Novel Dominant Rhodopsin Mutation Triggers Two Mechanisms of Retinal Degeneration and Photoreceptor Desensitization

Abstract

A variety of rod opsin mutations result in autosomal dominant retinitis pigmentosa and congenital night blindness in humans. One subset of these mutations encodes constitutively active forms of the rod opsin protein. Some of these dominant rod opsin mutant proteins, which desensitize transgenic Xenopus rods, provide an animal model for congenital night blindness. In a genetic screen to identify retinal degeneration mutants in Drosophila, we identified a dominant mutation in the ninaE gene (NinaE^pp100) that encodes the rhodopsin that is expressed in photoreceptors R1-R6. Deep pseudopupil analysis and histology showed that the degeneration was attributable to a light-independent apoptosis. Whole-cell recordings revealed that the NinaE^pp100 mutant photoreceptor cells were strongly desensitized, which partially masked their constitutive activity. This desensitization primarily resulted from both the persistent binding of arrestin (ARR2) to the NINAE^pp100 mutant opsin and the constitutive activity of the phototransduction cascade. Whereas mutations in several Drosophila genes other than ninaE were shown to induce photoreceptor cell apoptosis by stabilizing a rhodopsin-arrestin complex, NinaE^pp100 represented the first rhodopsin mutation that stabilized this protein complex. Additionally, the NinaE^pp100 mutation led to elevated levels of G_qα in the cytosol, which mediated a novel retinal degeneration pathway. Eliminating both G_qα and arrestin completely rescued the NinaE^pp100-dependent photoreceptor cell death, which indicated that the degeneration is entirely dependent on both G_qα and arrestin. Such a combination of multiple pathological pathways resulting from a single mutation may underlie several dominant retinal diseases in humans.

Introduction

Retinitis pigmentosa (RP) is a group of genetic disorders that exhibits abnormal electroretinograms and gradual vision loss attributable to the death of the rod and then cone photoreceptors in humans (Rattner et al., 1999; Phelan and Bok, 2000). Several X-linked and autosomal dominant and recessive forms of RP have been identified (Wang et al., 2001), with rhodopsin mutations being a prevalent cause of autosomal dominant RP (Kaushal and Khorana, 1994; Gal et al., 1997). Rhodopsin is a seven transmembrane, G-protein-coupled receptor that is activated by a photon of light to initiate the phototransduction cascade. Various models describe dominant rhodopsin-mediated degeneration mechanisms, including the following: (1) defective maturation and/or trafficking of rhodopsin (Roof et al., 1994; Sung et al., 1994), (2) constitutively active rhodopsin and persistent stimulation of the phototransduction cascade (Robinson et al., 1992; Dryja et al., 1993; Rao et al., 1994), and (3) abnormal interactions between rhodopsin and the phototransduction components (Min et al., 1993; Li et al., 1995; Rim and Oprian, 1995).

The Drosophila ninaE gene encodes the rhodopsin protein (NINAE) that is expressed in six (R1-R6) of the eight adult photoreceptor cells in each ommatidium (O'Tousa et al., 1985; Zuker et al., 1985). The NINAE protein is activated by blue light, which stimulates the dgq-encoded G_qα subunit of the heterotrimeric G-protein (Lee et al., 1990, 1994; Scott et al., 1995). The photoactivated metarhodopsin is inactivated by phosphorylation and arrestin (ARR2) binding (Byk et al., 1993; Dolph et al., 1993; Kiselev and Subramaniam, 1994; Alloway and Dolph, 1999). Orange light photoconverts metarhodopsin to rhodopsin, which dissociates from arrestin and is dephosphorylated by the rdgC-encoded Ca²⁺-dependent serine/threonine phosphatase (Byk et al., 1993). In the absence of arrestin dissociation, the persistent NINAE-arrestin complexes are endocytosed and induce apoptosis of photoreceptor cells (Alloway et al., 2000; Kiselev et al., 2000). Recessive ninaE mutants exhibit a defective light response and photoreceptor cell death (Leonard et al., 1992). Dominant ninaE mutations were identified as suppressors of either rdgC-mediated (Kurada and O'Tousa, 1995) or rdgB-dependent (Colley et al., 1995) retinal degeneration. The rdgB gene encodes a novel phosphatidylinositol transfer protein that is required for the light response and photoreceptor viability (Vihtelic et al., 1991, 1993). All of the previously isolated dominant ninaE mutants are defective in the maturation and/or trafficking of rhodopsin in the endoplasmic reticulum (ER) (Colley et al., 1995; Kurada et al., 1998). Thus, dominant ninaE mutations likely mimic the degeneration mechanisms underlying rhodopsin-mediated autosomal dominant RP in humans.

We report a new dominant ninaE mutant (NinaE^pp100) that exhibits retinal degeneration and constitutively active and strongly desensitized photoreceptor cells. The photoreceptor cell desensitization and death resulted from both the NINAE^pp100 mutant rhodopsin forming a stable complex with arrestin and the persistent localization of the G_qα in the cytosol. Genetic experiments reveal that the arrestin and G_qα mechanisms are mutually exclusive. Furthermore, the predominant localization of an active G_qα protein in the cytosol stimulates photoreceptor cell death through a novel mechanism.

Materials and Methods

Isolation of the dominant NinaE^pp100 mutant. Wild-type males were starved for 6 hr and then fed 25 mm ethyl methanesulfonate (Sigma, St. Louis, MO) in a 1% sucrose solution overnight (Lewis and Bacher, 1968). The males were mated en masse to SM1/Sco; TM2/Sb virgin females, and the F1 progeny were raised in constant light for 10 d. The F1 flies were screened for the absence of a wild-type deep pseudopupil, which is a virtual image of the rhabdomeres from several adjacent ommatidia (Franceschini, 1972).

Characterizing the retinal degeneration phenotype. Flies were raised under constant light and scored daily for the presence of the deep pseudopupil as described previously (Paetkau et al., 1999). The average percentage of flies possessing a wild-type deep pseudopupil and SDs were determined from at least four replicates (100-150 flies per trial) of each genotype and age. Flies raised in constant darkness were collected, aged until a designated day, scored for the presence or absence of a wild-type deep pseudopupil, and then discarded.

For light and electron microscopy of retinal sections, flies were raised in a 12 hr light/dark cycle to the desired age and then decapitated. The heads were bisected, fixed, and embedded in Polybed 812 as described previously (Lee et al., 1994). Sections for light and electron microscopy were generated and processed as described previously (Paetkau et al., 1999).

