(Received for publication, August 17, 1995; and in revised form, December 12, 1995)
From the
Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses.
ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level.
Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.
Virtually all eukaryotic cells utilize the phosphoinositide (PI) ()signal transduction pathway to mediate such diverse
cellular processes as metabolism, secretion, and cell growth and
proliferation. Many types of more specialized functions, including
muscle contraction, fertilization, sensory perception, and long term
potentiation in neurons, are also subserved by PI signaling (reviewed
by Berridge(1993)). The transduction cascade is initiated when
phosphatidylinositol 4,5-bisphosphate (PIP
), a minor plasma
membrane lipid, is hydrolyzed into two second messenger molecules,
inositol 1,4,5-trisphosphate (IP
) and diacylglycerol (DAG).
IP
releases calcium from intracellular stores (Berridge,
1993) whereas DAG is an activator of protein kinase C (Nishizuka,
1988). Hydrolysis of PIP
is catalyzed by the phospholipase
C family of effector enzymes. The various transmembrane receptors known
to activate these enzymes are responsive to hormones,
neurotransmitters, growth factors, or external stimuli such as light
and chemical ligands (Berridge, 1993).
PLCs have been isolated from
a variety of mammalian tissues (reviewed by Rhee et
al.(1989)), and complementary DNAs encoding some of these
proteins, as well as complementary DNAs encoding two Drosophila PLCs and a yeast PLC (Takehiko et al., 1993), have been
cloned and sequenced (see Meldrum et al. (1991) for
references). Based on deduced amino acid sequences and immunological
data, PLCs have been grouped into three classes: ,
, and
(Rhee et al., 1989) that differ in primary structure,
molecular weight, and mode of regulation. Activation of
class
PLCs is mediated through a G-protein-dependent mechanism, whereas
PLCs are activated by tyrosine residue phosphorylation (Morris et
al., 1990). All known eukaryotic PLCs have two regions of
extensive amino acid sequence homology called Box X and Box Y, which
are approximately 120 and 150 amino acids in length, respectively (Rhee et al., 1989). This sequence conservation, along with in
vitro biochemical data (Bristol et al., 1988), suggests
that Boxes X and Y contain the catalytic site of these enzymes.
Sequence homology and in vitro biochemical data have also been
used to localize, tentatively and broadly, additional functional
domains along the primary sequence of some
and
PLCs
(Bairoch and Cox, 1990; Bristol et al., 1988; Cifuentes et
al., 1993; Crooke and Bennett, 1989; Park et al., 1993;
Schnabel et al., 1993; Wu et al., 1993a, 1993b).
Much, however, remains to be determined about the structure-to-function
relationship and regulation of this ubiquitous family of effector
enzymes.
Since the mid-1980s, many studies have suggested that
invertebrate phototransduction is mediated by a PI signaling cascade
(reviewed by Bacigalupo et al.(1990) and Pak and
Shortridge(1991)). Analysis of Drosophila norpA (no
receptor potential) mutants has provided the most direct evidence
for the role of this pathway in phototransduction (reviewed by Pak and
Shortridge(1991)). The norpA gene encodes a -class PLC
(Bloomquist et al., 1988) that is predominantly expressed in
the rhabdomeres of the compound eye (Schneuwly et al., 1991).
It was one of the first PLC cDNA sequences to be elucidated.
Photoreceptors in severely affected norpA mutants show
essentially no electrophysiological response to light stimuli, and
therefore the flies are blind. Thus, the phototransduction cascade in Drosophila begins when light converts rhodopsin into its
activated form, metarhodopsin. Metarhodopsin activates a G-protein
which, in turn, activates the PLC effector enzyme to hydrolyze
PIP
. Specific roles for the PIP
hydrolysis
products, IP
and DAG, have not yet been clarified in the Drosophila phototransduction cascade. Nevertheless, calcium,
presumably released by IP
binding to its receptor, is
thought to be involved in photoexcitation and adaptation (Devary et
al., 1987) (reviewed by Bacigalupo et al. (1990)),
whereas an eye-specific protein kinase C, perhaps activated by DAG,
appears to be required for adaptation through negative regulation
(Hardie et al., 1993).
