JBC Connect with Cosmo for Collagen Detection

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M408683200 on September 23, 2004

J. Biol. Chem., Vol. 279, Issue 48, 50342-50349, November 26, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/48/50342    most recent
M408683200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Peshenko, I. V.
Right arrow Articles by Dizhoor, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Peshenko, I. V.
Right arrow Articles by Dizhoor, A. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis*

Igor V. Peshenko, Elena V. Olshevskaya, and Alexander M. Dizhoor, Martin and Florence Hafter professor of pharmacology{ddagger}

From the Hafter Research Laboratories, Pennsylvania College of Optometry, Elkins Park, Pennsylvania 19027

Received for publication, July 30, 2004 , and in revised form, September 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Guanylyl cyclase-activating proteins (GCAPs) are calcium sensor proteins of the EF-hand superfamily that inhibit retinal photoreceptor membrane guanylyl cyclase (retGC) in the dark when they bind Ca2+ but activate retGC when Ca2+ dissociates from GCAPs in response to light stimulus. We addressed the difference in exposure of GCAP-2 structure to protein kinase and a protease as indicators of conformational change caused by binding and release of Ca2+. We have found that unlike its homolog, GCAP-1, the C terminus of GCAP-2 undergoes phosphorylation by cyclic nucleotide-dependent protein kinases (CNDPK) present in the retinal extract and rapid dephosphorylation by the protein phosphatase PP2C present in the retina. Inactivation of the CNDPK phosphorylation site in GCAP-2 by substitutions S201G or S201D, as well as phosphorylation or thiophosphorylation of Ser201, had little effect on the ability of GCAP-2 to regulate retGC in reconstituted membranes in vitro. At the same time, Ca2+ strongly inhibited phosphorylation of the wild-type GCAP-2 by retinal CNDPK but did not affect phosphorylation of a constitutively active Ca2+-insensitive GCAP-2 mutant. Partial digestion of purified GCAP-2 with Glu-C protease revealed at least two sites that become exposed or constrained in a Ca2+-sensitive manner. The Ca2+-dependent conformational changes in GCAP-2 affect the areas around Glu62 residue in the entering helix of EF-hand 2, the areas proximal to the exiting helix of EF-hand 3, and Glu136–Glu 138 between EF-hand 3 and EF-hand 4. These changes also cause the release of the C-terminal Ser201 from the constraint caused by the Ca2+-bound conformation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Photoisomerized rhodopsin triggers hydrolysis of cGMP through the activation of the G-protein transducin, which stimulates cGMP phosphodiesterase and thus causes cGMP-gated channels to close (see Refs. 1–3 for review). Rods and cones rapidly recover to their resting potential after excitation induced by a brief non-saturating exposure to light. They also adapt to a constant background illumination by decreasing the amplitude and accelerating the kinetics of their electrical responses to a standard test flash (see Refs. 1–3 for review). Reopening of the cGMP-gated channels during recovery and light adaptation requires acceleration of cGMP synthesis by guanylyl cyclase, a process controlled by the level of intracellular free Ca2+. In the dark, Ca2+ extruded from the photoreceptor outer segment by a Na+/K+,Ca2+ exchanger re-enters the outer segment through the open cGMP-gated channels so that the free intracellular outer segment Ca2+ concentrations reach 250 nM in mammals and 350–450 nM in lower vertebrates (4–6). In the light, cGMP is hydrolyzed, and these channels close while the exchanger continues to extrude Ca2+ ions from the photoreceptor outer segment; therefore, Ca2+ concentrations decrease nearly 10-fold (4–7). Ca2+-binding proteins GCAP-11 and GCAP-2 activate retGC1 and retGC2 (and their homologs found in photoreceptors of different vertebrate species) in a Ca2+-sensitive manner (see Refs. 8–11 for review). GCAPs are a separate subfamily within the broader family of neuronal calcium sensor proteins that includes many recoverin-like proteins. The recoverin-like neuronal calcium sensor proteins have four EF-hand-type Ca2+-binding domains and an N-terminal Gly residue acylated with fatty acid residues. Only three of the EF-hand structures in GCAPs are true Ca2+-binding domains in which Ca2+ sensitivity is adjusted to the range within which the intracellular free Ca2+ varies between light and dark by intracellular free Mg2+ (12). The N-terminal EF-hand-related motif EF-1 cannot bind Ca2+ but strongly contributes to the GCAP interaction with retGC (13, 14).

The partial solution structure of the Ca2+-bound GCAP-2 established by Ames et al. (15) resembles those of recoverin and neurocalcin and consists of two globular halves connected by a flexible "hinge" region. The NMR structure was established only for the Ca2+-loaded form of GCAP-2 and does not include the C-terminal portion of the protein; therefore, the exact conformational changes that occur when dissociation of the Ca2+ ions causes GCAP-2 to transition into the activator conformation are not immediately apparent.

In our present study, we used the accessibility for the CNDPK phosphorylation of a Ser201 residue proximal to the C terminus of GCAP-2 and limited proteolysis by protease V8 to assess the conformational changes in GCAP-2 related to the binding and release of Ca2+. Our data indicate that the C terminus of GCAP-2 undergoes a conformational Ca2+-dependent switch that releases the C terminus from a constraint created by the Ca2+-loaded structure of GCAP-2 and changes the exposure of the regions between EF-hands 2 and 3 and between EF-3 and -4.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cyclic Nucleotide-dependent Protein Kinases—Purified cGMP-dependent kinase II (PKG II) and cAMP-dependent protein kinase (PKA) were kindly provided by Dr. Michael Uhler (University of Michigan). Purified cGMP-dependent protein kinase isoform I{alpha} (PKG I{alpha}) was from Calbiochem/EMD (San Diego, CA).

Recombinant myristoylated bovine and mouse GCAP-2, GCAP-1, and neurocalcin were expressed from pET11d vector (Novagen/EMD) in a BLR(DE3)pLysS Escherichia coli strain harboring yeast N-myr-istoyltransferase and purified as described previously in full detail (12, 16, 17).

Site-directed Mutagenesis—All mutations were incorporated into bovine GCAP-2 cDNA by PCR using the splicing by overlap extension technique and Pfu DNA polymerase (Stratagene) as described previously (18, 19). All mutant cDNAs where then sequenced using Cy5-modified dideoxynucleoside triphosphates, a ThermoSequenase kit, and an SEQ 4 x 4 automated sequencer (Amersham Biosciences). The C{Delta}1GCAP-2 mutant lacked the last nine amino acid residues. The Ca2+-insensitive GCAP-2, EF2/3/4, contains single substitutions in all three EF-hands that prevented high-affinity Ca2+ binding and was expressed and purified as described previously in detail (18, 19).

