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INTRODUCTION |
The InaD
(inactivation-no-after potential
D) gene is preferentially expressed in the compound
eye and was isolated by subtractive hybridization (1). Molecular
characterization of InaD indicates that it encodes a protein
of 674 amino acid residues that contains five distinct
PSD95/dlg/zonular occludens-1
(PDZ)1 domains (2, 3). PDZ
domains are protein-protein interaction motifs of 80-100 residues and
are implicated in clustering and localization of receptors and channels
(4, 5). In Drosophila photoreceptors, INAD has been shown to
interact with carboxyl-terminal sequences of three key components of
the visual cascade leading to the formation of a macromolecular
signaling complex (6-11). These INAD-interacting proteins include TRP
(transient-receptor-potential), NORPA (no-receptor-potential
A), and eye-PKC. Eye-PKC is involved in a negative
regulation of visual transduction (12, 13), a G protein-coupled
phospholipase C
-mediated process that converts the signal of light
leading to depolarization of photoreceptors (14, 15). Negative
modulation of visual signaling by eye-PKC in vivo is
dependent on its interaction with INAD (11). Previously we showed that
eye-PKC associated with PDZ2 of INAD (11). Other reports have
implicated PDZ3 or PDZ4 in the eye-PKC interaction as well (9, 16).
Structural studies of PDZ domains in PSD95, human homologues of
dlg, and calcium/calmodulin-dependent serine
protein kinase revealed that PDZ domains consist of six
-sheets and two
-helices forming a six-strand
-sandwich
structure (17-19). With some exceptions (6, 20), most PDZ domains bind
to the last 3-4 amino acids at the carboxyl-terminal tail of target
proteins (21). Based on the target or ligand sequences, PDZ domains can
be subdivided into two classes: type I and type II (21). Type I PDZ
domains recognize ligands that contain either a Ser or Thr at the
2
position. In contrast, type II domains bind to ligands containing a
bulky residue such as Phe or Tyr at the corresponding position (19, 21). Almost all PDZ-interacting ligands have a hydrophobic residue (Ile, Leu, or Val) at the carboxyl terminus (position 0) (2-5, 8).
X-ray crystallographic studies revealed that the tetrapeptide ligand
anchors to a groove formed between the second
-strand and the second
-helix of the PDZ domain (17, 19). Each PDZ domain appears to
recognize a unique carboxyl-terminal sequence (21).
Because the carboxyl-terminal of eye-PKC (11) contains a type I PDZ
ligand, we sought to identify a type I domain in INAD. Sequence
alignment with the third PDZ domain (PDZ3) of PSD95, a type I domain,
indicates that PDZ2 is the only type I domain in INAD. PDZ3 of PSD95
interacts with a tetrapeptide, Gln-Thr-Ser-Val (17), and the side
chains of Gln
3 form hydrogen bonds with those of Ser and
Asn in PDZ3 of PSD95. In contrast, the corresponding residue of
Gln
3 in eye-PKC is Ile
3, a residue with a
hydrophobic side chain. We explored how a different residue at the
3
position of the target may interact with the different type I PDZ
domains. We also performed site-directed mutagenesis by modifying
Ile
3 of eye-PKC and investigated the contribution of this
residue in the PDZ2 recognition. To explore the basis of the type I
interaction, we mutated a conserved His in the second
-helix of
PDZ2. This His has been implicated in the interaction with Ser/Thr at
the
2 position (17). Interestingly, we found that substitution of
His310 with a Leu resulted in enhanced eye-PKC interaction,
whereas replacement with Arg led to a reduction of association.
To gain insight into the regulation of InaD expression, we
analyzed the genomic structure of InaD and mapped the
transcription start site by RACE. The InaD gene contains
nine exons. Interestingly, we found SNPs in the coding exons leading to
substitutions in three residues of PDZ2. We analyzed these variant PDZ2
and show that two modified PDZ domains display an increase in eye-PKC binding.
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EXPERIMENTAL PROCEDURES |
Materials--
Ficoll, 5-bromo-4-chloro-3-indolyl phosphate, and
nitroblue tetrazolium were obtained from Research Organic, Inc.
(Cleveland, OH). Phenol and deoxynucleotide triphosphates were
purchased from U. S. Biochemicals. Chloroform and other chemicals were
from Fisher. Nitrocellulose filters were from Schleicher & Schuell. DNA
size markers were purchased from Life Technologies, Inc. Restriction enzymes and modifying enzymes such as T4 DNA ligase were obtained from
New England Biolabs (Beverly, MA), Stratagene (La Jolla, CA), or
Promega (Madison, WI). RNasin and pGEMEX1 were obtained from Promega.
