(Received for publication, July 18, 1995; and in revised form, August 3, 1995)
From the
Two vertebrate photoreceptor-specific membrane guanylyl
cyclases, RetGC-1 and RetGC-2, are activated by a soluble 24-kDa
retinal protein, p24, in a Ca-sensitive manner
(Dizhoor, A. M., Lowe, D. G., Olshevskaya, E. V., Laura, R. P., and
Hurley, J. B.(1994) Neuron 12, 1345-1352; Lowe, D. G.,
Dizhoor, A. M., Liu, K., Gu, O., Laura, R., Lu, L., and Hurley, J.
B.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5535-5539).
The primary structure of bovine p24 has been derived from peptide
sequencing and from its cDNA. p24 is a new EF-hand-type
Ca
-binding protein, related but not identical to
another guanylyl cyclase-activating protein, GCAP (Palczewski, K.,
Subbaraya, I., Gorczyca, W. A., Helekar, B. S., Ruiz, C. C., Ohguro, H.
Huang, J., Zhao, X., Crabb, J. W., Johnson, R. S., Walsh, K. A.,
Gray-Keller, M. P., Detwiler, P. B., and Baehr, W.(1994) Neuron 13, 395-404) and other members of the recoverin family of
Ca
-binding proteins. Antibodies against a truncated
fusion protein and against a p24-specific synthetic peptide
specifically recognize retinal p24 on immunoblot. Both antibodies
inhibit activation of photoreceptor membrane guanylyl cyclase by
purified p24. p24 is found only in retina, and it copurifies with outer
segment membranes. Immunocytochemical analysis shows that it is present
in rod photoreceptor cells. An immobilized antibody column was used to
purify p24 from a heat-treated retinal extract. Purified p24 appears on
SDS-polyacrylamide gel electrophoresis as a homogenous protein not
contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca
inhibits this activation with an EC
near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293
cells effectively stimulates photoreceptor guanylyl cyclase. These
findings demonstrate that p24, like GCAP, imparts Ca
sensitivity to photoreceptor membrane guanylyl cyclase. We propose that
p24 be referred to as GCAP-2 and that GCAP be referred to as GCAP-1.
Light triggers hydrolysis of cyclic GMP and closure of
cGMP-gated cation channels in photoreceptor outer segment plasma
membranes (OS) ()(reviewed in Stryer(1991), Lagnado and
Baylor(1992), and Yarfitz and Hurley(1994)). In darkness, these
channels allow Ca
influx, but light-induced closure
of the channels lowers free intracellular Ca
because
Ca
is extruded from the OS by a light-independent
Na
/K
, Ca
exchanger.
The most recent estimate of the magnitude of this effect is that light
lowers free intracellular Ca
from a dark level of 500
nM to as low as 50 nM (Gray-Keller and Detwiler,
1994). The decrease in free Ca
concentration allows a
soluble activator protein to stimulate a membrane guanylyl cyclase (GC)
(Lolley and Racz, 1982; Koch and Stryer, 1988). Two membrane GCs are
present in photoreceptor cells, RetGC-1 and RetGC-2 (Shyjan et
al., 1992; Lowe et al., 1995). When cloned and expressed
in HEK293 cells, both cyclases can be stimulated by a soluble protein
purified from retina (Dizhoor et al., 1994; Lowe et
al., 1995). This stimulation occurs only at free Ca
below 200 nM. RetGC-1 was also purified from bovine
retina (Koch, 1991; Hayashi and Yamazaki 1991; Margulis et
al., 1993), and both RetGC-1 (Goraczniak et al., 1994;
Yang et al., 1995) and RetGC-2 (Yang et al., 1995)
were cloned from bovine and rat retinal cDNA libraries. Other types of
membrane GCs have been well characterized (reviewed in Garbers and
Lowe(1994)). They are regulated by small peptide ligands that bind to
their extracellular domains. So far, there is no reported evidence that
small peptides regulate RetGC-1 or RetGC-2 (Shyjan et al.,
1992; Yang et al., 1995).