Photoreceptor outer segment membranes were isolated under infrared illumination from frozen dark-adapted bovine retinas (Lawson and Lawson) using sucrose density gradient centrifugation and were washed in low salt buffer to remove endogenous GCAPs as described previously (12, 16).

Soluble Retinal Extract (S100)—Fresh bovine eyes obtained at a local slaughterhouse were dark-adapted for 2 h on ice, dissected under infrared light, and homogenized in 0.5 ml of buffer A (5 mM sodium phosphate buffer, pH 7.2, 50 mM NaCl) per retina on ice by 10 strokes in a Dounce homogenizer. The homogenate was centrifuged at 20,000 x g at 4 °C, and the supernatant was collected and centrifuged in 200-µl aliquots in a Beckman A100 rotor for 30 min at 80,000 rpm. The supernatant fraction, S100, was collected and used immediately or divided into aliquots, frozen in dry ice, and stored at -70 °C. Typical total protein concentration in the S100 fraction was near 40 mg/ml

Phosphorylation Assay—Unless indicated otherwise, a typical phosphorylation reaction contained 30 mM Tris/HCl, pH 7.5, 5 mM MgCl2, 5 mM sodium phosphate buffer, pH 7.5, 6 mM dithiothreitol, 0.1 mM EGTA, 10 µM ATP or 10 µM ATP{gamma}S (Sigma), ~10 µCi of [32P]- or [35S]ATP{gamma}S (PerkinElmer Life Sciences), and 300 µM 8-Br-cGMP (Sigma). After incubation at 37 °C, the reaction was stopped by adding 2 volumes of a Laemmli sample SDS buffer containing 4 mM EGTA and was boiled for 5 min. Phosphorylated proteins were then separated by SDS-PAGE in 15% polyacrylamide gel, fixed in 7.5% acetic acid, 10% methanol solution, stained with Coomassie Blue, destained in 7.5% acetic acid, 10% methanol, and covered with thin polyethylene film exposed on Kodak Bio Max or X-Omat AR at room temperature. Adding EGTA in the sample buffer was required to prevent the appearance of multiple bands of GCAPs because of Ca2+ binding during PAGE.

The thiophosphorylation reaction contained 50 mM Tris/HCl, pH 7.6, 2 mM EGTA, 5 mM MgCl2, 0.5 mM 8-Br-cGMP, 10 µg/ml leupeptin, 20,000 units of PKG I{alpha}, 5.0 mM ATP{gamma}S, and 60 µM GCAP-2. The reaction was incubated at 23 °C for 2 h and then incubated overnight at 4 °C. After incubation, the reaction mixture was dialyzed against 10 mM Tris/HCl, pH 7.5.

Protein Phosphatase Assay—Wild-type GCAP-2 was 32P-labeled in the presence of PKG I{alpha} as described above, except instead of quenching the reaction by adding SDS sample buffer, the unincorporated label was removed by gel filtration on Sephadex G-50. The phosphatase reaction typically contained 1 µg of [32P]GCAP-2 (~1 µCi/µg), 10 µl of S100 protein fraction in 5 mM MgCl2, 20 mM Tris 7.5, and 6 mM dithiothreitol in a total volume of 20 µl. Fenvalerate, okadaic acid, and microcystine were added from 100x concentrated stock solution in Me2SO, and the corresponding volume of Me2SO was added in control samples even though Me2SO had no noticeable effect on the phosphatase activity because of low concentrations. After incubation at 37 °C for 15 min. the reaction was stopped by adding 2 volumes of a Laemmli sample SDS-buffer containing 4 mM EGTA and was boiled for 3 min followed by SDS-PAGE and radioautography as described above.

Immunoblot—Proteins were transferred from SDS-polyacrylamide gel onto Immobillon P membrane (Millipore) for 20 h at 60 V, stained for GCAP-2 using polyclonal rabbit antibody {Delta}NP24 (20), and developed using alkaline phosphatase-conjugated goat anti-rabbit IgG (ICN/Cappel).

Glu-C Proteolysis—The reaction contained 20 µl of 10 mM Tris/HCl, pH 7.5, 10 mM 2-mercaptoethanol, either 500 µM CaCl2 or 100 µM EDTA, 46 µg of purified GCAP-2, and 0.18 µg of Staphylococcus aureus protease V8 (Sigma). The components were mixed at room temperature and incubated for 7 min at 37 °C, and the reaction was stopped by heating to 100 °C for 5 min. 10-µl aliquots from each reaction were then loaded on a capillary C4 column. Peptides eluted with a gradient of acetonitrile in 0.05% trifluoroacetic acid were directly infused into a Sciex API III electrospray mass spectrometer and scanned for positive ions with m/z values between 600 and 1200. Mass spectrometry data were processed using Sciex MacProMass and MacSpec software.

RetGC Assay—All experiments were conducted under infrared light as described (12). The assay mixture (25 µl) contained 30 mM MOPS/KOH, pH 7.2, 60 mM KCl, 5 mM NaCl, 1 mM dithiothreitol, 2 mM Ca2+/EGTA buffer, 1 mM free Mg2+, zaprinast and dipyridamole (25 µM each), leupeptin and aprotinin (10 µg/ml each), 0.3 mM ATP, 4 mM cGMP, 1 mM GTP, ~1 µCi of [{alpha}-32P]GTP, 0.1 µCi of [8-3H]cGMP, 1 µl of washed bovine outer segment membranes (3.7 mg/ml rhodopsin), and purified recombinant GCAP-2 or its mutants as indicated in the figure legends. The products of the reaction were analyzed by TLC using fluorescent plastic polyethyleneimine cellulose as described (17).

Ca2+/EGTA buffers were prepared as described (21), and the final free Ca2+ and Mg2+ concentrations in the reaction mixture were calculated using the algorithm of Marks and Maxfield (22). Ca2+/EGTA buffers were calibrated using Ca2+ fluorescence dyes Fluo-3 and X-rhod FF (Kd of 325 nM and 17 µM, respectively) (Molecular Probes, Eugene, OR).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
According to the NMR spectroscopy study by Ames et al. (15), the overall folding of Ca2+-loaded GCAP-2 resembles recoverin, a Ca2+-binding protein that affects rhodopsin kinase activity (23) and neurocalcin (24). All attempts to solve the three-dimensional structure of the Ca2+-free activator form of GCAP-2 were unsuccessful, so there is no direct structural information available on how Ca2+ release or binding can trigger conformational changes in the GCAP-2 molecule. Moreover, the C-terminal portion of GCAP-2 was not resolved in the NMR structure of Ca2+-loaded GCAP-2. Reversible dimerization of Ca2+-free GCAP-2 (25) poses an additional challenge for the study because binding and release of Ca2+ not only affects the conformation of GCAP-2, but also alters GCAP-2/GCAP-2 interaction. To assess the possible structural change between Ca2+-bound and Ca2+-free GCAP-2, we studied the effects of Ca2+ on site-specific phosphorylation and limited proteolysis of GCAP-2.

