Identification of a Domain in Guanylyl Cyclase-activating Protein 1 That Interacts with a Complex of Guanylyl Cyclase and Tubulin in Photoreceptors*

Alexander SchremDagger §, Christian LangeDagger §, Michael Beyermann, and Karl-Wilhelm KochDagger parallel

From the Dagger  Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Postfach 1913, D-52425 Jülich, Germany and the  Forschungsinstitut für Molekulare Pharmakologie, Alfred-Kowalke-Strasse 4, D-10315 Berlin, Germany

    ABSTRACT
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Abstract
Introduction
References

The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.

    INTRODUCTION
Top
Abstract
Introduction
References

Rod and cone photoreceptor cells respond to light by closure of cGMP-gated ion channels in the plasma membrane of the outer segment. Light triggers an amplifying cascade of enzymatic reactions leading to the hydrolysis of the intracellular messenger cGMP (1, 2). A further consequence of illumination is the decrease in the cytoplasmic [Ca2+]. Changes in [Ca2+] are sensed by Ca2+-binding proteins that control the activity of several proteins in the enzyme cascade (3, 4). Recovery from the light response requires the termination of all activating steps in the cascade and the resynthesis of cGMP. Synthesis of cGMP is catalyzed by a membrane-bound retina-specific guanylyl cyclase in rod outer segments (ROS-GC).1 Two isoforms of this enzyme are expressed in photoreceptor cells (5-11). They are members of the family of receptor guanylyl cyclases (12, 13). Their mechanism of activation is different from other family members: Ca2+-binding proteins act on an intracellular domain of ROS-GC (14-16, 17), whereas other membrane guanylyl cyclases are activated by extracellular peptide hormones (12, 13). The activity of the ROS-GC forms is regulated by a photoreceptor-specific Ca2+-binding protein named guanylyl cyclase-activating protein (GCAP) that is also expressed in two isoforms (GCAP-1 and GCAP-2) (18-22). These proteins activate ROS-GC to restore the depleted cGMP pool after illumination. In the dark, when the [Ca2+ ] is high (500 nM), ROS-GC activity is low, and it is stimulated at least 10-fold during the light response, when the [Ca2+] drops to <= 100 nM. Both GCAP forms are heterogeneously acylated at the N terminus by C14:0, C14:1, C14:2, and C12:0 fatty acids. Although the N-terminal fatty acyl group may be important for regulation of ROS-GC, it is probably not essential for membrane binding (18, 20, 21, 23, 24). Several experiments have indicated that GCAP-1 is associated with rod outer segment membranes or ROS-GC independent of [Ca2+] (16, 24). Ca2+-loaded GCAP-1 competes with a constitutively active GCAP-1 mutant for binding to ROS-GC (25), which also led to the conclusion that GCAP-1 forms a stable complex with ROS-GC independent of [Ca2+]. In contrast, GCAP-2 does show a Ca2+ sensitivity of its membrane association; it binds more efficiently to membranes at [Ca2+] < 200 nM (23).

Knowledge about the precise interaction domains in GCAP-1 and GCAP-2 is incomplete or nonexistent. So far, peptide competition studies have indicated that the N-terminal 20 amino acids of GCAP-1 are involved in regulation of ROS-GC (18, 24). In addition, two peptides (Glu57-Lys86 and Asp37-Asp163 in GCAP-1) including the first and third functional Ca2+-binding motifs (EF-hands) inhibited stimulation of ROS-GC (24). A 25-amino acid truncation at the N terminus of GCAP-1 abolished the ability to increase ROS-GC activity almost completely (24).

It was the aim of our project to identify the domain(s) in GCAP-1 that is directly involved in the interaction with ROS-GC. We synthesized an oligopeptide library of overlapping peptides spanning the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays and column affinity binding studies. By this approach, we found that ROS-GC and tubulin bind as a complex to a 15-amino acid peptide of GCAP-1. Coimmunoprecipitation studies demonstrated the presence of an interacting complex of ROS-GC, GCAP-1, and tubulin independent of [Ca2+].