DNA isolation and sequencing. Genomic DNAs from wild-type and NinaE^pp100 mutant flies were isolated as described previously (Ashburner, 1989). Wild-type and mutant ninaE genes were PCR amplified using the 5′ AGG ATC CAA TGG AGA GGT ACG ATC GGT GAA TCC AC and 5′ GGT TGT GGA TCC AAA GAA TTT ATG CC primers that mapped to nucleotides -8 and 1518 of the ninaE gene, respectively (O'Tousa et al., 1985). Both PCR primers contain engineered BamHI sites at their 5′ ends (in bold), and the first primer also deletes a BamHI site (underlined) in the first ninaE intron. The PCR reactions contained 3 μg of genomic DNA, 1× native Pfu buffer, 2.5 mm of each dNTP, and 250 pmol of each primer. After incubating at 96°C for 7 min, 1 μl of native Pfu DNA polymerase (Stratagene, La Jolla, CA) was added and amplified through 30 cycles of 96°C for 1 min, 54°C for 1 min, and 72°C for 4 min, followed by a 10 min extension at 72°C. The PCR products were size fractionated on a Tris acetate-EDTA agarose gel, purified with the Wizard PCR prep resin (Promega, Madison, WI), and blunt-end ligated into the pZero 2.1 vector (Invitrogen, Carlsbad, CA). The plasmid DNA was purified with Strataclean Resin (Stratagene), precipitated, resuspended in H₂O, and sequenced using the Sequenase Quick Denature Kit (US Biochemicals, Cleveland, OH), with primers generated from the published ninaE genomic sequence (O'Tousa et al., 1985).

In vitro mutagenesis. The Pfu-generated wild-type ninaE gene was digested from pZero 2.1 with BamHI, gel purified, and cloned into the BamH1 site of the PK- ATG- vector, which contains the 3.2 kb ninaE promoter and a 0.7 kb ninaE poly(A⁺) tail (Kurada and O'Tousa, 1995; Shetty et al., 1998). The NinaE^pp100 mutation was introduced into the wild-type ninaE gene by in vitro mutagenesis in the PK- ATG- vector using the Quick Change Site-Directed Mutagenesis kit (Stratagene) and the complementary primer pairs 5′ TCA ACT GCA TGA GAC TGT TCA AGT 3′ and 5′ ACT TGA ACA GTC TCA TGC AGT TGA 3′ (the site of the nucleotide change is in bold). The PCR reaction contained 50 ng of template DNA, 125 ng of each primer, 1× buffer, 10 mm of each dNTP, and 1 μl of Pfu DNA polymerase. The PCR amplification protocol was 95°C for 30 sec and then 12 cycles of 95°C for 30 sec, 60°C for 1 min, 68°C for 2 min, and a final 15 min extension at 68°C.

Germ-line transformation. A 5.4 kb KpnI fragment containing the ninaE promoter, the in vitro mutagenized ninaE genomic DNA, and the ninaE poly(A⁺) tail was gel purified and cloned into the KpnI site of the pCaSpeR-4 transformation vector (Ashburner, 1989). The sequence of the entire ninaE coding region was confirmed in this vector. This in vitro mutagenized DNA was germ-line transformed with the Δ2-3 P-element (ratio of 6:1) using standard techniques (Spradling, 1986).

Electrophysiology. Electroretinograms (ERGs) were performed as described previously (Paetkau et al., 1999). ERG traces were recorded using a MacAdios II analog-to-digital converter using SuperScope II software on a Macintosh IIx computer (Apple Computers, Cupertino, CA).

To measure light-induced currents (LICs), orange light (OG 590 Ditric edge filter) from a xenon high-pressure lamp (75 W) was delivered to isolated ommatidia via the objective lens (60× Olympus Optical, Tokyo, Japan) and attenuated up to seven orders of magnitude by neutral density filters. The maximal luminous intensity of the orange light at the level of the ommatidia was ∼3.0 log units above the intensity required for a half-maximal response of the R1-R6 photoreceptors. The effective intensity of the white light without the orange filter was determined by comparing the light intensities with and without the orange filter, which were required to elicit a small response of the same amplitude in wild-type flies. In Figure 4, the intensity of the white light stimulus was converted to the effective relative intensity of orange light.

Figure 4.

LICs of NinaE^pp100 mutants exhibit a highly reduced sensitivity to light relative to wild-type flies. A, Top row, LIC of a wild-type photoreceptor cell in response to dim (log I = -5.0) and more intense (log I = -1.0) orange light pulses indicated by horizontal bars. The response to the dim light shows the typical current fluctuations attributable to the summation of quantum bumps. The response to intense light shows the initial transient phase that declines to a small steady-state phase attributable to light adaptation. Note the different current scales below all of the responses. A, Middle row, LIC of a NinaE^pp100 photoreceptor cell in response to intense orange light (log I = 0) and more intense white light expressed in equivalent orange light intensity (log I = 3). No bump noise is observed. A, Bottom row, LIC of an arr2^Y20STOP NinaE^pp100 photoreceptor cell in response to medium intensity orange light (log I = -3) and more intense white light expressed in equivalent orange light intensity (log I = 1). Note the appearance of bump noise at the lower light intensity. The slower LIC waveforms at both light intensities are typical for the arr2 mutant phenotype. B, Families of intensity-response functions (i-log I curves) obtained from wild-type, NinaE^pp100, and arr2^Y20STOP mutant photoreceptors. The graph plots the peak current amplitudes of the responses (in picoamperes) for wild-type (filled triangles), NinaE^pp100 (filled circles), and arr2^Y20STOP (open circles) mutant photoreceptors. Only a few of the NinaE^pp100 mutant cells responded to the intense orange light stimulus. The light intensity range above log I = 0 was obtained with white light that was converted to equivalent orange intensity (see Materials and Methods). The arr2^Y20STOP cells possessed i-log I curves that were indistinguishable from wild type.

Dissociated ommatidia were prepared from either newly eclosed adult flies (<1 hr after eclosion) or late pupae as described previously (Hardie, 1991). Whole-cell patch-clamp recordings were performed as described previously (Hardie and Minke, 1992; Peretz et al., 1994). Recordings were made at 21°C using patch pipettes of 5-10 MΩ pulled from fiber-filled borosilicate glass capillaries. Series resistance of 7-14 MΩ was carefully compensated (>80%) during all experiments. Signals were amplified with an Axopatch-1D or 2B (Axon Instruments, Foster City, CA) patch-clamp amplifier, sampled at 2 kHz, and filtered below 1 kHz.

Immunoblots of NINAE, ARR2, and G_qα proteins. Immunoblots of rhodopsin expression in flies 4-6 hr old were performed as described previously (Milligan et al., 1997). Head protein homogenates were electrophoresed, electrotransferred to Hybond polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Piscataway, NJ), and incubated with the 4C5 anti-rhodopsin monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). The 4C5 monoclonal antibody was detected with HRP-conjugated goat anti-mouse antibodies (1:5000) and ECL reagents (Amersham Biosciences). Band intensity was quantified on a scanning laser densitometer (Bio-Rad, Hercules, CA) and normalized to a standard curve generated by a dilution series of head protein extract, with five independent blots analyzed.

The ARR2 protein was isolated under dim red light from white-eyed wild-type and mutant fly heads that were 4 hr after eclosion. The heads were placed in 20 μl of PBS, pH 7.4, illuminated at the desired wavelength for 2 min, homogenized in darkness, and centrifuged with 15,000 × g. The supernatant fraction was transferred to a new Eppendorf tube (Eppendorf Scientific, Westbury, NY), and the pellet was resuspended in 20 μl of PBS. SDS extraction buffer (20 μl) was added to both fractions and incubated at 45°C for 1 hr before electrophoresing one head equivalent of cytosolic and membrane protein extracts through a 7-15% gradient SDS-PAGE gel. After electrotransfer, the PVDF membranes were incubated with a mixture of the 4C5 anti-rhodopsin monoclonal antibody and anti-ARR2 rabbit polyclonal antisera (Alloway et al., 2000; Kiselev et al., 2000) and detected with a mixture of HRP-conjugated mouse anti-rabbit and rabbit anti-mouse antibodies and the ECL system (Amersham Biosciences), with five independent blots analyzed.

