Cloning, sequencing, and expression of a 24-kDa Ca(2+)-binding protein activating photoreceptor guanylyl cyclase.

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(2+)-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(2+)-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 recovering family of Ca(2+)-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 homogeneous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca2+ inhibits this activation with an EC50 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 Ca2+ 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.

, and Yarfitz and Hurley (1994)). In darkness, these channels allow Ca 2ϩ influx, but light-induced closure of the channels lowers free intracellular Ca 2ϩ because Ca 2ϩ is extruded from the OS by a light-independent Na ϩ /K ϩ , Ca 2ϩ exchanger. The most recent estimate of the magnitude of this effect is that light lowers free intracellular Ca 2ϩ from a dark level of 500 nM to as low as 50 nM . The decrease in free Ca 2ϩ 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 2ϩ 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 2ϩ -binding proteins from retina stimulate photoreceptor GC at low Ca 2ϩ concentrations. A 21-kDa Ca 2ϩ -binding protein referred to as GCAP 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 2ϩ -binding protein present in photoreceptor cells. It is related to, but distinct from, GCAP and other members of the recoverin family of Ca 2ϩ -binding proteins. Highly purified p24, free from GCAP contamination, stimulates photoreceptor membrane GC at low Ca 2ϩ . 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).

MATERIALS AND METHODS
Isolation of OS and p24-Outer segments were isolated from frozen bovine retinas using a sucrose floatation technique described by McDowell and Kuhn (1977) with modifications. p24 was isolated from a crude retinal extract using heat denaturation, phenyl-Sepharose chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and reverse phase HPLC as described (Dizhoor et al., 1994). The only modification was that 5 mM CaCl 2 was added, and NaCl was omitted during homogenization of retinas.
Sequencing of p24-Purified p24 had a blocked N terminus, which made it unavailable for direct Edman degradation, so it was cleaved by trypsin and cyanogen bromide (CNBr). Purified p24 was transferred onto Millipore Immobilon-PSQ membranes (Matsudaira, 1987), reduced and alkylated with isopropylacetamide (Krutzsch and Inman, 1993), followed by digestion in 25 l of 0.1 M ammonium bicarbonate, 10% acetonitrile with 0.2 g of modified trypsin (Promega) at 37°C for 17 h (Henzel et al., 1994). The solution was concentrated in a Speed-Vac and injected onto a C18 0.22 ϫ 100-mm capillary column (LC Packings, Inc.). Peptides were eluted using a linear gradient of 0 -80% acetonitrile (solution A contained 0.1 aqueous trifluoroacetic acid, and solution B was acetonitrile containing 0.07% trifluoroacetic acid) at a flow rate of 3.5 l/min and detected by absorbance at 195 nm. Purified p24 was cleaved in 50 l of 0.1 N HCl at 45°C for 3 h using a single crystal of CNBr. The peptides were electroblotted onto polyvinylidene difluoride membrane after separation in a Tris-tricine SDS gel. Automated protein sequencing was performed on a model 470A Applied Biosystems sequencer equipped with an on-line PTH analyzer using modified cycles (Henzel et al., 1994). Sequence interpretation was performed on DEC Alpha (Henzel et al., 1987). Additional sequencing was performed by mass spectrometry of Lys-C fragments of p24. HPLC-purified peptides were mixed with ␣-cyano-4-hydroxycinnamic acid saturated in 50% acetonitrile and 2% trifluoroacetic acid. Mass spectra were obtained with a Vestec (Houston, TX) LaserTec Research laser desorption linear time-of-flight mass spectrometer equipped with a 337 nM VSL-337 ND nitrogen laser (Laser Science, Inc.). (Henzel et al., 1990(Henzel et al., , 1993. Molecular Cloning of p24 cDNA-The substantial length of the sequenced protein fragment allowed us to use short degenerate oligonucleotides as primers for polymerase chain reaction (PCR) to generate a long probe. Two primers were derived from the amino acid sequence of p24. Forward primer FB, 5Ј-CA(A/G)TA(T/C)GT(A/G/T/C)GA(A/G)AT-GTT-3Ј, corresponded to a peptide QYVEAMF, and a reverse primer RE, 5Ј-CC(T/C)TG(T/C)TC(A/G/T/C)GC(T/C)TC(A/G/T/C)AC(T/C)TC-3Ј, corresponded to peptide EVEAEQQG. Those were used for high stringency PCR to generate a PCR product to probe a cDNA library. A gt11 bovine retinal cDNA library (a gift from Dr. D. Oprian) was used as a template for the PCR reaction. A 246-base pair product was produced, subcloned into EcoRI-BamHI sites of BlueScript phagemide (Stratagene), and sequenced. This sequence was compared to the known peptide fragment sequences. The insert was excised with EcoRI/ BamHI, purified by electrophoresis, and used to synthesize a random primer-labeled radioactive probe. This probe was used to screen a bovine retinal gt10 library (a gift from Dr. J. Nathans). High stringency screening (5% mismatch) of 800,000 plaques identified six positive clones. The sequence of the p24 cDNA fragment encoding amino acids from Glu 26 to Phe 204 was derived from the longest clone of cDNA after subcloning it into BlueScript. In all six gt10 clones, the 5Ј-coding region for p24 was truncated. The region encoding M1-E26 was derived from cDNA amplified by 5Ј-rapid amplification of cDNA ends (Life Technologies, Inc.). Poly(A)-containing mRNA was isolated from fresh bovine retinas using a Life Technologies, Inc. mRNA purification kit. Approximately 100 ng of mRNA was reverse transcribed using reverse primer GSP-1, 5Ј-ATATATGGATCCTTAGAGCCCTCAGAACATGG-CACTTT-3Ј. The cDNA was polycytidylated and amplified using two consecutive amplifications with reverse primers GSP-2 (5Ј-ATATAA-GAATTCTCCACACTGCAGGCTTTCTTC-3Ј) and GSP-3 (5Ј-ATATAA-GAATTCCCATTGCGGTCCTTGTCGTAGATCT-3Ј). The final product was subcloned into the BlueScript vector and sequenced. It had more than 230 base pairs overlap with cDNA cloned from the cDNA library, which confirmed the identity of the amplified fragment as a 5Ј-region of p24 cDNA.