    MATERIALS AND METHODS

Synthesis of an Oligopeptide Library-- Peptides spanning the whole amino acid sequence of GCAP-1 were synthesized using a Multipin peptide synthesis kit (Chiron Mimotopes) according to the manufacturer's recommendations. Each peptide was 12 amino acids long and overlapped with the preceding one by 10 amino acids. All peptides were obtained in their amide form at the carboxyl terminus. Fmoc-protected amino acids were attached via carbodiimide chemistry. After 12 cycles of coupling, the N terminus of each peptide was acetylated by incubating with 3% acetanhydride and 0.5% N-ethyldiisopropylamine in dimethylformamide for 100 min. For synthesis of the myristoylated form of peptide 1, the acetylation was omitted. Instead, myristic acid was coupled to the peptide's free N terminus using the same chemistry as for coupling Fmoc-protected amino acids. Deprotection, cleavage, precipitation, and washing of peptides were done as recommended by the manufacturer. Lyophilized peptides were analyzed by high performance liquid chromatography. Peptide 37/38 and the GCAP-2 and the recoverin peptides were synthesized in larger amounts as described (26).

Peptide Affinity Chromatography-- Peptides were coupled to activated thiol-Sepharose 4B (Amersham Pharmacia Biotech) via a thiol group according to the manufacturer's description. The peptide-Sepharose was equilibrated with 1 mM dodecyl maltoside, 50 mM KCl, 20 mM Hepes-KOH (pH 7.4) and incubated with an extract of membrane and/or cytoskeletal proteins from rod outer segments (ROS). ROS and the extract were obtained as described (5, 20). Briefly, ROS (9-13 mg/ml rhodopsin) were diluted 30-fold with a low salt buffer (10 mM Hepes-KOH (pH 7.4), 0.1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM DTT) and centrifuged at 30,000 × g for 30 min. Washed ROS membranes were homogenized and solubilized in 5% Triton X-100, 4 mM MgCl2, 10 mM Hepes-KOH (pH 7.4), and 1 mM DTT. Subsequent centrifugation removed the majority of membrane proteins, including rhodopsin and the cGMP-gated channel (present in the Triton X-100 supernatant). The pellet was solubilized with 20 mM dodecyl maltoside, 20 mM Hepes (pH 7.4), 1 M KCl, and 1 mM DTT and centrifuged. This extract (dodecyl maltoside extract) was enriched in ROS-GC and contained tubulin, actin, and a few other unidentified proteins in minor quantities. Before application to the column, the extract was adjusted to 1 mM dodecyl maltoside, 50 mM KCl, and 20 mM Hepes (pH 7.4). The extract was incubated with the Sepharose at 4 °C for at least 2 h. The unbound fraction was collected, and the Sepharose was washed with 1 mM dodecyl maltoside, 20 mM Hepes-KOH (pH 7.4), and 0.5 M NaCl (10-fold the volume of the column) and subsequently with the same buffer containing 1 M NaCl. Salt was removed by washing with 20 mM Hepes (pH 7.4). Bound proteins were eluted with 0.5% SDS and 20 mM Hepes-KOH (pH 7.4). The Triton X-100 extract (5) was also used to test binding of tubulin to peptide columns in the absence of ROS-GC.

Guanylyl Cyclase Assay-- The activity of ROS-GC was determined as described previously (5, 20). In all experiments, the incubation volume was 50 µl. Added peptides were dissolved in 0.5% Me2SO and 0.05% Tween 20, which caused little interference with the guanylyl cyclase activity. Controls were incubated under identical buffer conditions. Free Ca2+ was adjusted by Ca2+/EGTA buffers as described (20).

