(Received for publication, January 11, 1996)
From the
In an approach directed to isolate and characterize key proteins
of the transduction cascade in photoreceptors using the
phosphoinositide signaling pathway, we have isolated the Calliphora homolog of the Drosophila InaD gene product, which in Drosophila InaD mutants causes slow deactivation of the light
response. By screening a retinal cDNA library with antibodies directed
against photoreceptor membrane proteins, we have isolated a cDNA coding
for an amino acid sequence of 665 residues (M = 73,349). The sequence displays 65.3% identity (77.3%
similarity) with the Drosophila InaD gene product. Probing
Western blots with monospecific antibodies directed against peptides
comprising amino acids 272-542
(anti-InaD-(272-542)) or amino acids 643-655
(anti-InaD-(643-655)) of the InaD gene product
revealed that the Calliphora InaD protein is specifically
associated with the signal-transducing rhabdomeral photoreceptor
membrane from which it can be extracted by high salt buffer containing
1.5 M NaCl. As five out of eight consensus sequences for
protein kinase C phosphorylation reside within stretches of 10-16
amino acids that are identical in the Drosophila and Calliphora InaD protein, the InaD gene product is
likely to be a target of protein kinase C. Phosphorylation studies with
isolated rhabdomeral photoreceptor membranes followed by InaD immunoprecipitation revealed that the InaD protein is a
phosphoprotein. In vitro phosphorylation is, at least to some
extent, Ca
-dependent and activated by phorbol
12-myristate 13-acetate. The inaC-encoded eye-specific form of
a protein kinase C (eye-PKC) is co-precipitated by antibodies specific
for the InaD protein from detergent extracts of rhabdomeral
photoreceptor membranes, suggesting that the InaD protein and
eye-PKC are interacting in these membranes. Co-precipitating with the InaD protein and eye-PKC are two other key components of the
transduction pathway, namely the trp protein, which is
proposed to form a Ca
channel, and the norpA-encoded phospholipase C, the primary target enzyme of
the transduction pathway. It is proposed that the rise of the
intracellular Ca
concentration upon visual excitation
initiates the phosphorylation of the InaD protein by eye-PKC
and thereby modulates its function in the control of the light
response.
Phototransduction by rhabdomeral photoreceptors, particularly of Drosophila compound eyes, has become an important model system for the ubiquitous phosphoinositide-mediated signal transduction. The progress achieved in this field is based on the powerful genetic and molecular biological techniques available for Drosophila, which have been successfully complemented by biochemical studies in other flies such as Calliphora and Musca. Despite the rapid progress that has been achieved in the understanding of sensory transduction mechanisms in recent years, the phototransduction cascade operating in this type of sensory cells has not yet been entirely resolved. In particular, the biochemical processes regulating the recovery and adaptation of the visual response in rhabdomeral photoreceptors are still obscure.
Extracellular Ca enters the photoreceptors through ion channels and is required
for rapid recovery of visual excitation (1, 2, 3, 4, 5) . A major
portion of the Ca
influx into the photoreceptor cell
appears to be carried by a Ca
-selective class of
channels that depend on, or may indeed be formed by, the transient
receptor potential (trp) (
)protein(6, 7) . The primary structure of
this trp protein was identified simultaneously by Wong et
al.(8) and Montell and Rubin(9) , and the trp gene product was shown to be localized to the rhabdomeral
photoreceptor membranes. Direct measurements of the extracellular
Ca
concentration revealed a decline of extracellular
Ca
upon illumination in the eyes of wild type flies,
which is significantly reduced in trp mutants (4) .
Signal transduction is also impaired in two other Drosophila mutants, inaC and InaD, which were originally
classified as inactivation-no afterpotential mutants by
Pak(10) . While the InaD gene product is an 80-kDa
protein of unknown function(11) , the inaC gene was
shown to encode an eye-specific protein kinase C
(eye-PKC)(12, 13) . Thus, it is tempting to assume
that the Ca
-dependent deactivation of the visual
response is controlled by phosphorylation of photoreceptor-specific
proteins associated with the rhabdomeral photoreceptor membrane.
Identified proteins that undergo light-dependent phosphorylation are
rhodopsin and arrestin
2(14, 15, 16, 17) . However, neither
rhodopsin nor arrestin 2 were found to be phosphorylated by the inaC protein because phosphorylation of activated rhodopsin is
not enhanced by Ca
(14, 15) , and the
Ca
-stimulated phosphorylation of arrestin 2 has been
shown to result from the activation of a
Ca
-calmodulin-dependent protein kinase(17) .