It was recently reported that two
Ca-binding proteins from retina stimulate
photoreceptor GC at low Ca
concentrations. A 21-kDa
Ca
-binding protein referred to as GCAP (Gorczyca et al., 1994; Palczewski et al., 1994) stimulates GC
activity in rod outer segment membranes. A different protein, p24,
which also stimulates GC in rod outer segment membranes, has been
purified from retina by a method different from the method used to
purify GCAP. It has also been shown that p24 stimulates recombinant
RetGC-1 and RetGC-2 (Dizhoor et al., 1994; Lowe et
al., 1995). In this report, we show that p24 is a novel
Ca
-binding protein present in photoreceptor cells. It
is related to, but distinct from, GCAP and other members of the
recoverin family of Ca
-binding proteins. Highly
purified p24, free from GCAP contamination, stimulates photoreceptor
membrane GC at low Ca
. Antibodies against p24 prevent
purified p24 from activating GC. We also demonstrate that recombinant
p24 expressed in HEK293 cells stimulates photoreceptor GC. We propose
to refer to GCAP as GCAP-1 and to p24 as GCAP-2 (guanylyl
cyclase-activating protein-2).
The second antibody, Np24,
was generated against an N-truncated recombinant fragment P52-F204 of
p24. The cDNA-encoding fragment Pro
-Phe
was
amplified by PCR using cloned p24 cDNA as a template. In the final
product Val
was substituted for Met encoded within a site
for NdeI. The DNA product was inserted into NdeI and BamHI sites of the expression vector pET15b (Novagene) and
expressed in an Escherichia coli strain BL21(DE3) to produce a
fusion protein linked at the N terminus to a 20-amino acid
His-Tag-containing peptide. Protein induced by
isopropyl-1-thio-
-D-galactopyranoside reacted with the
antibody P24SVE and demonstrated a Ca
-dependent shift
of electrophoretic mobility in SDS-PAGE. This protein was insoluble. It
could be solubilized in 6 M urea and purified on a
Ni
-bound His-bond column (Novagen). All buffers for
the purification contained 6 M urea. Purified protein was
dialyzed against 20 mM phosphate buffer, pH 7.5, containing
100 mM NaCl. The main part of the protein precipitates during
this procedure. Protein was solubilized again at pH 9.5, and more than
50% of it remained soluble after subsequent dialysis at pH 8. Antibody
was produced in rabbits and purified on the recombinant
Pro
-Phe
fragment cross-linked to
CNBr-activated Sepharose 4B at pH 8.3. Unreacted protein was removed by
extensive washing with 100 mM Tris buffer, pH 10.5. The
affinity column was neutralized to pH 8 and used for purification of
the antibody. The column was stable at 4 °C and efficient for
purification of up to 10 mg of antibodies from 30 ml of immune serum.
The antibody strongly reacted with p24 on immunoblot and was suitable
for immunocytochemical analysis. Only a trace of cross-reactivity of
Np24 antibody with recombinant GCAP was found using immunoblot. To
compete away this residual cross-reactivity, a soluble recombinant
N-truncated fragment Asp
-Gly
of GCAP was
expressed in E. coli as a His-Tag fusion protein using pET15b
vector and purified on a His-bond column as above.
Recombinant GCAP and anti-GCAP antibody UW14 were provided by K. Palczewski (University of Washington).
