From the Department of Ophthalmology/Kresge Eye
Institute and § Department of Pharmacology, Wayne State
University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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Photoreceptor membrane guanylate cyclases (RetGC) are regulated by calcium-binding proteins, GCAP-1 and GCAP-2. At Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, guanylate cyclase-activating protein (GCAPs) activate RetGC, and at free Ca2+ concentrations above 500 nM, characteristic of dark-adapted photoreceptors, GCAPs inhibit RetGC. A mutation, Y99C, in human GCAP-1 was recently found to be linked to autosomal dominant cone dystrophy in a British family (Payne, A. M., Downes, S. M., Bessant, D. A. R., Taylor, R., Holder, G. E., Warren, M. J., Bird, A. C., and Bhattachraya, S. S. (1998) Hum. Mol. Genet. 7, 273-277). We produced recombinant Y99C GCAP-1 mutant and tested its ability to activate RetGC in vitro at various free Ca2+ concentrations. The Y99C mutation does not decrease the ability of GCAP-1 to activate RetGC. However, RetGC stimulated by the Y99C GCAP-1 remains active even at Ca2+ concentration above 1 µM. Hence, the cyclase becomes constitutively active within the whole physiologically relevant range of free Ca2+ concentrations. We have also found that the Y99C GCAP-1 can activate RetGC even in the presence of Ca2+-loaded nonmutant GCAPs. This is consistent with the fact that cone degeneration was dominant in human patients who carried such mutation (Payne, A. M., Downes, S. M., Bessant, D. A. R., Taylor, R., Holder, G. E., Warren, M. J., Bird, A. C., and Bhattachraya, S. S. (1998) Hum. Mol. Genet. 7, 273-277). A similar mutation, Y104C, in GCAP-2 results in a different phenotype. This mutation apparently does not affect Ca2+ sensitivity of GCAP-2. Instead, the Y104C GCAP-2 stimulates RetGC less efficiently than the wild-type GCAP-2. Our data indicate that cone degeneration associated with the Y99C mutation in GCAP-1 can be a result of constitutive activation of cGMP synthesis.
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INTRODUCTION |
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The second messenger of phototransduction, cGMP, is synthesized in photoreceptors by two retinal guanylate cyclases, RetGC-1 and RetGC-2 (also referred to as ROSGC-1 and -2 or GC-E and GC-F, respectively) (2-10). RetGC1 are regulated by two homologous Ca2+-binding proteins, GCAP-1 and GCAP-2 (3, 4, 10-13).
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 they become closed in the light, because illumination stimulates cGMP hydrolysis by phosphodiesterase. Ca2+ is constantly extruded from the OS by a light-independent Na+/K+, Ca2+ exchanger, therefore interruption of Ca2+ influx through the channels decreases the intracellular free Ca2+ concentration (10, 14, 15), and that stimulates cGMP resynthesis in photoreceptors (10, 16). This Ca2+ feedback mechanism is essential for the recovery and light adaptation of photoreceptors (10).
RetGC itself is not sensitive to Ca2+, but it can interact with Ca2+ sensor proteins, GCAP-1 and GCAP-2 (3, 4, 11-13). A unique property of GCAPs is that they can be either activators or inhibitors of RetGC (17): at Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, GCAPs activate the cyclase, and at free Ca2+ concentrations above 500 nM, characteristic of dark-adapted photoreceptors, GCAPs inhibit RetGC. GCAP-1 and GCAP-2 have four EF-hand Ca2+-binding domains, and GCAPs can be turned into constitutive activators of RetGC by mutations that inactivate the ability of their EF-hands to bind Ca2+ (17-18).
The intracellular level of cGMP may be important not only for the phototransduction, but also for the viability of photoreceptors. Several types of rod or cone degeneration have been linked to the mutations in those photoreceptor proteins that regulate either synthesis or hydrolysis of cGMP (19-23). Recently Payne et al. (1) described a new case of human autosomal dominant cone dystrophy associated with a point mutation in GCAP-1 gene. In this paper we present the evidence that this mutation, Y99C, causes a dramatic change in Ca2+ sensitivity of GCAP-1. As a result, RetGC stimulated by the Y99C GCAP-1 remains active even at high free Ca2+ concentrations. We also demonstrate that the corresponding mutation in GCAP-2 produces a different effect. Our data indicate that dominant cone degeneration associated with the Y99C substitution in GCAP-1 can be caused by permanent activation of cGMP synthesis.