[32P]dCTP was from PerkinElmer Life Sciences.
Genomic Library Screening--
A Drosophila genomic
library prepared from Canton S strain in Charon 4 vector (22) was
plated. Filter replicas of the library were probed with radioactively
labeled InaD in a hybridization solution (5× SSCP, 0.5%
SDS, 10 mM EDTA, 5× Denhardt's solution) at 65 °C for
16 h. Subsequently filters were rinsed with a buffer containing
0.1× SSC and 0.1% SDS at 65 °C for 2 h (23). Positive plaques
were identified by autoradiography and purified. The EcoRI inserts containing the InaD gene were identified and
subcloned into the pBluescript KS vector (Stratagene) for sequence analysis.
DNA Sequencing--
The nucleotide sequence was determined
either by the dideoxy chain termination method (24) using the Sequenase
kit (Amersham Pharmacia Biotech) or by automatic DNA sequencing using
ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready
reaction kit (PerkinElmer Life Sciences). For DNA obtained by PCR at
least four to six independent subclones were sequenced.
Isolation of Fly Genomic DNA--
Total genomic DNA was isolated
as described previously (1). Briefly, 50 flies were gently homogenized
in 300 µl of buffer (100 mM NaCl, 100 mM
Tris, pH 7.6, 100 mM EDTA, 0.5% SDS). The homogenates were
incubated at 65 °C for 30 min, and 8 M potassium acetate
(one sixth volume) was added. The mixture was incubated on ice for 20 min, and the supernatant was recovered following centrifugation. RNase
A (50 µg) was added to the supernatant to hydrolyze RNA. The mixture
was subject to phenol/chloroform extraction to remove proteins. Ethanol
was added to the supernatant to precipitate genomic DNA.
Isolation of RNA and Synthesis of First Strand cDNA--
Fly
heads from various strains were isolated, and total RNA were extracted
according to Chomczynski and Sacchi (25). Total RNA was precipitated by
ethanol and quantified by spectrophotometry. To generate first strand
cDNA, 20 µg of total RNA were added to a 25-µl reverse
transcription reaction using the reverse transcription system
(Promega). Following incubation at 42 °C for 1 h, the reaction was terminated by addition of 75 µl of 10 mM EDTA (pH
8.0). An aliquot of cDNA (1-2 µg of RNA equivalent) was used as
templates for PCR analysis.
Polymerase Chain Reaction--
PCR was used to amplify genomic
DNA as well as cDNA using InaD-specific primers. A
negative control containing no added templates was also performed to
assure the specificity of amplifications. The amplified fragments were
subcloned into pCR2.1 using the TOPO TA Cloning system (Invitrogen),
and recombinant plasmids were purified and used for sequencing. The
experimental conditions for PCR (30 cycles) were denaturation at
94 °C for 30 s and annealing at 50 °C for 30 s followed
by extension at 72 °C for 2-3 min. The reaction mixture (50 µl)
contained 50-100 ng of each primer, 1 µg of total genomic DNA (or
first strand cDNA from 1-2 µg total RNA) as templates, 0.2 mM dNTP, and 2.5 units of Taq DNA polymerase (PerkinElmer Life Sciences), in a buffer containing 10 mM Tris (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2. Primers for InaD genomic and cDNA amplification were primers b and d (see Fig.
4A). Primer sequences for the amplification of the eye-PKC
carboxyl tail were TTC GAA TTC ATG GCA GGT (5') and TAT GGA TCC TTA AAT
GAT GGT TAT AAA CTC (3').
Rapid Amplification of cDNA Ends--
RACE was performed
using the Marathon cDNA amplification kit
(CLONTECH). Briefly, total RNA from
Drosophila head was primed with a modified
oligo(dT)30 primer to generate first strand cDNA by
avian myeloblastosis virus reverse transcriptase. The first strand
cDNA was then used as templates for the second strand cDNA synthesis mediated by RNase H, Escherichia coli DNA
polymerase, and E. coli DNA ligase. After ligating with an
adaptor primer, the double-strand cDNA was used as templates for
PCR using a gene-specific primer, b (see Fig. 4A) and AP1
primer that anneals to the adaptor primer. A second PCR reaction using
the initial PCR mixture as templates and a different set of primers,
primer a (see Fig. 4A) and AP1, was followed. Two antisense
primers, a and b (see Fig. 4A), were used for the 5' RACE,
and two sense primers, d and e (see Fig. 4A), were used
for 3' RACE. All four InaD-specific primers have melting
temperatures of 72 °C. The DNA fragments obtained from the nested
PCR amplification were subcloned and sequenced. Primer sequences for
InaD are CTT GTC CAG GGT CAC CAT GTG AAT (primer a), CAG CAG
CAT GTC GCC CAC TTT CA (primer b), TCA AGC AGC GAG GAT GGT TCA GTT
(primer d), and GGC ATG TGC GTC AAG CCC ATC AA (primer e).