The G_qα-protein was quantitated in different subcellular fractions as described previously (Kosloff et al., 2003). Wild-type and NinaE^pp100 flies were dark adapted for 24 hr and then subjected to either blue light illumination (490 nm) or continued darkness for 10 min at 22°C. The heads were homogenized at 4°C in the dark in hypotonic medium (20 mm HEPES buffer, pH 7.6, containing 15 mm N-ethylmaleimide, 20 μg/ml leupeptin, 1 μg/ml pepstatin A, and 0.35 mg/ml o-phenantroline). The samples were centrifuged at 15,800 × g for 15 min at 4°C to separate the membrane pellet from the soluble proteins. The pellet was washed and centrifuged, and the supernatants were combined. Ultracentrifugation at 150,000 × g for 30 min did not change the G_qα distribution between the fractions. The proteins were precipitated in 5% TCA, electrophoresed on a 10% SDS-PAGE, and electrotransferred to nitrocellulose using a semidry transfer apparatus (Bio-Rad). The nitrocellulose was blocked and incubated with a rabbit anti-G_qα polyclonal antibody raised against the decapeptide of the C terminus of the protein. The nitrocellulose was incubated with anti-rabbit polyclonal antiserum and detected with ECL Amersham Biosciences reagents according to the instructions of the manufacturer and quantitated with Fuji LAS-1000. The amount of G_qα in each fraction was calculated as a percentage of the total G_qα in the pellet and supernatant, with four independent blots analyzed.

GTPase assay. GTP hydrolysis of Drosophila head extracts under various light conditions were performed essentially as described previously (Blumenfeld et al., 1985).

Results

The NinaE^pp100 mutant exhibits a dominant retinal degeneration and a recessive electrophysiological defect

In a screen to identify Drosophila mutants that lacked a deep pseudopupil, we identified a dominant retinal degeneration mutation that mapped <1 cM from the ninaE gene, which encodes the rhodopsin protein expressed in photoreceptor cells R1-R6 (O'Tousa et al., 1985; Zuker et al., 1985). To determine whether NinaE^pp100 was a ninaE allele, we examined the ERGs of flies that were ∼12 hr after eclosion (before any signs of degeneration). White-eyed wild-type flies exhibited a prolonged depolarizing afterpotential (PDA) after a blue light converts a large fraction of rhodopsin to metarhodopsin (Fig. 1A) (Hillman et al., 1983; Dolph et al., 1993). The white-eyed ninaE^I17/ninaE^I17 null mutant lacked a PDA and possessed small light response amplitudes (Fig. 1B), which were attributable to the opsins expressed in photoreceptor cells R7 and R8. White-eyed NinaE^pp100/ninaE⁺ flies exhibited a wild-type ERG light response and a PDA (Fig. 1C), whereas NinaE^pp100/NinaE^pp100 flies lacked the PDA (Fig. 1E). Thus, the NinaE^pp100 flies exhibited a recessive mutant ERG phenotype. White-eyed NinaE^pp100/ninaE^I17 flies also lacked the PDA (Fig. 1D), which confirmed that NinaE^pp100 was a ninaE allele. Immunoblots revealed that the NinaE^pp100 mutant gene expressed ∼20% of the rhodopsin relative to the ninaE⁺ gene (Fig. 1F). We sequenced the ninaE gene from the NinaE^pp100 mutant and identified a G-to-A transition mutation, which changed the glycine at amino acid 299 to arginine (Fig. 1G).

Figure 1.

NinaE^pp100 is a ninaE allele. We recorded the ERG light response of wild-type (A), ninaE^I17/ninaE^I17 (B), NinaE^pp100/ninaE⁺ (C), NinaE^pp100/ninaE^I17 (D), and NinaE^pp100/NinaE^pp100 (E) flies at ∼12 hr after eclosion. Each fly was stimulated for 2 sec with orange light (or), 2 sec with blue light (bl), and a final 2 sec with orange light, as shown below D. Wild-type flies exhibited a PDA in response to blue light (A, C), which was absent in ninaE mutant flies (B, D, E). Calibration for ERG responses is shown below D. F, Immunoblot of rhodopsin expression relative to wild-type flies for ninaE^I17/ninaE^I17 flies (undetectable), ninaE⁺/ninaE^I17 heterozygotes (62% ± 4%), NinaE^pp100/NinaE^pp100flies (21%±5.0%), NinaE^pp100/ninaE⁺flies (75%±4.7%),andNinaE^pp100/ninaE^I17 flies (12% ± 3.1%). G, The putative amino acid sequence of the NINAE opsin protein is shown as a single letter amino acid code. The N and C termini are labeled (NH₂ and COOH, respectively), with the relative orientation of the intracellular and extracellular faces of the membrane labeled. The NINAE^pp100 mutation (G299R) is shown.

We examined the retinal degeneration that underlies the deep pseudopupil loss in the NinaE^pp100 mutant. A 2-d-old wild-type retina possessed a regular repeating pattern of ommatidia (Fig. 2A, left column), whereas the ultrastructure of a 10-d-old retina revealed large R1-R6 rhabdomeres surrounding the central R7 rhabdomere (Fig. 2A, right column). In contrast, the 2-d-old NinaE^pp100/ninaE⁺ retina possessed a few holes and reduced or missing rhabdomeres (Fig. 2B). Electron microscopy of the 10-d-old NinaE^pp100/ninaE⁺ retina revealed degenerating R1-R6 photoreceptors (darkly stained) and phagocytosed photoreceptors (Fig. 2B, asterisks). To prove that the G299R mutation caused the dominant NinaE^pp100 mutant phenotype, we created three independent transgenic lines that contained a wild-type ninaE gene that was in vitro mutagenized to introduce the G299R mutation. At both the light and electron microscope level, all three transgenic lines (ninaE⁺, P[ninaE-G299R]/ninaE^I17) (Fig. 2C) exhibited a dominant degeneration phenotype that was indistinguishable from the NinaE^pp100/ninaE⁺ flies. Therefore, the G299R mutation accounted for the dominant NinaE^pp100 degeneration phenotype.

Figure 2.

The NinaE^pp100 retinal degeneration phenotype. The histology of wild-type (ninaE⁺/ninaE⁺; A), NinaE^pp100/ninaE⁺ (B), ninaE⁺, P[ninaE-G299R]/ninaE^I17 transgenic flies (C), and NinaE^pp100/NinaE^pp100 flies (D) were examined using light microscopy (2 d after eclosion; left column) and electron microscopy (10 d after eclosion; right column). The dominant NinaE^pp100 (B) degeneration phenotype was characterized by loss of rhabdomeres, presence of intracellular vacuoles, and phagocytosis of photoreceptors (asterisks). The transgenic flies (C) exhibited a degeneration that was indistinguishable from the dominant mutant phenotype, which confirmed that the G299R mutation caused the dominant degeneration. The homozygous NinaE^pp100 degeneration (D) was characterized by a rapid loss of the rhabdomeres, increased number of large intracellular vesicles (arrows), phagocytosis of the photoreceptor cells (asterisk), and cytoplasmic condensation (arrowhead). Scale bars (in A): left, 10 μm; right, 1 μm (shown in the wild-type sections and are the same for the subsequent sections).