Accordingly, the target proteins of eye-specific protein kinase C have
yet to be specified.
In an attempt to identify proteins that are part of the biochemical pathway in rhabdomeral photoreceptors, we have used an immunological approach to isolate Calliphora cDNA clones, which encode photoreceptor membrane proteins. Antibodies directed against purified rhabdomeral membranes (18) were generated and employed for the isolation of genes encoding rhabdomere-specific proteins. By this approach, we have cloned the Calliphora homologs of the Drosophila InaD, inaC, and trp genes. In the present paper we show that the InaD gene product is associated with the rhabdomeral photoreceptor membrane and that it is a putative substrate of eye-PKC. We also provide for the first time evidence for an interaction among eye-PKC, InaD protein, trp protein, and the norpA- (no receptor potential A) encoded phospholipase C.
Polyclonal anti-InaD antibodies were generated as follows:
a DNA fragment encoding the 23 C-terminal amino acids of the Calliphora InaD protein was amplified by polymerase chain
reaction from cloned cDNA using sequence-specific primers. The
polymerase chain reaction product was cloned into the expression vector
pQE40 (Qiagen, Hilden/Germany) in frame with six His codons and the
dihydrofolate reductase gene. Fusion proteins were expressed in Escherichia coli M15 (pREP4), extracted with urea and purified
on Ni-agarose columns according to the
manufacturer's instructions. Purified fusion proteins were
dialyzed against phosphate-buffered saline (137 mM NaCl, 3
mM KCl, 8 mM Na
HPO
, 2 mM KH
PO
, pH 7.2) and used for the
immunization of a rabbit (200 µg of protein/injection). Antibodies
were purified from the antiserum by affinity chromatography on HiTrap
columns (Pharmacia, Freiburg/Germany), which had previously been
coupled with 1 mg of the antigen as described by the manufacturer. The
purified antibodies are hereafter referred to as
anti-InaD-(643-665), according to the InaD peptide from which they were raised. A second anti-InaD antibody (anti-InaD-(272-542)) was generated and
purified in the same way by using a recombinantly expressed peptide
comprising amino acids 272-542 of the Calliphora InaD protein.
For the production of anti-Calliphora trp antibodies a partial Calliphora cDNA clone encoding the
C-terminal half of the trp protein was expressed in E.
coli M15 (pREP4), and the expression product was used as an
antigen. Antibodies directed against the Calliphora inaC protein and the -subunit of the eye-specific G-protein were
raised against bovine serum albumin-coupled synthetic peptides
(CYMNPEFITMI and QNALKEFNLG, respectively), which correspond to the
C-terminal region if these proteins. Antibodies directed against the Drosophila norpA-encoded phospholipase C and against the
-subunit of the G-protein, which also detect the corresponding Calliphora proteins, were a generous gift of R. Shortridge (20) and J. B. Hurley (21) .
We obtained 14 cDNA
clones coding for the Calliphora homolog of InaD and
determined the nucleotide sequence of the longest cDNA for both
strands. This clone contained a 201-base pair 5`-untranslated region, a
1995-base pair open reading frame encoding a polypeptide of 665 amino
acids (M = 73,349), and a 194-base pair
3`-untranslated region. The translation initiation site was assigned
arbitrarily to the first AUG of the open reading frame at nucleotide
202, which is preceded by a stop codon at nucleotide 196, and fits well
with the consensus sequence for translation initiation sites in Drosophila, (C/A)AA(A/C)AUG (26) . Alignment of the
deduced amino acid sequence of the Calliphora cDNA clone with
the Drosophila InaD sequence (Fig. 1a) shows
that the two proteins display 65.3% overall amino acid identity and
77.3% similarity if conservative substitutions are taken into account.
Furthermore, both proteins share similar biophysical characteristics, i.e. the same predicted isoelectric point of 8.6, a high
abundance of basic (Lys, Arg, His) and acidic (Asp, Glu) amino acids
that together comprise more than 30% of the polypeptide, and similar
hydrophilicity profiles (Fig. 1b) that reveal no
stretches of hydrophobic sequences of 20 or more residues in length.
