Mapping Functional Domains of the Guanylate Cyclase Regulator Protein, GCAP-2*

Elena V. OlshevskayaDagger , Sergei BoikovDagger , Alexander ErmilovDagger , Dmitri Krylov§, James B. Hurley§, and Alexander M. DizhoorDagger parallel

From the Dagger  Department of Ophthalmology/Kresge Eye Institute and the  Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201 and the § Department of Biochemistry and HHMI, University of Washington, Seattle, Washington 98195

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Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.

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Guanylate cyclase activating proteins (GCAPs)1 are Ca2+-binding proteins that mediate regulation of cGMP synthesis by Ca2+ in retinal rods and cones. Ca2+ enters outer segments (OS) of vertebrate photoreceptors through cGMP-gated Na+/Ca2+ channels in the plasma membranes. These channels are open in the dark, but light closes them by stimulating cGMP hydrolysis. Channel closure lowers the intracellular free Ca2+ concentrations in rods from 700 nM to near 30 nM (1) because Ca2+ is removed from the OS by a light-independent Na+/K+, Ca2+ exchanger (2-4). The decrease in free Ca2+ concentration stimulates cGMP synthesis (5). cGMP, is synthesized in photoreceptors by two membrane guanylate cyclases, RetGC-1 and RetGC-2 (also referred to as ROSGC-1 and -2, or CG-E and GC-F, respectively) (5-11). The RetGCs are regulated by two homologous, but distinct Ca2+-binding proteins, GCAP-1 and GCAP-2 (6, 12-15) (Fig. 1). When free Ca2+ concentrations decrease from 700 nM to below 50 nM, GCAP-1 and GCAP-2 (6, 7, 9-14) activate membrane RetGC. GCAP-1 and GCAP-2 each have four EF-hand-like domains including three functional EF-hands that bind Ca2+ ions. At free Ca2+ concentrations below 100 nM, characteristic of light-adapted rods, GCAPs activate the cyclase, but at free Ca2+ concentrations above 600 nM (characteristic of dark-adapted photoreceptors) GCAPs inhibit it (16, 17). Inactivation of EF-hands makes GCAPs constitutive activators of RetGC (16, 17).

Despite their functional and structural homology, GCAP-2 and GCAP-1 appear to be different in several aspects. 1) The only significant sequence homology between GCAP-1 and GCAP-2 is within their EF-hands. 2) A naturally occurring point mutation Y99C in GCAP-1 that was linked to human cone degeneration (18) causes a constitutive Ca2+ insensitive activation of RetGC (19, 20), while the corresponding mutation in GCAP-2 causes partial inhibition of its activity without affecting its Ca2+ sensitivity (19). 3) N-terminal fatty acylation is reportedly essential for the regulatory properties of GCAP-1 (21), but it is not essential for GCAP-2 (22). 4) EF-hand 3 in GCAP-2 contributes Ca2+ sensitivity to RetGC regulation relatively weakly compared with EF-2 and EF-4 (16), whereas EF-3 plays a more significant role in GCAP-1 (17). 5) Krishnan et al. (23) reported that GCAP-1 and GCAP-2 may interact with two different sites on RetGC.

The goal of this study was to define major regulatory domain(s) of GCAP-2 using its deletion and chimera mutants. We find that in addition to the previously characterized EF-hand Ca2+-binding domains, there are three regions in GCAP-2 that cannot be deleted or substituted without loss of function. One of these regions determines that the Ca2+-free form of GCAP-2 activates, and the Ca2+-bound form inhibits RetGC activity in photoreceptor membranes.

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Site-directed Mutagenesis (Fig. 2)-- Chimera DNAs were constructed by using polymerase chain reaction (PCR) and "splicing by overlap extension." DNA fragments with chimeric termini were first amplified by PCR using Pfu polymerase (Stratagene), appropriate cDNAs (neurocalcin-delta cDNA was a gift from D. Ladant) as templates and PCR primers based on the sequences of GCAP-2, neurocalcin, or recoverin cDNAs. The fragments were purified from agarose gel using QIAEX II resin (QIAGEN) and spliced together by a second round of PCR according to the splicing by overlap extension technique (24). The resulting DNA fragments were ligated into the NcoI/BamHI sites of the pET11d vector (Novagen). DNA sequence of the resulting constructs were verified by an automated DNA sequences using the ABI Prism system (Perkin-Elmer). Deletion mutants were constructed by a similar approach except the second round of PCR was only used to splice fragments of DNA in order to introduce the internal deletions.

Expression of Recombinant Proteins-- Wild type and deletion mutants of bovine GCAP-2 and GCAP-2/neurocalcin chimeric proteins were expressed in E. coli from pET11d vector (Novagen) according to the procedures described previously in full detail (22) except that we used the BLR(DE3)pLysS Escherichia coli strain (Novagen). Wild type GCAP-2 and recoverin/GCAP-2 chimeras were also expressed in transfected HEK 293 cell culture as described previously (14). Bovine recoverin and neurocalcin-delta were expressed in E. coli according to Refs. 25 and 26 except that we used the BLR(DE3) E. coli strain (Novagen) instead of BL21(DE3). Recombinant GCAP-2 and chimera proteins expressed in E. coli were purified using gel filtration chromatography as described previously in detail (22). Recombinant recoverin and neurocalcin were purified using phenyl-Sepharose (Pharmacia) chromatography (25, 26). The recombinant proteins were verified for the presence of N-myristoylation by reversed-phase high performance liquid chromatography and electrospray mass spectrometry as described previously (27). All chimeras were predominantly in myristoylated form except for the chimeras XX and XXI.