In addition to the electrophysiological phenotype of norpA mutants, the photoreceptor rhabdomeres of these flies manifest light-dependent degeneration. It was first discovered in Drosophila that mutations in some genes encoding proteins involved in phototransduction can result in retinal degeneration (reviewed by Pak(1994)). Similarly, mutations in genes implicated in the mammalian phototransduction pathway can result in retinal degeneration. For example, some cases of retinitis pigmentosa, a heterogeneous class of human diseases involving retinal degeneration, are caused by mutations in the rod opsin gene (see, e.g., Dryja et al.(1990)).
Since Drosophila is readily amenable to molecular genetic analysis
and its visual system can be examined by a wide variety of analytical
techniques, the phototransduction pathway in Drosophila provides an excellent model system in which to study -class
PLCs and PI-mediated signal transduction. Furthermore, analysis of
degeneration in norpA mutants may help elucidate some of the
mechanisms of degeneration caused by defective signal transduction
pathways. In this work we have characterized eight norpA mutants by molecular, electrophysiological, and biochemical
analyses to gain better understanding of PLC function.
R7 and R8 photoreceptor responses were tested by first saturating the R1-R6 photoreceptor response with a maximal prolonged depolarizing afterpotential (PDA) induced with a strong blue light stimulus and then applying a second blue light stimulus during the PDA. The response to the second blue stimulus, seen superimposed on the R1-R6 PDA, is due to the responses of R7 and R8 photoreceptors (Minke et al., 1975).
Relative quantities of the NorpA protein detected on Western blots were determined by laser densitometry. Biomax MR film (Eastman Kodak Co.) was exposed to Western blots stained with LumiGLO peroxidase substrate. Bands on the exposed film corresponding to the 130-kDa protein, the expected size of the norpA-encoded protein (Bloomquist et al., 1988), were scanned using an Ultrascan XL 2222-020 laser densitometer with internal digital integrator (LKB). The area under the density curve (peak area) was then calculated for each 130-kDa band. A standard curve was constructed by plotting the peak area of the 130-kDa band against the corresponding quantity of protein homogenate loaded onto a SDS-polyacrylamide gel. Each Western gel was loaded with wild-type or mutant protein homogenate so that the peak area of the corresponding 130-kDa band fell in the linear region of the standard curve.
The specific activity of NorpA was determined by first converting
the emission activity (in dpm/(mg
min)) of the reaction
supernatant to curies (Ci/(mg
min)), using the conversion
factor (2.22
10
dpm/Ci), and then to specific
activity (nmol of [
H]PIP
cleaved/(mg
of total protein homogenate
min)) using the conversion factor
provided by the supplier, 8 Ci/mmol. As addressed under
``Discussion,'' approximately 1% of the wild-type total
activity was attributed to nonspecific PLC activity and subtracted from
the total activity for the purpose of specific activity calculations.
In addition, because the amount of the NorpA protein is reduced in P57, P16, and P79 mutants in comparison to
wild type, any reduction in specific activities in these mutants, as
calculated above, reflects both a reduction in the amount and a change
in the activity of the NorpA protein. To compare the NorpA activities
in the mutants independent of the reduction in amount, specific
activities of these mutants, as calculated above, were divided by the
ratio between the amount of the NorpA protein in mutants and that in
wild type (Fig. 4) to obtain specific activities per milligram
of ``equivalent total eye protein,'' having the same
concentration of the NorpA protein as in wild type.
Figure 4:
Comparison of the amount of the NorpA
protein in mutants and wild type. Vertical bars indicate the
peak area of the bands corresponding to the 130-kDa protein on Western
blots in arbitrary units. Peak areas of the 130-kDa protein band from
equal quantities of total-protein homogenate from norpA mutant
eyes or eya heads are compared to homogenate from wild-type
eyes. Vertical bars are the average of six determinations
± S.E. WT, wild-type; P57, etc., norpA mutant, etc.; eya, eyes absent mutant.
Typical wild-type ERGs evoked by our standard stimulus protocol are shown in the fifth set of traces in Fig. 1(only the responses to the white and first orange stimuli are shown for clarity; see ``Materials and Methods''). Although several types of cells contribute to the ERG, the dominant component is generated primarily by photoreceptor cells. Two ERG parameters were examined in this work, the amplitude and initial slope. The amplitude (V) is defined as the peak response evoked by any given stimulus, and the initial slope is measured from the initiation of the response to half the amplitude. Both of these parameters are stimulus intensity-dependent and increase with increasing stimulus intensity until response saturation. Fig. 2A shows the amplitude (V) plotted against the log of the stimulus intensity (log I), and Fig. 2B shows the initial slope of the response plotted against log I (wild type is the uppermost curve in both figures). Wild-type flies also exhibit a PDA in response to strong white or blue light stimuli (e.g. log I = -1 in Fig. 1). This protracted response, which can persist well after the termination of the light pulse, is due to the incomplete inactivation of metarhodopsin by arrestin when a substantial amount of rhodopsin is photoconverted to metarhodopsin (Byk et al., 1993; Dolph et al., 1993). Reconversion of metarhodopsin to rhodopsin with an orange light stimulus will terminate the PDA (e.g. the wild-type response to log I = -1 in Fig. 1).