Figure 1:
Comparison of the primary structure of
the Drosophila and Calliphora InaD protein. a, alignment of the deduced amino acid sequences of the Drosophila and Calliphora InaD gene products. Amino
acids are shown in single-letter code. Amino acid identities and
similarities between the two proteins are indicated by vertical
bars and points, respectively. Boxed serine and
threonine residues represent potential protein kinase C phosphorylation
sites (PKC), and boxed asparagine residues indicate
potential N-glycosylation sites (pGS). Two conserved
repeats are underlined. G-(Q/M) repeats that are present only
in the Drosophila InaD protein between amino acids 141 and 155
are indicated by a dotted line. A lysine/glutamate-rich
cluster is labeled by stars. b, hydrophilicity
profile (window size: 7) of the Drosophila and Calliphora
InaD protein according to Kyte and Doolittle(46) . The arrow depicts the position of Met, which is
replaced by a lysine in InaD
mutants. Bars indicate the position of lysine/glutamate-rich
clusters.
Two repeats of 40 amino acids (underlined in Fig. 1a) that were shown to share limited sequence
homology with the Drosophila disc-large (Dlg), the rat
post-synaptic density protein (PSD95), the vertebrate tight junction
protein ZO-1, and the human ROS protein (see (11) and
references therein), are highly conserved (90% similarity) between the Calliphora and Drosophila InaD proteins, implying a
common functional role within the family of proteins that contain these
repeats. On the other hand, repeats consisting of Gly-(Gln/Met), which
are present in the Drosophila InaD sequence between amino
acids 142 and 158, are not found in the Calliphora sequence.
Indeed, the region between residues 106 and 183 is the least conserved
part of the two proteins. The only common feature within this region is
the relatively high abundance of glutamine residues. Another striking
sequence motif, which is present in the Calliphora and the Drosophila InaD protein, is the highly hydrophilic cluster of
lysine and glutamate residues between amino acids 454 and 473
(indicated by bars in Fig. 1b). While
potential phosphorylation sites of cAMP- and cGMP-dependent protein
kinase and tyrosine kinase present in the Drosophila sequence
at Thr and Tyr
, respectively, are not found
in the Calliphora sequence, eight potential phosphorylation
sites of protein kinase C are conserved (Fig. 1a).
Finally, two potential glycosylation sites at Asn
and
Asn
(Drosophila) or Asn
and
Asn
(Calliphora) are found at similar positions
in both sequences.
Figure 2:
Immunoblot analysis of the InaD protein. Retinal proteins were separated by SDS-PAGE and stained
with Coomassie Blue (left panel) or transferred onto a
polyvinylidene difluoride membrane for immunological detection with
antibodies directed against the C-terminal region of the InaD protein (anti-InaD-(643-665)) or a peptide
comprising amino acids 272-542
(anti-InaD-(272-542)). Membrane and soluble proteins
from one Calliphora retina were analyzed in lanes 1 and 2, respectively. Proteins extracted with 1
SDS-PAGE buffer from purified photoreceptor membranes of 10 Calliphora eyes were loaded on lane 3. Lanes
4-6 show rhabdomeral proteins obtained after subsequently
extracting purified photoreceptor membranes with a low salt buffer
containing 3 mM EGTA (lane 4) with a high salt buffer
containing 1.5 M NaCl (lane 5) and with 1
SDS-PAGE buffer (lane 6).
Figure 3:
Co-immunoprecipitation of the InaD, inaC, trp, and norpA proteins. Rhabdomeral proteins were extracted with Triton X-100
buffer, immunoprecipitated with anti-InaD-(272-542) and
subjected to SDS-PAGE and Western blot analysis. a,
Coomassie-stained gel showing the proteins extracted with Triton X-100
buffer from rhabdomeral membranes of 10 eyes (lane 1), the
proteins that are not immunoprecipitated by
anti-InaD-(272-542) (lane 2; equivalent of 10
eyes), and the proteins present in the immunoprecipitates (lane
3; equivalent of 30 eyes). Lanes 4 and 5 show
control experiments. When protein A/G-agarose beads without antibody
were used in the immunoprecipitation experiment, no immunoprecipitates
were obtained (lane 4). On lane 5 anti-InaD-(272-542) eluted from protein A-Sepharose
beads was loaded in order to identify protein bands, which represent
the antibodies (indicated by bracket). b, Western
blots of the proteins shown in lanes 1-3 of panel a were probed with anti-InaD-(643-665) and with
antibodies specific for the trp, norpA, and inaC proteins and for the - and
-subunits of an eye-specific
G-protein (G
and G
,
respectively).