Because the NinaE^pp100 mutant exhibited a recessive ERG light response defect, we examined the histology of NinaE^pp100 homozygotes. The 2-d-old NinaE^pp100/NinaE^pp100 retina exhibited an increased number of large intracellular vesicles, more ommatidial disorganization, and fewer rhabdomeres relative to NinaE^pp100/ninaE⁺ (Fig. 2D,B, respectively). The ultrastructure of a 10-d-old NinaE^pp100/NinaE^pp100 ommatidium revealed that the R1-R6 rhabdomeres were more disorganized and smaller than the central R7 rhabdomere, and many large intracellular vesicles (Fig. 2D, arrows) were present within some of the R1-R6 photoreceptors. Some photoreceptors were being phagocytosed, and others exhibited a decreased cytoplasmic volume (Fig. 2D, asterisks, arrowhead, respectively). Thus, the NinaE^pp100 mutation caused a dominant retinal degeneration that was enhanced in the NinaE^pp100 homozygote.

The NinaE^pp100 mutant exhibits a light-enhanced retinal degeneration

We examined the effect of light on the NinaE^pp100 retinal degeneration to differentiate between various potential degeneration mechanisms. We used the deep pseudopupil as a rapid and noninvasive technique to assay the integrity of the retina and photoreceptor cells (Franceschini, 1972). The NinaE^pp100 mutant exhibited a dominant deep pseudopupil loss that was slightly faster in constant light than in constant darkness (Fig. 3, NinaE^pp100/ninaE⁺, open circles, filled circles, respectively). The absence of any wild-type NINAE⁺ protein in the NinaE^pp100/NinaE^pp100 flies dramatically increased the onset and rate of deep pseudopupil loss relative to NinaE^pp100/ninaE⁺ (Fig. 3). Furthermore, the NinaE^pp100/NinaE^pp100 degeneration time course exhibited a significantly faster deep pseudopupil loss in constant light relative to constant darkness (Fig. 3, open squares, filled squares, respectively). The NinaE^pp100/ninaE^I17 flies exhibited a deep pseudopupil loss that mimicked the NinaE^pp100/NinaE^pp100 degeneration time course (data not shown). The ninaE^D1 mutation, which is a serine-to-phenylalanine change at position 137 in the third transmembrane domain, causes a slow dominant retinal degeneration by blocking rhodopsin maturation in the endoplasmic reticulum (Kurada and O'Tousa, 1995; Kurada et al., 1998). The ninaE^D1 mutation suppressed the rapid NinaE^pp100-dependent deep pseudopupil loss (Fig. 3, NinaE^pp100/ninaE^D1, triangles). Therefore, the NinaE^pp100 mutation must cause its rapid degeneration through a mechanism subsequent to the ninaE^D1 block of rhodopsin maturation in the ER (Kurada and O'Tousa, 1995).

Figure 3.

Rates of NinaE^pp100 deep pseudopupil loss. We examined the time course of deep pseudopupil loss of NinaE^pp100/NinaE^pp100 flies (squares), NinaE^pp100/ninaE⁺ flies (circles), and NinaE^pp100/ninaE^D1 flies (triangles) under constant light and constant darkness (open and filled symbols, respectively). The homozygous NinaE^pp100 mutants exhibited an earlier and more rapid deep pseudopupil loss relative to the heterozygous NinaE^pp100 mutants. Furthermore, both the homozygous and heterozygous NinaE^pp100 mutants exhibited more rapid deep pseudopupil loss in constant light relative to constant darkness. The ninaE^D1 mutation strongly suppressed the rapid NinaE^pp100 deep pseudopupil loss (triangles). The percentages of flies retaining a deep pseudopupil on each day were plotted against their age, with each symbol representing the average from four independent trials of 100-150 flies per trial.

The NinaE^pp100 mutant is strongly desensitized

We performed whole-cell recordings on the homozygous NinaE^pp100 mutant before photoreceptor degeneration to elucidate the underlying physiological defect. The wild-type LIC to dim orange light stimulus showed current fluctuations that are attributable to the summation of the quantum bumps (Fig. 4A, top left panel), whereas a bright orange light stimulus invoked an initial transient phase that declined to a small steady-state phase attributable to light adaptation (Fig. 4A, top right panel). The LIC from one of the most sensitive NinaE^pp100 mutant cells to intense orange light elicited only a small response that lacked large current fluctuations (Fig. 4A, middle left panel). Furthermore, the intense white light required to elicit a sizable response in the NinaE^pp100 mutant (Fig. 4A, middle right panel) was approximately four orders of magnitude brighter than the stimulus eliciting a similar response amplitude in a wild-type cell. The need for a more intense light stimulus to induce a wild-type-sized LIC response in NinaE^pp100 suggested that the NinaE^pp100 mutant possessed a highly reduced sensitivity to light. The individual NinaE^pp100 cells exhibited a large variability in their sensitivity to light, with only a fraction of the light-sensitive NinaE^pp100 mutant cells possessing the maximal peak response amplitude (data not shown). Because this amplitude was similar to that of wild-type flies, the mutant cells must contain a sufficient number of light-sensitive rhodopsin molecules. It was shown previously that the high gain of the phototransduction cascade resulted in the ninaE^P332 mutant exhibiting a shift of ∼3.5 orders of magnitude in the intensity-response function (V-log I curve) and a wild-type peak amplitude of the receptor potential, although it had approximately three orders of magnitude less rhodopsin than wild-type flies (Johnson and Pak, 1986).

To confirm the reduced light sensitivity of the NinaE^pp100 cells, we plotted the intensity-response function (i-log I curve) for several different individual NinaE^pp100 mutant and wild-type cells (Fig. 4B). There was a large difference in the light intensity range at which wild-type and the NinaE^pp100 photoreceptors responded, with a considerable fraction of mutant cells either not responding to the maximal white light intensity or only exhibiting minimal response amplitudes. This data clearly demonstrated that a main electrophysiological phenotype of the NinaE^pp100 mutant relative to wild type is a large decrease in the sensitivity to light.

The NinaE^pp100 desensitization is partially rescued by elimination of arrestin

One mechanism that could underlie the NinaE^pp100 reduced light sensitivity is persistent binding of arrestin (ARR2) to NINAE^pp100. Persistent arrestin binding was shown previously to result in retinal degeneration of several Drosophila mutants (Alloway et al., 2000; Kiselev et al., 2000) and the transgenic mouse model expressing the K296E mutant human opsin (Li et al., 1995). Whole-cell recordings on the arr2^Y20STOP NinaE^pp100 double mutant (Fig. 4A, bottom left panel) revealed the bump noise that was also observed in wild-type flies (Fig. 4A, top left panel), but absent in NinaE^pp100 (Fig. 4A, middle left panel). Although the arr2^Y20STOP NinaE^pp100 double mutant revealed a large increase in light sensitivity relative to NinaE^pp100, it was still significantly less sensitive than wild type (Fig. 4A, bottom right panel). We compared the intensity-response function (i-log I curve) of the arr2^Y20STOP mutant photoreceptors with wild-type and NinaE^pp100 cells and found that the family of arr2^Y20STOP responses was nearly identical to wild type (Fig. 4B). Thus, the ARR2 protein specifically interacted with the NINAE^pp100 mutant rhodopsin to decrease the sensitivity to light. However, this interaction failed to account for the entire range of reduced sensitivity of the NinaE^pp100 cells.