RetGC Activity Assay-- Washed bovine outer segment membranes depleted of endogenous activator and containing both RetGC-1 and RetGC-2 were prepared, reconstituted with recombinant GCAPs, and assayed as described previously (6, 22). Experiments were conducted under infrared light using two 15 W safety lights equipped with Kodak number 11 infrared filters at a distance of 50 cm and Excalibur dual high performance GEN II+ tube (PVS-5c) goggles. A typical reaction mixture contained 5 µM GCAP-2 or its chimera constructs in 25 µl of 50 mM MOPS-KOH (pH 7.5), 60 mM KCl, 8 mM NaCl, 10 mM MgCl2, 2 mM Ca/EGTA buffer, 10 µM each of dipyridamole and zaprinast, 1 mM ATP, 1 mM GTP, 4 mM cGMP, 1 µCi of [alpha -32P]GTP, 0.1 µCi of [3H]cGMP, and washed bovine outer segment membranes (3.5 µg of rhodopsin). Reaction mixtures were incubated for 12 min at 30 °C, heated for 2 min at 95 °C, chilled on ice, centrifuged for 5 min at 10,000 × g, and analyzed by TLC on polyethylenimine cellulose plastic-backed plates with fluorescent background (Merck). After development in 0.2 M LiCl, cGMP spots were visualized under UV illumination, cut, eluted with 1 ml of 2 M LiCl, shaken with 10 ml of an Ecolume scintillation mixture (ICN) and both 3H and 32P radioactivity were counted. [3H]cGMP was used as an internal standard to ensure the absence of cGMP hydrolysis by phosphodiesterase. In all experiments time course of reaction was linear in time and less than 10% of GTP substrate was converted into cGMP. Basal activity of RetGC in different preparations of washed OS membranes typically varied between 2.5 and 5 nmol of cGMP/min/mg of rhodopsin. In the absence of protein activators the difference between RetGC basal activity measured at 6 nM and 1 µM free Ca2+did not exceed 20%. Data that are shown in every figure pertain to a single experiment using one preparation of washed OS membranes, they are representative of two or three similar independent experiments.

Recombinant Bovine RetGC-1 (ROSGC1, GC-E) and RetGC-2 (ROSGC2, GC-F)-- These clones were expressed in HEK293 cells as described previously for human RetGC-1 and -2 (6, 7). Bovine cDNA clones in pcDNA3.1 vector (23) were a gift from Dr. R. Sharma. DNA constructs were amplified in E. coli in the presence of ampicillin and purified using a Promega plasmid DNA purification kit. Cells were propagated in RPMI 1640 media supplemented with calf serum and antibiotics in 100-mm diameter culture dishes until approximately 60% confluent and then transiently transfected with 10-15 mcg of DNA/plate using a conventional calcium phosphate precipitation. Membranes were isolated from homogenized cells and reconstituted with GCAP-2 essentially as described (6, 7). Basal activity of recombinant RetGC-1 and RetGC-2 was less than 0.05 nmol/min/mg of protein. Nonspecific background produced by boiled membranes (near 500 disintegrations/min) was subtracted. Because activity of recombinant cyclases was much lower than that of native RetGC in washed OS membranes (6, 7), time of incubation was increased to 30 min. Approximately 1% of total GTP was converted into cGMP after stimulation of recombinant RetGC-1 and RetGC-2 with GCAP-2 during the reaction.

Ca/EGTA Buffers-- The buffers were prepared according to Ref. 28.

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Rationale for Mutagenesis and Analyses-- GCAP-1 and GCAP-2 are overall 40% identical (13, 14). However, most of the sequence identity between GCAPs derives from their EF-hands and there is almost no sequence identity in their N and C termini (Fig. 1A). GCAPs belong to a distinct group of recoverin-like Ca2+-binding proteins (29) that are distantly related to calmodulin. Recoverin-like proteins have four helix-loop-helix Ca2+-binding domains, EF-hands, usually named EF-1 through EF-4. Amino acids that form Ca2+-binding loops are shown in boxes in Fig. 1A. Unlike calmodulin, the EF-hand loop sequence in EF-1 in all of the currently characterized recoverin-like proteins is disrupted and cannot function as a Ca2+-binding center. Like other recoverin-like proteins, GCAPs also have a signal for the N-terminal fatty acylation (13, 14). The sequence identity between neurocalcin and GCAP-2 is almost as high as between GCAP-2 and GCAP-1. Recoverin is nearly 28%, and neurocalcin is nearly 39% identical to bovine GCAP-2. Yet unlike GCAPs neither neurocalcin (Fig. 1B) nor recoverin (as indicated in the inset to Fig. 6A, and in the accompanying article, Ref. 36) can regulate RetGC activity.