Antibodies Against p24-Anti-peptide antibody P24SVE was generated in rabbits against a synthetic peptide Cys 131 -Leu 155 coupled to m-maleimidobenzoyl-N-hydroxysuccinimide ester-activated keyhole limpet hemocyanin (Pierce). The final antibody was affinity purified on a Sepharose-coupled peptide. This antibody effectively recognized p24 on immunoblot but did not react with paraformaldehyde fixed frozen bovine retina sections.
The second antibody, ⌬Np24, was generated against an N-truncated recombinant fragment P52-F204 of p24. The cDNA-encoding fragment Pro 52 -Phe 204 was amplified by PCR using cloned p24 cDNA as a template. In the final product Val 51 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 2ϩ -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 2ϩ -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 52 -Phe 204 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 110 -Gly 205 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).
Immunocytochemistry-Cryosections of paraformaldehyde-fixed bovine retina were a gift from Dr. Ann Milam (University of Washington). Sections were air dried for 40 min, blocked in a solution of 1% horse serum in phosphate-buffered saline containing 0.1% Triton X-100 for 1 h, incubated overnight at 4°C with primary ⌬Np24 antibody (0.25 g/ml), washed with phosphate-buffered saline 2 ϫ 15Ј, incubated with fluoroisothiocyanate-labeled goat anti-rabbit IgG (Cappel) for 1 h at room temperature, and washed with phosphate-buffered saline and photographed using a Nikon fluorescence microscope (objective ϫ20 or ϫ40). For competition experiments, the antibody was preincubated for 30 min at room temperature with 2 M recombinant fragment Pro 52 -Phe 204 of p24 or recombinant recoverin or 4 M N-truncated recombinant GCAP. Both preincubated and non-preincubated antibody solutions were centrifuged for 10 min at 10,000 ϫ g, 4°C, prior to using them on retinal sections.
Immunoblot-After electrophoresis in 15% SDS-PAGE, proteins were transferred onto nitrocellulose membrane and probed with anti-p24 antibodies. Bovine neurocalcin, recoverin, and hippocalcin were expressed in E. coli as described in (Teng et al., 1994). The blot was developed using alkaline phosphatase-conjugated secondary antibody with a mixture of nitro blue tetrazolium and 5-bromo-4-chloro-indolylphosphate as a substrate.
To start the reaction, 5 l of substrate solution containing 5 mM GTP, ϳ1 Ci of [␣-32 P]GTP (Amersham) was added. This solution also contained 20 mM cGMP, ϳ100,000 dpm of [8-3 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 ϫ g for 10 min, and 8-l aliquots were loaded onto polyethyleneimine cellulose 20 ϫ 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 3 H and 32 P radioactivity was counted in each vial in 10 ml of Ecolume liquid scintillator. [ 3 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. Nonspe- FIG. 1. A, primary structure of a novel 24-kDa Ca 2ϩ -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 26 and Phe 204 was found in cDNA clone 9 -3 from a bovine retinal cDNA library. Region M1-F41 was encoded by a cDNA product of 5Ј-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 , 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. cific background of 32 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 [ 32 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 M free Ca 2ϩ and was directly proportional to the amount of OS membranes.
Expression of p24 in HEK293 Cells-The expression construct was made by ligation of a full-length coding region of p24 cDNA into EcoRI and BamHI sites of the pH␤APr vector under control of the human ␤-actin promoter. The construct was propagated in E. coli strain TB-1 and purified using QIAGEN plasmid DNA purification columns. HEK293 cells were transfected using calcium-phosphate-precipitated DNA. 10 g of DNA was used to transfect cell cultures at 50% confluence. After the transfection, cells were grown for 72 h until almost confluent. Control cells were treated simultaneously without adding the expression DNA construct. Before harvesting, the cells were briefly washed with an extraction buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl 2 , 10 mM ␤-mercaptoethanol), mechanically removed from the plate, and homogenized in 2 ml of the extraction buffer using a Dounce homogenizer. The homogenate was centrifuged at 80,000 rpm in a Beckman TLA100.3 rotor for 10 min. Equal volume aliquots of soluble and membrane fractions were loaded onto 15% SDS-PAGE, electroblotted onto nitrocellulose, probed with ⌬Np24 antibody, and developed by peroxidase-conjugated goat anti-rabbit antibody with ECL substrate (Amersham) by exposing the blot onto X-Omat film (Kodak). The level of expression of p24 was not high enough to quantify it by Coomassie staining. Before the GC assay, the soluble extracts from both control and p24-expressing 293 cells were concentrated to 10 mg/ml of total protein, and 5 l from each extract were reconstituted with washed OS membranes.
Ca-EGTA Buffers-Ca-EGTA buffers were prepared from solutions of EGTA (Sigma) and EGTA saturated with CaCl 2 (Fluka) by pH titration in strict accordance with the method of Tsien and Pozzan (1989). Free Ca 2ϩ concentrations were calculated using a multi-factor program (Marks and Maxfield, 1991) and verified by Ca 2ϩ -electrode and by titration with Rhod-2 fluorescent dye (Calbiochem).

