ca
and GCAP-2 suggest that they
function in the recovery of photoreceptors from photoexcitation
(Dizhoor et al., 1994; Lowe et al., 1995).
A
factor that influences the Ca-sensitive stimulation
of OS GCs is ATP. It has been reported that ATP or nonhydrolyzable ATP
analogues potentiate the Ca
-sensitive stimulation of
OS GCs in whole OS and in washed OS reconstituted with GCAP-1 (Gorczyca et al., 1994b). Both RetGC-1 and RetGC-2 are members of the
membrane GC family that includes the natriuretic peptide receptor-GCs
(NPR-A/GC-A and NPR-B/GC-B), the heat stable enterotoxin or guanylin
receptor-GC (StaR/GC-C), and the sea urchin sperm GCs (Shyjan et
al., 1992; Garbers and Lowe, 1994; Lowe et al., 1995). It
has been clearly shown that the stimulation of other members of this
family is influenced by adenine nucleotides. The stimulation of NPR-A
requires (Chinkers and Garbers, 1991; Marala et al., 1991),
and the stimulation of StaR is prolonged by (Vaandrager et
al., 1993a) the presence of ATP or nonhydrolyzable ATP analogues.
Each member of the membrane GC family is a type I transmembrane
protein that has a ligand-binding extracellular domain (ECD) linked by
a single transmembrane domain (TMD) to an intracellular domain (ICD).
Within the ICD the membrane-proximal region is homologous to protein
kinases (KHD). Adjacent to the KHD is a small domain that is likely to
form an amphipathic -helix and for which a clear role in the
dimerization of the NPR-A intracellular domain has been established
(Wilson and Chinkers, 1995). The C-terminal portion of the ICD contains
the cyclase catalytic domain. Based on the homology between RetGC-1,
NPR-A, NPR-B, and StaR and on hydropathy analysis, putative assignments
have been made for the extracellular and intracellular domains of
RetGC-1 (see Fig. 1) (Shyjan et al., 1992; Lowe et
al., 1995; Wilson and Chinkers, 1995). The orientation of RetGC-1
in membranes has yet to be determined experimentally.
Figure 1:
Schematic representation of the RetGC-1
constructs used in this study. Assignments of domains and their
boundaries are based on the alignment of RetGC-1 with NPR-A, NPR-B, and
StaR. SP, signal peptide; ECD, extracellular domain; TMD, transmembrane domain; KHD, kinase homology
domain; CAT, cyclase catalytic domain. DD indicates a
putative dimerization domain. The 41 amino acids shown to be the
minimum requirement for dimerization of NPR-A (Wilson and Chinkers,
1995) correspond to Ile-Leu
of
RetGC-1 and share 46% identity and 71% homology. Furthermore, helical
wheel analysis of this stretch of amino acids for NPR-A (Wilson and
Chinkers, 1995) and RetGC-1 shows that it is likely that both sequences
form an amphipathic
-helix. CT indicates a hydrophilic C
terminus for which there is no counterpart in the NPRs. StaR, however,
has a hydrophilic C-terminal extension, but it is longer than and has
little homology to that of RetGC-1. ICD indicates the entire
intracellular region. The bar denotes the approximate location
of the Ala
-Gln
peptide used to
generate the RetGC-1-IC antibody.
Previously
characterized members of the membrane GC family are activated by the
binding of peptide ligands to their extracellular domains (reviewed by
Garbers(1992) and Garbers and Lowe(1994)). For example, the membrane GC
NPR-A is stimulated by the binding of atrial natriuretic peptide to its
ECD. In contrast, the Ca sensitivity of RetGC-1 and
RetGC-2 activity stimulated by GCAP-2 (Lowe et al., 1995), the
absence of a signal peptide in the GCAP-2 primary sequence (Dizhoor et al., 1995), and the presence of
Ca
-binding sites on GCAP-2 all suggest that
regulation of RetGC-1 and RetGC-2 by GCAP-2, Ca
, and
ATP occurs in the cytoplasm. To determine experimentally if GCAP-2 acts
either through the predicted ICD of RetGC-1 or through the ECD, we
expressed deletion mutants of RetGC-1 lacking the ECD or both the TMD
and ECD. Our results show that RetGC-1 is regulated by GCAP-2 through
the ICD and that removal of the ECD and TMD does not have a significant
effect on regulation by GCAP-2, Ca
, and ATP.