Figure 4:
Identification of phosphorylated InaD protein. In order to demonstrate phosphorylation of the InaD gene product, proteins of dark-adapted rhabdomeral membranes that
remained in the membrane fraction after extraction with low salt buffer
containing 3 mM EGTA were phosphorylated in the presence of 2
µCi of [-
P]ATP as described under
``Experimental Procedures.'' After phosphorylation the InaD protein was extracted from the membrane with high salt
buffer. Membrane proteins that were not extracted under these
conditions were solubilized with SDS-PAGE buffer. The aliquots
equivalent to purified photoreceptor membranes of 10 retinae for the
SDS extract or 40 retinae for the high salt extract were subjected to
SDS-PAGE. a, Coomassie-stained protein pattern and
corresponding autoradiograph of the SDS-extracted proteins (lane
1) and the high salt-extracted proteins (lane 2). b, immunoblot of the same extracts as shown in panel a probed with anti-InaD-(643-665) and corresponding
autoradiograph.
Figure 5:
Immunoprecipitation of phosphorylated InaD protein. Proteins of blue light-illuminated rhabdomeral
membranes were phosphorylated in the presence of 2 µCi of
[-
P]ATP as described under
``Experimental Procedures.'' Then the rhabdomeral membrane
proteins were extracted with Triton X-100 buffer and subjected to
immunoprecipitation with anti-InaD-(272-542). Proteins
of the Triton X-100 extract (lane 1), of the supernatant
containing the nonprecipitated proteins (lane 2), and of the
immunoprecipitates (lane 3) were resolved by SDS-PAGE and
subjected to Western blot analysis and autoradiography. The
Coomassie-stained protein gel, the immunoblot probed with
anti-InaD-(643-665), and the autoradiograph of the
immunoblot are shown from left to right.
Since eye-PKC
co-immunoprecipitating with the InaD protein is assumed to be
a Ca-dependent protein kinase, we tested the effect
of Ca
on the phosphorylation of the InaD protein and compared its phosphorylation with phosphorylation of
rhodopsin and arrestin, which has been studied previously (14, 15, 16, 17) (Fig. 6).
Lowering of internal Ca
by the addition of 2 mM EGTA to the phosphorylation assay significantly reduced the
incorporation of radioactive phosphate into the InaD protein
as compared with standard phosphorylation assays performed at a
calculated free Ca
concentration of 60
µM. Under these conditions, the phosphorylation of two
other proteins involved in signal transduction is affected by lowering
the free Ca
concentration. First, the amount of
phosphorylated arrestin 2 associated with the rhabdomeral photoreceptor
membrane is decreased. This finding is consistent with earlier reports
indicating that arrestin 2 is phosphorylated by a
Ca
-calmodulin-dependent protein kinase(17) .
Secondly, the phosphorylation of rhodopsin is affected by calcium
concentration. There the amount of radioactive phosphate attached to
metarhodopsin is reduced in the presence of Ca
,
presumably due to dephosphorylation by a Ca
-dependent
rhodopsin phosphatase(27, 28) . We also investigated
whether the light conditions to which the photoreceptor membranes were
subjected before the reaction was started, might affect the
incorporation of phosphate into the InaD protein. Under the in vitro conditions used here, the already intensively studied
light activation of rhodopsin phosphorylation (14, 15) was reproduced, but light dependence of InaD protein phosphorylation was not revealed (Fig. 6a, lanes 1 and 2 and lanes
3 and 4). Thus, we can exclude the possibility that
activated rhodopsin (metarhodopsin), present in the membranes, directly
promotes (or suppresses) the phosphorylation of the InaD protein. Furthermore, the addition of soluble proteins did not
enhance the incorporation of phosphate into the InaD protein,
but rather suppressed its phosphorylation (Fig. 6a, lanes 5-8). This indicates (i) that the protein kinase
that catalyzes InaD phosphorylation resides in the
photoreceptive membrane, and (ii) that soluble cofactors are not
required for InaD protein phosphorylation.