C-terminal Ser201 in GCAP-2 Undergoes Phosphorylation by CNDPK
The RRKSAMF sequence at the C terminus of the GCAP-2 molecule (20) is a typical consensus recognition site for both cGMP- and cAMP-dependent kinases, so three different purified CNDPKs can phosphorylate the C-terminal Ser in GCAP-2 in vitro (Fig. 1, A–D). Purified PKG I phosphorylates GCAP-2 with low efficiency in the absence of added cyclic nucleotide, and the addition of cGMP enhances the phosphorylation (Fig. 1B, lanes b and c). GCAP-1 lacks such a sequence (26), and we did not observe phosphorylation of GCAP-1 by CNDPK under the same conditions (Fig. 1, A and B, lane e). Unlike wild-type GCAP-2, a chimera mutant, in which the C-terminal region downstream of EF-4 was substituted with the corresponding fragment from the related recoverin-like protein neurocalcin (19), did not undergo phosphorylation under these conditions (not shown). Deletion (mutant C{Delta}1) of the last nine amino acid residues, including Ser201, from GCAP-2 (19) also completely abolished phosphorylation (Fig. 1, A and B, lane d). Apparently, the C terminus in GCAP-2 was the only part in GCAP-2 that was phosphorylated. To ensure that the CNDPK site Ser201 was the only site of phosphorylation in GCAP-2, we substituted Ser201 with either Gly or Asp and found that both mutations completely abolished 32P incorporation in GCAP-2 (Fig. 1C). In the presence of N-myristoyltransferase, GCAP-2 is expressed in E. coli predominantly as a fatty acylated protein (16); however, a variable small fraction of GCAP-2 that remains non-acylated is often present in such preparations and appears in SDS-PAGE as a minor band just above the acylated GCAP-2 (16). When both myristoylated and non-myristoylated forms are present in preparation of GCAP-2, they incorporate the 32P label catalyzed by purified PKA and PKG (Fig. 1D). Hence, myristoylation is not required for GCAP-2 to become a substrate for CNDPK.



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 1.
Purified CNDPK phosphorylates Ser201 in GCAP-2. A, Coomassie stain of proteins in phosphorylation mixtures separated by 15% SDS-PAGE. The strong upper band corresponds to purified bovine serum albumin (BSA) added to the phosphorylation reaction as the internal negative control. B, radioautograph of 32P phosphorylation products (duplicate from A). The molecular mass (MW) markers (Bio-Rad, broad range) shown in lane a are 200, 115, 97, 67, 43, 31, 21, 14, and 6 kDa. The position of GCAPs in the gel is indicated by the asterisk. The minor band, ~1 kDa above the main GCAP band, in every case corresponds to non-myristoylated GCAP. The 50-µl reaction mixture contained purified PKG I{alpha} (1,000 units, Calbiochem), 40 µg of bovine serum albumin, and 20 µg of purified recombinant WT GCAP-2 (b and c), partially purified C{Delta}1GCAP-2 mutant (d), or purified recombinant GCAP-1 (e). c–e, 150 µM cGMP was added; b, no cGMP added. After incubation for 30 min at 37 °C, each reaction mixture was diluted with Laemmli buffer, and 20 µl were loaded on SDS-PAGE and analyzed as described under "Experimental Procedures." C, lack of PKG I{alpha}-dependent phosphorylation in GCAP-2 with Ser201 substituted by Asp (a) or Gly (b). c, non-substituted Ser201 GCAP-2 as a control. D, radioautograph of WT GCAP-2 phosphorylated in vitro by 1 µl each of PKG II (a), PKA (b), and PKG Ia (c). E, radioautograph of recombinant GCAP-2 phosphorylated by soluble retinal CNDPK in retinal extract as a function of Mg2+ concentration. a–e, 1.5 µg of WT GCAP-2; f–i,1 µgofC{Delta}1GCAP-2. The reaction contained 10 µl of retinal S100 fraction and 100 µM 8-Br-cGMP. F, GCAP-2 phosphorylation by S100 soluble retinal fraction requires cyclic nucleotide. A radioautograph of 1 µg of WT GCAP-2 phosphorylated in the presence of 10 µl of S100 fraction in the presence (a) or in the absence (b)of100 µM 8-Br-cGMP is shown. c, no exogenous GCAP-2 was added. d, purified [32P]GCAP-2 labeled in vitro by PKG I{alpha}, as in A, used as a protein marker. G and H, PKG I-dependent phosphorylation does not affect electrophoretic mobility of bovine or mouse GCAP-2. G, bovine GCAP-2 (2 µg) that contains 50% phosphorylated myristoylated GCAP-2 migrates as a single band (b). The extent of GCAP-2 phosphorylation by PKG I was determined from the specific activity of the 32P label incorporated into 75 µM GCAP-2 from 0.75 mM ATP containing 10 µCi of [{gamma}-32P]ATP (300 Ci/mmol). H, left panel (Coomassie Blue stain) shows protein molecular mass markers (a), 14 µg of non-phosphorylated recombinant myristoylated mouse GCAP-2 (mouse GCAP-2 migrates faster than bovine GCAP-2) (b), and 32P-phosphorylated GCAP-2 (c). The position of the main protein band is marked with an arrow covered with radioactive ink. Right panel, radioautograph of the same gel.