Figure 1: Electroretinogram recordings. Typical ERG traces from a wild-type fly and four norpA mutant flies. Responses elicited by 4 (log I = -6, -5, -3, and -1) of the 7 stimulus intensities tested on each fly are shown superimposed. Each trace consists of the responses to the white light stimulus and the first orange light stimulus of the stimulus protocol (see ``Materials and Methods'' for details). A 5-mV calibration pulse is shown 1 s before the onset of the light stimulus.
Figure 2:
Dependence of ERG parameters on stimulus
intensity. A, ERG amplitude versus log stimulus
intensity. The smooth curves were fit to the data points using the
equation, V/V = I
/(I
+
), where V = the ERG
amplitude at any given intensity, V
= the
maximum obtainable amplitude at the highest intensities, I = stimulus intensity,
= the stimulus intensity
at half-maximal amplitude, i.e.
V
, and n =
parameter adjusted to obtain a best fit (n = 0.45 and
0.56 for wild type and norpA
,
respectively). B, stimulus intensity dependence of the initial
slope of the ERG. The initial slope was determined by measuring the
slope of the trace from the beginning of the response to
V
. This region of the ERG trace is
essentially linear in all recordings. In both A and B, data were obtained from wild type and eight norpA mutants each carrying a different allele. Each data point
represents an average of measurements from six flies. The error flags
represent S.E. values. P57, etc. = norpA
, etc.
mutants.
In comparison to wild-type
flies, ERGs of norpA mutants display both reduced amplitude
and much slower response kinetics (see examples in Fig. 1).
Although the amplitude generally increases with the stimulus intensity,
as in wild type, it is substantially reduced in comparison to wild type
at each intensity ( Fig. 1and Fig. 2A). In
addition, norpA mutants display slow developing responses,
particularly at low stimulus intensities (log I =
-6 to -5) ( Fig. 1and Fig. 2B).
While the initial slope increases with increasing stimulus intensity,
it never reaches the level attained by the wild-type ERG at any
intensity ( Fig. 1and 2B). This general trend is seen
even in the mild norpA mutant, P57, although the
effect is not as marked ( Fig. 1and Fig. 2B).
Two mutants, P16 and P79, differ from the others in
that, at the highest stimulus intensities (log I =
-2 to 0), both V and the initial slope actually begin to
decrease (Fig. 2, A and B). The eight mutants
may be ranked, in order of decreasing magnitude, with respect to both
amplitude and initial slope, as follows: wild type > norpA > norpA
norpA
> norpA
norpA
norpA
norpA
> norpA
. Thus,
the mutants appear to fall into four groups. Group 1 consists of P57, which responds with a V and initial slope that
are somewhat reduced compared to wild type. The amplitude of P57 also saturates at a lower stimulus intensity (log I = -3 as compared to -2 in wild type). Group 2
includes P16 and P79, which are similar to each other
and have responses that are smaller and slower than either wild type or P57. Mutants P12, P42, P45, and P76 comprise group 3 and are comparable to each other, having
very reduced amplitudes and initial slopes. The final group consists of P24, which shows virtually no response at any stimulus
intensity.
All of these norpA mutants also exhibit a very prolonged response to white light stimuli of even low intensity (Fig. 1). This prolonged response can persist long after the termination of the stimulus but differs from the PDA of wild-type flies in that it can be generated by a low intensity stimulus and is not terminated by orange light.