Site-directed Mutagenesis--
Site-directed mutagenesis was
performed using the overlap extension method as described (26).
Fusion Protein Expression in Bacteria--
Carboxyl-terminal
tails of wild-type eye-PKC (641) and PDZ2 (206) of INAD were
expressed as fusion proteins of glutathione S-transferase
(GST). Briefly, recombinant pGEX4T1 plasmids were transformed into
E. coli HMS174. Overnight cultures (1 ml) were prepared from
a single colony and used to inoculate a 50-ml LB broth containing
ampicillin. The cultures were grown at 37 °C for 2-3 h until the
density of bacterial cultures (OD600) reached 0.6-0.7. The
expression of fusion proteins was initiated by the addition of
isopropyl-1-thio-
-D-galactopyranoside (final
concentration, 0.1-1 mM) (27). The cultures were harvested
3 h following induction, and bacterial pellets were collected by
centrifugation. Bacterial lysates containing the fusion protein were
prepared by resuspending the pellets in binding buffer (50 mM K3PO4, pH 7.0, 150 mM KCl, 10 mM MgCl2, 10% glycerol,
1% Triton X-100 plus a mixture of protease inhibitors) followed by
repeated sonication.
Radiolabeling of Proteins--
Recombinant plasmids containing
target cDNA in pGEMEX1 (Promega) were constructed and used as
templates for T7 RNA polymerase-dependent transcription.
Incorporation of [35S]methionine into T7 gene 10 fusions
containing PDZ2 (206), the carboxyl-terminal tail of eye-PKC
(562), and PDZ4 (485) was accomplished by in vitro
transcription and translation using the TNT-coupled reticulocyte lysate
system (Promega) (6). Briefly, 25 µl of reticulocyte lysates were
added for a 50-µl reaction that contained [35S]methionine (40 µCi), circular plasmid templates
(0.2-1 µg), RNasin (40 units), and T7 RNA polymerase (1 µl).
In vitro translated radiolabeled proteins were analyzed by
SDS/PAGE or used directly for pull-down assays.
GST Fusion Protein Pull-down Assays--
Bacterial lysates
containing similar amounts (5 µg) of GST fusion proteins or GST were
incubated with radiolabeled target proteins (10-20-fold excess) in
binding buffer at 4 °C for 1 h. The reaction mixture (55 µl)
was transferred to an Eppendorf tube containing10 µl of
glutathione-Sepharose beads (Amersham Pharmacia Biotech) prewashed with
binding buffer. Incubation proceeded for 1 h at 4 °C with
constant agitation for binding of GST fusion proteins to the beads. The
supernatant of the mixture was removed, and an aliquot (5%) was
analyzed by SDS/PAGE. The beads were rinsed with binding buffer (100 µl) three times to remove nonspecific binding. The GST fusion protein
with bound radioactive proteins was eluted with SDS/PAGE loading buffer
and analyzed on SDS/PAGE. Radioactivity was detected by autoradiography
or with a PhosphorImager (445SI, Molecular Dynamics). Affinity of
interaction was determined by the amount of bound radioactive probes.
Nonspecific binding to GST was used as a negative control.
Western Blot Analysis--
Western blotting was performed using
alkaline phosphatase-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories). The presence of antigens was visualized
upon staining with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue
tetrazolium. Polyclonal anti-GST antibodies were purchased from
Transduction Laboratories (San Diego, CA).
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RESULTS |
PDZ2 Is a Type I Domain and Interacts with an
Xaa-(Ser/Thr)-Xaa-(Val/Leu/Ile) Sequence--
Previously our
laboratory reported that PDZ2 interacted with the carboxyl-terminal
sequence of eye-PKC (11). However, Tsunoda et al. (9) showed
that retinal eye-PKC associated with PDZ4 by pull-down assays. Xu
et al. (16) suggested the involvement of PDZ3 and PDZ4 in
the eye-PKC association by heterologous expression and
co-immunoprecipitation. Questions remain as to which PDZ domains are
important for the eye-PKC association. Importantly, both Xu et
al. (16) and Adamski et al. (11) pointed out the
involvement of the carboxyl tail of eye-PKC; a point mutation that
converted the last residue of eye-PKC, Ile, to an Asp, led to a drastic reduction of the INAD interaction. To investigate which PDZ domain of
INAD associates with eye-PKC, we examined the carboxyl-terminal tail of
eye-PKC. Eye-PKC terminates with Ile-Thr-Ile-Ile, consistent with an
interaction with a type I PDZ domain. The molecular basis of the
interaction between a type I PDZ domain and a carboxyl-terminal sequence has been revealed in the x-ray crystallographic studies of
PDZ3 of PSD95 and a target peptide (17).