We extended this analysis by plotting the peak amplitude versus log of the relative light intensity from in vivo ERG recordings and confirmed that the arr2^Y20STOP NinaE^pp100 double mutant possessed an increased light sensitivity relative to the NinaE^pp100 mutant (Fig. 5A). Although these observations strongly suggested that arrestin contributed to the reduced light sensitivity of the NinaE^pp100 mutant, Figures 4A (bottom row) and 5A revealed that the arr2^Y20STOP NinaE^pp100 double mutant remained less sensitive to light relative to wild-type flies. This remaining desensitization in the arr2^Y20STOP NinaE^pp100 fly may be attributable to either the NINAE^pp100 molecules being constitutively active or a reduced interaction between NINAE^pp100 and the heterotrimeric G-protein.

Figure 5.

The NinaE^pp100 mutation induces constitutive activity of the light-sensitive channels. A, In vivo ERG recordings of intensity-response functions measured in wild-type (filled circles; n = 6), NinaE^pp100 (filled squares; n = 8), and arr2^Y20STOP NinaE^pp100 (filled triangles; n = 6). The arr2^Y20STOP mutation increased the NinaE^pp100 sensitivity to light ∼10-fold. B, Histograms plotting the percentage of NinaE^pp100 (n = 33 adult flies and 10 pupae) and arr2^Y20STOP NinaE^pp100 (n = 23, all pupae) cells showing constitutive activity in the dark. The arr2^Y20STOP mutation increased the fraction of NinaE^pp100 cells exhibiting constitutive activity. C-F, Families of current traces elicited by a series of 20 mV voltage steps from -100 to +80 mV (bottom row). A wild-type photoreceptor in the dark exhibits only a small leak current (C). Both NinaE^pp100 and arr2^Y20STOP NinaE^pp100 pupal photoreceptors possess inward and outward rectifying currents in the dark (D, E, respectively). Addition of 20 μm La³⁺ to the external medium of the same arr2^Y20STOP NinaE^pp100 pupal cell almost completely abolished the current (F). Very similar results were obtained when adult (newly eclosed) flies were used, except that, at negative holding potentials, the inward currents were smaller. Calibration is shown under the wild-type traces (C) and is the same for D-F.

To investigate whether the NINAE^pp100 molecules were constitutively active and induced persistent opening of the light-activated channels, we recorded whole-cell membrane currents to voltage steps in the dark (Fig. 5C-F). Wild-type cells revealed no significant currents under these conditions (Fig. 5C). However, small but significant (Yoon et al., 2000) currents were recorded from both NinaE^pp100 and arr2^Y20STOP NinaE^pp100 mutant cells (Fig. 5D,E, respectively). The characteristics of these currents and their ability to be blocked by a low concentration of La³⁺ (Fig. 5F) suggested that they originate from constitutively open transient receptor potential (TRP) channels (Hardie and Minke, 1994), which are the major light-activated channels (for review, see Minke and Hardie, 2000). These constitutive currents were recorded in 58% of the NinaE^pp100 cells and 74% of the arr2^Y20STOP NinaE^pp100 double mutant photoreceptors (Fig. 5B). Because the arr2^Y20STOP mutant cells failed to exhibit any significant whole-cell membrane currents to voltage steps in the dark (data not shown), the arr2^Y20STOP mutation only contributed to the constitutive opening of the TRP channels in the presence of the NINAE^pp100 mutant rhodopsin (Fig. 5B). Although arrestin partially masked the production of the constitutive current, possibly through a stable NINAE^pp100-ARR2 complex, it is not clear why some of the mutant cells were not constitutively active. This may be related to variability in the level of rhodopsin among different NinaE^pp100 cells, which is consistent with the large variability in the light sensitivity between different NinaE^pp100 mutant cells (Fig. 4B).

The dominant NinaE^pp100 mutant requires both G_qα and ARR2 proteins for rapid degeneration

It was shown previously that the norpA, rdgB, and rdgC mutations produced a persistent NINAE-ARR2 complex that induced photoreceptor cell apoptosis (Alloway et al., 2000; Kiselev et al., 2000). Because the electrophysiology suggested that a stable NINAE^pp100-ARR2 complex was present, we examined whether loss of the ARR2 protein suppressed the rapid NinaE^pp100-dependent degeneration. At 10 d after eclosion, wild-type photoreceptors possessed large rhabdomeres (Fig. 6A), whereas a 4-d-old NinaE^pp100 mutant possessed significantly smaller and disorganized rhabdomeres and signs of photoreceptor cell death (Fig. 6D). The arr2^Y20STOP null mutant possessed slightly smaller and irregular-shaped rhabdomeres relative to wild type (Fig. 6B), which is consistent with the light-dependent deep pseudopupil loss described previously (Alloway and Dolph, 1999). The 4-d-old arr2^Y20STOP NinaE^pp100 double mutant also exhibited small and abnormally shaped rhabdomeres (Fig. 6E), similar to the arr2^Y20STOP mutant. However, neither arr2^Y20STOP nor arr2^Y20STOP NinaE^pp100 exhibited the massive rhabdomere loss and photoreceptor cell death that occurred in the NinaE^pp100 mutant. This suggested that a stable NINAE^pp100-ARR2 complex induced both photoreceptor desensitization and cell death. Furthermore, the time course of deep pseudopupil loss revealed that the arr2^Y20STOP mutation significantly slowed, but did not abolish, the NinaE^pp100 degeneration in both constant light and constant darkness (data not shown). Thus, the NINAE^pp100-ARR2 complex was not the sole cause of retinal degeneration, and an additional independent mechanism must participate in generating the NinaE^pp100 mutant phenotypes.

Figure 6.

The rapid NinaE^pp100 degeneration requires both the ARR2 and G_qα proteins. Wild-type (Oregon-R; A, J), NinaE^pp100 (D), arr2^Y20STOP (B), dgq¹ (C), arr2^Y20STOP NinaE^pp100 (E, H), dgq¹; NinaE^pp100 (F, I), and dgq¹; arr2^Y20STOP NinaE^pp100 (G) flies were raised for 4 d (B, D, E), 10 d (A, C, F), 20 d (H, I), or 40 d (G, J) sectioned and examined by electron microscopy. Although both the arr2^Y20STOP and dgq¹ mutants exhibit smaller, disorganized and occasional missing rhabdomeres relative to wild type, they are significantly more intact than the NinaE^pp100 mutant. Both arr2^Y20STOP NinaE^pp100 and dgq¹; NinaE^pp100 exhibit less rhabdomere loss and photoreceptor degeneration than NinaE^pp100 at 4 and 10 d, respectively. Whereas arr2^Y20STOP NinaE^pp100 and dgq¹; NinaE^pp100 possess massive degeneration at 20 d, the dgq¹; arr2^Y20STOP NinaE^pp100 triple mutant exhibits only very minor abnormalities in the rhabdomere. Scale bar (in A): A-J, 1 μm. K, The time course of deep pseudopupil loss of various genotypes [NinaE^pp100 flies (squares), dgq¹ flies (circles), and dgq¹; NinaE^pp100 double mutants (triangles)] raised in either constant light or constant darkness (open and filled symbols, respectively). The percentages of flies retaining a deep pseudopupil on each day were plotted against their age, with each symbol representing the average from four independent trials of 100-150 flies per trial.