Expression and Purification of GCAP-1-- The coding sequence of bovine GCAP-1 was amplified from a full-length cDNA clone (20) via polymerase chain reaction using the following primers: 5'-sense primer, CAGGGATCCACCATGGGGAACATTATGGAC; and 3'-antisense primer, AGGGAATTCTATCAGCCGTCGGCCTCCGCG. The product obtained after 15 cycles of polymerase chain reaction was subcloned into pBluescript SK vector via the introduced BamHI and EcoRI sites. One of the resulting clones was sequenced to exclude that errors had been introduced by the polymerase chain reaction.

The GCAP-1/pBluescript plasmid was linearized by digestion with NcoI, and the ends were filled in with Klenow polymerase. The GCAP-1 coding sequence was then released by digestion with EcoRI and inserted between the NdeI and EcoRI restriction sites of the pET21a(+) expression vector (Novagene).

GCAP-1 protein was expressed from GCAP-1/pET21a(+) in an Escherichia coli BL21(DE3) strain carrying the plasmid pBB131 (a kind gift of Dr. J. Gordon) encoding for yeast N-myristoyltransferase. Cells were grown at 37 °C to an A600 of ~0.5 in LB medium containing 100 µg/ml ampicillin and 30 µg/ml kanamycin and then supplemented with 50 µg/ml myristic acid. Thirty minutes after addition of myristic acid, the expression of GCAP-1 was induced by addition of isopropyl-1-thio-beta -D-galactopyranoside to a final concentration of 0.5 mM. Expression was allowed to proceed for 4 h. Cells from 1 liter of expression culture were collected by centrifugation, resuspended in 250 ml of 50 mM Tris-HCl (pH 8), and stored frozen at -20 °C until further use. The thawed cell suspension was incubated at 30 °C for 30 min in the presence of 0.1% Tween 20, 100 µg/ml lysozyme (SERVA), and 10 units/ml DNase I (TaKaRa). Lysis was terminated by addition of 1 mM DTT and 0.1 mM phenylmethylsulfonyl fluoride. The insoluble fraction was collected by centrifugation at 370,000 × g for 20 min and solubilized by stirring in 250 ml of buffer containing 6 M guanidin chloride, 0.1 mM phenylmethylsulfonyl fluoride, and 2.5 mM DTT for 1 h at room temperature. The resulting suspension was filtered and dialyzed at 4 °C against three changes of 10 mM sodium phosphate buffer (pH 7) containing 1 mM EDTA and 1 mM DTT, followed by two changes of 10 mM sodium phosphate buffer containing 200 µM CaCl2 and 1 mM DTT. Precipitated material was removed by centrifugation at 370,000 × g for 20 min. The supernatant was concentrated using Centriplus devices (Amicon, Inc.) with a molecular mass cutoff of 10 kDa. The concentrate was applied to a Superdex 75 XK16/60 gel filtration column (Amersham Pharmacia Biotech) in 10 mM sodium phosphate (pH 7), 100 mM NaCl, and 200 µM CaCl2. Fractions containing recombinant GCAP-1 were pooled and bound to ceramic hydroxylapatite type I (Bio-Rad) in an HR5/5 FPLC column (Amersham Pharmacia Biotech). Elution was performed by a 0-100% gradient of 300 mM sodium phosphate (pH 7) in 10 mM sodium phosphate (pH 7) and 200 µM CaCl2. Recombinant GCAP-1 was obtained in >90% purity and stored at -80 °C.