 
PKA, PKG II, and, at a lower level, PKG I have been reported in photoreceptors (27, 28). Consistent with that report, we have found that the retinal extract contains soluble GCAP-2 kinase activity stimulated by cGMP or its slow hydrolyzable analog, 8-Br-cGMP (Fig. 1, D and F). To minimize potential interference from phosphorylation of the endogenous GCAP-2, we extracted soluble retinal proteins (S100 fraction) at normal ionic strength when GCAP-2 remained mostly associated with the membrane fraction (16). Although the S100 fraction contains an ~33-kDa endogenous protein (presumably phosducin) that is highly phosphorylated by the CNDPK (27, 29), this band migrates substantially higher than GCAP-2 and therefore does not interfere with the analysis. Using 8-Br-cGMP produces more stable and reproducible results because the S100 fraction, even isolated from the dark-adapted retina, contains substantial traces of cGMP phosphodiesterase activity (not shown). In the presence of cyclic nucleotide, purified recombinant GCAP-2 becomes phosphorylated by the S100 fraction. The Ser201 in GCAP-2 was the only site for the protein kinase activity present in the S100 fraction, and it was phosphorylated only by CNDPK. Removing the CNDPK site from GCAP-2 either by deletion or a by the point mutation S201D (Fig. 1, C and E) completely suppresses phosphorylation of GCAP-2 by the retinal extract, as does the removal of CNDPK activator (Fig. 1F). Although in other proteins phosphorylation can alter their electrophoretic mobility, phosphorylation of Ser201 in GCAP-2 (either of bovine or mouse origin) does not affect its electrophoretic mobility in denaturing SDS gel (Fig. 1, G and H).

Dephosphorylation of GCAP-2—GCAP-2 can be phosphorylated by CNDPK present in the S100 fraction, but the efficiency of phosphorylation in that case is drastically lower (>10-fold lower, data not shown) compared with purified CNDPK because the S100 fraction also contains a protein phosphatase (Fig. 2). Purified 32P-labeled GCAP-2, phosphorylated by PKG I{alpha} and added to the retinal S100 fraction, becomes rapidly dephosphorylated (Fig. 2, A and D). The loss of radioactivity was due to phosphatase activity, not protein degradation, because leupeptin and aprotinin did not prevent the loss of the 32P label from prephosphorylated GCAP-2 (not shown), and the integrity of GCAP-2 was not compromised after incubation with S100 (Fig. 2B).



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 2.
Dephosphorylation of phospho-Ser201 in GCAP-2 by retinal protein phosphatase. A, radioautograph of recombinant-purified [32P]GCAP-2 incubated with the retinal S100 fraction in the absence (a) or in the presence (b–f) of protein phosphatase inhibitors. Lanes a*, b*, c*, d*, e*, and f*, heat-inactivated S100; b and b*, 5 µM okadaic acid; c and c*, 5 µM fenvalerate; d and d*, 75 µM bromotetramisole; e and e* -100 µM microcystine; f and f*, 1 mM EGTA in the presence of 7 mM MgCl2. B, the loss of 32P from phosphorylated GCAP-2 in the presence of retinal S100 fraction is not because of GCAP-2 proteolysis. An immunoblot of GCAP-2 after 15 min of incubation in the absence (a) or in the presence (b) of the S100 fraction is shown. C, EDTA inhibits GCAP-2 dephosphorylation by the retinal protein phosphatase. A radioautograph of [32P]GCAP-2 after incubation for 0–45 min in the presence of the retinal S100 fraction containing 2 mM EDTA (left) or 5 mM MgCl2 (right) is shown. D, the time course of [32P]GCAP-2 dephosphorylation by retinal protein phosphatases in the absence ({blacktriangleup}) or in the presence of 100 µM microcystine ({blacksquare}), 5 µM fenvalerate ({triangleup}),7 mM MgCl2,1 mM EGTA mixture (•), or 2 mM EDTA ({circ}). The densities of [32P]GCAP-2 bands from radioautographs after incubation with S100 fraction were measured by densitometry (MultiAnalyst, Bio-Rad) and plotted relative to the 100% intensity at "0 time" sample containing heat-inactivated S100 fraction.

 
Inhibitors of protein phosphatases PP1, PP2A, and PP2B (such as Fenvalerate, microcystine, and okadaic acid) do not affect dephosphorylation of [32P]GCAP-2, even at concentrations high enough to completely inhibit these protein phosphatases (Fig. 2). Similarly, neither bromotetramisole (an alkaline phosphatase inhibitor) nor the removal of Ca2+ by EGTA inhibits dephosphorylation. Inhibition of the [32P]GCAP-2 dephosphorylation occurred only when Mg2+ was removed from the reaction mixture by EDTA. EDTA is the only inhibitor of the fourth type of protein phosphatase, the Mg2+-dependent phosphatase PP2C, which is also present in the retina (30) (Fig. 2, A, C, and D). Phospho-GCAP-2 appears to be the first PP2C-specific substrate identified in photoreceptors. The only covalent modification in GCAP-2 isolated from bovine retinas was fatty acylation (16), and the presence of potent phosphatase PP2C in retinal extract (30) may account for the lack of phosphorylated GCAP-2 after its isolation from the retina. Therefore, it remains unclear to what extent GCAP-2 can be phosphorylated in vivo.

Phosphorylation of GCAP-2 and RetGC Regulation
ATP is known to potentiate the stimulation of retGC by GCAP-2 in vitro (31–34). The light-activated photoreceptor-specific phosphodiesterase PDE6 can hydrolyze both cGMP and cAMP, and Ca2+-dependent regulation of adenylyl cyclase can regulate cAMP levels in rods (29, 35, 36). Therefore, either PKA or PKG could potentially affect retGC activity or Ca2+ sensitivity via phosphorylation of GCAP-2. However, neither inactivation of the phosphorylation site in GCAP-2 (Fig. 3) nor inclusion of CNDPK and its activators directly into the retGC assay reaction (not shown) had an effect on retGC regulation. Only a small change (<15%) in maximal activity of retGC was observed when Ser201 was substituted with either Gly or negatively charged Asp residues. Yet the potentiating effect of ATP on retGC activity remained present in both GCAP-2 mutants (Fig. 3). To completely exclude the possibility that PP2C, which is present in outer segment membranes, interferes with the analysis, we used modification by a thiophosphate group that can be transferred to proteins by protein kinases but cannot be removed by protein phosphatases. Prior to use in retGC assay, GCAP-2 was incubated with PKG I{alpha} and ATP{gamma}S, after which 92% of GCAP-2 became unavailable for subsequent phosphorylation using [{gamma}-32P]ATP (Fig. 4A). Neither the affinity for guanylyl cyclase nor the Ca2+ sensitivity of the cyclase regulation by GCAP-2 changed after the Ser201 thiophosphorylation of GCAP-2 (Fig. 4, A and B). Apparently, the effect of ATP on retGC activation is because of either ATP binding by retGC (33) or phosphorylation of retGC itself (37) but not because of phosphorylation of GCAP-2. Phosphorylation by PKG does not affect Ca2+-sensitive dimerization of GCAP-2 either (25) (Fig. 4, C and D). Distribution of [32P]GCAP-2 in the fractions eluted from the gel filtration column demonstrates that the amount of radioactivity follows the protein profiles. The phosphorylated GCAP-2 elutes predominantly as a monomer in the presence of Ca2+ but distributes almost equally between the monomer and the dimer in the presence of EGTA.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 3.
Activation of retGC in vitro in outer segment membranes by GCAP-2 and its mutants. RetGC activity stimulation by WT GCAP-2 is shown at 10 nM free Ca2+ (•, {circ}), S201D GCAP-2 ({triangleup}, {blacktriangleup}), and S201G GCAP-2 ({diamond}, {diamondsuit}) in the absence (•, {blacktriangleup}, {diamondsuit}, solid lines) and in the presence ({circ}, {triangleup}, {diamond}, dashed lines) of 0.5 mM ATP. Inset, Coomassiestained gel after SDS-PAGE of purified S201G GCAP-2 (1.8 µg) (a), S201D (2 µg) (b), and WT GCAP-2 (2.4 µg)(c).