To start the
reaction, 5 µl of substrate solution containing 5 mM GTP,
1 µCi of [
-
P]GTP (Amersham) was
added. This solution also contained 20 mM cGMP,
100,000
dpm of [8-
H]cGMP (Amersham), and 0.5 mM ATP. Each reaction mixture was incubated in a closed Eppendorf
tube for 10 min at 30 °C and then heated for 2 min at 100 °C to
stop the reaction. The reaction tubes were centrifuged at 10,000
g for 10 min, and 8-µl aliquots were loaded onto
polyethyleneimine cellulose 20
20-cm plastic Polygram TLC
plates (Alltech) with 1/2-inch intervals (14 samples per plate). The
TLC plates were air dried and developed first in water and then in 0.2 M LiCl. cGMP spots visualized using UV illuminator were cut
from the plate, placed into 20 ml of scintillation vials containing 1
ml of 2 M LiCl, and shaken for 10 min at room temperature on a
rotary shaker. Both
H and
P radioactivity was
counted in each vial in 10 ml of Ecolume liquid scintillator.
[
H]cGMP radioactivity was used as an internal
standard in each sample to ensure the absence of cGMP hydrolysis in the
course of the reaction. Nonspecific background of
P in
cGMP spots was controlled in each set of experiments using reaction
mixtures inactivated by heat denaturation prior to the addition of
radiolabeled substrate and was found to be insignificant compared to
the amount of [
P]cGMP synthesized by activated
GC.
GC activity was found to be linear within at least the first 15
min of the reaction, both in the absence and in the presence of
affinity-purified or expressed p24, both at 15 nM and 1
µM free Ca and was directly proportional
to the amount of OS membranes.
Figure 1:
A, primary structure
of a novel 24-kDa Ca-binding protein. Peptides of p24
purified from retina were generated by cyanogen bromide cleavage
(CNBr14kd CNBr20kd and CNBrCaBP) and tryptic digest (T7, T12, T21, T23)
and sequenced by Edman degradation. The sequence between Glu
and Phe
was found in cDNA clone 9-3 from a
bovine retinal cDNA library. Region M1-F41 was encoded by a cDNA
product of 5`-rapid amplification of cDNA ends (see ``Material and
Methods'' for the details). B, p24 belongs to the family
of recoverin-like proteins. The sequence of p24 was aligned to
sequences of GCAP (Palczewski et al., 1994), bovine
neurocalcin (Okazaki et al., 1992), rat hippocalcin (Kobayashi et al., 1992), and recoverin (Dizhoor et al., 1991;
Hurley et al., 1993). Amino acids identical to p24 are shadowed. The recombinant N-truncated fragment marked
Np24 (underlined) and a synthetic peptide specific for
p24 marked as P24SVE were used to produce antibodies against p24.
EF-hand-like regions are indicated as ef-1-ef-4. EF-hand motif
symbols are as follows: o, oxygen containing amino acid side
chain; j, Ile, Val, or Leu; e, glutamic acid; g, glycine; *, any amino acid.
p24 is 41, 38, and 29% identical to bovine GCAP, neurocalcin, and recoverin, respectively. Like other members of this family, p24 has a consensus sequence for N-terminal myristoylation. It has yet to be determined if it is heterogenously fatty acylated-like recoverin (Dizhoor et al., 1992) and other photoreceptor proteins (Johnson et al., 1994).
Figure 2:
Antibodies against p24. A,
reactivity with related Ca-binding proteins.
Ca
-binding proteins were transferred from SDS-PAGE
onto nitrocellulose membrane and probed with
Np24 antibodies (0.25
µg/ml). Upper panel: a, recombinant rat
hippocalcin (0.1 µg); b, recombinant bovine neurocalcin
(0.1 µg); c, recombinant bovine recoverin (1 µg); d, purified retinal p24 (0.1 µg). B and C, specificity of anti-p24 antibodies for p24 compared to
GCAP. B, antibody P24SVE (
0.5 µg/ml) was used to
stain an immunoblot containing 0.5 µg of recombinant GCAP (left) or p24 (right). C,
Np24 antibody
(0.25 µg/ml) was used to stain an immunoblot containing 0.5 µg
of recombinant GCAP (a, c, e) or
affinity-purified p24 (b, d, f). The
Np24 antibody was also preincubated for 10 min with 2 µM of N-truncated recombinant p24 (c, d) or 4
µM of N-truncated recombinant GCAP (e, f).
