Dominant cone and cone-rod dystrophies: functional analysis of mutations in retGC1 and GCAP1

38 HUNT ET AL

decrease because export by the Na+, K+, Ca2+ exchanger continues. This reduced [Ca2+]free results in the activation of retinal guanylate cyclase (retGC) by activating proteins (GCAPs). The increased conversion of GTP to cGMP leads to a restoration of cGMP levels to their dark level.

Changes in the regulation of cGMP are associated with mutations in all three components of this process. The rd mutation in the mouse is caused by a mutation in PDE (Pittler & Baehr 1991) and disease-causing mutations in both the a and b subunit genes have been reported in human (McLaughlin et al 1993, Dryja et al 1999). Mutations in GCAP result in dominant cone dystrophy (Payne et al 1998, Wilkie et al 2001) whereas mutations in retGC1 are responsible for recessive Leber congenital amaurosis (LCA1) (Perrault et al 1996) and dominant cone-rod dystrophy (Kelsell et al 1998).

The recovery process of photoreceptors after light exposure is mediated via a

change in intracellular [Ca2+]free in response to a drop in cyclic GMP level, both fundamental secondary messengers in photoreceptors. Several mechanisms, highly

conserved across species, have developed to maintain a low [Ca2+]free within photoreceptors. In the dark state, the [Ca2+]free in photoreceptor outer segments rises to around 500 nM due to the in£ux of Ca2+ through open cGMP-gated Na+/ Ca2+ channels (Dizhoor & Hurley 1999). Exposure to light leads to the hydrolysis of cGMP by PDE (via the rhodopsin-mediated cascade), and a subsequent decrease

in [Ca2+]free to approximately 30 nM due to the continued e¥ux of Ca2+ through light-independent Na+/K+/Ca2+ exchangers (Baylor 1996, Pugh 1996). This

decrease in [Ca2+]free stimulates an increase in cGMP, synthesized by retinalspeci¢c guanylate cyclase, in a feedback mechanism vital to the process of light adaptation and the recovery of the dark state.

The retGCs are not directly sensitive to changes in [Ca2+]free; Ca2+ exerts its regulatory e¡ect via specialized Ca2+ binding proteins known as guanylate

cyclase activating proteins (GCAPs). Three mammalian isoforms have been identi¢ed, GCAP1 (Palczewski et al 1994), GCAP2 (Lowe et al 1995, Gorczyca et al 1995) and GCAP3 (Haeseleer et al 1999). GCAPs belong to a subgroup of the neuronal-speci¢c Ca2+ binding proteins, which includes recoverin, all of which incorporate four variably functional repeats of the EF hand domain (Polans et al 1996), a helix-loop-helix structural domain with a selectively high a⁄nity for Ca2+ binding. In the case of the GCAPs, only EF2, EF3 and EF4 are functional. Numerous studies have demonstrated that GCAPs function to mediate the Ca2+-sensitive synthesis of cGMP by retGC, activating

photoreceptors respectively (Pugh et al 1997). This distinguishes the GCAPs from other Ca2+ binding proteins, which typically activate e¡ector proteins in their Ca2+-loaded forms.

The three GCAP isoforms have all been localized to the retina in various species, with GCAP1 present in higher concentrations in cone than in rod outer segments (Gorczyca et al 1995, Cuenca et al 1998), GCAP2 localizing to rods (Lowe et al 1995), cone inner segments (Otto-Bruc et al 1997) and layers of the inner retina (Cuenca et al 1998), and GCAP3 speci¢c to cones (Imanishi et al 2002). To date two dominant cone dystrophy mutations have been identi¢ed in GUCA1A, the gene encoding GCAP1, a Tyr99Cys substitution in EF3 (Payne et al 1998) and a Glu155Gly substitution in EF4 (Wilkie et al 2001).

Mutations in GUCY2D, the gene encoding retGC1, have been shown to be responsible for LCA1 (Perrault et al 1996), the most severe form of inherited retinopathy with total blindness or greatly impaired vision recognized at birth or in early infancy. These mutations show a recessive pattern of inheritance with no reported heterozygous e¡ects. In addition, mutations causing autosomal dominant cone-rod dystrophy have been identi¢ed in this gene. In contrast however to the LCA1 mutations that are found in most regions of the gene, the autosomal dominant cone-rod dystrophy mutations all share a common feature of the substitution of Arg838 (Payne et al 2001).

Experimental procedures

RetGC1 activity

Mutant and wild-type retGC1 activity was assayed as described by Wilkie et al (2000). Point mutations were introduced into a retCC1 cDNA cloned in pBluescript using the Altered Site kit (Promega). The wild-type and mutant copies were then subcloned into the expression vector pRC-CMV (Invitrogen) and used for the transient transfection of HEK 293T cells. RetGC1 was isolated in the form of a membrane preparation. Measurement of cyclase activity followed the radioassay of Dizhoor et al (1995). Basal activity and activity stimulated by wild-type and mutant GCAP1 were measured in reaction bu¡ers containing a range of [Ca2+]free.