Western analysis was used to determine
the level of NorpA expression in total eye protein homogenates from the
mutants. Polyclonal antiserum generated against the amino-terminal
region of the NorpA protein (see ``Materials and Methods'')
detects a major protein of approximately 130 kDa on Western immunoblots
of total head-protein homogenates from wild-type flies (Zhu et
al., 1993) and eya mutants (Fig. 3) or of total
eye-protein homogenates of wild-type flies and norpA mutants P16, P79, and P57 (Fig. 3). The size
of the detected protein is consistent with the predicted size of NorpA
from the work of Bloomquist et al.(1988), and the protein is
not detected in strongly affected norpA mutants. However, more
lightly stained, smaller proteins are also visible in some lanes of Fig. 3. Since the additional bands do not appear in any of the
lanes where the 130-kDa protein is not detected, they are most likely
related to the 130-kDa NorpA protein. For example, they could result
from alternative splicing of norpA transcript (Zhu et
al., 1993) or from degradation of the 130-kDa protein. An equal
quantity of wild-type or mutant total eye protein homogenate was loaded
into each lane of the Western blot shown in Fig. 3. Since the
NorpA protein is much more highly expressed in wild type, a given
quantity of total protein homogenate contains much more of this protein
in wild type than in mutants. Consequently, the 130-kDa protein and the
smaller proteins are especially prominent in this lane. NorpA
expression is reduced to approximately 9, 9, 18, and 13% of wild type
in P16, P79, P57, and eya,
respectively (Fig. 4). Neither the 130-kDa band nor any smaller
bands are detected in P12, P24, P42, P45, or P76 mutants (Fig. 3). Low expression
of the 130-kDa protein in eya heads (13%) compared to
wild-type eyes is consistent with the previous reports that NorpA
expression is predominantly in the compound eye (see Yoshioka et
al.(1985), Schneuwly et al.(1991), and Zhu et
al.(1993)).
Figure 3:
Western blot of total protein homogenates.
An equal quantity of protein homogenate from dissected eyes of
wild-type or norpA mutants flies, or isolated heads of eya flies was loaded on a 7% SDS-polyacrylamide gel. WT,
wild-type; P12, etc., norpA mutants, etc.; eya, eyes absent mutant.
Next, we molecularly characterized the eight norpA alleles to determine if a mutation has been introduced
into their coding sequences that might cause the reduced NorpA
expression and observed ERG phenotype. DNA sequencing of the PCR
products spanning the coding region (see ``Materials and
Methods'') revealed 13 polymorphic differences between the
published Canton-S wild-type cDNA sequence (Bloomquist et al.,
1988) and the Oregon-R wild-type sequence, obtained in the present
work. One of the polymorphisms results in an amino acid substitution
(Arg-446 His; numbering follows Bloomquist et
al.(1988)). All of the polymorphisms were also found in the mutant
sequences, which were induced in the Oregon-R background. Each norpA allele, however, also contains a nucleotide difference
not found in the Oregon-R wild-type sequence. Each of these mutations
is expected to result in an alteration of the encoded amino acid
sequence upon translation. Allele P57 contains a guanine (2955
in the numbering system of Bloomquist et al.(1988)) to adenine
transition, which results in a missense mutation and the substitution
of a glycine (768) with an aspartic acid. P16 and P79 are identical mutations containing a cytosine(1733) to thymine
transition which creates a missense mutation resulting in the
substitution of arginine 361 with cysteine. Alleles P12, P42, P45, and P76 each contain a cytosine to
thymine transition (nucleotides 1370, 2516, 2896, and 2468,
respectively) that creates a premature termination codon, which is
expected to result in early truncation of the protein product. Finally,
allele P24 has a 28-base pair deletion (nucleotides
2710-2737), which causes a reading frameshift, resulting in the
substitution of 24 amino acid residues, followed by a premature
termination codon. Fig. 5summarizes these data.
Figure 5:
Expected protein product from each norpA mutant. Schematic representation of the primary
structure of the expected protein product from wild-type flies and norpA mutants. Numbers below the figures are amino
acid residue positions based on the numbering system of Bloomquist et al.(1988). Arrows indicate the position of amino
acid substitutions found in the missense mutants. WT, wild
type; P12, etc., norpA mutant,
etc.
The extent
to which these mutations affect the enzymatic activity was investigated
by in vitro PLC enzyme assay of total eye-protein homogenate.
Eye homogenate from wild-type flies produces a emission activity
of approximately 82
10
dpm/(mg
min) of PLC
activity under our reaction conditions yielding specific activity of
4.6 nmol of [
H]PIP
cleaved/(mg
min) (see ``Materials and Methods''). Before any
corrections are applied, P57 and P16/P79 eye protein
homogenates have specific activities of approximately 4.7% (0.22 nmol
[
H]PIP
cleaved/(mg
min)) and
2% (0.091 nmol [
H]PIP
cleaved/(mg
min)) of the wild-type activity, respectively (Fig. 6).