We aligned PDZ3 of PSD95 with five PDZ domains of INAD and found that
only PDZ2 resembles a type I domain (Fig.
1A) because several critical
residues involved in target binding are conserved. In particular, the
His residue located at the beginning of the second
-helix (
B) is
conserved in PDZ2 of INAD, His310. The N-3 nitrogen of
His310 is likely to be involved in hydrogen bonding with
the hydroxyl side chain of Ser/Thr at the
2 position of the target
(Fig. 1B). The presence of (Ser/Thr)
2 is a
hallmark of the type I ligand. Consistently, eye-PKC contains Thr at
the
2 position for an interaction with PDZ2; none of the other PDZ
domains of INAD contain a His at the corresponding position. Another
conserved residue in PDZ2 includes Arg254 implicated in
binding to the terminal carboxyl group of the target (Fig. 1). By
analogy, the hydrophobic pocket for binding to the terminal hydrophobic
residue of eye-PKC, Ile0, is contributed by
Leu260, Leu262, Leu264, and
Phe317 from PDZ2 (Fig. 1B). Overall, PDZ2 of
INAD and PDZ3 of PSD95 share 28% sequence identity.

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Fig. 1.
PDZ2 of INAD is a prototypical PDZ domain
containing a conserved His in the second
-helix. A, sequence alignment of
PDZ2 with the third PDZ domain of PSD95. Amino acid identities are
shown in bold type. The six -strands
( A- F) and two -helices
( A and B) are
boxed and labeled. The carboxylate binding loop
is indicated with an arrow. Three residues with observed
polymorphism in INAD are labeled with asterisks. Key amino
acids involved in binding to target are numbered.
B, a hypothetical chemical interaction between PDZ2 and the
carboxyl-terminal tail of eye-PKC. Amino acids in PDZ2 are labeled with
single-letter codes. The last four residues of eye-PKC,
Ile-Thr-Ile-Ile, are critical for binding to PDZ2.
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It is noteworthy that PDZ3 of PSD95 associates with a target sequence,
Gln-Thr-Ser-Val. The polar side chain of Gln at the
3 position,
specifically the free carbonyl group, forms hydrogen bonds with the
polar side chains of Asn326 and Ser339 of PDZ3
(17). In contrast, PDZ2 of INAD recognizes eye-PKC containing a
hydrophobic residue, Ile, at the
3 position. The side chain of Ile
does not participate in hydrogen bonding. Consistently, the
corresponding residues in PDZ2 for bonding with Ile
3 are
Ala263 and Ala278 (Fig. 1), whose side chains
also do not form hydrogen bonds. The divergence of these two residues
predicted to interact with Ile
3 of eye-PKC further lends
support for the role of PDZ2 in interacting with the carboxyl terminus
of eye-PKC.
Site-directed Mutagenesis of Eye-PKC Tail Sequence--
To
investigate whether Ile
3 of eye-PKC is involved in the
PDZ2 interaction, we generated and characterized two point mutants. The
codon of Ile
3 was replaced with that of Glu
(Ile
3
Glu) or Lys (Ile
3
Lys) by
site-directed mutagenesis. Wild-type and modified eye-PKC cDNAs
were used to generate radiolabeled fusion protein for PDZ2 binding. As
shown in Fig. 2, we detected a drastic
reduction of the PDZ2 interaction in these two mutants. In particular,
the Glu substitution (Ile
3
Glu) displayed a total
loss of the association (Fig. 2A, lane 4, and
Fig. 2B). These findings indicate that Ile
3 of
eye-PKC is critically involved in the PDZ2 interaction; substitutions with charged residues almost abolished the association.

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Fig. 2.
Modified eye-PKC containing substitution at
the 3 position displays a reduction of the INAD association.
A, a pull-down assay to examine PDZ2 binding to wild-type or
mutant eye-PKC. Top panel, an autoradiogram showing the
amount of 35S-labeled PKC retained following incubation
with GST (lanes 1, 3, and 5) or
GST-PDZ2 fusion protein (lanes 2, 4, and
6). Radioactive probes (5% of input) in the reaction are
shown (middle panel). The amount of GST and GST-PDZ2 in the
reaction was determined by Western blotting (bottom panel).