Because the electrophysiology revealed that the NINAE^pp100 mutant protein was constitutively active, we examined whether activation of the light-sensitive channels induced the retinal degeneration phenotype. It was demonstrated previously that the early-onset retinal degeneration in the rdgA^BS12 mutant, which possesses constitutively active light-sensitive channels, was significantly slowed by the trp³⁰¹ mutation (Raghu et al., 2000). We examined whether mutants blocking three different points in the phototransduction cascade could suppress the rapid NinaE^pp100-dependent degeneration. The mutations we tested corresponded to the dgq (which encodes the G_qα that is stimulated by light-activated metarhodopsin), norpA (which encodes the phospholipase C that is activated by G_qα), and trp genes (which encodes the major light-activated channel). The norpA^p24 mutant exhibited no signs of retinal degeneration when raised for 6 d in constant darkness (Fig. 7B). In contrast, the NinaE^pp100 mutant possessed the previously described degeneration phenotype after 6 d in constant darkness (Fig. 7C). Although norpA^p24; NinaE^pp100 exhibited reduced and misshapen rhabdomeres after 6 d in constant darkness (Fig. 7D), it lacked the severe degeneration that was present in the NinaE^pp100 mutant. This suggests that norpA^p24 slightly delayed the NinaE^pp100 degeneration. Similarly, the trp³⁰¹ mutation only slightly suppressed NinaE^pp100 degeneration relative to the arr2^Y20STOP mutation (Fig. 7E). Thus, constitutive activation of the phototransduction cascade is only a minor component of the NinaE^pp100 degeneration mechanism.

Figure 7.

The norpA^p24 and trp³⁰¹ mutations only weakly suppress the rapid NinaE^pp100 retinal degeneration. Wild-type (Oregon-R; A), norpA^p24 (B), NinaE^pp100 (C), and norpA^p24; NinaE^pp100 (D) flies were raised for either 10 d (A) or 6 d (B-D), sectioned, and examined by electron microscopy. Although the norpA^p24; NinaE^pp100 double mutant exhibits rhabdomere shrinkage and disruption of the cell body relative to norpA^p24, it lacks the massive cell degeneration characteristics observed in NinaE^pp100. Scale bar (in A): A-D, 1 μm. E, The time course of deep pseudopupil loss of various genotypes [NinaE^pp100 flies (squares), trp³⁰¹ flies (circles), and NinaE^pp100trp³⁰¹(triangles)]raised in either constant light or constant darkness (open and filled symbols, respectively). The percentages of flies retaining a deep pseudopupil on each day were plotted against their age, with each symbol representing the average from four independent trials of 100-150 flies per trial.

Surprisingly, a mutation in the dgq gene, which encodes the G_qα subunit target for metarhodopsin (Lee et al., 1990, 1994; Scott et al., 1995), suppressed the NinaE^pp100 deep pseudopupil loss substantially better than the trp³⁰¹ mutation (Figs. 6K, 7E, respectively). At 10 d after eclosion, the dgq¹ mutant possessed only a few missing rhabdomeres relative to the massive rhabdomere loss and photoreceptor cell death in NinaE^pp100 (Fig. 6C,D, respectively). The ultrastructure of a 10-d-old dgq¹; NinaE^pp100 double mutant exhibited only minor photoreceptor abnormalities that were similar to the dgq¹ mutant (Fig. 6F,C, respectively). Furthermore, the time course of deep pseudopupil loss in the dgq¹; NinaE^pp100 double mutant was similar to the dgq¹ mutant in both dark and light-raised flies, which was significantly slower than the NinaE^pp100 mutant (Fig. 6K). However, both the dgq¹; NinaE^pp100 and arr2^Y20STOP NinaE^pp100 double mutants still exhibited a slow retinal degeneration (Fig. 6E,F,K). After 20 d, the dgq¹; NinaE^pp100 and arr2^Y20STOP NinaE^pp100 double mutants exhibited massive photoreceptor cell death (Fig. 6I,H, respectively) relative to an equivalent aged wild-type retina (Fig. 6J). Surprisingly, the dgq¹; arr2^Y20STOP NinaE^pp100 triple mutant exhibited only very minor rhabdomeric abnormalities after 40 d (Fig. 6G). The absence of significant cell death in this 40-d-old triple mutant relative to either of the 20-d-old double mutants and the minor suppression of NinaE^pp100 degeneration by the norpA^p24 and trp³⁰¹ mutants revealed that both the ARR2 and G_qα proteins, but not activation of the light-sensitive channels, play major roles in the rapid NinaE^pp100-dependent retinal degeneration.

The dominant NINAE^pp100 protein causes a constitutive disassociation and translocation of the heterotrimeric G-protein

Whole-cell recordings revealed that the NinaE^pp100 mutant photoreceptors were constitutively active (Fig. 5B,D), but the weaks uppression of the NinaE^pp100-dependent retinal degeneration by norpA or trp suggested that constitutive activation of the light-sensitive channels was not required for the rapid NinaE^pp100-dependent retinal degeneration. We examined the extent of the constitutive activation by comparing the GTPase activity in wild-type and NinaE^pp100 mutant flies. Whereas we found high levels of light-dependent GTPase activity in wild-type flies, significantly lower light-dependent GTPase activity levels were measured in the NinaE^pp100 mutant (Fig. 8A), which suggested that a large fraction of the G_qα-protein was not activated in the NinaE^pp100 mutant. We demonstrated previously that the G_qα-protein translocates from the rhabdomeric membrane to the cytosol during blue light illumination (Kosloff et al., 2003). Therefore, we examined whether the G_qα-protein in the constitutively active NinaE^pp100 mutant photoreceptors was predominantly in the cytosol. In wild-type flies, ∼80% of the G_qα was associated with the membrane fraction in the dark (Fig. 8B), which represented the GDP-bound G_qα in the heterotrimer. During illumination, ∼70% of the G_qα translocated into the cytosol (Fig. 8B). In contrast, ∼70% of the G_qα was present in the cytosol of the NinaE^pp100 mutant, regardless of illumination (Fig. 8B). This persistent and abnormal distribution of the G_qα-protein in the cytosol could partially account for both the absence of light-dependent GTPase activity and the desensitization of the NinaE^pp100 mutant photoreceptor cell.

Figure 8.