Primary Structure of p24 (GCAP-2)-
We determined the primary structure of p24 by Edman degradation of peptides cleaved from purified p24 (Dizhoor et al., 1994) and by sequence analysis of p24 cDNA (Fig. 1A). p24 is a novel Ca 2ϩ -binding protein most closely related to a family of recoverin-like proteins within the EF-hand protein super family. Fig. 1B demonstrates the close similarity between several recoverin-like proteins. p24 has four EF-hand-like motifs, but the first two align poorly with the consensus motif for EF-hands. Only EF-hands 3 and 4 are likely to bind Ca 2ϩ . A consensus glycine is substituted with asparagine in the second potential EF-hand, and two oxygen-containing amino acid residues are missing from the first EF-hand-related motif.
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 acylatedlike recoverin (Dizhoor et al., 1992) and other photoreceptor proteins .
Tissue Specificity of p24-We produced two types of antibodies that specifically recognize p24. The first, ⌬Np24, was raised against a large 152-amino acid fragment Pro 52 -Phe 204 expressed in E. coli as a His-Tag fusion protein. The second antibody, p24SVE, was generated against a synthetic peptide, Cys 131 -Leu 155 , highly specific for p24 and significantly different from the corresponding region in GCAP (Fig. 1B). Both antibodies were raised in rabbits and affinity purified. They reacted with HPLC-purified p24 and did not react with recombinant bovine recoverin, neurocalcin, or hippocalcin ( Fig. 2A). Special attention was paid to the possibility of cross-reactivity with GCAP. P24SVE does not react with recombinant GCAP on immunoblots (Fig. 2B). Only very weak cross-reactivity with GCAP was detected with the ⌬Np24 antibody on immunoblots. This was completely suppressed by pre-adsorption of ⌬Np24 antibody with a recombinant fragment (Asp 110 -Gly 205 ) of GCAP (Fig. 2C).
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 GCstimulating activity was not detected in those tissues (Dizhoor et al., 1994).
Immunolocalization of p24-We originally isolated p24 from  1 g). B and C, specificity of anti-p24 antibodies for p24 compared to GCAP. B, antibody P24SVE (ϳ0.5 g/ml) was used to stain an immunoblot containing 0.5 g of recombinant GCAP (left) or p24 (right). C, ⌬Np24 antibody (0.25 g/ml) was used to stain an immunoblot containing 0.5 g of recombinant GCAP (a, c, e) or affinity-purified p24 (b, d, f). The ⌬Np24 antibody was also preincubated for 10 min with 2 M of N-truncated recombinant p24 (c, d) or 4 M of N-truncated recombinant GCAP (e, f).