To determine whether the ECD or TMD of RetGC-1 play a role in
stimulation of GC activity by GCAP-2, deletion mutants were constructed
that lacked either the ECD (ECD RetGC-1) or both the ECD and TMD
domains (ICD RetGC-1) of RetGC-1 (Fig. 1). We then determined if
the truncated proteins could be regulated in a
Ca
-sensitive manner by GCAP-2. Both recombinant and
purified retinal GCAP-2 were used to carry out these experiments. We
also compared the effect of ATP on the truncated and wt forms of
RetGC-1 to further gauge how these truncations affected the regulation
of RetGC-1 activity.
Figure 2:
Expression and
Ca-sensitive stimulation of wt RetGC-1 and
ECD
RetGC-1 by GCAP-2. A, membranes from HEK 293 cells transiently
transfected with pRC CMV, pRC CMV
ECD RetGC-1, or pRC CMV wt
RetGC-1 were washed in 0.4 M NaCl, mixed with Laemmli sample
buffer, electrophoresed on a 7.5% SDS gel, and transferred to a
nitrocellulose membrane. Immunoblot analysis was carried out with the
RetGC-1-IC antibody to demonstrate expression of the recombinant
proteins. pRC CMV (B), pRC CMV
ECD RetGC-1 (C),
or pRC CMV wt RetGC-1 recombinant membranes (D) were assayed
for GC activity as described under ``Materials and Methods''
in the presence of BSA in 7 nM Ca
(lanes
a), 1.2 µM recombinant GCAP-2 in 7 nM Ca
(lanes b), or 1.2 µM recombinant GCAP-2 in 1.2 µM Ca
(lanes c). The bars represent the mean of
duplicate data points from a single experiment with the range indicated
by the error bars. The data shown are representative of six
independent experiments.
Figure 3:
Adenine nucleotides potentiate the
activation of ECD RetGC-1 and wt RetGC-1 by GCAP-2. GC activity
was measured for wt RetGC-1 (A) and
ECD RetGC-1 (B) as described under ``Materials and Methods.''
Recombinant membranes were washed in 0.4 M NaCl and assayed in
the presence of BSA (lanes a); 1.5 µM recombinant
GCAP-2 (lanes b); BSA and 0.5 mM ATP (lanes
c); 1.5 µM recombinant GCAP-2 and 0.5 mM ATP (lanes d); or 1.5 µM recombinant GCAP-2 and 0.5
mM AMP-PNP all in 7 nM Ca
(lanes e). All assay points contained a final free
Ca
concentration of 7 nM. The bars represent the mean of duplicate data points with the range
indicated by the error bars. The data shown are from one
experiment and is representative of six experiments with wt RetGC-1 and
two experiments with
ECD RetGC-1.
To examine whether the ECD influences the effect of ATP, we
examined the concentration dependence of the ATP effect on wt RetGC-1
and ECD RetGC-1 (Fig. 4). No significant difference in ATP
dependence was detected, indicating that the ECD is not involved in the
mechanism by which ATP potentiates activation of RetGC-1. For both wt
RetGC-1 and
ECD RetGC-1, the effect of ATP reaches a maximum near
0.5 mM ATP then decreases. The decrease in activity above 0.5
mM ATP may be due to competition for binding at the catalytic
site of RetGC-1 between ATP and the substrate, GTP. The nucleotide TTP
had no effect on the stimulation of catalytic activity by GCAP-2 (data
not shown).
Figure 4:
Concentration dependence of the effect of
ATP on stimulation of wt RetGC-1 and ECD RetGC-1 by GCAP-2.
Membranes washed with 0.4 M NaCl were assayed in the presence
of 1.5 µM recombinant GCAP-2 and 1 mM EGTA at the
indicated concentrations of ATP. The ordinate displays fold
potentiation of GCAP-2 stimulated GC activity by ATP. This was
calculated by subtracting basal GC activity (BSA) from the sample
activity and dividing by activity in the absence of ATP. The values for
the basal and stimulated activity (without ATP) from the two
experiments are reported under ``Materials and Methods.'' The
results shown for both wt RetGC-1 and
ECD RetGC-1 are the means of
the fold potentiation from two experiments, each experiment carried out
with duplicate data points. Error bars indicate the standard
deviation of the fold stimulation calculated for each of the four data
points.