The subcellular distribution of the G_qα-protein is altered in the NinaE^pp100 mutant relative to wild type. A, The light-dependent GTPase activity (picomoles of phosphate per milligram of total protein per minute) was determined by subtracting the GTPase activity measured during orange light illumination from the GTPase activity measured during blue light illumination in the same experiment (n = 4). The histogram shows negligible blue light-stimulated GTPase activity in the NinaE^pp100 mutant relative to wild-type flies. Furthermore, both the NinaE^pp100 mutant and wild-type flies exhibited the same low level of GTPase activity in orange light (data not shown). B, The subcellular distribution of the G_qα-protein was examined in wild-type (ninaE⁺) and NinaE^pp100 mutant heads. The percentage of total G_qα-protein in the supernatant was determined in both light and dark conditions by subcellular fractionation and immunoblots. Although light treatment induced the translocation of G_qα to the cytosol in wild-type flies, light did not alter the percentage of G_qα-protein in the NinaE^pp100 supernatant (n = 4). Furthermore, the stable percentage of G_qα in the NinaE^pp100 supernatant was equivalent to the percentage of G_qα in the light-stimulated wild-type supernatant.

The formation of a stable rhodopsin-arrestin complex is required for the rapid photoreceptor degeneration

The ability of the arr2^Y20STOP mutation to significantly reduce the desensitization of the NinaE^pp100 photoreceptor and suppress the NinaE^pp100 degeneration suggested that the ARR2 protein persistently bound the NINAE^pp100 mutant protein (Alloway et al., 2000; Kiselev et al., 2000). ARR2 is predominantly a soluble protein when rhodopsin is not photoactivated (such as during or after orange light illumination) (Fig. 9). However, during blue light conversion of rhodopsin to metarhodopsin, ∼80% of ARR2 protein binds metarhodopsin in the membrane (Fig. 9) (Byk et al., 1993). The ARR2 binding to the membrane is rhodopsin dependent, because only low levels of ARR2 are detected in ninaE^I17 null mutant membrane fractions under either light condition (Fig. 9). In contrast, a significant percentage of ARR2 remained associated with the NinaE^pp100 membrane, regardless of the light conditions (Fig. 9). The lower percentage of ARR2 in the newly enclosed NinaE^pp100 membrane relative to the wild-type membrane after blue light illumination is consistent with the ∼80% reduction of NINAE protein in the NinaE^pp100 mutant compared with wild type (Fig. 1F). Thus, the role of ARR2 in the rapid degeneration and strong desensitization in the NinaE^pp100 mutant is through its persistent binding to NINAE^pp100.

Figure 9.

The ARR2 protein is persistently bound to the NINAE^pp100 mutant rhodopsin. The subcellular distribution of the ARR2 protein was examined in wild-type (ninaE⁺) and mutant (ninaE^I17, NinaE^pp100, and dgq¹; NinaE^pp100) heads (<4 hr after eclosion). A, The percentage of total ARR2 protein in the membrane pellet was determined using either 2 min of orange (or) or 2 min of blue (bl) light illumination, followed by subcellular fractionation and immunoblots (n = 5). The NinaE^pp100 mutant had the same percentage of ARR2 protein in the pellet, regardless of the light conditions. This stable association was significantly more than that observed in the ninaE^I17 null mutant and significantly less than the wild-type flies under blue light illumination. B, A representative immunoblot of the subcellular distribution of ARR2 and NINAE proteins in ninaE^I17, NinaE^pp100, ninaE⁺, and dgq¹; NinaE^pp100 fly heads. The detection of NINAE in only the membrane pellet fraction demonstrates the completeness of removing the membrane from the soluble fractions.

The NinaE^pp100 mutant degenerates through two independent pathways that involve the abnormal distribution of the ARR2 and G_qα

Because both the dgq¹ and arr2^Y20STOP mutations significantly suppressed the rapid NinaE^pp100-dependent retinal degeneration (Fig. 6), we examined whether the G_qα-protein was required for the persistent association of ARR2 with the NINAE^pp100 protein. We found that the percentage of ARR2 protein present in the membrane fraction of dgq¹; NinaE^pp100 double mutants was essentially the same as in NinaE^pp100 mutant flies (Fig. 9). Because the loss of the G_qα protein did not reduce the level of the persistent NINAE^pp100-ARR2 complexes, the dgq¹ suppression of NinaE^pp100 degeneration must be arrestin independent. This demonstrates that G_qα is involved in a novel mechanism for the rapid NinaE^pp100 degeneration, which may involve its predominant and persistent localization in the cytosol. Furthermore, the complete suppression observed in the 40-d-old dgq¹; arr2^Y20STOP NinaE^pp100 triple mutant clearly demonstrates that both the G_qα and ARR2-dependent degeneration pathways are both necessary and sufficient to account for the entire NinaE^pp100-dependent degeneration process.

Discussion

The NinaE^pp100 mutant exhibits a novel retinal degeneration phenotype

We isolated a novel dominant rhodopsin mutation, NinaE^pp100, that requires the presence of both G_qα and arrestin to induce a rapid degeneration. Transgenic expression of an in vitro-generated glycine-to-arginine mutation at position 299 conclusively demonstrated that this is the molecular cause of the dominant NinaE^pp100 mutant phenotype (Fig. 2C). Previously, several dominant rhodopsin mutations were isolated that blocked rhodopsin trafficking and maturation out of the endoplasmic reticulum and slowly degenerated (Colley et al., 1995; Kurada and O'Tousa, 1995; Kurada et al., 1998). Several lines of data demonstrated that the NinaE^pp100 mutation did not affect rhodopsin trafficking and maturation. First, the ninaE^D1 mutation, which is one of the most severe of the previously isolated dominant rhodopsin mutations (Kurada and O'Tousa, 1995; Kurada et al., 1998), suppressed the rapid NinaE^pp100 degeneration (Fig. 3). Therefore, the NINAE^pp100 protein must exert its effect subsequent to the ninaE^D1 trafficking defect. Second, NinaE^pp100/ninaE⁺ flies lacked immature forms of rhodopsin on immunoblots (Fig. 1F). Thus, both wild-type and NINAE^pp100 mutant proteins must complete their maturation, although the NINAE^pp100 protein was present at ∼20% of the NINAE⁺ protein level (Fig. 1F). Third, electron microscopy independently immunolocalized both epitope-tagged NINAE⁺ and epitope-tagged NINAE^pp100 in the rhabdomeres of NinaE^pp100/ninaE⁺ flies (data not shown), which confirmed that both forms reach the rhabdomere. Therefore, NinaE^pp100 represents a new Drosophila model for dominant rhodopsin mutations.

