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J Biol Chem, Vol. 274, Issue 36, 25583-25587, September 3, 1999

Dimerization of Guanylyl Cyclase-activating Protein and a Mechanism of Photoreceptor Guanylyl Cyclase Activation^*

Elena V. Olshevskaya, Alexandre N. Ermilov, and Alexander M. Dizhoor^§¶

From the  Department of Ophthalmology/Kresge Eye Institute and ^§ Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Ca²⁺-binding guanylyl cyclase-activating proteins (GCAPs) stimulate photoreceptor membrane guanylyl cyclase (retGC) in the light when the free Ca²⁺ concentrations in photoreceptors decrease from 600 to 50 nM. RetGC activated by GCAPs exhibits tight dimerization revealed by chemical cross-linking (Yu, H., Olshevskaya, E., Duda, T., Seno, K., Hayashi, F., Sharma, R. K., Dizhoor, A. M., and Yamazaki, A. (1999) J. Biol. Chem. 274, 15547-15555). We have found that the Ca²⁺-loaded GCAP-2 monomer undergoes reversible dimerization upon dissociation of Ca²⁺. The ability of GCAP-2 and its several mutants to activate retGC in vitro correlates with their ability to dimerize at low free Ca²⁺ concentrations. A constitutively active GCAP-2 mutant E80Q/E116Q/D158N that stimulates retGC regardless of the free Ca²⁺ concentrations forms dimers both in the absence and in the presence of Ca²⁺. Several GCAP-2/neurocalcin chimera proteins that cannot efficiently activate retGC in low Ca²⁺ concentrations are also unable to dimerize in the absence of Ca²⁺. Additional mutation that restores normal activity of the GCAP-2 chimera mutant also restores its ability to dimerize in the absence of Ca²⁺. These results suggest that dimerization of GCAP-2 can be a part of the mechanism by which GCAP-2 regulates the photoreceptor guanylyl cyclase. The Ca²⁺-free GCAP-1 is also capable of dimerization in the absence of Ca²⁺, but unlike GCAP-2, dimerization of GCAP-1 is resistant to the presence of Ca²⁺.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Photoexcitation of photoreceptors results in hydrolysis of cGMP and closure of the cGMP-gated Na⁺/Ca²⁺ channels, thus causing both hyperpolarization of the photoreceptor membrane and decrease in the intracellular free Ca²⁺ concentrations (1-4). Lowering of the intracellular Ca²⁺ concentrations caused by illumination stimulates cGMP synthesis by guanylyl cyclase (retGC)¹ that contributes to recovery from photoexcitation and to light adaptation of photoreceptors (5, 6). Recoverin-like Ca²⁺-binding proteins, GCAP-1 and GCAP-2, mediate Ca²⁺ sensitivity of the cyclase in vertebrate retinas (7-10) so that GCAPs activate the cyclase when the free concentrations of Ca²⁺ decrease below 100 nM (a characteristic of light-adapted photoreceptors), but they do not stimulate the cyclase in the dark when the free Ca²⁺ concentrations exceed 500 nM (6, 11). GCAP-2 is highly abundant in mammalian rods, while GCAP-1 is highly expressed in cones (9, 10, 12, 13). The cDNA for the third homologue of GCAPs has been recently cloned from a human retinal cDNA library, while it was absent form retinal cDNA libraries of other vertebrate species (14).