The remaining five mutants have roughly similar activity levels of
0.9-1.5% (0.039-0.067 [
H]PIP
cleaved/(mg
min)) that of wild type. Total head
homogenate from the eya mutant has about 12% (0.56 nmol of
[
H]PIP
cleaved/(mg
min)) of
the wild-type eye homogenate PLC activity.
Figure 6:
Phospholipase C activity in protein
homogenates. The bar graph represents PLC activity from norpA mutant eye homogenates or eya head homogenates.
Activities in equivalent quantities of eye or head homogenate are
presented. Vertical bars are shown in units of nmol of
[H]PIP
cleaved(mg of total protein
homogenate
min of reaction time) (nmol of
[
H]PIP
cleaved/(mg
min)). The
wild-type activity level is 4.6 ± 1 nmol
[
H]PIP
cleaved/(mg
min). No
corrections have been applied to any of the values shown. Each bar
graph represents average ± S.E. from six determinations. P57, etc., norpA
mutant, etc.; eya, eyes absent mutant.
To establish definitively
that mutations in the norpA gene are entirely responsible for
the norpA phenotype, we have rescued the norpA null mutant by P-element-mediated germline
transformation (Rubin and Spradling, 1982; Spradling and Rubin, 1982)
(see ``Materials and Methods''). Similar experiments have
also been reported by McKay et al. (1995). Western analysis
showed that the expression of the NorpA protein in transgenic flies is
at least as great as in wild-type flies (Fig. 7, A and B).
Figure 7:
Analysis of norpA transformants. A,
Western blot of an equal quantity of total eye protein homogenate from
wild-type and transformant flies. Same procedure was used as in the
Western blot shown in Fig. 3. B, peak area of the band
corresponding to the 130-kDa protein in total eye homogenates of norpA
transformants compared to that in
equal amounts of wild-type eye homogenates. C, ERG recording
from a norpA
transformant compared to
those of wild type and a norpA
mutant,
using a white stimulus of log I = -2. D,
ERG recordings showing the response to a blue light stimulus given
during the blue light-induced PDA as a test for R7 and R8 function. WT, wild-type; P24 trans, w
norpA
transformants.
ERG responses from transformed flies are also similar to those of wild-type flies in both amplitude and time course (Fig. 7C). However, further examination reveals a difference between wild type and transformant in the responses of a minority subset of photoreceptor cells. Each ommatidium in the Drosophila compound eye contains a total of eight photoreceptors in three subtypes: R1-R6, the major subtype, and two minor subtypes, R7 and R8. The ninaE promoter used to drive the expression of the NorpA protein encoded in the P-element construct (see ``Materials and Methods'') will express the protein in R1-R6 photoreceptor cells only (O'Tousa et al., 1985; Zuker et al., 1985). Therefore, it is expected that transformation will rescue photoreceptors R1-R6, but not R7 or R8. Although the ERG responses from R7 and R8 are usually masked by the much larger response from R1-R6 photoreceptors, their response can be isolated by inducing a maximal PDA in R1-R6 using strong blue light to saturate and inactivate these photoreceptors (Minke et al., 1975). Neither R7 nor R8 is inactivated with blue light since they contain rhodopsins that absorb maximally at wavelengths distinct from that of the R1-R6 rhodopsin (Harris et al., 1976; Hardie and Kirschfeld, 1983; Montell et al., 1987; Zuker et al., 1987). Consequently, if a second blue stimulus is applied during the PDA, any response observed would be generated from R7 and R8 photoreceptors and would be seen superimposed on the R1-R6 PDA (Minke et al., 1975). Fig. 7D shows that R7 and R8 photoreceptors respond to the second blue stimulus in wild-type flies but not in transgenic flies.
In addition to the electrophysiological defect, norpA mutants undergo a slow, light-dependent degeneration of the
photoreceptor that is particularly evident in rhabdomeres (Meyertholen et al., 1987; Pak, 1994). Rhabdomere degeneration in
transgenic flies was investigated by ultrastructural comparison of
ommatidia from wild-type, norpA and transformed
animals. Transverse thin sections were cut through the compound eyes of
both newly eclosed and 6-week-old flies and examined by electron
microscopy. Ommatidia from newly eclosed P24 mutant and
transformant flies show R1-R6 rhabdomeres that are similar in
size and pattern to those of wild-type flies. The R7 rhabdomere is also
present (Fig. 8, panels A, C, and E).