Immunoprecipitation-- Protein A-Sepharose was equilibrated with 100 mM Tris (pH 8.0). Polyclonal anti-GCAP-1 serum from immunized rabbit (20) was adjusted to 100 mM Tris (pH 8.0) and incubated with protein A-Sepharose for 1 h at room temperature. Afterward, the Sepharose was washed with 40 ml of 100 mM Tris (pH 8.0) and with 40 ml of 10 mM Tris (pH 8.0) and equilibrated with 0.2 M sodium borate buffer (pH 9.0). The Sepharose beads were resuspended in 40 ml of sodium borate, and 1.1 g of dimethyl pimelimidate was added to the suspension. After incubation for 30 min at room temperature, the reaction was stopped with 0.2 M ethanolamine. Coimmunoprecipitation of GCAP-1 and ROS-GC was done as follows. Soluble ROS proteins were separated from the membranes by centrifugation of a 5-fold diluted suspension of ROS in 10 mM Tris (pH 7.5) (45,000 rpm for 15 min; Beckman TLA-45). The supernatant was concentrated with a Centricon device (cutoff = 10 kDa), adjusted to 150 mM KCl, and incubated with the anti-GCAP antibody-Sepharose equilibrated in the same buffer (200 µl of supernatant and 100 µl of Sepharose). The unbound fraction was separated from the beads by a quick spin using 0.45 micropure separators (Amicon, Inc.). Pelleted ROS membranes (see above) were resuspended in 10 mM Tris (pH 7.5); solubilized in 500 µl of buffer containing 2% Triton X-100, 0.5 M KCl, and 20 mM Tris (pH 7.4); and centrifuged for 15 min at 45,000 rpm. The supernatant was incubated with the anti-GCAP antibody-Sepharose beads. Samples were run in parallel with either 1 mM CaCl2 or 1 mM EGTA present, including all subsequent washing steps. Samples were incubated for 10 min at room temperature, for 30 min at 4 °C, and for 10 min at room temperature and then spun. The filtrates were collected (unbound fractions), and the beads were washed with 0.2% Triton X-100, 0.15 M KCl, and 20 mM Tris (pH 7.5). These washing cycles were done five times and were followed by a washing step with 10 mM Tris (pH 7.5). Bound proteins were eluted first with 100 mM glycine (pH 2.5) and second with 2% SDS, 5 mM EDTA, and 20 mM Tris (pH 7.5).

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-- SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described (20). The polyclonal anti-ROS-GC antibody (27) was diluted 1:5000; the polyclonal anti-GCAP-1 antibody (20) was diluted 1:10,000; and the monoclonal anti-alpha -tubulin antibody (Sigma) was diluted 1:50,000. The secondary horseradish peroxidase-coupled antibodies were diluted 1:5000. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).

    RESULTS

Identification of an Interaction Domain in GCAP-1-- Ninety-six peptides encompassing the entire amino acid sequence of GCAP-1 were tested at a concentration of 0.1 mM for their efficiency to inhibit the ROS-GC activity in the presence of GCAP and 10-9 M Ca2+. Most peptides caused a weak inhibition from 0 to 10% (Fig. 1). A few peptides even showed a small stimulatory effect (see, for example, peptides 8-10). The most effective peptides were peptides 37 and 38 (74MEYVAALSLVLKGK87); they caused 30-40% inhibition (Fig. 1). Peptides of intermediate efficiency (20-30%) were peptides 1 (amino acids 2-15), 15 and 18 (amino acids 30-47), and 44 and 45 (amino acids 88-102). Peptides that displayed inhibition >= 20% were further examined at increasing concentrations (0.1-0.3 mM). The results for peptides 1, 38, and 44 are shown in Fig. 2A (peptide 36 was chosen as a control). The normalized ROS-GC activity was lowered to 30% with 0.3 mM peptide 38. Less strong, but still significant were the inhibitory effects of peptides 1 and 44 (60-70% normalized ROS-GC activity) and peptide 37 (60-70% normalized ROS-GC activity) (data not shown). Peptides 15, 18, and 45 were of similar efficiency as peptide 44 (data not shown). A small effect was seen with peptide 36 (Fig. 2A). Because native GCAP-1 contains a myristoyl group at the N terminus, we synthesized a myristoylated and a nonmyristoylated form of the N-terminal peptide. The nonmyristoylated peptide did not change the ROS-GC activity (data not shown), whereas the myristoylated peptide displayed the modest inhibitory effect shown in Fig. 1 (peptide 1). Basal ROS-GC activity in washed ROS membranes depleted of GCAP was not influenced by most peptides, with the exception of peptides 45-47, which showed a weak inhibition of 20% (data not shown). In conclusion, peptides 37 and 38 were the most effective in interfering with ROS-GC activity. In further studies, we used a peptide that encompassed the sequences of both peptides 37 and 38 (peptide 37/38) (Fig. 3A).