The mechanisms underlying the highly reduced sensitivity to light in the NinaE^pp100 mutant and its implications on congenital night blindness (CNB) in humans

The strongly desensitized NinaE^pp100 photoreceptor was characterized by the following: (1) the intense light required to elicit a small-amplitude response that lacked the typical bump noise (Fig. 4A), (2) the increased light intensity necessary to reach the saturated amplitude (Fig. 4B), and (3) the shift of the intensity-response relationship toward higher light intensity levels (Figs. 4B, 5A). Although either the 80% decrease in the number of rhodopsin molecules (Fig. 1F) or the 80% decrease of G_qα-protein associated with the membrane (Fig. 8B) would reduce the NinaE^pp100 light sensitivity, they are insufficient to account for the observed four orders of magnitude reduction. A more significant mechanism is the constant state of light adaptation that results from the persistent NINAE^pp100-ARR2 complex (Fig. 9). Normally, rhodopsin is present at a 5:1 molar ratio to ARR2 (Dolph et al., 1993). However, the NINAE^pp100 mutant protein is expressed at only 20% of the wild-type level of rhodopsin in wild-type photoreceptors (Fig. 1F), which yields an ∼1:1 molar ratio with ARR2. Thus, if 30-40% of the ARR2 is associated with the NinaE^pp100 membrane, approximately an equivalent percentage of the NINAE^pp100 mutant protein is likely in a NINAE^pp100-ARR2 complex. This would correspond to ∼35% of the NINAE^pp100 being present in the persistent complex, which corresponds to 6-8% of the rhodopsin level in wild-type photoreceptors. This complex could account for the shift in the intensity-response curve toward brighter light (Fig. 5A). The fact that the light response in a fraction of mutant cells reached a normal peak amplitude at intense lights (Fig. 4B) is consistent with a significant number of light-sensitive rhodopsin molecules (i.e., ∼10³-10⁵) (Johnson and Pak, 1986) not being bound by arrestin. The NINAE^pp100-ARR2 complex would also account for the minimal light-stimulated GTPase activity in the NinaE^pp100 mutant photoreceptor cell. However, genetic elimination of ARR2 only partially increased the light sensitivity of the mutant NinaE^pp100 (Figs. 4A, 5A), which could be explained by two different mechanisms. First, constitutively active NINAE^pp100 would continually open the light-sensitive channels (Fig. 5), which leads to a persistent Ca²⁺ influx and photoreceptor desensitization. Second, activated G_qα-protein may stimulate the internalization of specific G-protein-coupled receptors in the absence of arrestin binding (Rochdi and Parent, 2003). Thus, constitutively active NINAE^pp100 would activate G_qα, which could drive internalization of both wild-type and NINAE^pp100 rhodopsin molecules to desensitize the photoreceptor cell.

This strong desensitization response, with only a minor effect on retinal degeneration, is similar to the human CNB phenotype (Dryja, 2000). One form of autosomal dominant CNB results from constitutively active forms of rod opsin (Jin et al., 2003). Three dominant rod opsin mutations were shown to cause a significant shift in the intensity-response relationship of transgenic Xenopus rods (Jin et al., 2003). The constitutive activity and desensitization observed in the NinaE^pp100 mutant suggests that the NINAE^pp100 mutant rhodopsin may serve as an important model for elucidating the molecular details of one form of autosomal dominant CNB.

A novel mechanism for dominant rhodopsin-mediated retinal degeneration

The NinaE^pp100 dominant mutation induces three different degeneration mechanisms. The first involves constitutive opening of the light-sensitive channels. The Drosophila rdgA^BS12 and trp^P365 mutations were shown to constitutively depolarize the photoreceptor cell and cause retinal degeneration (Raghu et al., 2000; Yoon et al., 2000). Furthermore, three different trp alleles (including trp³⁰¹) significantly slowed the rapid rdgA^BS12 retinal degeneration, which confirmed that the rdgA^BS12 degeneration was dependent on the constitutive opening of the light-activated channels (Raghu et al., 2000). However, the constitutive current observed in the NinaE^pp100 mutant was approximately fivefold smaller than that found in the Drosophila rdgA^BS12 and trp^P365 mutants (Raghu et al., 2000; Yoon et al., 2000) and was not found in all of the cells (Fig. 5B). Furthermore, the weak suppression by the norpA^p24 and trp³⁰¹ mutations on the rapid NinaE^pp100 degeneration (Fig. 7D,E, respectively) confirmed that the constitutive opening of the light-sensitive channels is a minor NinaE^pp100 degeneration mechanism.

The second degeneration mechanism involves the formation of a stable NINAE^pp100-ARR2 complex. A variety of Drosophila mutations were shown previously to induce retinal degeneration by generating a stable NINAE-ARR2 complex that was endocytosed in a clathrin-dependent manner (Alloway et al., 2000; Kiselev et al., 2000). However, NinaE^pp100 is the first Drosophila rhodopsin mutation that persistently targeted ARR2 to the membrane in all light conditions (Fig. 9). On the basis of the arr2^Y20STOP mutation suppressing the NinaE^pp100 degeneration (Fig. 6), the NINAE^pp100-ARR2 complex accounts for a major NinaE^pp100 degeneration mechanism. The K296E opsin mutation in transgenic mice exhibited an analogous dominant photoreceptor degeneration phenotype (Robinson et al., 1992; Li et al., 1995; Rim and Oprian, 1995). Although the K296E mutant opsin constitutively activated the heterotrimeric G-protein in cell culture, it failed to constitutively stimulate the phototransduction cascade in the mouse photoreceptor because it was persistently phosphorylated and bound by arrestin. This stable rhodopsin-arrestin complex may be a common stimulator of photoreceptor apoptosis for constitutively active rhodopsin mutations rather than persistent activation of the phototransduction cascade.

The third and novel Drosophila degeneration mechanism requires G_qα but not activation of the visual transduction cascade, because neither the norpA^p24 nor trp³⁰¹ mutations suppressed NinaE^pp100 degeneration to the same extent as dgq¹ (Figs. 7, 6, respectively). Furthermore, the inability of the dgq¹ mutation to disrupt the stable NINAE^pp100-ARR2 complex (Fig. 9) and the consistent presence of 70% of the G_qα-protein in the cytosolic fraction of both NinaE^pp100 and arr2^Y20STOP NinaE^pp100 mutants (Fig. 8B and data not shown, respectively) demonstrated that the G_qα- and ARR2-dependent degeneration mechanisms were independent. The light-independent localization of most of the G_qα-protein to the NinaE^pp100 cytosol is consistent with the constitutively active NINAE^pp100 stimulating G_qα dissociation from Gβγ and the subsequent depalmitoylation of G_qα (Kosloff et al., 2003). The cytosolic localization and the markedly decreased light-dependent GTPase activity in the NinaE^pp100 mutant (Fig. 8) suggests that the NINAE^pp100 protein either diminishes the interaction between the cytosolic G_qα-GTP with the membrane-associated norpA-encoded phospholipase C effector molecule, which also functions as a GTPase activating protein (Cook et al., 2000), or suppresses the reassociation of the cytosolic G_qα with the membrane. Because the loss of G_qα-protein significantly slowed the NinaE^pp100 degeneration, the persistent cytosolic localization of G_qα induces the degeneration rather than its absence from the membrane.

Whereas most previously characterized retinal degeneration models identified a single major pathway, the NinaE^pp100 mutant identified two major degeneration mechanisms, the stable NINAE^pp100-ARR2 complex and the G_qα-dependent pathway. The ability of ninaE^D1 to prevent the rapid NinaE^pp100-dependent degeneration (Fig. 3) is consistent with the requirement of NINAE^pp100 to reach the rhabdomere and interact with the ARR2 and G_qα proteins. Recently, knock-out mice revealed that light-induced degeneration also involved two mechanisms (Hao et al., 2002), one requiring the transducin α subunit and the other involving photoactivated metarhodopsin and/or its bleaching intermediates (Hao et al., 2002). Thus, the NinaE^pp100 mutant may reveal not only the complex interactions between constitutive activation and desensitization but also the multiplicity of mechanisms that may be involved in some of the rhodopsin-dependent degeneration mutations.