At 6 weeks of age, however, P24 flies completely lack
R1-R6 rhabdomeres but retain the R7 rhabdomeres (Fig. 8, panel D). The transformants, on the other hand, retain
R1-R6 rhabdomeres in a size and pattern that appear similar to
wild type. The R7 rhabdomere is also present; however, it was not
possible to determine from these data whether R7 is showing any signs
of degeneration or not (Fig. 8, panel F).
Figure 8: Rhabdomeral structure in newly eclosed and 6-week-old wild-type flies, P24 mutants, and P24 transformants. Electron micrographs were taken of thin sections cut transversely through the eye. A, a newly eclosed wild-type fly; B, a 6-week-old wild-type fly; C, a newly eclosed P24 fly; D, a 6-week-old P24 fly; E, a newly eclosed P24 transformant; F, a 6-week-old P24 transformant. R1, etc., indicate rhabdomere 1, etc. Scale bar, 2 µm.
In all eight norpA mutants characterized in this
study, both the level of in vitro PLC activity and the amount
of the NorpA protein expressed are found to be drastically reduced ( Fig. 4and Fig. 6). Moreover, the severity of the ERG
phenotype ( Fig. 1and Fig. 2) is roughly correlated with
the levels of NorpA expression and PLC activity. As noted under
``Results,'' the eight norpA mutants
examined fall into four groups on the basis of the ERG phenotype. In
order of increasing severity, they are: Group 1, P57; Group 2, P16 and P79; Group 3, P12, P42, P45, and P76; and Group 4, P24. Each of
these mutants falls into exactly the same group when examined on the
basis of levels of NorpA protein expression or PLC activity, except
that these measurements are not sensitive enough to distinguish Group 3
from Group 4. Thus, P57 (Group 1), with a NorpA level of
18% (Fig. 4) and PLC activity level of
5% that of wild
type (Fig. 6), is the least affected. The next in order of
severity are P16 and P79 (Group 2), with a protein
level of
9% of wild type and PLC activity level of
2%. For
the remaining five mutants (Groups 3 and 4), the NorpA expression
cannot be detected by Western blot analysis (Fig. 3), and the
PLC activity level is uniformly about 1.0-1.5% of the wild-type
level (Fig. 6).
Generally, ERG responses observed in the
mutants are much larger and more robust than might be expected from the
levels of NorpA expression and PLC activity. For example, in P57 just 18% of the wild-type level of the NorpA protein (Fig. 4) and 5% of the wild-type level of PLC activity (Fig. 6) are enough to generate a response that, although
reduced from that of wild type, is large and rapid ( Fig. 1and Fig. 2). Thus, at log I = 0, V obtained
from P57 is approximately 72% of that of wild type (Fig. 2A), and the initial slope of the response is 69%
that of wild type (Fig. 2B). Even in Group 3 mutants,
in which the NorpA level is undetectable and the in vitro PLC
activity is just 1.0-1.5% that of wild type, distinctly
recognizable ERG responses are present, although much reduced in
amplitude (13% of wild type at I = 0) and very
slow and prolonged in time course.
Since ERG responses are present
in Group 3 mutants, some NorpA protein is likely to be present, even
though it is not detected on Western blots. Calculations based on
standard curves, plotting the peak area (see ``Materials and
Methods'') of the 130-kDa (NorpA protein) band versus the
quantity of eye homogenate loaded onto a gel, show that as much as 2%
of the wild-type NorpA protein could be present but not detected on
Western blots (data not shown). However, even the maximum possible
amount of the NorpA protein that could be present in these mutants (2%)
is still much lower than what the ERG amplitude (13%) generated in
these mutants might suggest. As for the 1.0-1.5% wild-type level
of PLC activity observed in these mutants, much of it is likely due to
nonspecific background activity. At least one other PLC isozyme is
known to be present in Drosophila heads in addition to the
NorpA protein (Shortridge et al., 1991; Toyoshima et
al., 1990). Since our PLC enzyme assay does not distinguish the
activities of different isozymes, a small amount of non-NorpA PLC
present in the eye homogenates could contribute to the observed PLC
activity. Approximately 1.0% of the wild-type activity is detected in
even norpA
, a mutant displaying virtually no ERG
response, lending further support to the suggestion that this level of
activity is largely or entirely due to non-NorpA PLC(s).