Figure 5:
Expression of ICD RetGC-1 and its
regulation by GCAP-2, Ca, and adenine nucleotides. A, HEK 293 cells were transiently transfected with either pRC
CMV or pRC CMV ICD RetGC-1. Membranes from these cells were washed in
0.4 M NaCl, mixed with Laemmli sample buffer, and
electrophoresed on a 10% SDS gel. After transfer to nitrocellulose,
immunoblot analysis was carried out with the RetGC-1-IC antibody. A
mass of 68,178 was calculated from the expected amino acid sequence of
ICD RetGC-1 and approximates the relative molecular weight of the
recombinant protein recognized by the antibody. B and C, GC activity was measured as described under
``Materials and Methods'' on cell homogenates from HEK 293
cells transiently transfected with pRC CMV or pRC CMV ICD RetGC-1.
Assays were carried out in the presence of BSA in 7 nM Ca
(lanes a), 3 µM recombinant GCAP-2 in 7 nM Ca
(lanes b), 0.5 mM ATP and 3 µM recombinant GCAP-2 in 7 nM Ca
(lane
c), 3 µM recombinant GCAP-2 in 1.2 µM Ca
(lane d), 0.5 mM ATP and 3
µM recombinant GCAP-2 in 1.2 µM Ca
(lane e), or 0.5 mM AMP-PNP
and 3 µM recombinant GCAP-2 in 7 nM Ca
(lane f). Cell homogenates were used
in these experiments because the ability of ICD Ret GC-1 to be
stimulated by GCAP-2 is lost in membranes washed with 0.4 M NaCl. The bars represent the mean of triplicate data
points with the standard deviation indicated by the error bars. The
data are representative of six independent
experiments.
Figure 6:
Comparison of the Ca
sensitivity of GCAP-2 activation for wt RetGC-1,
ECD RetGC-1, and
ICD RetGC-1. GC activity was measured on HEK 293 cell homogenates as
described under ``Materials and Methods.'' Assays of wt
RetGC-1 (closed triangle),
ECD RetGC-1 (open
circle), and ICD RetGC-1 (open square) in the presence of
retinal GCAP-2 and 0.5 mM ATP were carried out at
Ca
concentrations ranging from 7 nM to 2.6
µM free Ca
. A Ca
-EGTA
buffering system was used to achieve the desired free Ca
concentrations. Duplicate data points are plotted from a single
experiment and are representative of two independent experiments. GC
activities were normalized for the
comparison.
Figure 7:
Concentration dependence of GC activation
by GCAP-2. The activity of wt RetGC-1, ECD RetGC-1, and ICD
RetGC-1 was measured in 1 mM EGTA, 0.5 mM ATP, and
the indicated concentrations of recombinant GCAP-2. GC activity was
plotted against the GCAP-2 concentration, and a curve was fit to each
plot using the equation v = [GCAP-2] V
/K
+
[GCAP-2] (Abelbeck Kaleidagraph software). Data shown are
from a single experiment and are representative of three independent
experiments. GC activity was normalized to the predicted V
. K
(± standard
error) values of 7.48 (± 0.59), 10.09 (± 0.88), and 2.28
(± 0.27) µM for wt RetGC-1,
ECD RetGC-1, and
ICD RetGC-1, respectively, were calculated from the above experiment.
Average K
values generated from this and two
additional experiments, both carried out with duplicate measurements at
each [GCAP-2], are reported in the results
section.
The data we present here show that the ECD and TMD of RetGC-1
are not required for its regulation by Ca and GCAP-2.