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Fig. 1.   A, sequence homology between GCAP-2 and other recoverin-like proteins. GCAP-2 from two different species, bovine (BGCAP2) and human (HGCAP2) (16, 30), are aligned with each other and with other recoverin-like proteins: bovine GCAP-1 (bGCAP-1, 13), bovine neurocalcin delta  (BovNCa, 25), and bovine recoverin (BovRv, 26). Amino acid residues identical between HGCAP1 and HGCAP2 and other proteins of this group are highlighted. EF-hand-related motifs, EF-1 through EF-4, are shown in boxes. B, unlike GCAP-2, closely related to it, the protein neurocalcin does not regulate RetGC. Washed OS membranes depleted of endogenous GCAPs were assayed for RetGC activity as a function of free Ca2+ concentrations in the absence () or presence of either GCAP-2 (open circle ) or neurocalcin ([/delta). See "Experimental Procedures" for details.

To define the regions of GCAP-2 containing specific structures that are essential for its ability to regulate RetGC, we produced and characterized deletion mutants of GCAP-2 as well as chimeric proteins in which various regions of GCAP-2 were substituted by the corresponding fragments of neurocalcin or recoverin (Fig. 2).


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Fig. 2.   Protein constructs used in this study. A, mutations in Ca2+-binding region (EF-2 through EF-4) of bovine GCAP-2. I, neurocalcin fragment Ala61-Phe169 substitutes for Ala57-Phe170 in GCAP-2; II, neurocalcin fragment Ala61-Glu146 substitutes for Ala57-Glu148 in GCAP-2; III, neurocalcin fragment Ala61-Met131 substitutes for Ala57-Leu127 in GCAP-2; IV, neurocalcin fragments Ala61-Asn81 and Lys118-Met131 substitute for Ala57-Phe78 and Asp113-Leu127 in GCAP-2, respectively; V, neurocalcin fragments Ala61-Asp81 and Lys118-Glu146 substitute for Ala57-Asp78 and Asp113-Glu148 in GCAP-2, respectively; VI, neurocalcin fragments Ala61-Asp81 and Lys118-Phe169 substitute for Ala56-Asp77 and Asp113-Phe170 in GCAP-2, respectively; VII, a fragment Val133-Gly141 in GCAP-2 is deleted. B, mutations in the C-terminal part of GCAP-2. VIII, recoverin fragment Glu199-Leu202 substitutes for Ser201-Phe204 in GCAP-2; IX, a GCAP-2 mutant that contains mutations E80Q and D158N (16), but unlike the (E80Q/D158N) Ca2+-insensitive mutant described in Ref. 16, it also has a frameshift resulting in an additional 25-amino acid fragment, SEGSKDPAANKARKEAELAAATAEQ, encoded by pET11d vector following Met203 of GCAP-2; X, GCAP-2 truncated downstream of Ser195; XI, GCAP-2 truncated downstream of Asn189; XII, GCAP-2 truncated downstream of Asn181; XIII, neurocalcin fragment Thr158-Phe192 substitutes for Glu159-Phe204 in GCAP-2; XIV, neurocalcin fragment Thr158-Asp186 substitutes for Glu159-Asn189 in GCAP-2; XV, GCAP-2 truncated at Gln140. C, mutations within the N-terminal part of GCAP-2. XVI, neurocalcin fragment Ala61-Gly78 substitutes for Ala57-Asn74 in GCAP-2; XVII, neurocalcin fragment Gly53-Asp81 substitutes for Phe49-Asp78 in GCAP-2; XVIII, recoverin fragment Met1-Ser10 substitutes for Met1-Ala10 in GCAP-2; XIX, neurocalcin fragment Met1-Ile27 substitutes for Met1-Leu24 in GCAP-2; XX, fragment Leu8-Leu18 is deleted from the neurocalcin part of the chimera protein XIX; XXI, in chimera protein XX a neurocalcin fragment Gly32-Gly78 substitutes for Lys30-Asn74 in GCAP-2; XXII, fragment Met1-Ala79 of GCAP-1 substitutes for Met1-Ala84 in GCAP-2. Chimera constructs VIII and XVIII were expressed in HEK 293 cells as described previously (14), all other constructs were expressed in E. coli as described in Ref. 22 and under "Experimental Procedures." Fragments of GCAP-2 were substituted between the indicated amino acid residues of GCAP-2. The diagrams representing GCAP-2 and neurocalcin are "aligned" at the EF-1 region.

In order to study the effects of mutations in GCAP-2 and GCAP-1, we used both native RetGC-containing OS membranes and recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells for the following reasons. 1) OS membranes are more likely to retain any additional unidentified components(s) that might be potentially significant for RetGC regulation in photoreceptors. 2) Relative contribution of RetGC-1 or RetGC-2 to the overall cGMP synthesis in photoreceptors membranes remains unknown. 3) It is impossible to exclude a potential existence of any additional RetGC homologs that can be present in photoreceptors. 4) Activity of recombinant RetGCs is 10-100-fold lower than RetGC activity in OS membranes. Therefore we consider OS membranes as a closer in vitro approximation to the physiological conditions in order for studying RetGC regulation by GCAPs. 5) However, even with all mentioned limitations for using recombinant cyclases for studying GCAP-2 mutants, the recombinant RetGC-1 and RetGC-2 demonstrate their basic ability to interact with GCAPs and therefore must reflect at least some of the regulatory properties of the native enzymes present in photoreceptors. Hence, we also demonstrate that the key GCAP-2 mutations affecting native RetGC activity in OS also affect individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.