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Fig. 1.   Inhibition of GCAP-1-dependent activation of ROS-GC at low [Ca2+] by peptides shown as percentage of inhibition. Peptides at 0.1 mM were added to suspensions of bovine ROS, and ROS-GC activity was measured at low [Ca2+] (~1 nM). Data represent the mean ± S.D. of two to four measurements.


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Fig. 2.   A, normalized ROS-GC activity at different peptide concentrations. Activity was measured at 1 nM Ca2+ in whole ROS. ROS-GC activity without peptide was set to 1. B, comparison of the inhibitory effect of peptides 37/38 (open circle ) and 38 () at increasing peptide concentrations. The IC50 values were 0.07 and 0.14 mM for peptides 37/38 and 38, respectively. Curves were fitted to a modified Hill equation. Data represent mean ± S.D. (triplicates).


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Fig. 3.   A, domain structure of GCAP-1 in the myristoylated (myr) form and localization of peptides 37/38 and 38. The nonfunctional EF-hand motif (EF1) is shown as shaded box; the three functional EF-hand motifs (EF2-EF4) are shown as black boxes. B, sequence alignment of the identified interaction domain in GCAP-1 (Phe73-Lys87) and the corresponding regions in GCAP-2 (Phe78-Thr92) (22), recoverin (Rec; Phe83-Lys97) (29), and calmodulin (CaM; Phe65-Thr79 (31). Identical amino acids are shaded. C, percentage inhibition of ROS-GC activity at low [Ca2+] by peptides from GCAP-1, GCAP-2, and recoverin (sequences shown in B) at 0.1 mM.

Inhibition of ROS-GC was saturable at a concentration of 0.3 mM with peptide 37/38 (73FMEYVAALSLVLKGK87), but the maximal inhibition did not exceed 80% (Fig. 2B). A shorter section of this putative interaction domain represented by peptide 38 inhibited ROS-GC to the same maximal extent. The IC50 value of 0.14 mM, however, was larger than the one observed for peptide 37/38 (0.07 mM). We conclude that the three additional amino acids (Phe73-Glu75) in peptide 37/38 determine the affinity of ROS-GC for GCAP-1. It is interesting that flanking regions of this domain (represented by peptides 36 (Fig. 2A) and 39 (data not shown)) showed only a marginal effect.

Comparison of the GCAP-1 Peptide with Related Peptides from GCAP-2 and Recoverin-- A peptide from recoverin related to peptide 37/38 caused only 15% inhibition of ROS-GC at low [Ca2+], whereas the related peptide from GCAP-2 showed 50% inhibition (GCAP-1 peptide 37/38 caused 75% inhibition (Fig. 3, B and C); all peptides at 0.1 mM). These results indicated that the GCAP-1 peptide (and the almost identical GCAP-2 peptide) acts specifically on ROS-GC and that the amino acid differences between recoverin and GCAP peptides are critical for the interaction with ROS-GC.

Inhibition by Peptides Is Reversible-- Recombinant GCAP-1 was added to a suspension of ROS in which ROS-GC was inhibited by either peptide 37/38 or 38. GCAP-1 at 12 µM could completely relieve the inhibition by both peptides. Addition of GCAP-1 to a ROS suspension without peptides did not change the maximal rate of ROS-GC activity (Fig. 4). This experiment clearly showed that the observed inhibition is caused by a direct interference of the peptides with the GCAP-1/ROS-GC interaction, which can be overcome by an additional amount of GCAP-1.


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Fig. 4.   Inhibition of ROS-GC activity by GCAP-1 peptides is reversible. ROS preparations were incubated at low [Ca2+]. The sample contained endogenous GCAP-1; additional GCAP-1 did not further increase maximal ROS-GC activity (black and white control columns). Peptides 38 and 37/38 at 0.1 mM suppressed the stimulation of ROS-GC activity. Addition of GCAP-1 compensated the inhibitory effects. The specific activity of ROS-GC is calculated in relation to the amount of rhodopsin present in the incubation mixture. Data show mean ± S.D.