GCAP-2 must exert its action through the intracellular domain of
RetGC-1. RetGC-1 associates with the OS membranes of photoreceptors, is
glycosylated, and is predicted from the cDNA to have a signal peptide
for membrane localization on the N terminus (Dizhoor et al. 1994; Koch et al., 1994; Shyjan et al., 1992;
Lowe et al., 1995). These properties of RetGC-1 suggest that
the ECD is either extracellular or intradiscal and that a new mechanism
for regulating membrane GCs from the cytoplasm has now been
established. This does not, however, preclude RetGC-1 from also having
a yet to be discovered extracellular ligand analogous to those of
NPR-A, NPR-B, and StaR.
We also show that ATP potentiates but is not necessary for the stimulatory effect of GCAP-2 on RetGC-1 and that neither the ECD nor TMD are necessary for this effect. The observation that AMP-PNP also potentiates stimulation of RetGC-1 by GCAP-2 indicates that the effect of ATP is not due to phosphorylation or ATPase activity. In vivo, a 2-3-fold increase in the rate of cGMP synthesis could potentially have a large effect on the inward current of a photoreceptor OS because cGMP binding to the cGMP gated channels is cooperative (Fesenko et al., 1985). However, it has yet to be experimentally determined whether intracellular ATP levels vary enough to regulate RetGC-1 activity in vivo.
Based on available published data, it appears that the nonobligatory effect of adenine nucleotides on RetGC-1 differentiate it from NPR-A, for which ATP is a necessary cofactor for stimulation by atrial natriuretic peptide (Chinkers and Garbers, 1991; Marala et al., 1991). In contrast, ligand activation of StaR activity does not require ATP but is potentiated approximately 2-fold by ATP in a manner similar to our findings with RetGC-1 (Vaandrager et al., 1993a). The activation of immunoaffinity-purified NPR-A and StaR by ATP and their extracellular ligands has been reported (Vaandrager et al., 1993b; Wong et al., 1995). This strongly suggests that ATP regulates both receptors through direct binding. Interestingly, both RetGC-1 and StaR lack a glycine-rich nucleotide-binding motif, which is conserved in protein kinases and in the KHDs of both NPR-A and NPR-B (Koller et al., 1992; Shyjan et al., 1992). Mutations in the glycine-rich sequence make NPR-A insensitive to ATP and atrial natriuretic peptide (Goraczniak et al., 1992; Duda et al., 1993). These data taken together suggest that the effect of ATP on membrane GCs may be mediated by binding of ATP to the KHDs.
It is unclear why the effect of ATP
we observe for washed OS membranes is not as pronounced as when we use
recombinant RetGC-1. One possibility is that the state of
phosphorylation of RetGC-1 influences the effect of adenine
nucleotides. Recently, it was reported that ATP has a 2-fold
stimulatory effect on Ca-sensitive GC activity in
fresh intact OS (Wolbring and Schnetkamp, 1995). However, the effect of
ATP was lost by washing the OS or by adding inhibitors of protein
kinase C. The ATP effect could be restored by treating washed OS with a
purified preparation of brain protein kinase C. Together with our data,
these findings suggest that phosphorylation by protein kinase C may be
a prerequisite for a noncatalytic role of ATP in the potentiation of
RetGC-1 activation. Different states of phosphorylation of OS GC and
recombinant RetGC-1 might explain the different magnitude of ATP
effects observed with recombinant RetGC-1 and OS GCs.
Although we
have shown that the intracellular domain of RetGC-1 is sufficient for
activation, we have not shown whether or not GCAP-2 functions through
direct binding to an intracellular domain of RetGC-1. The
reconstitution of Ca sensitive regulation of
recombinant RetGC-1 using recombinant GCAP-2 supports this model but
does not rule out the involvement of an additional factor found in both
OS and in HEK 293 cell membranes. For example, if the detergent
insolubility of both OS and recombinant RetGC-1 is indicative of a
cytoskeletal association, then we cannot rule out the involvement of a
cytoskeletal protein as an intermediate for stimulation by GCAP-2. It
also remains to be determined through which intracellular subdomain(s)
of RetGC-1 that GCAP-2 transduces its direct or indirect stimulatory
effect on catalytic activity.
RetGC-2 is another photoreceptor specific membrane GC that has been shown to be stimulated by GCAP-2 in vitro (Lowe et al., 1995). It is highly homologous to RetGC-1, and together these proteins may define a new subfamily of membrane GCs that respond to intracellular activators.