The Region between EF-2 and EF-3 Determines Whether the Ca2+ Bound Form of GCAP-2 Activates or Inhibits RetGC-- For other known EF-hand proteins, such as calmodulin, it is their Ca2+-loaded form that regulates an effector protein (29). In contrast, the apo-form of GCAP-2 and GCAP-1 activates RetGC, and the Ca2+-loaded form inhibits RetGC (16, 17). Apparently, exposure of regulatory domains in GCAPs is regulated by Ca2+ binding to the EF-hand domains (16). Mutations in EF-hands that hamper Ca2+ binding can lock GCAP-2 into its activator conformation thus preventing the Ca2+-dependent transition into the inhibitory conformation (16). The greatest homology between GCAPs and neurocalcin is within the central part of the molecule that contains all three functional EF-hands: EF-2, -3, and -4 (Fig. 1A). We refer to that region as the "Ca2+-binding core region." We examined the ability of that region of neurocalcin to substitute for the corresponding region of GCAP-2 by constructing and analyzing the chimeric proteins shown in Fig. 2 (I-III). Surprisingly, these chimeras do not activate RetGC at low Ca2+ concentrations. Instead, they activate RetGC only when free Ca2+ concentrations exceed 200 nM (Fig. 3, A and B). Unlike wild type GCAP-2 which is Ca2+-sensitive activator of RetGC, these chimerical proteins are Ca2+-dependent activators of RetGC.


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Fig. 3.   Ca2+ sensitivity of RetGC regulation in OS membranes is determined by the region that includes EF-2 and EF-3. A, substitution of the Ca2+-binding core region in GCAP-2 with that from neurocalcin results in Ca2+-dependent activation of RetGC. Cyclase activity is shown as a function of free Ca2+ in the absence () or presence of chimeras I (), V (open circle ), or VI (diamond ). B, the region in GCAP-2 that determines the "sign" of Ca2+ sensitivity is located between Phe78 and Asp113 of GCAP-2. RetGC activity in the absence () or presence of wild type GCAP-2 (open circle ), chimera III (black-triangle), or chimera IV (Delta ). Inset, unlike wild type GCAP-2, chimeras I, II, and III fail to stimulate RetGC at 12 nM Ca2+. C, RetGC activity in the absence () and presence (circo]) of GCAP-2 deletion mutant VII. Protein constructs are described in the legend to Fig. 2. See "Experimental Procedures" for other details.

It should be noted that chimeras I-III are unable to activate RetGC at low Ca2+ not because they are simply denatured, or cannot bind Ca2+. Fig. 3 shows that they are able to activate RetGC, but that their Ca2+ sensitivity has been reversed (Fig. 3, A and B). Data for construct II are not shown, but they are similar to the results with chimeras I and III. These results imply that the Ca2+-binding core region of GCAP-2 is not only responsible for binding Ca2+, but also determines the direction of the switch between the "inhibitor" and "activator" conformations of GCAP-2.

The neurocalcin structure responsible for this paradoxical effect appears to be the region between EF-2 and EF-3. In order to verify this, we substituted the neurocalcin sequence between EF-2 and EF-3 in chimera III with the corresponding fragment Phe78-Asp113 from GCAP-2 (numbers are given according to the GCAP-2 sequence). The resulting mosaic (chimera IV) demonstrates Ca2+ sensitivity of RetGC typical for GCAP-2 (Fig. 3B). The same GCAP-2-specific fragment, Phe78-Asp113, when it is inserted into constructs I and II creates mosaic chimera protein constructs V and VI in which the length of neurocalcin-specific fragments is extended toward the EF-4 (Fig. 2A). Unlike chimeras I-III, chimeras V and VI demonstrate normal regulation of RetGC by Ca2+ (Fig. 3A). We therefore conclude that the direction of the Ca2+-regulated switch between activator and inhibitor conformations in GCAP-2 is determined by the GCAP-2-specific amino acid sequence(s) located between Phe78 and Asp113. Interestingly, in a related protein, recoverin, this region appears to act as a swivel or a "hinge" between two halves of the molecule (33). It is therefore possible that in GCAP-2 it may also form a similar structure between regulatory domains and thus may determine the directions of intramolecular rearrangements caused by Ca2+ binding. However, the three-dimensional structures of Ca2+-free and Ca2+-bound GCAP-2 will be needed to confirm this possibility.

Other regions of the GCAP-2 Ca2+-binding core structure can be substituted with the corresponding fragments of neurocalcin (Fig. 3, chimera V and VI), or partially deleted (chimera VII, Fig. 3C) and this does not cause complete loss of function or reverse Ca2+ sensitivity of RetGC regulation. This shows that except for the presence of functional EF-hands, no essential GCAP-2-specific functions are exclusively determined by the GCAP-2 amino acid sequence located between Glu56 and Asp77 or between Arg114 and Phe170. The region Arg114-Phe170 includes a part of the EF-3 and EF-4 Ca2+-binding domains. Also, the distance between EF-3 and EF-4 in GCAP-2 is longer than in other recoverin-like proteins, including GCAP-1 (Fig. 1A). Nevertheless, the difference in distance between EF-3 and EF-4 does not seem to be a critical characteristic of the GCAP-2 molecule as a RetGC regulator. An 8-amino acid fragment from that part of the molecule can be deleted virtually without inactivation of GCAP-2 (Fig. 3C). However, some other parts of these regions in GCAP-2, in particular, the entering helix of EF-4 are most likely to contribute to the proper folding and activity of GCAP-2 molecule because chimeric proteins V and VI have 2.5-3-fold lower maximal level of RetGC activation and more than 3-fold increase in EC50 values compared with wild type GCAP-2 (data not shown).