Influence of Peptides on the Ca2+ Regulation of ROS-GC-- We next examined whether inhibitory peptides could change the [Ca2+] at which the activation of ROS-GC was half-maximal. The inhibitory effect of peptides 37/38 and 38 (0.1 mM peptide) was studied within the physiologically important range of [Ca2+] (Fig. 5). Peptide 37/38 was more efficient than peptide 38, consistent with the results in Fig. 2B. Peptides at 0.1 mM did not significantly change the Ca2+ regulation of ROS-GC activity (Fig. 5, inset). The activation of ROS-GC was half-maximal at 60 nM Ca2+ and cooperative (nH = 1.6) independent of peptide addition. The EC50 of 60 nM is at the lower range of reported values in the literature (50-280 nM) (12). ROS-GC activity at high [Ca2+] was not influenced by these peptides. Basal ROS-GC activity in ROS membranes depleted of GCAP was also not influenced at high or low [Ca2+]. Therefore, peptides 37/38 and 38 interfere with the GCAP-1/ROS-GC interaction and not with basal ROS-GC activity.


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Fig. 5.   Ca2+-dependent regulation of ROS-GC in the presence of peptides 37/38 (open circle ) and 38 () at 0.1 mM and without peptides (control; black-down-triangle ). The EC50 values are 70, 54, and 60 nM for peptides 38 and 37/38 and the control, respectively. The specific activity of ROS-GC is calculated in relation to the amount of rhodopsin present in the incubation mixture. The inset shows the normalized ROS-GC activity for the incubation with and without peptides. Maximal ROS-GC activity at low [Ca2+] was set to 1. Data show mean ± S.D. (triplicates).

Binding of ROS-GC and Tubulin to a Peptide Affinity Column-- We tested direct binding of ROS-GC to the most effective GCAP peptide (peptide 37/38). Peptide 37/38 was covalently coupled to an activated thiol-Sepharose matrix via an additional N-terminal cysteine and a glycine spacer (37/38-Sepharose). The 37/38-Sepharose was incubated with an extract containing ROS-GC and other ROS membrane and cytoskeletal proteins. Two controls were used, Sepharose columns to which either cysteine (Cys-Sepharose) or peptide 53 (53-Sepharose) was coupled. GCAP-1 peptide 53 (102CIDRDELLTIIR113) was chosen as a control because it did not show a significant effect in our screening assay (Fig. 1) and could be coupled to the column matrix via its terminal cysteine. No protein was retained by these columns (Fig. 6A, bound, C and 53 lanes), and the unbound fractions of the Cys-Sepharose and 53-Sepharose were identical in their protein composition when compared with the starting material (C and 53 lanes). Occasionally, some proteins bound nonspecifically to these columns (data not shown). Two major polypeptides of 112 and 55 kDa, respectively, were bound to the 37/38-Sepharose. They were completely removed from the ROS extract (Fig. 6A, nonbound, 37/38 lane) and could be eluted from the Sepharose by 0.5% SDS (bound, 37/38 lane). Western blotting of the unbound and bound fractions revealed that the band at 112 kDa is ROS-GC (Fig. 6B), whereas the protein at 55 kDa was identified as tubulin (Fig. 6D, DE, B lane). A few other proteins appeared in both the bound and unbound fractions of the 37/38-Sepharose. Therefore, binding of these proteins to the 37/38-Sepharose was not specific, whereas it was for ROS-GC and tubulin. Tubulin in a control extract without ROS-GC (Fig. 6C, TE) did not bind to the 37/38-Sepharose (Fig. 6D, TE, NB lane). Sometimes tubulin was lost, probably due to aggregation when ROS-GC was not present.