Figure 6:
Ca-dependent
phosphorylation of the InaD protein. a, purified
photoreceptor membranes of 10 Calliphora retinae were
phosphorylated in the presence of 2 mM EGTA (lanes 1, 3, 5, and 7) or 60 µM calculated free Ca
(lanes 2, 4, 6, and 8). In lanes 5-8 the
membranes were reconstituted with a soluble extract obtained from six Calliphora retinae. Before starting phosphorylation by adding
2 mM ATP, samples were either kept under dim red light (r, lanes 1, 2, 5, and 6)
or illuminated with blue light for 2 min in order to convert 70%
rhodopsin to metarhodopsin (panel b, lanes 3, 4, 7, and 8). The phosphorylation reactions
were carried out for 5 min in the dark. Migration of molecular weight
standards is indicated on the right; arrows on the left show the position of the InaD protein (InaD), arrestin 2 (Arr2), and opsin. b,
time course for the incorporation of radioactive phosphate into the InaD protein. For comparison, time courses of phosphorylation
of arrestin 2 and opsin are shown. Phosphorylation reactions were
performed in the absence (
), or presence (o) of Ca
as described in panel a for lanes 3 and 4, respectively. At the indicated times, the reactions were
terminated by adding 5
SDS-PAGE buffer. All samples were
subjected to SDS-PAGE as described under ``Experimental
Procedures,'' and protein phosphorylation was detected by
autoradiography or quantified by using a phosphor
imager.
Time courses of
the protein phosphorylation revealed similar phosphorylation kinetics
for the InaD protein and for opsin with no further increase in
net phosphate incorporation 10 min after the reactions were started (Fig. 6b). Arrestin 2 phosphorylation, described as the
most rapid protein phosphorylation observed in Drosophila eyes(29) , saturated 2 min after starting the reactions.
The Ca dependence of the phosphate incorporation into
the InaD protein, arrestin 2, and metarhodopsin is evident
throughout the entire phosphorylation time course, except in the
initial phase of metarhodopsin phosphorylation (Fig. 6b). The Ca
-enhanced
phosphorylation of the InaD gene product suggested that this
reaction is catalyzed either by a protein kinase C or by a
Ca
-calmodulin-dependent protein kinase. In order to
discriminate whether the InaD protein is phosphorylated by a
protein kinase C or a Ca
-calmodulin-dependent protein
kinase, protein kinase C was hyperactivated using a phorbolester or
specifically inhibited with bisindolylmaleimide I. As shown in Fig. 7, the addition of phorbol 12-myristate 13-acetate to the
phosphorylation reaction enhances the phosphate incorporation into the InaD protein by 25%. In the presence of bisindolylmaleimide I InaD protein phosphorylation is reduced by 25%. These effects
are statistically significant (see legend of Fig. 7), and they
are comparable with those observed in studies with other photoreceptor
membrane proteins, for example the protein kinase C-dependent
phosphorylation of bovine rhodopsin(30) . The protein kinase C
activator and inhibitor used here had no significant effect on the
phosphorylation of arrestin 2 and opsin (Fig. 7), indicating
that the addition of the phorbolester or of bisindolylmaleimide I
modulated specifically the phosphorylation of the InaD protein
but did not generally enhance or quench the phosphorylation of
rhabdomeral proteins.
Figure 7: Effect of phorbolester and the protein kinase C inhibitor bisindolylmaleimide I on the phosphorylation of the InaD protein. Phosphorylation of blue light-illuminated photoreceptor membranes was carried out under standard conditions (control), in the presence of 1 µM phorbol 12-myristate 13-acetate (PMA), and in the presence of 0.2 µM bisindolylmaleimide I (BIM). Samples were subjected to SDS-PAGE, and phosphorylation of the InaD protein (InaD), arrestin 2 (Arr2), and opsin was quantified using a phosphor imager. Data represent mean ± S.E. of four separate experiments. Statistical analysis using Student's t test shows that the effects of PMA and BIM on the phosphorylation of the InaD protein are significant (PMA-treated versus control: t = -2.56, p = 0.042; BIM-treated versus control: t = 2.72, p = 0.034). Phosphate incorporation into arrestin 2 and opsin shows no significant differences upon PMA or BIM treatment.