In light of
the above discussion, a specific activity level of 0.053 nmol of
[H]PIP
cleaved/(mg
min) (1.1%
wild type activity) was considered a background PLC activity and
subtracted from those of wild type, P57 and P16/P79 presented under ``Results'' and in Fig. 6. After
this correction, the NorpA specific activities in total eye protein
homogenates of wild type, P57, and P16/P79 are
approximately 4.6, 0.16, and 0.04 nmol of
[
H]PIP
cleaved/(mg
min) total
eye protein. However, the amount of the NorpA protein is reduced by
factors of 0.18 and 0.09, respectively, in the total eye proteins of P57 and P16/P79 relative to that of wild type (Fig. 4). To assess reduction in specific activities of the
mutant NorpA proteins independent of their reduction in the amount, the
above specific activities were divided by the reduction factors, 0.18
and 0.09. With these corrections, the specific activities for P57 and P16/79 are approximately 0.9 and 0.4 nmol of
[
H]PIP
cleaved/(mg
min)
equivalent total eye protein, respectively, where equivalent total eye
protein represents one having the same concentration of NorpA protein
as in wild type (see ``Materials and Methods'').
The above considerations, taken together, lead to the conclusion that the NorpA PLC need be activated by only a few percent of its normal capacity to yield a large and rapid light response from the photoreceptor. In the case of rhodopsin, activation of less than 1% of its normal amount is known to generate a maximal response of the photoreceptor (see, e.g., Johnson and Pak(1986) for Drosophila). In the case of vertebrates, this phenomenon has been attributed to the amplification of the response in the phototransduction cascade (see, e.g., Stryer(1983) and Lolley and Lee(1990)), i.e. each photoactivated rhodopsin molecule activates many G-protein molecules, and each activated effector molecule (cGMP phosphodiesterase, in the case of vertebrates) hydrolyzes many cGMP. The present results suggest that similar amplification occurs at, or subsequent to, the effector molecule level in the PLC-based phototransduction cascade, i.e. each activated PLC molecule may generate many second messenger molecules to produce a sizable response even when the amount of PLC activated is very small.
Let us
now consider the molecular defect identified in each of the norpA alleles and how it may relate to the ERG defect. In P16 and P79, the amino acid substitution occurs at an
arginine residue (Arg-361), which is equivalent to Arg-358 in
PLC-1, a bovine brain PLC
(Katan et al., 1988; Suh et al., 1988), and is conserved in Box X of all known
eukaryotic PLCs (Fig. 5). Arg-361 is located within a cluster of
several other highly conserved, contiguous residues that may be part of
the catalytic site of the enzyme (Crooke and Bennett, 1989). Moreover,
cysteine, which replaces arginine 361 in P16 and P79,
is among the most reactive of amino acids. These data suggest that the
mutation would have a significant effect on the function of the NorpA
protein.
In contrast, the glycine residue substituted in P57 (Gly-768 Asp; Fig. 5) is located outside of both Box
X and Box Y and is conserved in the
and
subclasses of PLC
only. It is, therefore, not likely to be directly involved in catalytic
function. Instead, it could possibly be involved in
and
class-specific regulation of the enzyme. The effect of this mutation on
the ERG is relatively mild compared to P16/P79.
The remaining five alleles (P12, P24, P42, P45, and P76) contain nonsense mutations that result in premature termination codons. If translated at all, it is expected that the protein product from these alleles would be truncated and functionally defective (Fig. 5). None of the mutants carrying these alleles show detectable NorpA protein on Western blots (Fig. 3). Nevertheless, at least some ERG response does occur in four of these mutants (P12, P42, P45, and P76), and the responses are similar to each other in amplitude and time course (Fig. 2) irrespective of the expected extent of protein truncation (Fig. 5). The observation suggests that the abnormal ERG response is not due to the expression of mutant NorpA protein per se. It is possible that read-through of the termination codon in these four alleles allows expression of a very low level of protein. Since each contains a single point mutation, read-through would result in a wild-type protein or protein that differs from wild type by a single amino acid. The premature termination codon, UAG, found in P12, P42, and P45 and UGA in P76 are not the most commonly utilized termination codons in Drosophila (Brown et al., 1990) and may be more likely to allow read-through than the more common UAA codon. Because of the amplification of transduction signal at the PLC level, previously discussed, a very low level of PLC expression due to read-through may be enough to produce the observed ERG response.