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Fig. 6.   Peptide affinity column chromatography. A dodecyl maltoside extract (DE) containing ROS-GC was incubated with three kinds of Sepharose: Cys-Sepharose (C lanes), 53-Sepharose (53 lanes), and 37/38-Sepharose (37/38 lanes). Unbound and bound fractions were electrophoresed, and the gel was stained with Coomassie Blue (A) or blotted afterward for immunostaining (B) using a polyclonal anti-ROS-GC antibody. C shows the distribution of tubulin and ROS-GC in Triton X-100-solubilized ROS membranes (TS lanes) and in Triton X-100 extracts (TE lanes) tested by immunoblotting. D shows the results from affinity chromatography of tubulin-containing extracts on the 37/38-Sepharose. Tubulin was found in the unbound (nonbound (NB lanes)) fraction when no ROS-GC was present, and it was found in the bound (B lanes) fraction when ROS-GC was present (DE).

Coimmunoprecipitation of GCAP-1, ROS-GC, and Tubulin-- The results of the peptide affinity chromatography suggested that ROS-GC and tubulin bind as a complex to GCAP-1. We tested this by coimmunoprecipitation using a polyclonal GCAP-1 antibody (20). This antibody, covalently coupled to protein A-Sepharose, specifically immunoprecipitated GCAP-1. GCAP-1 was partially eluted from the beads by glycine; the remaining GCAP-1 was eluted by SDS (data not shown). Both ROS-GC and tubulin coimmunoprecipitated with GCAP-1 (Fig. 7, A and B, SDS elution). Tubulin could be partially eluted by glycine, whereas ROS-GC needed an SDS buffer for elution. The presence of either 1 mM CaCl2 or 1 mM EGTA did not change the binding of the ROS-GC·tubulin complex to GCAP-1. Thus, the interaction of the ROS-GC·tubulin complex with GCAP-1 seemed to be independent of Ca2+. A small amount of ROS-GC and tubulin was not immunoprecipitated (Fig. 7, A and B, nonbound); however, GCAP-1, ROS-GC, and tubulin were not bound to protein A-Sepharose without immobilized GCAP-1 antibody (c lanes). These results confirmed that ROS-GC and tubulin form a complex with GCAP-1.


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Fig. 7.   Coimmunoprecipitation experiments. A cytoplasmic extract of ROS proteins was incubated with anti-GCAP antibody beads to allow binding of GCAP-1 to the antibody. Solubilized ROS membranes (RM lanes) were incubated with the beads either with 1 mM CaCl2 (Ca lanes) or with 1 mM EGTA (EGTA lanes). Bound fractions were collected by elution with glycine or SDS. Unbound and bound samples were analyzed by Western blotting. Blots were probed with antibodies against ROS-GC (A) and tubulin (B). Control incubations (c lanes) were performed with protein A-Sepharose beads without attached antibodies.


    DISCUSSION

We have identified a 15-amino acid region located C-terminally from the second EF-hand (Phe73-Lys87) in GCAP-1 as the main interaction domain (Fig. 3) for ROS-GC. The peptide interfered with the GCAP-1-dependent activation of ROS-GC at low [Ca2+], which indicates that it critically disturbs the Ca2+-dependent activation of ROS-GC by GCAP-1. We cannot exclude that the peptides also compete with the action of GCAP-2. However, based on other reports (21, 28) and our previous observations (20), GCAP-1 is the major component of bovine ROS homogenates. We compared the region with regions adjacent to the second EF-hand in GCAP-2, recoverin, and calmodulin, three other Ca2+-binding proteins involved in regulation of phototransduction (21, 22, 29, 30). GCAP-1 and GCAP-2 are almost identical in this region and, not surprisingly, showed the same inhibition of ROS-GC activity (Fig. 3, B and C). The related peptide from recoverin was much less effective. Sequence identity between GCAP-1 and recoverin is only found at 8 out of 15 positions. Significant differences in recoverin compared with GCAP-1 are Lys84 (corresponding to Met74 in GCAP-1) and the stretch of five amino acids at His91-Ala95 (Ser81-Lys85 in GCAP-1), which is more hydrophobic in GCAP-1. We conclude that the differences in amino acid sequences in the compared regions are essential for different target recognition of recoverin and GCAP-1.