B, a histogram depicting relative binding of two mutant
eye-PKC affecting Ile 3 in the carboxyl terminus. The
amount of radioactivity recovered was quantitated by PhosphorImager and
relative level of binding plotted (n = 3). Wild-type
eye-PKC exhibits a stronger interaction with PDZ2. In contrast,
substitutions of Ile 3 with either Glu (Ile 3
Glu) or Lys (Ile 3 Lys) led to a drastic reduction
of the association.
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Site-directed Mutagenesis of INAD PDZ2--
As mentioned above,
type I PDZ domains usually contain a basic residue, either His or Arg,
at the beginning of
B (Fig. 1A) for binding to
(Ser/Thr)
2 of type I targets. In contrast, type II
targets contain either a bulky hydrophobic or Tyr at the
2 position
that interacts with a hydrophobic residue (e.g. Val) in the
corresponding
B position of type II PDZ domains. We examined the
involvement of His310 in
B of PDZ2 by amino acid
replacement followed by pull-down assays. When His was substituted by
Arg (H310R), the interaction with wild-type eye-PKC was reduced to
about 50% (Fig. 3). Interestingly, the
Leu substitution (H310L) led to a 1-fold increase in the eye-PKC interaction. We also tested the eye-PKC binding to PDZ4 of INAD and
showed a much weaker binding compared with that of PDZ2 (Fig. 3).

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Fig. 3.
Mutations at the histidine residue in the
second -helix of PDZ2 affect the eye-PKC
interaction. A, a pull-down assay to examine the
eye-PKC binding to wild-type (from W1118), modified PDZ2,
and PDZ4. Top panel, an autoradiogram showing the amount of
35S-labeled PDZ2 or PDZ4 retained following incubation with
GST (lanes 1, 3, 5, and 7)
or GST-PKC fusion protein (lanes 2, 4,
6, and 8). Radioactive PDZ2 probes (5%) in each
reaction are shown (middle panel). The amount of GST and
GST-PKC used for the pull-down assay was determined by Western blotting
(bottom panel). B, the relative level of eye-PKC
binding to modified PDZ2 or wild-type PDZ4 compared with wild-type PDZ2
from W1118 is shown in a histogram (n = 3).
Substitution of His310 with a Leu led to an increase in
eye-PKC binding, whereas the Arg substitution resulted in about 50%
reduction. PDZ4 of INAD exhibited a weaker interaction with eye-PKC,
compared with PDZ2. The enhanced binding of PDZ2 (H310L) was abolished
by a second mutation converting Arg308 to a Gly.
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Both His and Arg have side chains capable of hydrogen bonding with
(Ser/Thr)
2; however, Arg has an aliphatic side chain
instead of an imidazole ring like His. It is likely that differences in
the side chains at the corresponding
B position lead to a change in
the eye-PKC interaction. When His310 was substituted by Leu
whose side chain does not support hydrogen bonding, an increased
affinity toward eye-PKC was observed. We proposed that polar side
chains from a neighboring residue of His310, such as
Arg308, can engage in hydrogen bonding with the hydroxyl
group of Thr
2 of eye-PKC to stabilize the interaction in
the H310L mutant. To investigate whether Arg308 is
important for the observed eye-PKC association, we substituted Arg308 with Gly in the H310L background (R308G,H310L). We
show that double mutants displayed a great reduction of the eye-PKC
association (Fig. 3B), suggesting that Arg308 is
essential for the eye-PKC interaction in the H310L mutant.
Genomic Structure of the InaD Gene--
We identified the
InaD gene and investigated its genomic organization. Genomic
clones were obtained either by screening a Drosophila
genomic library or by gene amplification via PCR (Fig. 4A). The InaD gene
was subjected to restriction enzyme mapping (Fig. 4A) and
nucleotide sequencing. Comparison of the genomic DNA (accession number
AF245280) with cDNA sequences provided the basis for the
exon/intron organization as shown in Fig. 4A (middle
panel). The coding sequence of InaD is contained within a 3-kilobase genomic fragment that has nine exons interrupted by eight
intervening sequences. All introns are rather small in size ranging
from 54 to 368 nucleotides and are flanked by conserved 5' and 3'
consensus sequences (28) at each end. The InaD gene product
is composed of five distinct PDZ domains (2, 3, 9, 11). To reveal
whether each PDZ domain is encoded within an exon, we projected the
location of introns in the translation product of InaD (Fig.
4A, bottom panel). We found both PDZ1 and PDZ2
are encoded by only one exon, whereas the remaining three PDZ domains
are encoded by two adjacent exons (Fig. 4A).

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Fig. 4.