In the case of P24, on the other hand, essentially no ERG response is observed (Fig. 2). P24 has a 28-base pair deletion that causes a reading frameshift, resulting in the substitution of 24 amino acids followed by a premature truncation of the protein. Read-through of the termination codon, therefore, would be in a wrong reading frame and would still result in a significantly altered, and presumably non-functional, protein product.
As discussed in the Introduction, the phototransduction cascade in Drosophila involves the activation of the NorpA protein (PLC) by photoactivated rhodopsin through a G-protein. Although details of the cascade have not yet been fully elucidated, calcium has been implicated in both photoexcitation and adaptation (Devary et al., 1987) (reviewed by Bacigalupo(1990)). Dark-adapted photoreceptors have a relatively low internal calcium concentration, which increases during the light response. This increase initially has a positive feedback effect on the transduction cascade facilitating the rapid onset of the light response. As the internal concentration continues to increase, however, this positive feedback transforms into negative feedback resulting in adaptation and rapid deactivation of the response (Hardie and Minke, 1993). The altered kinetics of the light response in norpA mutants may be due to the failure of this sequential positive then negative feedback effect of cytosolic calcium.
Since the amount of NorpA protein is reduced while the size of the rhabdomeres, before their degeneration, is similar to wild type in the mutants (Fig. 8), whatever PLC molecules are present would be distributed at a lower density. This reduced PLC density, along with the decreased specific activity, would result in a diminished and, perhaps, slower activation of the effector enzyme at any given stimulus intensity, resulting in a diminished and slower rise in cytosolic calcium during the light response. Without the rapid calcium kinetics, the initial positive feedback mechanism would be attenuated, severely reducing the the rapid initial phase of the light response in mutants, as seen in the ERG responses of mutants (Fig. 1). With increasing stimulus intensities, however, the slope of the initial phase, although clearly not wild type, does increase in mutants ( Fig. 1and 2B). It may be that at higher stimulus intensities, the PLC is activated at a sufficiently high rate to activate the positive feedback mechanism even in the mutants.
In addition to the reduced amplitude and initial slope, the light response in norpA mutants is very prolonged relative to wild-type responses (Fig. 1). The response, in some cases, is maintained up to several minutes after the end of the light stimulus. This prolonged response differs from the PDA of wild-type flies in that it is not terminated by orange light and is generated by even low stimulus intensities. It is possible that normal adaptation or deactivation does not occur in the mutants because the concentration of cytosolic calcium does not reach the level required for activation of the negative feedback mechanism. An eye-specific protein kinase C has recently been implicated in adaptation (Hardie and Minke, 1993) and deactivation (Ranganathan et al., 1991; Smith et al., 1991). This protein kinase C may not be activated properly at the severely reduced calcium and/or DAG concentrations generated in norpA mutants.
The ERG recorded from transgenic norpA flies
carrying the cloned wild-type norpA coding sequence (Fig. 7C) shows that the PLC encoded by the cloned
sequence is sufficient to restore the normal phototransduction process.
Since the ninaE promoter (O'Tousa et al., 1985;
Zuker et al., 1985) used in the transformation experiments
drives the expression of the cloned PLC coding sequence in R1-R6
photoreceptors only, only R1-R6 photoreceptors respond to light
normally in transgenic flies, while R7 and R8 photoreceptors do not
respond (Fig. 7D).
In addition to the ERG phenotype,
strong norpA alleles cause light-dependent degeneration of
photoreceptors (Meyertholen et al., 1987) (reviewed by
Pak(1994)). As with the ERG phenotype, it might be expected that
R1-R6 rhabdomeres would be rescued from degeneration in the
transformants whereas the R7 rhabdomeres would not. As shown in Fig. 8, R1-R6 rhabdomeres, which degenerate completely in norpA by 6 weeks, appear normal in transgenic
flies at the same age. On the other hand, because R7/8 rhabdomeres
degenerate slowly even in strong norpA mutants, it was not
possible to make a reliable assessment of their rescue, or the lack
thereof, in the 6-week-old transgenic flies examined. Taken together,
the above results establish definitively that mutations in the norpA gene are solely responsible for both the ERG and
degeneration phenotypes of norpA mutants.