Other regions that were less effective in the competition studies might also participate in controlling ROS-GC activity. For example, a myristoylated N-terminal peptide significantly decreased ROS-GC activity, whereas a nonmyristoylated form failed to show an effect. This result is in agreement with two previous studies. One study showed that a myristoylated peptide from the N terminus decreased GCAP-1-stimulated ROS-GC activity (18); in the other study, it was reported that an N-terminally truncated mutant of GCAP-1 was unable to stimulate ROS-GC (24). The myristoylated N terminus of GCAP-1 might be involved in locating GCAP-1 close to the membrane in the right orientation to interact with ROS-GC. Otto-Bruc et al. (24) identified three larger regions in GCAP-1 as putative interaction domains. These regions correspond to peptides 1-7, 27-38, and 68-75. Although our screening assay narrowed down two of their regions to a stretch of 15 amino acids (peptides 1 and 37/38), we did not observe any significant inhibitory effect with peptides from the C-terminal part (peptide 68-75 in Fig. 1). Incubation with a peptide mixture that covered this entire region also showed no significant inhibitory effect (data not shown). One explanation for these two different results is that the interaction domain represented by this C-terminal region needs a correct folding, which might be present in the larger peptide of Otto-Bruc et al. (24).

We also demonstrated direct binding of ROS-GC to peptide 37/38 by affinity chromatography. ROS-GC bound to this peptide in a complex with tubulin. The same complex formation was observed when ROS-GC was coimmunoprecipitated with GCAP-1. These two independent experiments strongly suggest that there exists a tight association of ROS-GC with the cytoskeletal protein tubulin.

Association of ROS-GC with the outer segment cytoskeleton has been noted by different investigators based on fractionation, solubilization, and overlay studies (5, 32, 33). So far, only actin has been identified as one of the interacting components (33). Our finding that tubulin and ROS-GC form a complex with GCAP-1 indicates that Ca2+/cGMP signaling in photoreceptor cells occurs in close proximity to cytoskeletal elements, but it is unclear whether cytoskeletal proteins are directly involved in enzyme regulation.

The immunoprecipitation studies also indicate that GCAP-1 and ROS-GC form a stable complex independent of the free [Ca2+]. This is consistent with previous cross-linking experiments in which GCAP-1 was found to be in close proximity to ROS-GC at high and low [Ca2+] (16). Competition experiments of Ca2+-loaded GCAP-1 with a constitutively active GCAP-1 mutant also showed that Ca2+-bound GCAP binds to ROS-GC (25). All these findings support a model in which a change in cytosolic [Ca2+] would be detected by GCAP-1 while it is bound to ROS-GC. Activation occurs by a conformational switch in GCAP-1 that is transmitted to the cyclase. Such a regulatory mechanism would be different from classical calmodulin-regulated systems, in which calmodulin controls the activity of its targets by Ca2+-dependent binding and dissociation (31), but the mechanism would resemble the regulation of calmodulin targets containing IQ motifs (34).

    ACKNOWLEDGEMENTS

We thank D. Höppner-Heitmann for excellent technical assistance and Dr. U. B. Kaupp for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by Grant Ko948/5-1 from the Deutsche Forschungsgemeinschaft (to K.-W. K).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These two authors contributed equally to this work.

parallel To whom correspondence should be addressed. Tel.: 49-2461-613255; Fax: 49-2461-614216; E-mail: k.w.koch{at}fz-juelich.de.

    ABBREVIATIONS

The abbreviations used are: ROS-GC, membrane-bound retina-specific guanylyl cyclase in rod outer segments; GCAP, guanylyl cyclase-activating protein; Fmoc, N-(9-fluorenyl)methoxycarbonyl; ROS, rod outer segment(s); DTT, dithiothreitol.

    REFERENCES
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Abstract
Introduction
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