Generation of recombinant wild-type and mutant GCAP1

A wild-type human GCAP1 cDNA clone used for the activity studies carried an arti¢cial Glu6Ser substitution to facilitate N-myristoylation of the protein. The Glu155Gly and Tyr99Cys mutations were introduced with the GeneEditor sitedirected mutagenesis kit (Promega). Mutant and wild-type GCAP1 was subcloned into pET3a (Novagen), expressed in E. coli strain BL21 and puri¢ed as described in Newbold et al (2001).

Results

Functional analysis of mutant GCAP1 activity

The binding of Ca2+ to the three EF hands of GCAP1 regulates its ability to

activate retGC1. Thus at [Ca2+]free below about 100 nM, retGC1 is activated by GCAP1 (Gorczyca et al 1994, Dizhoor et al 1994) but at micromolar

concentrations it is inhibited (Rudnicka-Nawrot et al 1998). Ca2+ titrations of retGC1 activity in the presence of wild-type and Glu155Gly mutant GCAP1 are

shown in Fig. 1A. With wild-type GCAP1, the [Ca2+]free for half-maximal activity (ED50) was estimated to be about 350 nM. With mutant GCAP1, however, the

curve shows very little inhibition of activity above 350 nM and even at 1mM, almost 70% of maximal activity persists, compared to about 10% with wild-type.

Using an equimolar mixture of wild-type and mutant GCAP1, activity at [Ca2+]free above 350 nM is again maintained; at 1mM almost 50% of maximal activity persists.

Thus the e¡ect of the mutation is to maintain high levels of cyclase activity over the whole physiologically relevant range of [Ca2+]free. This e¡ect is dominant in that it persists even in the presence of wild-type GCAP1 protein.

The Tyr99Cys mutation in GCAP1 has been shown to result in a similar constitutive activation of retGC1 at high [Ca2+]free (Sokal et al 1998, Dizhoor et al 1998). Comparison of the magnitude of the e¡ects of the two mutations on retGC activity (Fig. 1B) shows that the Glu155Gly mutation is more severe than Tyr99Cys in that the failure to inhibit activity at high [Ca2+]free is more pronounced.

Functional analysis of mutant retGC1 activity

A series of point mutations at codon 838 were made in the retGC1 cDNA to generate the Arg838Ser/His/Cys mutations identi¢ed in the dominant cone-rod dystrophy patients (Kelsell et al 1998, Payne et al 2001). These were transiently transfected into HEK 293-T cells for expression and the activities of the recombinant proteins assayed in vitro. The three mutants all showed a somewhat depressed basal activity. However, when activated with recombinant 8 mM human GCAP1, the activities of the mutants were similar to wild-type (Fig. 2). The major di¡erence in the activity of the mutant cyclases lies in their sensitivity

to increasing [Ca2+]free. At [Ca2+]free5100 nM, the mutants show a similar activity to wild-type but as the [Ca2+]free increases, the cyclase activity remains elevated compared to wild-type and this persists beyond the normal physiological range

of 100^500 nM (Fig. 3).

Domain structure

The retGC1 protein comprises ¢ve domains, an extracellular domain, a transmembrane domain, a kinase homology domain, a dimerization domain and

FIG. 2. Basal and stimulated activities of wild-type and mutant retGC1. (A) Basal activity with zero free Ca2+. (B) Activity with 8 mM GCAP1 with zero free Ca2+. Cyclase activity is expressed as percentage basal activity of wild-type.

a cyclase domain. A signi¢cant feature of the distribution of mutations within the gene is that those causing recessive LCA1 are found in each domain except the dimerization domain, whereas the dominant cone-rod dystrophy mutations are restricted to a single codon within this domain.

The retGC1 dimers that form the functional protein interact via a coiled coil formed between the dimerization domains of the two subunits (Ramamurthy et al 2001). Coiled-coils share a characteristic heptad repeat where the residues at position 1 and 4 are hydrophobic (Fig. 4). Hydrophobic interactions between the two a-helices occur every fourth residue of the sequence. This is frequently leucine, thereby giving rise to a hydrophobic core which maintains the structure. The COILS2 program (http://tofu.tamu.edu/Pise/5.a/coils2.html) predicts a coiled coil between residues 815^844 involving four heptads, which is broken by Arg838. Replacement of this residue with either Cys, Ser or His increase the probability of the coiled-coil structure continuing for further turns. The e¡ect of this is to increase the stability of the structure such that it resists disruption via the binding of Ca2+- loaded GCAP1.

FIG. 4. Probability of forming and extending an a-helical coiled-coil region in dimerization domain of wild-type and mutant retGC1.