Genomic organization of the InaD
locus. A, genomic structure of the
InaD locus. Shown at the top is the restriction
map of a genomic clone. The direction of transcription is indicated as
an arrow above. The locations of four oligonucleotide
primers (a, b, c, and d)
used for RACE (see "Experimental Procedures") are indicated below
as arrows. The restriction map of the InaD
cDNA is shown in the middle panel. The location of eight
introns is indicated as triangles, and the sizes are indicated. The
translation product of InaD is depicted below the
cDNA, and each of the five PDZ domains is labeled as a filled
square. The locations of amino acid residues that show
polymorphism are marked with asterisks. RI,
EcoRI; RV, EcoRV; bp, base pair;
nt, nucleotides. B, ethidium bromide staining of
a DNA gel containing DNA fragments obtained by RACE. Lane 1,
initial 5' RACE PCR reaction using AP1 and b primers; lane
2, a nested PCR using the initial PCR mixtures as templates with
AP1 and a primers resulted in a 0.6-kilobase 5' RACE product;
lane 3, initial 3' RACE PCR reaction (using AP1 and d
primers); lane 4, a nested PCR (using AP1 and e primers)
gave rise to a 1.0-kilobase 3' RACE product. DNA molecular mass
standards (lane M) are shown on the left.
C, nucleotide sequence of the 5'-UTR and regulatory sequence
of the InaD gene. Transcription initiation site of the
InaD gene (+1) is localized about 157 nucleotides upstream
of the translation initiation ATG. The translated exon sequence is
shown in bold type. The putative cis element for
photoreceptor-specific expression is boxed and is 40 nucleotides 5' upstream of the transcription initiation site. The
underlined sequences are parts of the promoter
sequences.
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To determine the transcription start site of the InaD gene,
we employed 5' RACE (Fig. 4B) followed by DNA sequencing.
Similarly, the 3'-untranslated region (UTR) was also determined (Fig.
4B). The InaD cDNA has 157 nucleotides in the
5'-UTR and 67 nucleotides in the 3'-UTR. We also sequenced several
InaD cDNAs from the expressed sequence tags collection
generated by the Berkeley Drosophila Genome Project (29).
Two of the longest cDNAs (accession numbers AA567782 and AA697690)
contain an additional 23 nucleotides extending beyond our
experimentally determined transcription initiation site (+1) (Fig.
4C). It is possible that multiple transcription start sites
of InaD are used. Alternatively, premature chain termination by reverse transcriptase during the synthesis of cDNA may have also
resulted in a shorter 5'-UTR.
The 5'-UTR and the adjacent upstream sequence of InaD are
shown in Fig. 4C. We found stretches of TA-rich sequences
upstream of the transcription initiation site (Fig. 4C) that
could serve as binding sites for general transcription factors (30).
Moreover, we also found sequences similar to the photoreceptor-specific cis-acting element, CTAATTGAATT (31), at 40 nucleotides
upstream of the putative transcription start site (Fig. 4C).
This sequence may be important for controlling the
photoreceptor-specific expression of InaD.
Polymorphisms in InaD--
We compared nucleotide sequences of
genomic DNA and cDNA and first noted an inconsistency in the codon
of amino acid 319 (Table I). Genomic DNA
obtained from the Canton S strain encodes a Ser (AGC) at 319 instead of
an Asn (AAC) as seen in several InaD cDNAs. This
discrepancy could be either post-transcriptional modifications such as
editing or single nucleotide polymorphisms (SNPs). To distinguish
between these two possibilities, we isolated and sequenced both genomic
and cDNA sequences from three laboratory strains including two
wild-type strains, W1118 and Oregon R, and
InaDp215 (Table I). If editing occurs at a given
codon, one expects that cDNA sequences will be different from
genomic sequences obtained from the same strain. However, we found both
genomic and cDNA sequences from each of these three strains are the
same at codon 319. In both W1118 and Oregon R, the codon
(AAC) at 319 encodes Asn. In contrast, in
InaDp215 and likely Canton S, Ser (AGC) was
encoded at 319 (Table I). The corresponding amino acid in
Calliphora INAD is Asn (32). This finding ruled out the
possibility of post-transcriptional modifications and provided support
for polymorphisms at residue 319.
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Table I
SNPs of the InaD locus in D. melanogaster
Genomic and cDNA sequences corresponding to residues 282, 319, and
333 of InaD from four strains of Drosophila
melanogaster are shown.