The C-terminal Region Thr158-Asn189 in GCAP-2 Contributes to Its RetGC Activation but Not to RetGC Inhibition-- The very C terminus of GCAP-2 is not required for activation or inhibition of RetGC. The last 4 amino acids can be substituted with the amino acids from the recoverin C terminus (Fig. 4). A random 25-amino acid sequence (adding an extra 10% to the length of GCAP-2, Fig. 2) can be attached to the C terminus of GCAP-2 without affecting RetGC activation (Fig. 4, chimera IX). Deletion of up to 9 amino acids from the C terminus does not completely prevent GCAP-2 from activating RetGC at low Ca2+ concentrations nor from inhibiting the cyclase in high Ca2+ (Fig. 5). However, longer deletions from the C terminus of GCAP-2 in mutants XI (Fig. 5A) and XII (data not shown) cause loss of its ability to activate RetGC. Consistent with this, GCAP-2 with the EF-4 Ca2+-binding loop substituted with neurocalcin EF-4 (Fig. 3, chimera VI) still activates RetGC while substitution of the additional C-terminal 18-amino acid sequence Val171-Asn189 adjacent to the EF-4 loop abolishes RetGC activation (Fig. 5B, lines XIII and XIV).


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Fig. 4.   The very C terminus of GCAP-2 is not required for RetGC stimulation. A, RetGC activity in the absence (a) or presence of wild type GCAP-2 (b), GCAP-2/recoverin chimera VIII (c and d) at 6 nM (a-c), or 2 µM (d) free Ca2+. B, RetGC activity at 6 nM free Ca2+ in the absence (f) or presence of wild type GCAP-2 (g), or chimera IX (h). Protein constructs are described in the legend to Fig. 2. See "Experimental Procedures" for other details.


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Fig. 5.   An EF-4 proximal region in GCAP-2 participates in RetGC activation at low Ca2+ but is not required for RetGC inhibition at high Ca2+. A, RetGC activity at 6 nM free Ca2+ in the absence (a) or presence of wild type GCAP-2 (b), GCAP-2 deletion mutants X (c) or XI (d), GCAP-2/neurocalcin chimeras XIII (e) or XIV (f), GCAP-2 deletion mutant XV (g). Inset, Ca2+-loaded C-terminal deletion and chimera mutants inhibit RetGC basal activity. B, Ca2+ sensitivity of RetGC activation in the absence () or presence of GCAP-2 truncated mutant X (open circle ), GCAP-2/neurocalcin chimera proteins XIII (Delta ) or XIV (). Protein constructs are described in the legend to Fig. 2. See "Experimental Procedures" for other details.

The N-terminal fragment adjacent to the EF-4 in neurocalcin is shorter than in GCAP-2. However, the following observations suggest that the C-terminal GCAP-2/neurocalcin chimera protein does not lose its activator capacity just because it is shorter than wild type GCAP-2. 1) The length of the C-terminal fragment adjacent to the EF-4 in GCAP-2 deletion mutant X is similar to that in XIII, yet the deletion mutant X activates RetGC while chimera XIII does not (Fig. 5A, B); 2) The C-terminal fragment in chimera XIV is 8 amino acid residues longer than in chimera XIII because it contains an additional fragment (Trp193-Phe204) from GCAP-2. Chimera XIV is therefore longer than deletion mutant X, yet it fails to activate RetGC (Fig. 5B). Importantly, all the deletion mutant and chimeras shown in Fig. 5 still inhibit RetGC basal activity at high Ca2+ concentrations.

In summary, the region Val171-Asn189 appears to contain GCAP-2-specific amino acids essential for activation of RetGC by apo-GCAP-2. Our results also show that there are no GCAP-2-specific sequences essential for RetGC inhibition by Ca2+-loaded GCAP-2 in this region. Almost 30% of the length of GCAP-2 from its C terminus does not appear to be absolutely essential for the inhibitory effect of Ca2+-loaded GCAP-2 on the basal activity of RetGC. Even the GCAP-2 deletion mutant, XV, truncated at Gln140 shortly after EF-3, still inhibits RetGC basal activity at high Ca2+ concentrations (Fig. 5).

Mutations within the N-terminal Region of GCAP-2-- The N terminus is the most variable part of GCAP-2. Its length and sequence varies considerably across animal species. The distance between the N-terminal glycine and EF-1 in bovine, mouse, chicken (16, 30, 31), and zebra fish2 GCAP-2 varies between 27 and 32 amino acid residues. Despite this variability it has been reported that removal of the first 8 amino acids from bovine GCAP-2 inactivates it (21). In contrast, our findings suggest that there is no GCAP-2-specific sequence essential for RetGC regulation in that region. That is consistent with the relatively high variability of the N-terminal GCAP-2 sequences in different animal species. A chimera XVIII in which the first 10 amino acids of GCAP-2 are substituted with the corresponding region from recoverin demonstrates normal regulation of RetGC in vitro (Fig. 6). It is likely that the effect observed in Ref. 21 was due to misfolding caused by excessive truncation of recombinant GCAP-2 rather than the absence of an essential GCAP-2 specific-sequence motif.