FIG. 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 g 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). a crude retinal extract (Dizhoor et al., 1994). However, immunoblot analysis demonstrates that p24 is also present in a preparation highly enriched in OS (Fig. 3). This suggests that a substantial fraction of this protein copurifies with OS. Direct immunocytochemical localization of p24 was done using affinity-purified antibody ⌬Np24, which specifically recognized outer and inner segments of photoreceptors on cryosections of fixed bovine retina. A weak staining of synaptic termini of photoreceptor cells was also detected (Fig. 4A). Similar results were obtained with fixed paraffin sections of retina (not shown). Preincubation of the antibody with recombinant fragment of p24 eliminated the signal from the photoreceptor cells (Fig. 4, B and E). Preincubating the antibody with a recombinant N-truncated fragment of GCAP did not block the signal in the photoreceptors (Fig. 4D). Neither full-length recombinant GCAP nor recoverin competed with the specific signal from photoreceptors (data not shown). Cones did not appear to stain with the antibody (Fig. 4, A and D). The most intense signal was detected in the outer segments and in the upper part of the inner segments of rods. Although this suggests that p24 is a rod-specific protein, the possibility that the epitope for antibody recognition is masked in cone cells cannot be excluded. Further studies using additional antibodies will be required to determine the exact localization of p24 in photoreceptor cells.
Antibodies Against p24 Inhibit Activation of GC-A crude heat-treated retinal extract containing p24 can be completely depleted of GC-stimulating activity by passing it through a column of immobilized affinity-purified antibody ⌬Np24 (Fig.  5A). The activator retained on the column can be eluted at low pH as an apparently homogenous 24-kDa protein (Fig. 5B). In our SDS gel system, p24 and GCAP have almost identical electrophoretic mobility. Therefore, we used antibodies against GCAP to examine our preparations for the presence of GCAP (Fig. 5C). A highly specific antibody against GCAP, UW14, strongly recognizes recombinant GCAP on an immunoblot (left lane) but does not detect any GCAP in our immunoaffinitypurified p24 (right lane). This preparation of p24 activates GC in washed OS membranes (Fig. 4D) with the same Ca 2ϩ sensitivity (EC 50 ϭ 200 nM) and cooperativity (Hill coefficient, 1.7) as HPLC-purified p24 (Dizhoor et al., 1994). Both anti-⌬Np24 and anti-p24SVE antibody efficiently inhibit activation of GC by purified p24 in vitro (Fig. 6). Since p24SVE antibody does not cross-react with GCAP (Fig. 2C), we can say with certainty that GC-stimulating activity in purified p24 preparations derives from p24 itself. This conclusion is consistent with the fact that GC-stimulating activity copurifies with p24 through several purification steps (Dizhoor et al., 1994;Lowe et al., 1995). Based on these results, p24 is a Ca 2ϩ -sensitive regulator of photoreceptor GC distinct from GCAP.
Recombinant p24 Stimulates Activation of GC in Vitro-Several attempts to express full-length p24 in E. coli gave us only insoluble protein (data not shown). However, we successfully expressed recombinant p24 in HEK293 cells. Fig. 7A demonstrates that p24 is expressed in p24 cDNA-transfected cells as a soluble protein. Only a small amount of it was found in the particulate fraction of homogenized cells. We compared extracts from p24 cDNA-transfected and control, mock-transfected cells for their ability to stimulate GC. As shown in Fig.  7B, only the extract containing recombinant p24 effectively activates GC in washed OS membranes.

DISCUSSION
The data presented in this paper demonstrate that p24 is a Ca 2ϩ -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 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 . 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 2ϩ -binding domains, es- FIG. 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 2ϩ . Washed OS membranes before (a) and after (b-d) addition of the following: 1 l of phenyl-Sepharose fraction (b), 1 l of flow-through fraction from the column (c), and 1 l 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 g of crude retinal protein extract (a), 30 g of the heat-treated extract (b), 30 g of phenyl-Sepharose fraction (c), or 2 g 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 g of recombinant GCAP (a) or immunoaffinity-purified p24 (b) was stained by anti-GCAP antibodies UW14 (dilution of antisera 1:5,000). D, Ca 2ϩ -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. q, OS membranes only (no p24 added); E, 200 nM immunoaffinity-purified p24 added.

FIG. 6. Antibodies against p24 inhibit GC activation in vitro.
The GC assay mixture contained washed OS membranes (a) and 0.2 g of affinity-purified p24 (b) or p24 preincubated for 10 min at room temperature with either 4 g of preimmune Ig (c) or 2.5 g of antibodies ⌬Np24 (d) or P24SVE (e).
FIG. 7. Recombinant p24 activates GC in washed OS membranes at low Ca 2ϩ . A, expression of recombinant p24 in HEK293 cells. The cells were transfected with p24 cDNA, and 15-l 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 2ϩ . Washed OS membranes were reconstituted with 50 g 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.
pecially 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 2ϩ 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 2ϩ 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 2ϩ -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" 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 . 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 2ϩ -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. 2 Further studies are required to establish the relationship between RetGC-1 and -2 and GCAP-1 and -2.