 



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 4.
Phosphorylation of GCAP-2 and retGC regulation. A, Ca2+ sensitivity of retGC activation in washed outer segment membranes by 5 µM thiophosphorylated (•) or non-thiophosphorylated ({circ}) GCAP-2. Inset, radioautograph of [32P]GCAP-2. Control (a) or thiophosphorylated (b) GCAP-2 was 32P-labeled using [{gamma}-32P]ATP. B, activation of retGC by thiophosphorylated (•) or non-thiophosphorylated ({circ}) GCAP-2 in the presence of 2 mM EGTA. C and D, phosphorylation by PKG does not interfere with Ca2+-sensitive dimerization of GCAP-2. Purified bovine myristoylated GCAP-2 was phosphorylated using [{gamma}-32P]ATP and purified PKG in the presence of EGTA as described under "Experimental Procedures." After phosphorylation, free [{gamma}-32P]ATP was removed by repeated dilution/concentration using a Millipore Ultrafree-4 cartridge, and the [32P]GCAP-2 was subjected to gel permeation chromatography on an Amersham Superdex 200 HR 10/30 column connected to a fast protein liquid chromatography system. Samples contained either 2 mM CaCl2 (C) or 2 mM EGTA (D). The elution buffer contained 10 mM Tris/HCl, pH 7.5, 10 mM NaCl, 60 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol, and either 500 µM CaCl2 (C) or 500 µM EGTA (D).

 
This result is consistent with our previous observation that small C-terminal deletions in GCAP-2 have only a minor effect on retGC activation (19) and argues against GCAP-2 phosphorylation playing a part in retGC regulation. Yet we cannot exclude the possibility that phosphorylation contributes to processes other than GCAP-2/retGC interaction because the C-terminal CNDPK site is preserved in all vertebrate species (38).

Conformational Change in GCAP-2 as a Function of Ca2+ Binding
Our preliminary experiments indicated that phosphorylation of Ser201 in GCAP-2 by the retinal CNDPK was suppressed when concentrations of free Ca2+ were above 1 µM in the reaction mixture (not shown). However, the efficiency of GCAP-2 phosphorylation by the crude retinal CNDPK is dramatically influenced by PP2C activity present in the retinal extract, which can potentially interfere with the interpretation of these data. Therefore, to avoid interference from the PP2C activity, we used [35S]ATP{gamma}S. Protein kinases can efficiently utilize this ATP analog as a substrate, whereas the resulting thiophosphoprotein is not recognized by protein phosphatases (39). By using this approach we found that retinal CNDPK indeed phosphorylates GCAP-2 in a Ca2+-sensitive manner (Fig. 5). GCAP-2 fully saturates with Ca2+ when free Ca2+ concentrations are above 1 µM, even in the presence of 5 mM MgCl2 (12, 15). The saturating free Ca2+ concentrations suppress GCAP-2 thiophosphorylation by the soluble retinal CNDPK (Fig. 5A, lanes a and b). S201D GCAP-2 was used as a control for the CNDPK-specific site (Fig. 5A, lane c), and both thiophosphorylation and phosphorylation of GCAP-2 by purified CNDPK were also inhibited (Fig. 5, B and C). In different experiments, the relative efficiency of the retinal CNDPK-dependent phosphorylation for the Ca2+-loaded GCAP-2 varied between 6 and 40% compared with the Ca2+-free GCAP-2, and on average, phosphorylation of the Ca2+-bound GCAP-2 was suppressed nearly 5-fold (Fig. 5D).



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 5.
Ca2+-sensitive GCAP-2 phosphorylation by CNDPK. A, 35S thiophosphorylation of GCAP-2 by soluble retinal extract. A radioautograph of 35S thiophosphorylation products is shown. 0.2 µg of GCAP-2 was incubated for 70 min in reaction mixture containing 2.5 µl of the retinal S100 fraction in the presence of 10 µCi of [35S]ATP{gamma}S (300 Ci/mmol) followed by SDS-PAGE. a and b, WT GCAP-2; c, S201D GCAP-2; d and e, EF2/3/4 GCAP-2; a, c, and d, <6 nM free Ca2+; b and e, 40 µM free Ca2+. B, 35S thiophosphorylation of WT GCAP-2 by 100 units of purified PKG I{alpha} at <6 nM (a) and 40 µM Ca2+ (b). C, 32P phosphorylation of purified WT GCAP-2 (a and b) and its calcium-insensitive EF2/3/4 mutant (c and d) by 100 units of PKG I{alpha} at <6 nM (a) and 40 µM Ca2+ (b). D, relative efficiency of WT (a and b) and EF2/3/4 (c and d) GCAP-2 phosphorylation and thiophosphorylation in the presence of EGTA (a and c) or 40 µM Ca2+ (b and d). The optical density of bands on the radioautograph from independent measurements using scanning densitometer and MultiAnalyst software is plotted relative to the density of the GCAP-2 band produced in the sample containing EGTA.

 
Was the effect of Ca2+ because of a change in the specificity of the CNDPK activity or the result of a conformational change in GCAP-2? To answer this question, we used the constitutively active GCAP-2 mutant EF2/3/4 (18). This mutant remains constitutively active even in the presence of Ca2+, because inactivation of all three active EF-hands by substitutions in the first or the last amino acid residues of a 12-amino acid Ca2+-binding loop of the EF-hands 2, 3, and 4 locks GCAP-2 in a Ca2+-free conformation (18). If the effect of Ca2+ was because of changes in CNDPK activity and specificity rather than related conformational changes in GCAP-2, then phosphorylation of the constitutive EF2/3/4 GCAP-2 ought to be as Ca2+-sensitive as the wild-type GCAP-2. Conversely, if the effect of EGTA and Ca2+ on the phosphorylation of GCAP-2 by CNDPK resulted mainly from the change in exposure of the Ser201 as a substrate for CNDPK, Ca2+ should have had, in contrast to wild-type GCAP-2, little effect on phosphorylation of the EF2/3/4 GCAP-2. Indeed, unlike the wild type, the constitutively active GCAP-2 mutant was equally well phosphorylated at both low and high Ca2+ concentrations (Fig. 5A, lanes d and e, and Fig. 5C, lanes c and d). We therefore conclude that the effect of Ca2+ on GCAP-2 phosphorylation directly reflects the difference in exposure of the Ser201 rather than the change in CNDPK activity by Ca2+.