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Fig. 6.   A chimera XVIII in which first the 10 amino acid residues of GCAP-2 are substituted with the corresponding fragment from recoverin is capable of regulating RetGC. A, washed OS membranes were reconstituted with soluble extracts (50 µg of total protein) from transfected HEK293 cells as described in detail in Ref. 14. RetGC activity was measured as a function of free Ca2+ concentrations. Protein extract from mock-transfected control cells (), cells transfected with wild type GCAP-2 cDNA (open circle ), or chimera protein XVIII-encoding DNA (black-triangle). Inset, unlike GCAP-2 or recoverin/GCAP-2 chimera XIII, recoverin does not activate RetGC. RetGC activity in washed OS membranes: in the absence (a) or presence (b and c) of 4 µg of recombinant recoverin (b) or GCAP-2 (c). B, a Hill plot of the data from panel A demonstrates that recoverin/GCAP-2 chimera XVIII regulates RetGC with the same Ca2+ cooperativity as wild type GCAP-2. Protein constructs are described in the legend to Fig. 2. See "Experimental Procedures" for other details.

Although we cannot conclude that the N terminus of GCAP-2 does not play a functional role in regulation of RetGC, it appears that the total length of the N-terminal region between Gly2 and Trp27 (numbers correspond to bovine GCAP-2 sequence) rather than the presence of any GCAP-2-specific sequence may be important for the proper activator conformation. We demonstrated this using two similar neurocalcin/GCAP-2 chimeras, XIX and XX. In chimera XIX the N-terminal sequence of GCAP-2 is substituted with the corresponding region from neurocalcin (Fig. 2). This chimera does not activate RetGC (Fig. 7A). However, the N terminus of this chimera not only has a sequence different from GCAP-2, but is also 3 amino acids longer than bovine GCAP-2 (Fig. 1A), which itself is the longest form of GCAP-2 among the reported animal species. The N termini of chicken, mouse, and human GCAP-2 are 3-5 amino acid residues shorter than bovine GCAP-2 (31). If we now delete 9 amino acids from the inside of the neurocalcin part of the chimera, the N terminus (MGKQNSEESTDFTEHEIQ) is shortened and it has very little homology with GCAP-2, but it will be still within the range in which GCAP-2 terminus varies among different species (Figs. 1 and 2). Fig. 7B demonstrates that such reduction in length substantially restores the ability of the chimera protein to activate RetGC (Fig. 7B). Interestingly, Ca2+-loaded forms of both chimeras, XIX and XX, are able to inhibit RetGC in OS (Fig. 7, A and B). We therefore conclude that the part of bovine GCAP-2 between its N terminus and Leu24 does not posses major regulatory properties that are specific to GCAP-2. Neither does the region between Phe49 and Phe78 (which includes part of EF-2) specifically determine the essential regulatory properties of GCAP-2. Partial (XVI, data not shown) or complete (chimera XVII) substitution of this region with the corresponding regions from neurocalcin does not eliminate the ability to regulate RetGC in a Ca2+-sensitive manner (Fig. 7C).


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Fig. 7.   Regulation of RetGC by N-terminal neurocalcin/GCAP-2 chimeras as a function of free Ca2+ concentrations. A, RetGC activity in the absence () or presence (open circle ) of chimera XIX. B, RetGC activity in the absence () or presence of chimeras XX (open circle ) or XXI (Delta ). C, RetGC activity in the absence () or presence of chimera XVII (). D, chimera XXI does not cause excessive hydrolysis of cGMP. Approximately 0.1 µCi of [3H]cGMP was added in every RetGC assay reaction mixture (see "Experimental Procedures") prior to incubation. After the incubation, 5-µl aliquots from each sample were subjected to thin-layer chromatography, and [3H]cGMP was recovered from PEI cellulose by elution in 1 ml of 2 M LiCl and counted in 10 ml of an Ecolume scintillation mixture (ICN). 100% level corresponds to the original amount of [3H]cGMP standard recovered from the reaction mixture that contained heat-inactivated OS membranes and was incubated in parallel with other samples. No significant cGMP hydrolysis was detected in the presence or absence of WTGCAP-2, neurocalcin, chimeras III, XIII, or XXI or any other chimera constructs used in this study (data not shown). Chimera constructs are described in the legend to Fig. 2.