Immunoblot analysis using both antibodies showed p24 immunoreactivity only in an extract from retina (Fig. 3) and not in extracts from kidney, liver, adrenal, lung, spleen, heart, or brain. This is consistent with our previous finding that GC-stimulating activity was not detected in those tissues (Dizhoor et al., 1994).
Figure 3:
Tissue specificity of p24. Homogenates of
different bovine tissues were subjected to electrophoresis in SDS-PAGE,
transferred onto nitrocellulose membrane, and probed with Np24.
Approximately 20 µg of total protein in extracts from heart (a), adrenal (b), kidney (c), lung (d), liver (e), brain (f), retina (g), and outer segments fraction (h). No signal was
found in the spleen (not shown). Essentially the same result was
obtained with P24SVE anti-peptide antibody (not
shown).
Figure 4:
Immunolocalization of p24 in bovine
retina. Cryosections of fixed bovine retina were probed with Np24
antibody (0.25 µg/ml) (A) or same antibody preincubated
with 2 µM purified N-truncated recombinant p24 (B). Panel C shows similar region in phase contrast
(objective,
20 for A-C). D,
photoreceptor cell layer at higher magnification (objective,
40)
stained with
Np24 preincubated with 4 µM recombinant
truncated GCAP for competition, E, same as D but
preincubated with 2 µM N-truncated recombinant p24 for
competition. The secondary antibody was labeled with
fluoroisothiocyanate. COS, cone outer segments; OS,
outer segments; G, ganglion cells; INL, inner nuclear
layer; IPL, inner plexiform layer; IS, inner
segments; ONL, outer nuclear layer; ROS, rod outer
segments; RIS, rod inner segments.
Figure 5:
Purification of p24 using immunoaffinity
chromatography on Np24 antibody column. A, binding and
elution of GC activator from the column. A heat-treated extract (HT) from retina partially enriched in GC activator by
phenyl-Sepharose chromatography (marked PS, 5 mg of total
protein) was loaded onto the anti-
Np24 antibody column and eluted
at pH 2.5. Fractions were tested for their ability to stimulate GC in
washed OS membranes at 7 nM free Ca
. Washed
OS membranes before (a) and after (b-d)
addition of the following: 1 µl of phenyl-Sepharose fraction (b), 1 µl of flow-through fraction from the column (c), and 1 µl of fraction eluted from the antibody column (d). B, copurification of p24 with the GC-stimulating
activity. At various steps of purification, fractions containing GC
activator were separated by electrophoresis in 15% SDS-PAGE and stained
with Coomassie Blue (30 µg of crude retinal protein extract (a), 30 µg of the heat-treated extract (b), 30
µg of phenyl-Sepharose fraction (c), or 2 µg of pH
2.5-eluted fraction from the anti-
Np24 antibody column (d)). C, immunoaffinity-purified p24 is not
contaminated with GCAP. Immunoblot containing 0.5 µg of recombinant
GCAP (a) or immunoaffinity-purified p24 (b) was
stained by anti-GCAP antibodies UW14 (dilution of antisera 1:5,000). D, Ca
-sensitive activation of OS GC by
immunoaffinity-purified p24. Washed OS membranes were incubated under
the conditions of the GC assay in the presence of Ca/EGTA buffer.
, OS membranes only (no p24 added);
, 200 nM
immunoaffinity-purified p24 added.
Figure 6:
Antibodies against p24 inhibit GC
activation in vitro. The GC assay mixture contained washed OS
membranes (a) and 0.2 µg of affinity-purified p24 (b) or p24 preincubated for 10 min at room temperature with
either 4 µg of preimmune Ig (c) or 2.5 µg of
antibodies Np24 (d) or P24SVE (e).