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Additional SNPs were also observed. In Oregon R, we found nucleotide
substitutions in residues 282 and 333. Pro282 is located in
the second PDZ domain, and it is substituted by Leu in the Oregon R
strain (Table I). The corresponding residue in Calliphora
INAD is Thr32. Lys333 is also localized in
PDZ2, and it is replaced by Gln in Oregon R. It is interesting to
detect substitutions in PDZ2 because PDZ2 is critically involved in the
eye-PKC interaction. It is likely that these changes may not lead to
any significant alteration in the folding of PDZ2, and consequently the
interaction with eye-PKC remains intact. Another possibility is that
there are concomitant changes in the carboxyl-terminal tail of eye-PKC, which compensate for these modifications in INAD.
To investigate whether any polymorphisms in eye-PKC exist, we amplified
and sequenced the genomic sequences encoding the last 139 amino acids
of eye-PKC from both the Oregon R and the
InaDp215strains. However, we did not observe any
SNPs for eye-PKC (562) (data not shown).
Interaction between Eye-PKC and PDZ2 Domain Variants--
We
investigated whether polymorphic PDZ2 domains display different
affinities toward eye-PKC. 35S-Labeled PDZ2 domains (N319S,
P282L, and Q333K) were generated for pull-down assays. The eye-PKC
interaction was compared with PDZ2 present in wild-type
W1118 and quantitated. As shown in Fig.
5B, we detected a stronger eye-PKC interaction in both PDZ2 (N319S) and PDZ2 (P282L); particularly PDZ2 (N319S) displayed a 2-fold increase in the eye-PKC binding. However, PDZ2 (Q333K) displayed binding similar to wild-type PDZ2.

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Fig. 5.
Enhanced eye-PKC association of two naturally
occurring PDZ2 variants. A, results of a pull-down
assay to examine eye-PKC binding by wild-type (PDZ2 from
W1118) and variant PDZ2 domains. Top panel, an
autoradiogram showing the amount of 35S-labeled PDZ2
retained following incubation with GST (lanes 1,
3, and 5) or GST-PKC fusion protein (lanes
2, 4, and 6). Radioactive probes (5% of
total) in the reaction are shown (middle panel). The amount
of GST and GST-PKC used for the pull-down assay was determined by
Western blotting (bottom panel). B, a histogram
depicts relative level of eye-PKC binding. Both PDZ2 (N319S) and PDZ2
(P282L) exhibited an enhanced eye-PKC association.
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DISCUSSION |
PDZ domains are protein-protein interaction motifs found in a
variety of signaling molecules. Prototypical PDZ domains recognize a
tetrapeptide ligand located at the carboxyl-terminal tail of the target
protein (17, 19). The PDZ-target interaction has been implicated in
subcellular localization and stabilization of receptors or other
interacting proteins (4, 5, 19). INAD contains five distinct PDZ
domains that are shown to associate with phospholipase C
, eye-PKC,
and TRP. Like INAD, proteins containing multiple PDZ domains are
capable of tethering several signaling proteins leading to the
formation of a signal transduction complex (transducisome or
signalplex). This clustering of signaling proteins facilitates
protein-protein interactions leading to fast kinetics of signaling
processes. Furthermore, restricted distribution of proteins may
contribute to specificity of signaling mechanisms, thereby preventing
undesired cross-talks. Thus, INAD serves to illustrate the importance
of scaffolding proteins in multi-component signaling processes such as
G protein-coupled pathways.
PDZ2 Interacts with the Carboxyl-terminal Tail of Eye-PKC--
We
previously showed that the last three residues of eye-PKC interact with
PDZ2 of INAD by protein overlay and yeast two-hybrid assays (11).
Reports by Xu et al. (16) also implicated the importance of
the carboxyl terminus of eye-PKC in the INAD association. The carboxyl
terminus of eye-PKC contains the motif Ile-Thr-Ile-Ile, which is known
to associate with a type I PDZ domain. However, there is a discrepancy
as to which PDZ domain of INAD is responsible for anchoring eye-PKC to
the complex (9, 16). Here we show that only PDZ2 is a type I domain
that contains a conserved His, His310, in the second
-helix. Based on the structure of a PDZ domain-ligand complex (17),
the side chain of this His is involved in hydrogen bonding with that of
Thr/Ser at the
2 position of the type I ligand. Indeed, we show that
Arg replacement of His310 in PDZ2 still promoted the
eye-PKC interaction, although the affinity was reduced to about 50% of
wild type. Arg is capable of hydrogen bonding similar to His; however,
it has an aliphatic side chain backbone instead of an imidazole ring.
Differences in structure of the side chain may lead to alteration in
the PDZ-target binding. Surprisingly, we detected an enhanced eye-PKC
interaction when His310 was substituted with a Leu in PDZ2.
As the side chain of Leu does not form hydrogen bonds, we speculated
that a neighboring basic residue (Arg, His, or Lys) might serve as an
alternative residue for anchoring Thr
2 of eye-PKC.