However, additional substitution of the region Lys29-Phr48 in GCAP-2 (that includes the "EF-1" motif) with the corresponding region of neurocalcin completely inactivates GCAP-2 (Fig. 7B, chimera XXI). It seems unlikely that the loss of function in this chimera results from major loss of protein structure caused by the sequence exchange because this substitution does not affect the size of the protein. Also, the neurocalcin sequence in this region has fairly high homology with GCAP-2 (Fig. 1A). GCAP-2 and GCAP-1 also show very little homology to each other within their N termini except for the region adjacent to EF-1 (which is highly homologous in all recoverin-like proteins including neurocalcin and GCAPs) (Fig. 1A). Yet unlike chimera XXI, chimera XXII does regulate RetGC even though the whole N-terminal part of GCAP-2 in chimera XXII is substituted by the corresponding amino acid sequence from GCAP-1 (Fig. 8). We conclude that the region Lys30-Phe48 contains GCAP-specific amino acid residues that play a key role in RetGC regulation both by apo- and Ca2+-loaded forms of GCAP-2. The only amino acid residues that are conserved in this region in both GCAP-1 and GCAP-2, but not in neurocalcin and other recoverin-like proteins are Lys30 and Phe48 (numbers according to GCAP-2 sequence). It is therefore possible that one or both of these residues participate in the interactions between RetGC and GCAP-2. Alternatively, they may provide for the intramolecular interactions in GCAP-2 essential for the proper conformation of regulatory sites within the GCAP-2 structure.


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Fig. 8.   Activation of RetGC by 2 µg of GCAP-1/GCAP-2 chimera protein XXII (open circle ) as a function of free Ca2+ concentrations (), no activator added. Protein construct is as described in the legend to Fig. 2. See "Experimental Procedures" for other details.

There is unambiguous evidence that GCAP-1 and -2 are present in photoreceptors and regulate RetGC (6, 13-15, 31, 32). And even though a priori we cannot rule out that GCAPs may also be involved in regulating some additional function(s) in photoreceptors, we would like to emphasize that all the results presented in this paper pertain solely to the effects of GCAP-2 mutants on stimulation or inhibition of cGMP synthesis, not cGMP hydrolysis. The activity of cGMP PDE in assay that contains washed membranes and PDE inhibitors, zaprinast and dipyridamole, under infrared illumination typically is insignificant. Moreover, we always directly monitor PDE activity in every assay in order to ensure the absence of cGMP hydrolysis that could potentially affect the recovery of cGMP synthesized by RetGC. Fig. 7D demonstrates that regardless of free Ca2+ concentrations used in this study, neither GCAP-2 nor its mutants affected the recovery of [3H]cGMP added as internal standard at the beginning of the reaction. Also, less than 10% of the total added GTP was utilized in the course of the reaction based on TLC analysis (data not shown). Therefore the effects of GCAP-2 mutants in the conditions of our assays were directly on RetGC activity and by the very design and conditions of the assay did not reflect the activity of the rhodopsin right-arrow GTPase right-arrow PDE cascade.

Mutations in GCAP-2 That Affect Native RetGC in OS Membranes Also Affect the Regulation of Recombinant RetGC-1 and RetGC-2-- Using recombinant RetGC-1 and -2 for studying RetGC regulation by GCAPs may have both limitations and advantages over using native RetGC in washed OS membranes for a number of reasons. Interpretation of the results using recombinant RetGCs can be potentially complicated by a possible lack of yet unaccounted factor(s) that may contribute to the activities of the native enzymes. It has been known, for example, that even purification native RetGC affects its ability to react with the Ca2+-sensor proteins (35). The inhibitory effect of WTGCAPs and their mutants on the total RetGC activity in OS membranes can be easily detected. However, the basal activity of each recombinant RetGC is approximately 100-fold lower than the basal RetGC activity in OS membranes (6, 7). That, again, might reflect the lack of some yet unidentified factor(s) responsible for higher basal level of RetGC or can be merely a result of lower efficiency of folding, post-translational modifications, and/or proper transport and incorporation of recombinant RetGCs into membranes compared with the native RetGC in the OS membranes. For this particular reason, we find it difficult to reliably study the inhibitory effects of GCAP-1 and GCAP-2 using recombinant cyclases. Also, the exact contribution of RetGC-1 and RetGC-2 to the overall RetGC activity in membranes has not yet been determined. And last, but not the least, it is impossible to exclude that there are additional isoforms of RetGC present in native membranes that are significant for the net cGMP synthesis.

Nevertheless, even after taking all these limitations into account, we think that recombinant RetGCs can be very useful tools in studying specific mechanisms of RetGC regulation. Therefore we also verified to what extent the effects of the key GCAP-2 mutants in the three most important regulatory regions in GCAP-2 reflects its ability to regulate recombinant RetGC-1 and RetGC-2. The results presented in Fig. 9 demonstrate that chimeras XIII and XXI that fail to activate RetGC in OS membranes are also inefficient in stimulating individual recombinant RetGC-1 and RetGC-2 (Fig. 9, A and C). Chimeric mutant III that reverses Ca2+ sensitivity of cGMP synthesis in OS membranes also stimulates recombinant RetGC-1 only at high Ca2+ concentrations (Fig. 9). The second consequence of this mutation is decreased efficiency of activation of both OS RetGC and RetGC-1 (Figs. 3 and 9). In the case of recombinant RetGC-2 its activity in the presence of chimera III was even lower so that it made it difficult to detect RetGC-2 activation by chimera III even at high Ca2+ (Fig. 9). However, mosaic chimera IV that activates OS RetGC in a Ca2+-sensitive manner also efficiently activates recombinant RetGC-2. Despite the much lower activity of recombinant RetGCs compared with the native total RetGC in OS membranes, these data allow us to expect that these three major regulatory regions in GCAP-2 are also engaged in regulating both RetGC-1 and RetGC-2 in vivo. However, we cannot conclude that the interactions between GCAP-2 and both cyclases are strictly identical. Such issue would require identification of the exact sites of protein-protein contacts between GCAP-2 and both cyclases which is beyond the scope of our present study.