This study describes the molecular and biochemical characterization of the Calliphora InaD protein. The experiments have been performed to understand the function of this protein in the deactivation of light-triggered responses of photoreceptor cells. Cumulative evidence suggests that the biochemical reactions involved in phototransduction are identical in Drosophila and Calliphora. The eyes of both species have the same morphological architecture, they contain photoreceptors with identical absorbance characteristics, and the photoreceptor cells respond to light stimuli in the same way. Biochemical studies show that identified proteins of the phototransduction pathway, including Rh1 opsin(31, 32, 33) , arrestin 2 (17, 27, 34) , and phospholipase C(35, 36) , perform identical functions in both species. However, these functionally homologous proteins are less conserved than they are between photoreceptors currently used as model systems of vertebrate phototransduction, for instance bovine, rat, and mouse. These differences in the overall homology allow us to identify conserved regions as probable sites of functional importance within the protein.
The sequence alignment of the Drosophila and Calliphora InaD proteins (see Fig. 1) highlights the weakly, as well as the highly, conserved regions of the protein sequences. The N-terminal region (amino acids 1-14 of the Drosophila sequence) and the stretch between amino acids 106 and 183 show little if any sequence homology, suggesting that these regions are functionally less important and were, therefore, subject to extensive mutation during the evolution of both fly species.
Other sites of the InaD protein are well conserved. With respect to the phosphorylation of the InaD protein investigated in the present study it is particularly striking that five out of eight conserved potential protein kinase C phosphorylation sites (at positions 19, 194, 329, 330, and 553) reside within stretches of 10-16 amino acids that are identical in the Drosophila and Calliphora InaD protein.
Despite the fact that there are some poorly conserved regions in the Drosophila and Calliphora InaD protein, the overall biophysical characteristics (for example the isoelectric point at 8.6, the high abundance of acidic and basic amino acids, and the hydrophilicity profile of both proteins) are nearly identical. The apparent molecular mass of about 75 kDa of the Calliphora InaD protein, as estimated by SDS-PAGE, fits the molecular mass calculated from the sequence data (73.4 kDa). The discrepancy between the calculated and apparent molecular mass of the Drosophila InaD protein (80 and 90 kDa, respectively) reported by Shieh and Niemeyer (11) is not evident in Calliphora. Due to the hydrophilic nature of the InaD protein, it has been proposed that the Drosophila InaD gene product is not an integral membrane protein(11) . Our results obtained with the Calliphora homolog of InaD are in agreement with this prediction.
In the present study we show that the InaD protein is
associated with the rhabdomeral photoreceptor membrane, from which it
is extracted by buffers of high ionic strength. The attachment to the
photoreceptive membrane may be crucial for InaD function,
because functional impairment of the Drosophila InaD mutant (InaD; (10) ) results from a single
point mutation in which a methionine (Met
), located
within a small stretch of hydrophobic amino acids, is replaced by
lysine(11) . In the Calliphora InaD protein leucine is
present at the corresponding position, indicating that Met
is not necessarily required for normal InaD function,
and may be exchanged with another hydrophobic amino acid. Distortion of
the hydrophobic character of the region by a highly polar amino acid,
for example lysine of InaD
, however, might lead
to the mutant phenotype, because the Met
to Lys mutation
may render a soluble InaD protein that is nonfunctional.
Alternatively, the nonpolar character of this region may be crucial for
hydrophobic protein-protein interactions. A significant contribution to
the hydrophilic character of the InaD protein results from a
conserved stretch of lysine and glutamate residues (see bars in Fig. 1b). Interestingly, similar
lysine/glutamate-rich clusters are found in the bovine and mouse rod
photoreceptor cGMP-gated channels(37, 38) . Analysis
of the structure-function relationship of cGMP-gated channels has not
yet established the function of this hydrophilic cluster.
The
biochemical experiments of the present study were designed to
investigate whether or not the function of the InaD gene
product might be controlled by phosphorylation. The striking
conservation of several putative protein kinase C phosphorylation sites
between the Drosophila and Calliphora InaD sequence (Fig. 1), the localization of the InaD protein and the inaC encoded eye-PKC in the rhabdomeral photoreceptor
membranes ( Fig. 2and (13) ), and, most importantly, the
co-immunoprecipitation of the InaD protein and eye-PKC (Fig. 3) suggest that the InaD protein is a likely
candidate for phosphorylation by eye-PKC. Moreover, Drosophila InaD and inaC mutants show a similar phenotype, which is
characterized by a defect in photoreceptor deactivation and by abnormal
light
adaptation(1, 2, 11, 13, 39) ,
indicating that the respective gene products are acting, or even
interacting, in closely related steps of the transduction cascade. The
phosphorylation studies presented here reveal that the InaD protein is a phosphoprotein ( Fig. 4and Fig. 5). The
Ca-dependence of the InaD protein
phosphorylation (Fig. 6) and the findings that the incorporation
of phosphate into the InaD protein is moderately enhanced in
the presence of a phorbolester and quenched by the protein kinase C
inhibitor bisindolylmaleimide (Fig. 7) are in line with the
assumption that this phosphorylation is catalyzed by eye-PKC.