GCAP-1 and GCAP-2 interact with the photoreceptor membrane guanylyl cyclases, retGC-1 and retGC-2 (also known as ROSGCs or GC-E and GC-F in Refs. 8 and 15-18), via cyclase intracellular domains (19-21). Several regions in GCAPs that contain amino acid sequences that are specific for GCAPs function as cyclase regulators have been identified using site-directed mutagenesis (22-24). Although the exact mechanism for retGC activation by GCAPs remains undetermined, it apparently involves dimerization of the cyclase. Consistently with other membrane guanylyl cyclases being active as dimers (25-27), retGCs in photoreceptors also form homodimers (28). Moreover, retGC subunits dimerize or at least come into closer contact when stimulated by GCAPs so that they can be chemically cross-linked (29). Here we report the evidence that GCAP-2 undergoes Ca²⁺-sensitive dimerization and that GCAP-2 dimerization correlates with its ability to activate retGC. We propose that the dimer of the Ca²⁺-free GCAP-2 acts as an adapter that controls dimerization of retGC. Similarly to GCAP-2 a substantial fraction of GCAP-1 also forms a dimer in the absence of Ca²⁺, but unlike GCAP-2 this dimerization apparently is not reversed by Ca²⁺ binding.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Recombinant wild type GCAP-2 (30), its constitutively active mutant (E80Q/E116Q/D158N) (31), and GCAP-2/neurocalcin chimeras (III, IV, XIII, and XIX, Ref. 22) were expressed in Escherichia coli and purified as described previously in detail (22, 30, 31). Recombinant myristoylated GCAP-1 was produced in E. coli and purified using chromatography on Sephacryl S-100 as described previously (23, 32). Neurocalcin was expressed in E. coli and purified using a phenyl-Sepharose chromatography column (22, 33). Based on SDS-PAGE, purity of the recombinant proteins was at least 90%. Recombinant proteins were injected in a volume of 200 µl into a Superdex 200 HR10/30 column (Amersham Pharmacia Biotech) using an automated fast protein liquid chromatography system and eluted at 0.5 ml/min in buffer A (20 mM Tris-HCl, 50 mM KCl, 10 mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol, pH 7.5) containing either 400 µM EGTA or 300 µM CaCl₂. The column was pre-equilibrated with 2 volumes of corresponding buffer between the runs. Free Ca²⁺ concentrations in the samples were adjusted prior to injection by adding EGTA or CaCl₂, respectively. The molecular weight standards for gel filtration included blue dextran (2,000 kDa), -amylase (200 kDa), alcohol dehydrogenase (150 kDa, Stokes radius: 45.5 Å), bovine serum albumin (66 kDa, Stokes radius: 36.1 Å), carbonic anhydrase (29 kDa, Stokes radius: 20.1 Å) and cytochrome c (12.4 kDa) (Sigma). The column was calibrated prior to the analysis and reproducibility of the calibration was tested between series of the experiments. It was virtually identical in the presence of either EGTA or CaCl_2.

Activation of retGC in washed outer segment membranes reconstituted with GCAPs was assayed under infrared illumination using [-³²P]GTP as a substrate and [³H]cGMP as an internal standard and analyzed using TLC on polyethyleneimine-cellulose plates (Merck) essentially as described in full detail in our previous publications (9, 30).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The three-dimensional structure of Ca²⁺-bound GCAP-2 was recently determined by Ames et al. (34) using NMR spectroscopy. The main chain conformation in GCAP-2 molecule is very similar to that of neurocalcin or recoverin (35-37), although unlike Ca²⁺-loaded neurocalcin GCAP-2, is a monomer. Consistently with the previous report by Vijay-Kumar and Kumar (37) on crystal structure of neurocalcin, in the presence of Ca²⁺, neurocalcin elutes from the Superdex 200 column as a dimer (molecular mass ~46 kDa, Fig. 1), while it becomes a monomer in the presence of EGTA (molecular mass ~22 kDa, Fig. 1). However, Ca²⁺ has an opposite effect on the chromatographic behavior of GCAP-2. We have found that in the absence of Ca²⁺, GCAP-2 can dimerize, while the presence of Ca²⁺ strongly inhibits its dimerization (Fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Neurocalcin undergoes dimerization in the presence, but not in the absence, of Ca2+. Aliquots of 2 mg/ml recombinant bovine neurocalcin  were analyzed by high-resolution gel filtration as described under "Experimental Procedures" in the presence of either 300 µM CaCl2 (line a) or 0.4 mM EGTA (line b). In the presence of Ca2+ neurocalcin elutes in a volume that corresponds to its dimer (peak D), and in the absence of Ca2+ it elutes as a 22-kDa monomer (peak M). The molecular mass values for both peaks of neurocalcin () were calculated from the plot shown in the inset, where  indicates positions of the corresponding molecular mass standards.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   GCAP-2 forms dimers in the absence of Ca2+. A, aliquots of 2 mg/ml myristoylated recombinant GCAP-2 (200 µl) were analyzed by high-resolution gel filtration as described under "Experimental Procedures" in the presence of either 300 µM CaCl2 (line a) or 0.4 mM EGTA (line b). In the presence of Ca2+ neurocalcin elutes in a volume that corresponds to its monomer (peak M), and in the absence of Ca2+ it elutes as both as a monomer and a ~60-kDa dimer (peak D). The molecular mass values for both peaks of GCAP-2 () in line b were calculated from the plot shown in the inset, where  indicates positions of the corresponding molecular mass standards. In B GCAP-2 peaks eluted from the column (marked with a thick horizontal bar in A, line b) were collected as 0.3-ml fractions and analyzed by SDS-PAGE. Fractions marked D (dimer) and M (monomer) were reconstituted with washed bovine rod outer segments membranes, and retGC activity was measured at 10 nM free Ca2+ concentration. Results are representative of six similar experiments using different preparations of GCAP-2. B, RetGC stimulation by peak fractions D and M eluted from the column, shown on Coomassie-stained gels after SDS-PAGE. For other details see "Experimental Procedures."

GCAP-2 has a K_d for Ca²⁺ near 300 nM (34), and activation of retGC by GCAP-2 is also inhibited by Ca²⁺ (EC₅₀ 200-300 nM; Refs. 8, 9, and 32). Therefore, at 300 µM free Ca²⁺ GCAP-2 is fully Ca²⁺-bound and completely incapable of activating the cyclase (see also inset in Fig. 3). At the saturating free Ca²⁺ concentration myristoylated wild type GCAP-2 elutes from the column as a protein with a Stokes radius of ~22 Å and apparent molecular mass of ~31 kDa (Fig. 2), approximately 7 kDa larger than its actual molecular mass (23,808 Da) determined by electrospray mass spectrometry (30).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Constitutively active E80Q/E116Q/D158N GCAP-2 mutant forms dimers both in the absence and in the presence of Ca2+. A, aliquots of 2 mg/ml myristoylated recombinant E80Q/E116Q/D158N GCAP-2 (31) were analyzed by high-resolution gel filtration as described under "Experimental Procedures" in the presence of either 300 µM CaCl2 (line a) or 0.4 mM EGTA (line b). In both cases the GCAP-2 mutant elutes both as a monomer (peak M) and a dimer (peak D). Inset, Ca2+ sensitivity of retGC in washed outer reconstituted with wild type GCAP-2 () or E80Q/E116Q/D158N GCAP-2 () assayed as described in Ref. 31. B, SDS-PAGE of the fractions eluted from the column as in A, line a. Fractions marked D and M correspond to the peaks in A. Minor upper band (Myr) corresponds to the nonmyristoylated GCAP-2 (30) that comigrates with the major myristoylated form (+Myr).

In addition to the "31-kDa" monomer peak (marked "M" in Fig. 2), the Ca²⁺-free GCAP-2 elutes as a peak that corresponds to its dimer (molecular mass ~57 ± 6 kDa, Stokes radius: ~34 Å, marked "D" in Fig. 2). The GCAP-2 dimer is relatively stable, otherwise it would have completely dissociated during the chromatography (~29 min). Dimerization of GCAP-2 in the presence of EGTA may also be one of the reasons why the Ca²⁺-free GCAP-2 exhibits a poor NMR spectrum (34). However, the dimer is not irreversible even in the presence of EGTA, because when the peak of dimer is collected and subjected to the second round of chromatography, GCAP-2 elutes again as two peaks, a dimer and a monomer (data not shown). Also, the dimer collected in the absence of Ca²⁺ dissociates during the subsequent round of chromatography in the presence of Ca²⁺ (not shown). Conversely, the Ca²⁺-loaded GCAP-2 monomer (as in Fig. 2A, line a) forms a dimer when rechromatographed in the presence of EGTA (Fig. 2A, line b).

Dimerization and dissociation of the GCAP-2 complex is a function of Ca²⁺ concentrations and cannot be attributed to differences in ionic strength, because concentrations of CaCl₂ and EGTA are insignificant compared with the total salt concentration in the elution buffer. It can neither be attributed to a nonspecific effect of divalent cations, because both elution buffers contain 10 mM MgCl₂. Importantly, we can also rule out a possibility that either of the peaks shown in Fig. 2A, line b, represents merely a misfolded inactive form of GCAP-2. When tested in retGC activation assay at low free Ca²⁺, the specific activity of GCAP-2 in each peak is similar (Fig. 2B). This demonstrates that each peak contains active GCAP-2 capable of reversible dimerization such that at low Ca²⁺ concentrations the equilibrium between the dimer and the monomer is shifted toward the formation of the dimer, and at high free Ca²⁺ it shifts toward the dissociation of the complex into Ca²⁺-loaded monomers.

The ability of the Ca²⁺-free GCAP-2 to dimerize gives rise to a possibility that GCAP-2 dimerization is directly involved in regulation of retGC. In order to verify this hypothesis, we compared several mutants of GCAP-2 that demonstrated distinct differences in their properties as retGC regulators. We have found that the ability of these mutants to activate retGC at low Ca²⁺ concentrations correlates with their dimerization (Figs. 3 and 4).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   The ability of various forms of GCAP-2 to dimerize at low free Ca2+ concentrations correlates with their ability to activate photoreceptor guanylyl cyclase. A, left panel, schematic representation of wild type GCAP-2 (line a) and GCAP/neurocalcin chimeras (22) (lines b-e). Line b, XIX, neurocalcin fragment M1-I27 substitutes for M1-L24 in GCAP-2; line c, XIII, neurocalcin fragment Thr158-Phe192 substitutes for Glu159-Phe204 in GCAP-2; line d, III, neurocalcin fragment Ala61-Met131 substitutes for Ala57-Leu127 in GCAP-2; line e, IV, neurocalcin fragments Ala61-Asp81 and Lys118-Met131 substitute for Ala57-Phe78 and Asp113-Leu127 in GCAP-2, respectively. Right panel, activation of retGC activity in washed outer segment membranes by the corresponding constructs (lines a-b) at 10 nM free Ca2+ compared with the basal activity in the absence of GCAPs (filled in black), assayed as described in Ref. 22. B, myristoylated recombinant wild type GCAP-2 (line a) and or its chimera mutants (lines b-e) were analyzed in the presence of 0.4 mM EGTA by high-resolution gel filtration as described under "Experimental Procedures" and in the legend to Fig. 2. Peaks corresponding to the wild type GCAP-2 dimer or monomer are marked D and M, respectively. Plus or minus on the right side of each protein construct indicates its ability to activate retGC at low Ca2+ concentrations. C, unlike GCAP-2, GCAP-1 forms stable dimers both in the presence and in the absence of Ca2+. Recombinant myristoylated GCAP-1 was loaded onto the column in the presence of either Ca2+ (trace a) or EGTA (trace b). The analysis was performed as described under "Experimental Procedures."

A constitutively active mutant of GCAP-2 (E80Q/E116Q/D158N, Ref. 31) that stimulates retGC in both low and high free Ca²⁺ also forms the dimer regardless of the free Ca²⁺ concentrations (Fig. 3A). The elution profiles of GCAP-2 (E80Q/E116Q/D158N) in the presence of 0.4 mM EGTA and in 300 µM Ca²⁺ are almost identical. Myristoylation of GCAP-2 that is apparently nonessential for regulation of retGC (30) is also nonessential for dimerization of GCAP-2, because a minor fraction of nonmyristoylated GCAP-2 commonly present in preparations of recombinant fatty acylated GCAP-2 (30) appears in peaks of GCAP-2 dimer as well as monomer (marked "Myr" in Fig. 3B).

In our recent study we constructed several chimera mutants of GCAP-2 (22). We have found that either the substitution of a large N-terminal fragment of GCAP-2 (chimera mutant XIX, see Ref. 22 for details) or the substitution of a region proximal to EF-hand 4 (chimera mutant XIII, Ref. 22) with the corresponding neurocalcin fragments interferes with activation of retGC. When tested for their ability to dimerize in the absence of Ca²⁺, neither of these chimeras exhibit a distinct dimer peak in the presence of EGTA (Fig. 4B). In our experiments the elution time for GCAP-2 dimer is approximately 29 min; therefore, even if these mutants were capable of dimerization, their dimers completely dissociated during the chromatography. Thus, our results demonstrate that the ability of these GCAP-2 mutants to dimerize at low Ca²⁺ concentrations is either completely lost or at least dramatically reduced compared to the wild type GCAP-2. Importantly, these GCAP-2 mutants retain their ability to bind retGC in the presence of Ca²⁺, because they are both capable of inhibiting retGC at high free Ca²⁺ concentrations (22).

Another GCAP-2 chimera protein whose central part was substituted with the corresponding neurocalcin fragment and that fails to activate retGC at low free Ca²⁺ concentrations (chimera III, Ref. 22) also fails to dimerize in the presence of EGTA (Fig. 4A, line d). However, if we insert into this chimera the exiting helix of EF-2 together with the entering helix of EF-3 derived from GCAP-2 (mosaic chimera IV, Ref. 22), that simultaneously restores the ability of the mosaic GCAP-2 chimera to activate retGC at low Ca²⁺ concentrations (22) and to dimerize in the presence of EGTA similarly to the wild type GCAP-2 (Fig. 4A, line e).

We propose that dimerization of GCAP-2 is what controls dimerization of retGC and thus contributes to the cyclase regulation by Ca²⁺ (Fig. 5). This model is based on the following observations: (i) catalytic domains of guanylyl cyclases are very similar to those of membrane adenylyl cyclase (38, 39) that form dimers (40), and peptide receptor membrane guanylyl cyclases also function as dimers (25). Likewise, retGCs form homodimers in photoreceptors in vivo (28). (ii) Activation of retGC by GCAPs stimulates cyclase dimerization or at least closer contacts between the subunits in retGC dimers (29). (iii) GCAP-2 is known to bind to retGC both in the presence and in the absence of Ca²⁺ (31, 41), but only the Ca²⁺-free GCAP-2 activates retGC, while the Ca²⁺-loaded GCAP-2 inhibits it. (iv) The ability of GCAP-2 and its mutants to activate retGC at low free Ca²⁺ concentrations correlates with its ability to dimerize, indicating that not only GCAP-retGC interaction, but also GCAP-GCAP interaction, is important for the regulation of the cyclase.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Putative mechanism of retGC activation via dimerization of GCAP-2. GCAP-2 can bind to the cyclase both when in Ca2+-loaded or Ca2+-free form. However, in the presence of Ca2+ the affinity between the two Ca2+-loaded GCAP-2 subunits is low; therefore the interaction between the catalytic subunits in retGC homodimer is weak and the basal activity of the enzyme is low. Dissociation of Ca2+ causes conformational changes in GCAP-2 that increase the affinity of GCAP-2-GCAP-2 complex. Dimerization of the Ca2+-free GCAP-2 molecules bound to the cyclase subunits promotes tighter dimerization of the cyclase subunits and thus activates cGMP synthesis. The process is reversed when the free Ca2+ concentrations increase to the level characteristic of dark-adapted photoreceptors, and GCAP-2 becomes Ca2+-bound.

Our general hypothesis is that retGC becomes activated because GCAP dimer acts as an adapter for dimerization of the cyclase. GCAP-2 can be always bound to the cyclase through interaction with its kinase homology and/or catalytic domains regardless of the free Ca²⁺ concentrations. However, when GCAP-2 is loaded with Ca²⁺ in the dark, it is unable to promote the formation of retGC dimers. Conformational changes in GCAP-2 caused by dissociation of Ca²⁺ result in strong GCAP-GCAP interaction such that a Ca²⁺-free GCAP-2 dimer brings the two subunits (or parts of the subunits) of retGC closer together and stabilizes the interaction between the catalytic domains in retGC subunits. Dimerization is reversed when the free Ca²⁺ concentrations increase in the dark. It cannot be excluded that cyclase subunits may also undergo reversible spontaneous dimerization in the absence of GCAPs in vitro, which may account for relatively high basal activity of retGC in washed photoreceptor membranes in the absence of GCAPs (8, 9). If Ca²⁺-loaded GCAP-2 monomers attached to the cyclase subunits interfere with spontaneous dimerization of retGC, it would explain why a fully Ca²⁺-loaded GCAP inhibits basal activity of retGC in vitro (31, 32).

It remains unclear whether or not such "dimer adapter" mechanism is applicable to retGC activation by GCAP-1. Similarly to GCAP-2, GCAP-1 also forms dimers in the absence of Ca²⁺ (Fig. 4C) and is apparently able to interact with similar peptide fragments derived from the cyclase sequence (42). Moreover, activation of retGC by GCAP-1 also results in tight dimerization of the cyclase or at least closer interaction between its subunits (29). Nevertheless, the ability of GCAP-1 to dimerize alone would not be sufficient to account for the activation of the cyclase. There is an obvious difference between GCAP-1 and GCAP-2 at high Ca²⁺ concentrations. The retention time for the Ca²⁺-free dimer of GCAP-1 is slightly different from that of the Ca²⁺-loaded dimer of GCAP-1, which suggests that Ca²⁺ affects conformation of the whole dimer, but apparently does not change the GCAP-1 dimer stability (Fig. 4C). Conformational changes in Ca²⁺-loaded versus Ca²⁺-free GCAP must play a critical role in the interaction between the GCAPs and the cyclase. However, only in case of GCAP-2 such changes also strongly affect stability of GCAP/GCAP dimer. That may account for the different specificity of GCAP-1 versus GCAP-2 relative to different isoforms of the cyclase (14, 20, 43) and may also contribute to the fact that similar mutations in GCAP-1 and GCAP-2 can result in different biochemical phenotypes (22-24, 31, 44).

We outline in the model described in Fig. 5 the simplest hypothesis that a dimer of GCAP activates RetGC catalytic domains by causing dimerization of the cyclase. It may be a complete dissociation/association of the cyclase subunits that regulates its activity. However, there are also indications that membrane guanylyl cyclases in photoreceptors may always exist as homodimers (28), and therefore it is possible that dimers of GCAPs can also affect the conformation of retGC dimers rather than the equilibrium between association and dissociation of the cyclase. Based on chemical cross-linking experiments (29), GCAP-activated retGC can either undergo association from two completely separate cyclase monomers or equally likely change the conformation of a pre-existing retGC dimer so that it results in a closer contacts between the two cyclase subunits. Conformational changes and dimerization of a Ca²⁺-free GCAP-2 (or just conformational changes in GCAP-1 dimer) may provide the necessary energy to cause either association of retGC subunits or the intramolecular rearrangements within the pre-existing "flexible" dimer of the cyclase. Also, GCAP dimer does not necessarily act as an immediate "bridge" between the two catalytic domains of the cyclase. Instead, it may cause conformational changes in other parts of the cyclase subunits, and these changes could be then transduced through the structure of cyclase subunits to their catalytic domains.

The overall three-dimensional structure of the Ca²⁺-bound monomer of GCAP-2 is similar to that of recoverin or neurocalcin (34-38). Neurocalcin dimers formation results from close interactions between EF-hands 2, 3, and 4 in its subunits. Hence, one could assume that the corresponding residues on the surface of GCAP-2 may also be involved in dimerization. However, there is an obvious difference between GCAP-2 and neurocalcin, since GCAP-2 forms relatively stable dimers only in the absence of Ca²⁺. Besides, the dimers of Ca²⁺-free GCAP-2 are less stable compared with Ca²⁺-loaded neurocalcin, because they partially dissociate during the time of chromatography (compare Figs. 1 and 2). Further study will have to determine what sites in GCAP-2 may play critical role in its dimerization and what sites are directly involved in interaction with the cyclase. It will also require additional study to determine how Ca²⁺ binding changes the kinetic parameters of the GCAP-2 dimerization.

	ACKNOWLEDGEMENT

We thank Sergei Boikov for technical assistance.

	FOOTNOTES

^* This work was supported by National Eye Institute Grant EY11522 (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.

^¶ Recipient of a career development award from Research to Prevent Blindness. 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@med.wayne.edu.

	ABBREVIATIONS

The abbreviations used are: retGC, photoreceptor membrane guanylyl cyclase; GCAP, guanylyl cyclase-activating protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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