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Fig. 9.   Effect of GCAP-2/neurocalcin chimeras on Ca2+ regulation of recombinant RetGC-1 and RetGC-2. Panels A and B, activity of recombinant RetGC-1, panels C and D, activity of recombinant RetGC-2. RetGC-1 and RetGC-2 were expressed in HEK293 cells and assayed as described under "Experimental Procedures" in the absence () or presence of 5 µM wtGCAP-2 (open circle ), 10 µM chimera XIII (), 10 µM chimera XXI (diamond ), 5 µM chimera III (black-square), or 10 µM chimera IV (black-triangle). Basal activities of RetGC-1 and -2 correspond to 0.04 nmol of cGMP/min/mg of protein, maximal wtGCAP-2 stimulated activity corresponds to 1.26 and 0.7 nmol of cGMP/min/mg of protein, respectively.

A Map of GCAP-2 Functional Domains (Fig. 10)-- Our previous and present observations reveal that there are six domains in the GCAP-2 primary structure that determine its most basic characteristics as a RetGC regulator. Three functional EF-hands (EF-2 through EF-4) are required for GCAP-2 to be a Ca2+ sensor within the submicromolar range of free Ca2+ concentrations. EF-2 and EF-4 contribute to its Ca2+ sensitivity more than EF-3 (16). It is possible that certain GCAP-2-specific amino acid residues within the EF-hands are essential for RetGC regulation. However, the exact amino acid sequences of the EF-hand Ca2+-binding loops can be partially (EF-2) or even completely (EF-4) changed as long as they do not deviate from the basic EF-hand loop motif (for the EF hand loop structure, see Ref. 34). Chimeras IV, V, and VI each have EF-2 and EF-3 loops partially derived from neurocalcin, and the entire EF-4 loop in chimera VI is substituted with the neurocalcin sequence, yet these chimeras are able to regulate RetGC in a Ca2+-sensitive manner.


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Fig. 10.   A map of functional domains in GCAP-2. Explanations are as described in the text.

The fundamental difference between GCAPs and other known EF-hand Ca2+-binding proteins is that the apo-form of GCAPs activates target enzyme. This means that in GCAPs Ca2+ binding is not required for RetGC activation, and only results in inhibition of RetGC. Binding of Ca2+ to the EF-hands of GCAP-2 drives a switch between the activator and inhibitor conformations. The direction of the switch appears to be controlled by the region Phe78-Asp113 located between the EF-2 and EF-3 Ca2+-binding domains. This "switch" domain determines that upon binding Ca2+ ions to the EF-hands, GCAP-2 undergoes an activator-to-inhibitor transition, but not vice versa. It should also be noted that since the corresponding chimeras are able to stimulate RetGC in high Ca2+, we cannot conclude from this study whether or not this domain may also contribute to the inhibition of basal activity of RetGC in Ca2+-loaded wild type GCAP-2.

There are two other domains in GCAP-2 that contain GCAP-2-specific amino acid sequences essential for native RetGC regulation: the C-terminal region, Val171-Asn189, adjacent to EF-4 loop and the N-terminal domain, Lys29-Phe48, that includes EF-1. The C-terminal domain adjacent to EF-4 is important for activation of RetGC by apo-GCAP-2, but it is not required for inhibition of the cyclase by Ca2+-loaded GCAP-2. The N-terminal region surrounding the EF-1 is essential for both activation and inhibition of RetGC. Even substitution of this region with the homologous sequence of neurocalcin disables regulation of RetGC. We speculate that this region of GCAP-2 is responsible for one of the primary interactions between GCAP-2 and the cyclase in both apo- and Ca2+-loaded forms of GCAP-2. Other domains may perform secondary interactions specifically required for RetGC activation or inhibition, or for conformational changes induced by Ca2+. In order to resolve these questions it will be necessary to define the specific roles of particular amino acid residues in these regulatory domains of GCAP-2 using directed mutagenesis and analysis of the three-dimensional structure of GCAP-2.

    ACKNOWLEDGEMENT

We thank Greg Niemi for mass spectrometry of GCAP-2 mutants.

    FOOTNOTES

* This work was supported by National Eye Institute Grants EY11522 (to A. M. D) and EY06641 (to J. B. H), and by a Career Development Award from Research to Prevent Blindness (to A. M. D.).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.

parallel To whom correspondence should be addressed: Dept. of Ophthalmology/Kresge Eye Institute, Wayne State University School of Medicine, 4717 St. Antoine, Detroit, MI 48201. Tel.: 313-577-1573; Fax: 313-577-7635; E-mail: adizhoor{at}med.wayne.edu.

2 C. Tucker and J. B. Hurley, unpublished data.

    ABBREVIATIONS

The abbreviations used are: GCAP, photoreceptor guanylate cyclase activating protein; EF-1-4, Ca2+-binding loops of EF-hands; OS, photoreceptor outer segment membranes; RetGC, photoreceptor membrane guanylate cyclases (other names are RosGC1 and ROSGC2 or GC-E and GC-F, respectively); PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; BNC, bovine neurocalcin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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