Despite this evidence, the data do not yet allow us to unequivocally
rule out the possibility of phosphorylation of the InaD protein by other protein kinases. Ca-dependent
phosphorylation of arrestin 2, reported to result from a
Ca
-calmodulin-dependent protein kinase(17) ,
is observed in parallel to the Ca
-dependent
phosphorylation of the InaD protein, indicating that the
corresponding protein kinase is present in the membrane preparation
used in the assays. Also, Matsumoto and
colleagues(17, 40, 41) reported on the
phosphorylation of an 80-kDa protein present in the photoreceptor cell
layer of Drosophila eyes. The molecular mass of this
phosphoprotein suggests that it might represent the Drosophila InaD protein. However, the phosphorylation of this Drosophila 80-kDa protein was shown to be activated by cAMP but not by
calcium(17) . At least the Calliphora InaD protein
lacks consensus sites for phosphorylation by a cAMP-dependent protein
kinase. In dark-adapted Drosophila eyes, this 80-kDa protein
is in the nonphosphorylated state, but it rapidly (within 3 s) becomes
phosphorylated when the flies are exposed to a 1-ms light
flash(41) . Furthermore, the light-dependent phosphorylation of
this protein is not observed in Drosophila norpA mutants(40) , indicating that it depends on the activation
of the phototransduction cascade and occurs downstream of the norpA-encoded phospholipase C.
Phosphorylation by protein
kinase C is shown to be involved in the desensitization of a number of
vertebrate G-protein-mediated transduction cascades, e.g. vertebrate phototransduction (30, 42) and
-adrenergic receptor signaling(43) . There desensitization
is achieved by a protein kinase C-dependent phosphorylation of the
respective receptor (rhodopsin or
-adrenergic receptor), which in
contrary to the phosphorylation by rhodopsin kinase or
-adrenergic
receptor kinase, occurs in the activated and the nonactivated state of
the receptor. Protein kinase C-mediated phosphorylation was shown to
uncouple the receptor from its G-protein (44) , thereby
terminating the signal response. The proposed deactivation of the
visual response via phosphorylation of the InaD protein by
eye-PKC would act at a different site of the transduction cascade.
Towards a model for the Ca-dependent response
inactivation in fly photoreceptor cells, we propose that the InaD protein is modulated via phosphorylation by eye-PKC, which itself
should be activated by the transient rise of the intracellular
Ca
concentration upon visual excitation.
Phosphorylated InaD protein in turn may be a subunit of, or
act on, a third protein, e.g. an ion channel, in order to
regulate Ca
influx into the cytosol. In this respect
it is important to note that the trp protein, which is
proposed to represent a novel Ca
channel responsible
for light-dependent inositol trisphosphate-mediated Ca
entry(6, 7) , co-immunoprecipitates with the InaD protein. Alternatively, the activated InaD protein could be part of a feedback control mechanism that acts on
upstream members of the transduction cascade. One of these may be the norpA-encoded phospholipase C(11) . Our finding that
key proteins of the phototransduction cascade investigated here
co-immunoprecipitate with the InaD protein may indicate that
proteins that provide a control mechanism of visual excitation are
associated into a functional protein complex.
In conclusion, we have
for the first time provided evidence that the Calliphora homolog of the InaD protein is phosphorylated by the inaC-encoded eye-PKC. InaD protein phosphorylation
may be part of the mechanism that regulates the deactivation of the
light response in invertebrate photoreceptors, in a way that is
distinct from the protein kinase C-mediated desensitization of
vertebrate phototransduction or -adrenergic receptor signaling.
However, a similar mechanism may operate in other vertebrate and
invertebrate signaling pathways in which trp homologs are used
as part of a store-operated Ca
entry(45) .
The nucleotide sequence from which the amino acid sequence reported in this paper was deduced has been submitted to the GenBank(TM)/EMBL Data Bank with accession number Z69883[GenBank].