To detect other major changes in GCAP-2 caused by binding and release of Ca2+, we performed partial proteolytic digestion of GCAP-2 by S. aureus protease V8. This was performed when <50% of the full-length protein underwent proteolytic cleavage. The products of the reaction were analyzed by liquid chromatography electrospray ionization mass spectroscopy (Fig. 6). We found major difference for the two sets of products with regard to Ca2+ sensitivity. The set of fragments generated by the cleavage of Glu136 or Glu138 and found in the presence of Ca2+ was virtually undetectable among the products of proteolysis of the Ca2+-free GCAP-2. These results indicate that the region between EF-hand 3 and EF-hand 4 (Fig. 7) becomes less constrained when EF-hands are filled with Ca2+. In contrast, the Glu62 located in the entering alpha helix of EF-hand 2 is only exposed when GCAP-2 undergoes the transition into its Ca2+-free (cyclase activator) conformation. The three possible scenarios through which Ca2+ may affect the exposure of the parts of GCAP-2 are discussed in the following paragraphs.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 6.
Fragments of limited V8 proteolysis generated in the presence and in the absence of Ca2+. A, fragments of different molecular masses eluted from the C4 column and identified by liquid chromatography electrospray ionization mass spectroscopy. The x axis is a time scale, and the y axis is relative intensity. Total counts for one ion species from the series obtained for each fragment is shown in parentheses. Red traces are fragments generated in the absence of Ca2+. Blue traces are fragments generated in the presence of a saturating Ca2+ concentration. Top to bottom, profiles extracted from chromatograms for each ion species (m/z): a, 885.20 (18H+); b, 880.20 (9H+); c, 857.80 (9H+); d, 1461.60 (5H+); and e, 919.00(18H+). B, the primary structure of GCAP-2 with the sites exposed for proteolysis and CNDPK phosphorylation in Ca2+-loaded (blue) and Ca2+-free (red) form. The diagram shows the cleavage pattern at the sites most available for the protease V8 in the absence (red) and in the presence (blue) of Ca2+. Asp and Glu residues in the GCAP-2 primary structure are shown as vertical lines above and below the bars representing protein fragments. The molecular masses of the fragments ± standard deviation are shown above each fragment. The calculated averaged isotopic masses are shown in parentheses next to the experimental values.

 



View larger version (61K):
[in this window]
[in a new window]
  		FIG. 7.
Putative conformational changes in GCAP-2 caused by binding and release of Ca2+. A, the C terminus in GCAP-2 is flexible and unstructured (15), but it is loosely constrained in Ca2+-bound conformation and remains largely inaccessible for CNDPK. Dissociation of Ca2+ from GCAP-2 results in a conformational change that promotes both the dimerization of GCAP-2 (25) and the release of its C terminus from the constraint created by protein structure. Other explanations are in the text under "Results and Discussion." B and C, change in exposure of Glu62, Glu136, and Glu138. B, Glu62, Glu136, and Glu138 side chains are shown space-filled in two projections based on three-dimensional partial NMR structure of the Ca2+-loaded GCAP-2 (Ames et al.) (15). The model was drawn using SPDB Viewer 3.7 software. Positions of Ca2+ ions within the Ca2+-binding loops of EF-hands 2, 3, and 4 are shown as green spheres. C, Glu62, shown with space-filled atoms in the entering helix of EF-hand 2, is likely to move away from the exiting helix of EF-hand 3 in the absence of Ca2+ and thus become exposed for proteolysis when GCAP-2 undergoes transition into the activator conformation.

 
Compartmentalization—GCAP-2 could associate with the membrane and/or the target cyclase in such a manner that in the Ca2+-bound form, the C terminus is protected from phosphorylation by proximity to the membrane. However, we are justified in ruling out this possibility because (a) the GCAP-2 phosphorylation and proteolysis experiments described in this paper were carried out using only soluble protein, and (b) Ca2+-loaded GCAP-2 tends to dissociate from the membrane more efficiently than the Ca2+-free form (16).

Ca2+-dependent Association of GCAP-2—Ca2+-dependent association of GCAP-2 with unknown soluble retinal protein(s) could hamper its interaction with CNDPK. This possibility cannot be completely ruled out in the case of a complex protein mixture such as the S100 fraction. Yet it remains highly unlikely, because we observed Ca2+ sensitivity of GCAP-2 phosphorylation even by purified CNDPK in the absence of other proteins in the reaction (Fig. 5, B and C). Therefore, the change in availability of the C-terminal Ser201 as a substrate for CNDPK reflects its constraint in the Ca2+-bound form and its release from constraint in Ca2+-free GCAP-2 (Fig. 7A).

Dimerization of GCAP-2—The Ca2+-free form of GCAP-2 can undergo dimerization, and the dimer dissociates upon its transition back into the Ca2+-loaded retGC inhibitor form (13, 25). Obviously, the exposure of some regions of the main and side chains in GCAP-2 can be affected by the GCAP-2 dimerization. In particular, the flexible portion of GCAP-2 structure between EF-hands 3 and 4 could become masked when it forms the Ca2+-free dimer. However, this seems an unlikely explanation, because even in the Ca2+-free form of GCAP-2, we observed a mixture of monomers and dimers at a nearly equal (or at least comparable) ratio. Should the Glu136–Glu138 region become constrained only as a result of dimerization, we would still expect almost one-half of the protein to undergo cleavage at these sites. Yet unlike Ca2+-bound GCAP-2, none of the corresponding peptides was detectable in the case of the Ca2+-free GCAP-2 (Fig. 6, left panel). Therefore, it is more reasonable to suggest that the flexible loop between EF-hands 3 and 4, well exposed in Ca2+-bound GCAP-2 structure (15), becomes constrained in both Ca2+-free monomers and dimers of GCAP-2. Even though dissociation of Ca2+ promotes GCAP-2 dimerization (25), its C-terminal Ser201 becomes more accessible for the CNDPK (Figs. 5 and 7).