Further mutational analysis of Arg308 confirmed this hypothesis.
How does Leu substitution of His310 in PDZ2 lead to an
increase in the eye-PKC interaction? As mentioned, the tail sequence of eye-PKC or ligand of PDZ2 is rather hydrophobic with three of the four
residues being Ile. The presence of an additional hydrophobic residue
in the binding pocket as in the Leu substituted PDZ2 may positively
increase the eye-PKC association via additional hydrophobic interactions, provided that a neighboring basic residue, such as
Arg308, can be substituted for His310. Based on
these findings, we suggest that the ligand binding pocket of PDZ
domains is flexible for allowing a slight rotation of ligand in the
binding pocket for optimal interaction. The presence of
Arg308 is a unique feature of PDZ2 because none of the four
PDZ domains of INAD contain a basic residue at the corresponding position.
We also examined the involvement of Ile
3 of eye-PKC,
because the residue at the
3 of the ligand also contributes to
specificity of PDZ-target interactions. We show that replacement of
Ile
3 with charged residues such as Glu or Lys led to a
drastic reduction of the PDZ2 association. However, substitution of Ile
with a hydrophobic residue, Phe, was tolerated without significantly
affecting the PDZ2 interaction as previously shown in the yeast
two-hybrid assay (11). Additional evidence supporting the contribution
of Ile
3 was obtained by examining the structure of PDZ3
of PSD95. PDZ3 associates with a target sequence containing Gln at
3.
The polar side chain of Gln
3 forms hydrogen bonds with
those of Asn326 and Ser339 in PDZ3 of PSD95. In
contrast, eye-PKC has an Ile at the
3 position, which contains a
hydrophobic side chain. Consistently, the projected residues for
interacting with Ile
3 in PDZ2 of INAD are
Ala263 and Ala278, which have hydrophobic side
chains. We conclude that the interaction between PDZ2 and the
carboxyl-terminal of eye-PKC belongs to that of the type I PDZ domain
and that basic residues flanking the critical His in the
B can be
substituted for this critical His for anchoring
(Ser/Thr)
2 of the ligand. Moreover, our results indicate
that PDZ2 of INAD prefers a hydrophobic residue at the
3 position of
the target.
Amino Acid Polymorphisms in PDZ2--
Nucleotide sequencing of
several wild-type InaD alleles revealed SNPs in three
residues of the InaD gene product. All three substitutions
are in PDZ2 including Leu substitution for Pro282 (P282L),
Ser substitution at Asn319 (N319S), and Gln substitution at
Lys333 (K333Q). K333Q is located beyond the sixth
-strand, N319S is in the linker region flanking the second
-helix
and the sixth
-strand, and P282L is located in the linker region
between the third
-strand and the first
-helix (Fig.
1A). These three residues have not been implicated in
critical bonding with the target sequence, but substitutions may affect
overall folding of PDZ2 leading to an alteration in the eye-PKC
interaction. Indeed, P282L and N319S display an increased affinity
toward eye-PKC in pull-down assays. P282L was detected in Oregon R,
whereas N319S was detected in the Canton S strain.
Both Canton S and Oregon R strains display wild-type electroretinograms
similar to another wild-type strain, W1118. It is likely
that a change of the INAD-PKC interaction results in subtle phenotype
that is detected only in sensitive assays such as patch clamp
recordings of dissociated photoreceptors. To date, there are no reports
on comparison of various wild-type responses using the patch clamp
analysis. It is also possible that the INAD-PKC interaction is optimal
in W1118 and that a further increase in affinity does not
result in any electrophysiological phenotype.
In summary, we evaluated the association between eye-PKC and PDZ2 and
conclude that PDZ2 is a type I PDZ domain interacting with a type I PDZ
ligand at the carboxyl tail of eye-PKC. The binding pocket of PDZ2 is
unique; Leu substitution of a conserved His leads to an increased
eye-PKC interaction, likely because of the use of a flanking Arg for
binding to (Ser/Thr)
2 of the target protein. We isolated
the InaD gene and found three SNPs in PDZ2 resulting in
amino acid substitution at residues 282, 319, and 333. PDZ2 containing
P282L or N319S polymorphism leads to an increased eye-PKC binding.
However, the electrophysiological phenotype associated with an enhanced
eye-PKC binding may be subtle and remains to be investigated. The role
of the INAD-PKC interaction may have a primary role in localization of
eye-PKC and targeting the kinase to its substrates. Because the
interaction between eye-PKC and INAD appears constitutive, a change of
affinities may not have an overt effect on the overall visual signaling.