Discussion

The major e¡ect of the retGC1 mutations is a reduced sensitivity to suppression of cyclase activity by Ca2+/GCAP1. The altered Ca2+ sensitivity is predicted to result in residual (constitutive) activity even at elevated [Ca2+]free, with consequent changes in the equilibrium of Ca2+ and cGMP concentrations. The three disease-

associated mutations are not entirely equivalent, with a shift in the [Ca2+]free for half maximal activation to higher concentrations in the order of Arg838Ser4

Arg838His4Arg838Cys. These results parallel the e¡ects of the mutations on the disease phenotype, with patients with the Arg838Ser mutation generally displaying more severe symptoms at an earlier age than those with the Arg838Cys or Arg838His mutations (Downes et al 2001).

Analysis of the sequence of the dimerization domain using the structure prediction program COILS reveals that the residue at position 838 is a key determinant of the extent of the coiled-coil structure responsible for holding together the active retGC1 dimer. Arg838 is predicted to disrupt the structure, limiting it to just four turns of each helix, whilst substitution with other residues

FIG. 5. Model of the regulation of wild-type and mutant retGC1 by GCAP1 under light and dark conditions of illumination. ED, extracellular domain; KHD, kinase homology domain; DD, dimerization domain; CAT, catalytic domain.

results in a higher probability that the structure will continue for further turns. Arg838Ser and Arg838His substitutions might be expected to have the largest impact on structure. The structural consequences of these substitutions has been examined in greater detail by Ramamurthy et al (2001) and a model to account for the e¡ect of Arg838 substitutions is presented in Fig. 5. In the dark when

intracellular Ca2+ is high, repulsion between Ca2+/GCAP1 monomer units forces the retGC1 dimer apart, inhibiting the cyclase activity. After light stimulation

when the [Ca2+]free falls, the GCAP1 dimer facilitates formation of the coiled-coil structure in the dimerization domain of retGC1 and enzyme activation occurs. The

e¡ect of substitutions at site 838 is therefore to stabilise the dimeric structure such that it is more resistant to disruption by Ca2+/GCAP1.

The Tyr99Cys and Glu155Gly mutations in GCAP1 also exert their e¡ect via a change in Ca2+ sensitivity and the magnitude of the e¡ect is very similar to the more severe retGC1 mutations. The mutations are in EF hands 3 and 4 respectively, the protein domains directly involved in Ca2+ binding. In both cases therefore, the number of Ca2+ binding sites is reduced from three to two and the reduced level of cyclase inhibition by the mutant GCAP1s at higher [Ca2+]free is therefore not surprising.

Photoreceptors contain two distinct forms of retinal guanylate cyclase, retGC1 and retGC2, and three forms of activating protein, GCAP1, GCAP2 and GCAP3. Various studies have revealed the presence of retGC1 (Liu et al 1994, Hallet et al 1996) and GCAP1 (Cuenca et al 1998) in both cone and rod photoreceptors, so di¡erential expression does not explain the restriction of cell loss to cones in the GCAP1 mutations. LCA1 is caused by recessive mutations in retGC1 (Perrault et al 1996) and the mouse knock-out of the orthologue of human retGC1 results in a distinct retinal pathology (Yang et al 1999), indicating in both cases that retGC2 does not protect photoreceptors from the damaging e¡ect of mutations in retGC1. Likewise, the introduction of a GCAP1 transgene (Howes et al 2002) into a mouse line lacking both GCAP1 and GCAP2 fully restores the normal £ash responses whereas Mendez et al (2001) have shown that GCAP2 does not restore the normal physiological response of rod photoreceptors to light £ashes. These results indicate that GCAP2 is relatively unimportant in phototransduction. What remains unclear however is why this reduced sensitivity to increasing

[Ca2+]free results in a cone dystrophy for the GCAP1 mutations but a cone-rod dystrophy for the retGC1 mutations.

What are the possible consequences of the reduced sensitivity of mutant GCAP1

and retGC1 to [Ca2+]free and how does it relate to the pathology of the cone and cone^rod dystrophies? The operating range of rods is substantially extended by the

regulation of the cyclase activity of retGC1 by GCAP1 in decreasing the £ash sensitivity in darkness and increasing the incremental £ash sensitivity in bright steady light. Reduced Ca2+ sensitivity of the mutant systems may serve to reduce this range and thereby account for the photophobia experienced by a number of our cone and cone-rod patients. The net result of the changes in Ca2+ sensitivity might be expected to be the maintenance of cGMP levels in the cell above that required to keep the cGMP-gated cation channels open, resulting in persistently high intracellular Ca2+ levels. Persistent elevated Ca2+ levels in cells tend to disrupt

the membrane potential of the mitochondrial outer membrane, leading to release of cytochrome C, with subsequent caspase activation and apoptosis (Green & Reed 1998). Treatment with Ca2+ channel blockers like L-cis-diltiazem which block both rod and cone cGMP-gated channels may therefore be bene¢cial. However, it is not possible to predict with any certainty the e¡ect on cellular cGMP and Ca2+ concentrations because it is unclear as to what extent the activity of other components of the phototransduction cascade might change in a compensatory manner. The generation of transgenic animals will be especially useful therefore in extending our understanding of this aspect of the aetiology of these disorders and this work is currently in progress.

Acknowledgments

This work was supported by a Programme Grant from the Wellcome Trust.

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