In the case of recoverin, binding of Ca2+ results in a calcium-myristoyl switch, during which the fatty acylated N-terminal segment of the molecule undergoes a Ca2+-dependent extrusion from a hydrophobic pocket (40). A similar process may also take place in the case of other recoverin-like proteins (24). Yet, both functional and structural analyses of GCAP-2 strongly indicate that Ca2+-dependent conformational changes in GCAP-2 are different from recoverin and do not include the N-terminal Ca2+-myristoyl switch (16, 41). Our data support the possibility that, instead, the C terminus in GCAP-2 undergoes a Ca2+-sensitive conformational switch. Although the flexibility and exposure of the C terminus is limited by the constraint created in the protein structure by Ca2+ binding to EF-hands, the constraint at the C-terminal Ser201 is released after dissociation of Ca2+ from GCAP-2 (Fig. 7). Hence, if a Ca2+-sensitive switch exists in GCAP-2, it is located at the C terminus of GCAP-2, which is opposite to the N-terminal Ca2+-myristoyl switch in recoverin that extrudes the N terminus of the protein when Ca2+ is bound (42). The NMR spectroscopy does not show the presence of stable long-range interactions at the C terminus (amino acids 189–204). This would indicate that the C terminus remains unstructured and flexible even in Ca2+-bound conformation, yet it is not extruded far enough from the protein core to become accessible for CNDPK.

Interestingly, neither the C terminus nor the flexible unstructured loop between EF-hands 3 and 4 observed in the NMR structure of Ca2+-bound GCAP-2 (15) is essential for retGC activation (19), so their exposure or constraint reflects the overall conformational change in GCAP-2 rather than playing an important role in cyclase regulation. In contrast to that role, the region between EF-hands 2 and 3 in GCAPs apparently plays an essential role in retGC regulation (13, 19, 43, 44), especially in the hinge region located between the exiting helix of EF-2 and the entering helix of EF-3 (15). Because the Glu62 residue located in the entering helix of EF-2 becomes exposed in Ca2+-free GCAP-2, the release of Ca2+ most likely destabilizes the interaction between the entering helix of EF-2 and exiting helix of EF-3, potentially making the hinge region more accessible for external protein-protein interactions and/or reflecting a motion between the two globular parts of GCAP-2. It has also been speculated that such a motion takes place in GCAP-1 during binding and dissociation of Ca2+ based on tryptophan fluorescence changes and tryptic digestion (45–47). Also, in Ca2+-free recoverin, the entering helix of EF-2 is solvent-exposed and structurally unstable. By contrast, in Ca2+-bound recoverin, this helix becomes highly stabilized by its interaction with the helices of EF-3 at the domain interface (48, 49). Similar Ca2+-induced changes may take place in GCAP-2.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant EY11522 from the NEI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Pennsylvania College of Optometry, 8360 Old York Rd., Elkins Park, PA 19027. Tel.: 215-780-1468; Fax: 215-780-1464; E-mail: adizhoor{at}pco.edu.