Figure 7:
Recombinant p24 activates GC in washed OS
membranes at low Ca. A, expression of
recombinant p24 in HEK293 cells. The cells were transfected with p24
cDNA, and 15-µl aliquots of extracted proteins were analyzed on
immunoblot probed with
Np24 antibodies as described under
``Materials and Methods.'' a, soluble fraction from
untransfected cells; b, membrane fraction from untransfected
cells; c, soluble fraction from p24cDNA-transfected cells; d, membrane fraction from p24 cDNA-transfected cells. B, GC activation by recombinant p24 at 7 nM free
Ca
. Washed OS membranes were reconstituted with 50
µg of total protein from soluble fraction of mock-transfected (a) or p24-expressing HEK293 cells (b), which were
assayed for GC activity. No extract was added in c.
The data presented in this paper demonstrate that p24 is a
Ca-binding protein that regulates photoreceptor
membrane GC. It is present in the outer and inner segments of
photoreceptor cells and, when expressed as recombinant protein in 293
cells, effectively stimulates photoreceptor GC in vitro.
Based on its primary structure, p24 is closely related to another
recently identified GC activator referred to as GCAP (Gorczyca et
al., 1994; Palczewski et al., 1994). p24 and GCAP are
both members of the recoverin family of EF-hand proteins, but they are
structurally and functionally more similar to each other than to other
members of the recoverin family. Therefore, we propose that GCAP be
referred to as GCAP-1 and p24 as GCAP-2. It has been proposed that the
very N-terminal domain of GCAP-1 participates in activation of GC
(Palczewski et al., 1994). However, GCAP-2 is clearly distinct
from GCAP-1 within the first 20 amino acid residues (Fig. 1B). This implies that a domain other than the
very N terminus is involved in GC activation. GCAP-1 and GCAP-2 are
most similar within the Ca-binding domains,
especially EF-2 and EF-3. However, this region is also highly conserved
in all recoverin-like proteins, including those that do not activate
GC. It is possible that the GC-activating domain is formed by a
tertiary structure that brings together remote elements of primary
structure rather than by a particular short region of amino acid
sequence.
GCAP-2 most likely binds Ca only in the
EF-3 and EF-4 sites. Amino acid residues in EF-1 and EF-2 of GCAP-2 do
not match well to the EF-hand consensus sequence. In EF-1, two
oxygen-containing residues (positions 35 and 41) of the consensus are
substituted with Cys and Phe, respectively. EF-2 has all the consensus
oxygen-containing amino acid residues, but a consensus Gly is
substituted with Asn at position 74. The number of Ca
binding sites and their affinities have to be determined
experimentally.
At the N terminus of GCAP-2, there is a motif known
to be recognized by N-myristoyl transferase. Two photoreceptor
Ca-binding proteins, recoverin and GCAP-1, are known
to be heterogenously fatty acylated at their N termini (Dizhoor et
al., 1992; Palczewski et al., 1994). This fatty acylation
plays an important role in a structure of recoverin referred to as the
``calcium-myristoyl switch'' (Zozulya and Stryer, 1992;
Dizhoor et al., 1993; Ames et al., 1995). Other
proteins from retina that have this motif are also heterogenously
acylated with C14 and C12 saturated and non-saturated fatty acids
(Johnson et al., 1994). A mass spectrometry analysis is being
done to verify if GCAP-2 is also heterogenously acylated.
Both
GCAP-1 and GCAP-2 are present in photoreceptor cells, but their precise
intracellular localization has not yet been established. Two
Ca-sensitive membrane cyclases, RetGC-1 and RetGC-2,
are also present in photoreceptor cells (Dizhoor et al., 1994;
Lowe et al., 1995). RetGC-1 is present in outer segments and
inner segment and appears more abundant in cones than in rods (Dizhoor et al., 1994; Liu et al., 1994). RetGC-2 protein in
photoreceptor cells has not been localized. GCAP-2 appears to be more
abundant in rods, whereas GCAP-1 immunoreactivity is both in rods and
cones. (
)Further studies are required to establish the
relationship between RetGC-1 and -2 and GCAP-1 and -2.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U32856[GenBank].