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EXPERIMENTAL PROCEDURES |
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Recombinant GCAP-1 and GCAP-2--
Recombinant GCAP-1 and GCAP-2
were expressed in Escherichia coli according to the
procedure described previously in detail (17, 24), except that we used
BLR(DE3)pLysS E. coli strain (Novagen) instead of
BL21(DE3)pLysS. Myristoylated GCAP-2 was expressed as described
previously (24). The N terminus of GCAP-1 is a poor substrate for yeast
N-myristoyltransferase (NMT; Ref. 25). Substitution D6S
makes it a better substrate (25), that allows us to produce GCAP-1,
which is >90% myristoylated and is fully capable of regulating RetGC
(Fig. 1). To make the GCAP-1 expression system, a cDNA encoding
GCAP-1 was isolated from a bovine retinal cDNA library (a gift from
Dr. D. Oprian, Brandeis University), amplified by polymerase
chain reaction using forward primer
AAAAAACCCATGGGGAACATTATGAGCGGTAAGTCGGTG and reverse primer ATATATGGATCCTTAAAGAGTAGGCAGTGAGCTCA. The resulting 0.65-kilobase pair
fragment was inserted into the NcoI/BamHI
restriction endonucleases sites of pET11d vector (Novagen) and
expressed under the lac-controlled T7 promoter in the
BLR(DE3)pLysS E. coli strain (Novagen) that harbored a
plasmid encoding yeast NMT (a gift from Dr. J. Gordon, Washington
University) as described previously (24). To produce Y C
substitutions fragments of GCAPs cDNAs were amplified by polymerase
chain reaction using Pfu polymerase (Stratagene) and spliced
by "splicing by overlap extension" (26). Pairs of primers encoding
the base substitutions were: GGTACTTCAAGCTCTGCGACGTGGACGGCAA and
TTGCCGTCCACGTCGCAGAGCTTGAAGTACC for making Y99C GCAP-1 and AGTGGACCTTCAAGATCTGCGACAAGGACCGCAA and
TTGCGGTCCTTGTCGCAGATCTTGAAGGTCCACT for making Y104C GCAP-2. Mutant
GCAP-1 and GCAP-2 were expressed using the same method (24).
Expressed proteins were purified as described previously (24) using
chromatography on Sephacryl S-100 column. Positions of the mutations
were verified by automated DNA sequencing (ABI Prizm, Perkin-Elmer).
Calculated average isotopic mass for the myristoylated Y99C used in
this study is 23,500.00. The actual average isotopic mass of purified
Y99C GCAP-1 found by electrospray mass-spectrometry was 23,500.0 ± 1.3. The nonmyristoylated form was undetectable.
RetGC Activity Assay--
Washed bovine OS membranes (containing
both RetGC-1 and RetGC-2) were prepared, depleted of endogenous GCAPs,
reconstituted with recombinant GCAPs, and assayed as described
previously (12, 24). The assay mixtures (25 µl) contained 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 [-32P]GTP, 0.1 µCi
of [8-3H]cGMP (NEN Life Science Products) and washed
bovine OS membranes (3.5 µg of rhodopsin). Reaction mixtures were
incubated under infrared illumination for 12 min at 30 °C. The
reaction was stopped by heating for 2 min at 95 °C. Samples were
chilled on ice, centrifuged, and analyzed by TLC using fluorescent
plastic-backed polyethylenimine cellulose plates (Merck). After
development in 0.2 M LiCl, cGMP spots were visualized under
UV illumination, cut, eluted with 1 ml of 2 M LiCl, mixed
with 10 ml of an Ecolume scintillation mixture, and both 3H
and 32P radioactivity were counted. [3H]cGMP
was used as an internal standard to ensure the absence of cGMP
hydrolysis by light-sensitive phosphodiesterase. Ca/EGTA buffers were
prepared according to (27).
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RESULTS AND DISCUSSION |
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Mutation Y99C Affects Ca2+ Sensitivity of GCAP-1-- GCAPs are highly conserved proteins (28). Human, mouse, and bovine GCAP-1 are virtually identical within their EF-hands regions, and this is also true for GCAP-2 (Ref. 19; also, see Fig. 1, top panel). When expressed as recombinant proteins, both GCAP-1 and GCAP-2 stimulate RetGC in a Ca2+-sensitive manner as it is shown in Fig. 1. It is also important to notice that GCAPs regulate RetGC within the submicromolar range of free Ca2+ concentrations. The exact free Ca2+ concentrations in rods and cones of mammals and humans have yet to be determined, but in dark-adapted resting rods of lower vertebrate, the free Ca2+ concentration is near 550 nM, and it decreases to near 50 nM (10) after strong illumination. Therefore we consider the submicromolar range of free Ca2+ as "physiologically relevant."
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The Y99C GCAP-1 Competes with the Wild Type GCAP-1 and GCAP-2-- Payne et al. (1) reported that the Y99C mutation in GCAP-1 gene had a dominant phenotype. The question is why does the presence of the normal allele(s) of GCAP(s) not protect cone cells from degeneration?
Even though the exact level of GCAP-1 and GCAP-2 expression in cones and rods has not been unambiguously defined, it has been well established that both GCAP-1 and GCAP-2 are expressed in photoreceptors (11-13, 29-32). Several antibodies were raised in different laboratories that could detect both GCAP-1 and GCAP-2 in rods (12, 30) and in cones (30, 31) (some conclusions about the distribution of GCAP-1 and GCAP-2 in rods versus cones (29) were at variance apparently because of the different masking of GCAP-2 epitopes in animal species (30)). Both immunocytochemical (13, 30-32) and in situ hybridization analyses (11) indicate that GCAP-1 is strongly expressed in cones. At the same time, Y99C mutation in GCAP-1 results only in cone dystrophy, and rods appear to be unaffected (1). This fact suggests that GCAP-1 is either not functioning in rods, or its concentration in rods is insignificant for RetGC regulation. It also strongly argues that GCAP-1 plays an important role in RetGC regulation in cones. On the other hand, GCAP-2 was initially found in rod outer segments (12). This localization of GCAP-2 in rods has been confirmed by other groups (30, 32). However, a lower level of GCAP-2 expression in cones has also been detected (30, 32). It is therefore possible that the normal alleles of GCAP-1 and GCAP-2 can both be present in the affected human cones along with the Y99C GCAP-1. Based on that assumption, we tested whether Y99C GCAP-1 could activate RetGC in the presence of both Ca2+-loaded GCAP-1 and GCAP-2 in vitro. We have found that the Y99C GCAP-1 efficiently competes with Ca2+-loaded GCAP-1 and GCAP-2 and prevents their inhibitory effect at free Ca2+ as high as 1 µM (Fig. 2). The addition of nonmutant GCAP-1 and GCAP-2 increases the EC50 for the RetGC activation by the Y99C GCAP-1, but it does not prevent RetGC from being activated by the mutant protein (Fig. 2A). The Y99C GCAP-1 stimulates RetGC in the presence of equimolar concentrations of either wild type GCAP-1 or GCAP-2 (Fig. 2B). The normal GCAP-1 and GCAP-2 are able to only partially decrease RetGC activity stimulated by the Y99C GCAP-1, at free Ca2+ above 1 µM. Therefore, given that the intracellular free Ca2+ in human photoreceptors in the dark is within the micromolar range (10), the Y99C mutation should be able to cause an excessive synthesis of cGMP in resting photoreceptors, even in the presence of normal GCAPs. That could explain the dominant phenotype of Y99C mutation in GCAP-1 found in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. James Hurley and Greg Niemi (University of Washington) for the electrospray mass-spectrometry analysis of the recombinant GCAPs. We are also grateful to the anonymous reviewer for stimulating criticism.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant EY11522 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.
¶ 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.
1 The abbreviations used are: RetGC, photoreceptor membrane guanylate cyclases; GCAP, guanylate cyclase-activating protein; NMT, N-myristoyltransferase; OS, photoreceptor outer segments; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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