1 The abbreviations used are: GCAP, guanylyl cyclase-activating protein; retGC, retinal photoreceptor membrane guanylyl cyclase; EF-1, -2, -3, and -4, the first, second, third, and fourth EF-hand domains, respectively, in GCAP primary structure; CNDPK, cyclic nucleotide-dependent protein kinases; PKG, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; ATP{gamma}S, adenosine 5'-O-(thiotriphosphate); MOPS, 4-morpholinepropanesulfonic acid; PP, protein phosphatase; WT, wild type; 8-Br-cGMP, 8-bromo-cyclic GMP. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Michael Uhler (University of Michigan) for the generous gifts of purified PKG II and PKA and for stimulating discussion. We also thank Irina Peshenko for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Pugh, E. N., Jr., and Lamb, T. D. (1993) Biochim. Biophys. Acta 1141, 111-149[Medline] [Order article via Infotrieve]
  2. Baylor, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 560-665[Abstract/Free Full Text]
  3. Pugh, E. N., Jr., Nikonov, S., and Lamb, T. D. (1999) Curr. Opin. Neurobiol. 9, 410-418[CrossRef][Medline] [Order article via Infotrieve]
  4. Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., and Fain, G. L. (2002) J. Physiol. (Lond.) 542, 843-854[Abstract/Free Full Text]
  5. Woodruff, M. L., Wang, Z., Chung, H. Y., Redmond, T. M., Fain, G. L., and Lem, J. (2003) Nat. Genet. 35, 158-164[CrossRef][Medline] [Order article via Infotrieve]
  6. Sampath, A. P., Matthews, H. R., Cornwall, M. C., Bandarchi, J., and Fain, G. L. (1999) J. Gen. Physiol. 113, 267-277[Abstract/Free Full Text]
  7. Gray-Keller, M. P., Detwiler, P. B. (1994) Neuron 13, 849-861[CrossRef][Medline] [Order article via Infotrieve]
  8. Pugh, E. N., Jr., Duda, T., Sitaramayya, A., and Sharma, R. K. (1997) Biosci. Rep. 17, 429-473[CrossRef][Medline] [Order article via Infotrieve]
  9. Dizhoor, A. M. (2000) Cell. Signal. 12, 711-719[CrossRef][Medline] [Order article via Infotrieve]
  10. Palczewski, K., Polans, A. S., Baehr, W., and Ames, J. B. (2000) BioEssays 22, 337-350[CrossRef][Medline] [Order article via Infotrieve]
  11. Haeseleer, F., Imanishi, Y., Sokal, I., Filipek, S., and Palczewski, K. (2002) Biochem. Biophys. Res. Commun. 290, 615-623[CrossRef][Medline] [Order article via Infotrieve]
  12. Peshenko, I. V., and Dizhoor, A. M. (2004) J. Biol. Chem. 279, 16903-16906[Abstract/Free Full Text]
  13. Ermilov, A. N., Olshevskaya, E. V., and Dizhoor, A. M. (2001) J. Biol. Chem. 276, 48143-48148[Abstract/Free Full Text]
  14. Krylov, D. M., Niemi, G. A., Dizhoor, A. M., and Hurley, J. B. (1999) J. Biol. Chem. 274, 10833-10839[Abstract/Free Full Text]
  15. Ames, J. B., Dizhoor, A. M., Ikura, M., Palczewski, K., and Stryer, L. (1999) J. Biol. Chem. 274, 19329-19337[Abstract/Free Full Text]
  16. Olshevskaya, E. V., Hughes, R. E., Hurley, J. B., and Dizhoor, A. M. (1997) J. Biol. Chem. 272, 14327-14333[Abstract/Free Full Text]
  17. Hurley, J. B., and Dizhoor, A. M. (2000) Methods Enzymol. 315, 708-718[Medline] [Order article via Infotrieve]
  18. Dizhoor, A. M., and Hurley, J. B. (1996) J. Biol. Chem. 271, 19346-19350[Abstract/Free Full Text]
  19. Olshevskaya, E. V., Boikov, S., Ermilov, A. N., Krylov, D., Hurley, J. B., and Dizhoor, A. M. (1999) J. Biol. Chem. 274, 10823-10832[Abstract/Free Full Text]
  20. Dizhoor, A. M., Olshevskaya, E. V., Henzel, W. J., Wong, S. C., Stults, J. T., Ankoudinova, I., and Hurley, J. B. (1995) J. Biol. Chem. 270, 25200-25206[Abstract/Free Full Text]
  21. Tsien, R., and Pozzan, T. (1989) Methods Enzymol. 172, 230-262[Medline] [Order article via Infotrieve]
  22. Marks, P. W., and Maxfield, F. R. (1991) Anal. Biochem. 193, 61-71[CrossRef][Medline] [Order article via Infotrieve]
  23. Flaherty, K. M., Zozulya, S., Stryer, L., and McKay, D. B. (1993) Cell 75, 709-716[CrossRef][Medline] [Order article via Infotrieve]
  24. Vijay-Kumar, S., and Kumar, V. D. (1999) Nat. Struct. Biol. 6, 80-88[CrossRef][Medline] [Order article via Infotrieve]
  25. Olshevskaya, E. V., Ermilov, A. N., and Dizhoor, A. M. (1999) J. Biol. Chem. 274, 25583-25587[Abstract/Free Full Text]
  26. 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[CrossRef][Medline] [Order article via Infotrieve]
  27. Lee, R. H., Brown, B. M., and Lolley, R. N. (1990) J. Biol. Chem. 265, 15860-15866[Abstract/Free Full Text]
  28. Gamm, D. M., Barthel, L. K., Raymond, P. A., and Uhler, M. D. (2000) Investig. Ophthalmol. Vis. Sci. 41, 2766-2773[Abstract/Free Full Text]
  29. Pagh-Roehl, K., Lin, D., Su, L., and Burnside, B. (1995) J. Neurosci. 15, 6475-6488[Abstract/Free Full Text]
  30. Klumpp, S., Selke, D., Fischer, D., Baumann, A., Muller, F., and Thanos, S. (1998) J. Neurosci. Res. 51, 328-338[CrossRef][Medline] [Order article via Infotrieve]
  31. Gorczyca, W. A., Van Hooser, J. P., and Palczewski, K. (1994) Biochemistry 33, 3217-3222[CrossRef][Medline] [Order article via Infotrieve]
  32. Laura, R. P., Dizhoor, A. M., and Hurley, J. B. (1996) J. Biol. Chem. 271, 11646-11651[Abstract/Free Full Text]
  33. Tucker, C. L., Laura, R. P., and Hurley, J. B. (1997) Biochemistry 36, 11995-12000[CrossRef][Medline] [Order article via Infotrieve]
  34. Yamazaki, A., Yu, H., Yamazaki, M., Honkawa, H., Matsuura, I., Usukura, J., and Yamazaki, R. K. (2003) J. Biol. Chem. 278, 33150-33160[Abstract/Free Full Text]
  35. Willardson, B. M., Wilkins, J. F., Yoshida, T., and Bitensky, M. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1475-1479[Abstract/Free Full Text]
  36. Yamazaki, A., Sen, I., Bitensky, M. W., Casnellie, J. E., and Greengard, P. (1980) J. Biol. Chem. 255, 11619-11624[Abstract/Free Full Text]
  37. Aparicio, J. G., and Applebury, M. L. (1996) J. Biol. Chem. 271, 27083-27089[Abstract/Free Full Text]
  38. Burgoyne, R. D., and Weiss, J. L. (2001) Biochem. J. 353, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  39. Gergely, P., Vereb, G., and Bot, G. (1976) Biochim. Biophys. Acta 429, 809-816[Medline] [Order article via Infotrieve]
  40. Ames, J. B., Ishima, R., Tanaka, T., Gordon, J. I., Stryer, L., and Ikura, M. (1997) Nature 389, 198-202[CrossRef][Medline] [Order article via Infotrieve]
  41. Hughes, R. E., Brzovic, P. S., Dizhoor, A. M., Klevit, R. E., and Hurley, J. B. (1998) Protein Sci. 7, 2675-2680[Abstract]
  42. Dizhoor, A. M., Chen, C.-K., Olshevskaya, E., Sinelnikova, V. V., Philippov, P. P., and Hurley, J. B. (1993) Science 259, 829-832[Abstract/Free Full Text]
  43. Li, N., Sokal, I., Bronson, J. D., Palczewski, K., and Baehr, W. (2001) Biol. Chem. 382, 1179-1188[CrossRef][Medline] [Order article via Infotrieve]
  44. Otto-Bruc, A., Buczylko, J., Surgucheva, I., Subbaraya, I., Rudnicka-Nawrot, M., Crabb, J. W., Arendt, A., Hargrave, P. A., Baehr, W., and Palczewski, K. (1997) Biochemistry 36, 4295-4302[CrossRef][Medline] [Order article via Infotrieve]
  45. Sokal, I., Otto-Bruc, A. E., Surgucheva, I., Verlinde, C. L. M. J., Wang, C.-K., Baehr, W., and Palczewski, K. (1999) J. Biol. Chem. 274, 19829-19837[Abstract/Free Full Text]
  46. Sokal, I., Li, N., Klug, C. S., Filipek, S., Hubbell, W. L., Baehr, W., and Palczewski, K. (2001) J. Biol. Chem. 276, 43361-43373[Abstract/Free Full Text]
  47. Rudnicka-Nawrot, M., Surgucheva, I., Hulmes, J. D., Haeseleer, F., Sokal, I., Crabb, J. W., Baehr, W., and Palczewski, K. (1998) Biochemistry 37, 248-257[CrossRef][Medline] [Order article via Infotrieve]
  48. Ames, J. B., and Ikura, M. (2002) Adv. Exp. Med. Biol. 514, 333-348[Medline] [Order article via Infotrieve]
  49. Ames, J. B., Hamasaki, N., and Molchanova, T. (2002) Biochemistry 417, 5776-5787

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/48/50342    most recent
M408683200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles