From the Emil-Fischer-Zentrum, Institut für
Biochemie, Friedrich-Alexander-Universität
Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany
and § Max-Planck-Institut für Hirnforschung,
Abteilung Neuroanatomie, Deutschordenstrasse 46, 60528 Frankfurt, Germany
Received for publication, May 24, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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The correct targeting of modifying enzymes to ion
channels and neurotransmitter receptors represents an important
biological mechanism to control neuronal excitability. The recent
cloning of protein kinase C-zeta interacting
proteins (ZIP1, ZIP2) identified new scaffolds linking the
atypical protein kinase PKC- Mammalian GABAC receptors are composed of three Recently, two proteins interacting with the TM3-TM4 loops of
GABAC receptor Screening a rat brain cDNA-library for proteins binding to the
GABAC receptor All experiments were performed in compliance with the guidelines
for the welfare of experimental animals issued by the Federal Government of Germany, the National Institutes of Health, and the Max
Planck Society.
Yeast Two-hybrid Techniques--
Unless specified otherwise, all
reagents were parts of the MATCHMAKER GAL4 Two-hybrid System 3 (Clontech, Palo Alto, CA). The large intracellular
loop between TM3 and TM4 of the rat GABAC receptor
To map interacting protein domains, PCR-amplified DNA fragments of
ZIP3, GABAC, and GABAA receptor subunits (see
Fig. 2 for details) were subcloned in bait or prey yeast vectors using
the EcoRI-XhoI sites. After generating individual
yeast strains expressing the constructs, protein-protein interactions
were analyzed by transforming bait strains with pGADT7-constructs and
prey strains with pGBKT7-constructs. Activation of the ADE2, HIS3, and
Pull-down Techniques--
Unless otherwise stated, all reagents
were purchased from Novagen (Madison, WI). The TM3-TM4 loops of the rat
GABAC receptor
The complete coding sequence of the rat Preparation of Protein Extracts from HEK-293 Cells and Rat
Brain--
Human embryonic kidney cells (HEK-293, ATCC CRL1573) were
transfected (45) with cDNAs encoding for T7-tagged ZIP3, the
Adult rat brains were homogenized on ice in 10 ml/mg tissue of
homogenization buffer I (0.32 M saccharose, 20 mM Tris-HCl, pH 7.4) containing 10 mg/ml DNaseI and
protease inhibitors (Roche Molecular Biochemicals) using a glass/Teflon
homogenizer and centrifuged at 30,000 × g for 30 min,
and the resulting crude membrane pellet was homogenized again in 5 ml/mg tissue of hypotonic homogenization buffer II (20 mM
Tris-HCl, pH 7.4, plus protease inhibitors) to release proteins inside
cytosolic vesicles. After centrifugation at 30,000 × g
for 30 min, the supernatant was separated completely from the pellet,
which was solubilized in 5 ml/mg tissue of buffer III (20 mM Tris-HCl, 200 mM NaCl, 1% Triton X-100, pH
7.4, plus protease inhibitors) for 2 h. Ultracentrifugation was
carried out at 100,000 × g for 1 h, and the
supernatant was saved (P2). For binding assays, ~5 mg of S1 and P2
protein fractions were incubated with beads coated with GST-ZIP3, the
His-tagged
Primary antibodies were used as follows for Western blotting: rabbit
anti-PKC- Immunocytochemistry--
Adult Wistar rats were anesthetized
with halothane and decapitated. The posterior eye cups with the retinas
attached were immersion-fixed for 15-30 min in 4% (w/v)
paraformaldehyde in phosphate buffer (PB; 0.1 M, pH 7.4).
The retinas were dissected and cryoprotected in 10% (w/v), 20% (w/v)
sucrose in PB for 1 h each, and in 30% (w/v) sucrose in PB
overnight at 4 °C. Pieces of retinas were mounted in freezing medium
(Reichert-Jung, Bensheim, Germany), sectioned vertically at a thickness
of 12 µm with a cryostat and processed for immunocytochemistry.
The following antibodies were used: a rabbit polyclonal antiserum
(1:100) recognizing the Isolation of RNA and Reverse Transcription-PCR--
Total
RNA was extracted from brain, lung, liver, kidney, and spleen using the
TRIZOL Reagent according to the manufacturer's protocol (Invitrogen)
and from cerebellum, cortex, hippocampus, olfactory bulb, retina,
thalamus, and spinal cord of an adult rat following a method described
by Chomczynski and Sacchi (46). For cDNA-synthesis, 3 µg of total
RNA were incubated in 20 µl of cDNA-synthesis buffer (in
mM: 50 Tris-HCl, 3 MgCl2, 75 KCl, 10 dithiothreitol, pH 8.3), 0.5 mM of each dNTP (Amersham
Biosciences), 250 ng of p(dN)6 (Boehringer, Mannheim,
Germany), 20 Units RNasin (Roche Molecular Biochemicals) and 400 units
of SuperscriptII RNaseH ZIP3 Represents a New C-terminal Splice Variant of the
PKC-
To analyze the distribution of ZIP3, total RNA from different tissues
was reverse-transcribed and subjected to PCR amplification. ZIP3 was
present at similar levels in all organs analyzed, including the brain
(Fig. 1B). Within the central nervous system, ZIP3 was abundantly expressed in spinal cord, thalamus, cortex, and the retina
(Fig. 1B, right panels). PCR products of similar
intensity for Mapping of Interacting Domains between ZIP3 and GABAC
Receptor
The specific interaction of ZIP3 with the
To map the location of amino acids that would act in combination with
the 10 amino acids of the
Next, regions of ZIP3 were analyzed for their capability to bind the
ZIP3 Is Able to Dimerize and to Interact with GABAC
Receptor
In a next step, we analyzed the capability of native proteins to
interact with ZIP3. Immobilized GST fusion proteins were incubated with
cytosolic (S1) or membrane (P2) protein fractions prepared from adult
rat brains. Consistent with our previous results, ZIP3 interacted
specifically with PKC-
Specific immunsera to detect native ZIP3 or the GABAC
receptor
In addition, we tested whether the full-length Indication for a
PKC-
ZIP3 might serve as a linker, bringing PKC- PKC-
In the rat central nervous system, the highest concentration of
GABAC receptor Increasing evidence underlines the importance of macromolecular
signaling complexes containing ion channels, neurotransmitters receptors, kinases, and phosphatases for the specific regulation of
neuronal excitability. The modulation of neurotransmitter receptors by
kinases and phosphatases represents an important mechanism to regulate
neuronal activity (54). However, factors controlling the specific
targeting of these enzymes are largely unknown. The recently discovered
ZIP proteins (38) form a new protein family and physically link the
atypical protein kinase C isoform PKC- Mapping of protein regions mediating the ZIP3/GABAC
receptor binding identified 10 amino acids that are conserved in the
TM3-TM4 loops of In this study, we identified a region of ZIP3, including the ZZ-zinc
finger domain that has been associated with protein-protein interactions (48), to be important for the binding to the To test this hypothesis directly, we competed the binding of the A prerequisite for the formation of the proposed ternary complex
composed of ZIP3, PKC- Earlier studies that analyzed the retinal distribution of PKC- PKC consensus sequences are present in the TM3-TM4 loops of rat In summary, we present ZIP3 as a new member of the PKC- to target proteins.
GABAC receptors are composed of three
subunits
(
1-3) that are highly expressed in the retina, where they are
clustered at synaptic terminals of bipolar cells. A yeast two-hybrid
screen for the GABAC receptor
3 subunit identified ZIP3,
a new C-terminal splice variant of the ZIP protein family. ZIP3 was
ubiquitously expressed in non-neuronal and neuronal tissues, including
the retina. The
3-binding region of ZIP3 contained a ZZ-zinc finger domain, which interacted with 10 amino acids conserved in
1-3 but
not in GABAA receptors. Consistently, only
1-3 subunits
bound to ZIP3. ZIP3 formed dimers with ZIP1-3 and interacted with
PKC-
and the shaker-type potassium channel subunit Kv
2. Different domains of ZIP3 interacted with PKC-
and the
3 subunit, and simultaneous assembly of ZIP3, PKC-
and
3 was demonstrated
in vitro. Subcellular co-expression of ZIP3 binding
partners in the retina supported the proposed protein interactions. Our
results indicate the formation of a ternary postsynaptic complex
containing PKC-
, ZIP3, and GABAC receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Aminobutyric acid
(GABA)1 is the most important
inhibitory neurotransmitter in the mammalian central nervous system
gating GABAA, GABAB, and GABAC
receptors. While GABAA/C receptors form ligand-gated ion
channels (1, 2), GABAB receptors couple to ion channels via
G-proteins (3). Unlike GABAA receptors, GABAC
receptors show no sensitivity for anesthetics and are predominantly expressed in the retina (4-6) where they participate in sharpening the
visual image by extracting spatial edges of neuronal representations (7). Furthermore, GABAC receptors were detected in the
superior colliculus (8-10), hippocampus (11, 12), cerebellum (13, 14),
lateral geniculate nucleus (15), and amygdala (16).
subunits (
1-3) that assemble into homo- and hetero-oligomers (17,
18). In the retina,
subunits are intensively clustered at bipolar cell terminals (19-21), while expression in other brain areas was significantly lower (22-25). N and C termini of
subunits are extracellular (26) and held in position by four transmembrane regions
(TM1-TM4). Between TM3 and TM4, a long intracellular loop contains
consensus sites for modulatory proteins, such as protein kinase C
(PKC), and activation of PKC down-regulated GABA-gated chloride
currents by receptor internalization (27-31).
1 and
2 subunits were identified. A
new C-terminal splice variant of the glycine transporter GLYT-1 bound
to
1 (32), while the microtubule-associated protein 1B (MAP-1B)
interacted with
1 and
2 and was co-localized with
GABAC receptors in the retina (33-35). Although MAP-1B
binds microtubuli, the protein was not essential to anchor
GABAC receptors at the cytoskeleton of bipolar cell
synapses since GABAC receptor expression in
MAP-1B-deficient mice was indistinguishable from wild-type animals
(36). Importantly, neither GLYT-1 nor MAP-1B interacted with the
GABAC receptor
3 subunit or GABAA receptors.
3 subunit, we identified ZIP3, a new
C-terminal splice variant of a protein family interacting with the
-isoform of protein kinase C (PKC-
; Refs. 37, 38). ZIP proteins
link PKC-
to shaker-type potassium channel
subunits (39) and
play an important scaffold role in the activation of the transcription factor NF-
B (40, 41). This study identified a new interaction between ZIP3 and GABAC receptors in vitro and
postulates the formation of a PKC-
/ZIP3/GABAC receptor
macromolecular complex.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
subunit (TM3-TM4, amino acids 344-445) was PCR-amplified and subcloned
in-frame into the EcoRI-BamHI site of the bait
vector pGBKT7-BD for expression as a GAL4 fusion protein. Yeast AH109 cells were sequentially transformed with the
3 bait vector and 1 mg
of a rat brain MATCHMAKER cDNA-library cloned in pGADT7-AD using
the lithium acetate method (42) and plated on media selecting for
reporter gene activations containing 10 mM
3-amino-1,2,4-triazole (3-AT, Sigma) to suppress background
growth. Yeast colonies were incubated for 4 days at 30 °C and
transferred to plates containing 5-bromo-4-chloro-3-indoxyl-
-D-galactopyranoside (Sigma)
to test
-galactosidase reporter gene activation. Prey plasmids were
isolated from positive yeast colonies using CHROMA SPIN-1000 columns
(Clontech), shuttled into Escherichia
coli DH5
(Invitrogen), and retransformed into yeast
cells together with pGBKT7-Lam encoding for human lamin C to test for
transactivation. Library inserts of positive, retested interactors were
sequenced (43) using an automatic DNA sequencer (AbiPrism 377, Applied
Biosystems, Foster City, CA) and analyzed with protein and nucleotide
databases of the National Center for Biotechnology Information (NCBI,
Bethesda, MD) using the Basic Local Alignment Search Tool (BLAST, 44).
-galactosidase reporter genes was analyzed on selection plates as
described above containing 3 mM or 10 mM 3-AT.
Transactivation of all constructs was tested using one of the two
positive control plasmids pGBKT7-53 or pGADT7-T of the MATCHMAKER GAL4
Two-hybrid System 3 (Clontech) encoding for the
murine tumor suppressor protein p53 and the SV40 large T-antigen,
respectively. Semi-quantitative intensities of protein-protein
interactions were calculated according to the "Yeast Protocols
Handbook" from Clontech (Palo Alto, CA) using o-nitrophenyl-
-D-galactopyranoside (Sigma) as
a substrate. Values are expressed as arbitrary
-galactosidase units
and represent the mean of the reporter gene activity of three yeast
colonies. Error bars are ± S.E.
1-3 subunits, the complete coding
sequences of the rat ZIP1-3, and of the rat shaker-type potassium
channel Kv
2 subunit were ligated in-frame to the coding sequence of
glutathione-S-transferase (GST) in pET-41 or fused to the
His tag of pET-30. The coding sequences of ZIP3 and PKC-
were tagged
with a T7-epitope by cloning in pET-21. Plasmids were transformed in
Escherichia coli BL21(DE3)pLysS, and protein expression was
induced by adding 1 mM
isopropyl-beta-D-thiogalactoside (Sigma). Fusion proteins
were purified under native conditions from frozen bacteria pellets by
incubating for 30 min in ice-cold lyses buffer (50 mM
NaH2PO4, 300 mM NaCl, pH 8.0)
containing 25 units/ml Benzonase (Novagen), 1 mg/ml Lysozyme (Sigma)
and a mixture of protease inhibitors (Roche Molecular Biochemicals),
and subsequent sonication (6 bursts for 10 s at 300 W).
Alternatively, the "BugBuster GST-Bind-Purification Kit" from
Novagen was used. GST- and His-tagged fusion proteins were immobilized
to glutathione-Sepharose or Ni-NTA beads that were pre-incubated with
0.1% Triton-X100 and 0.1% bovine serum albumin in the binding
buffers for GST beads (in mM: 4.3 Na2HPO4, 1.47 KH2PO4,
137 NaCl, 2.7 KCl, pH 7.3) or Ni-NTA agarose (in mM: 500 NaCl, 20 Tris-HCl, 5 imidazole, pH 7.9). The protein concentration of
the coated beads was estimated from a Coomassie Brilliant Blue R-250
(Serva, Heidelberg, Germany)-stained SDS-PAGE. Similar concentrations
of immobilized fusion proteins were incubated with the cytosolic
fraction of E. coli expressing T7-tagged ZIP3 or PKC-
for
2 h at 4 °C under slow agitation, followed by four washes (GST:
4.3 Na2HPO4, 1.47 KH2PO4, 137 NaCl, 2.7 KCl, 0.1% Triton X-100,
pH 7.3; Ni-NTA: 500 NaCl, 20 Tris-HCl, 60 imidazole, 0.1% Triton
X-100, pH 7.9; all concentrations in mM). To obtain comparable conditions in competition experiments, E. coli
protein extracts of similar protein concentrations, as measured at 280 nm, were used. The total volume of these samples was adjusted to 500 µl (defined as 100%) by adding protein extract of non-transfected E. coli BL21(DE3)pLysS. Bound proteins were eluted by
boiling in SDS sample buffer, separated by SDS-PAGE, and analyzed by
Western blotting using a monoclonal anti-T7 immunserum and the enhanced chemiluminescence system (ECL; Amersham Biosciences).
3 subunit was ligated in
pCR3.1 (Invitrogen) by PCR cloning techniques. The protein was
synthesized using the T7 promoter of the plasmid according to the
manuals provided with the RiboMAX RNA Production-Kit (Promega) and the
Flexi Rabbit Reticulocyte Lysate System (Promega) in the presence of
2.4% canine pancreatic microsomal membranes vesicles (Promega) and 0.8 mCi/ml (specific activity >1 mCi/mmol) [35S]methionine
(Amersham Biosciences). The reaction mixture was incubated with ZIP3
immobilized on glutathione-Sepharose in the presence of 0.1% Triton
X-100. Bound proteins were washed as described above and separated by
SDS-PAGE. Gels were dried and exposed to Hyper film (Amersham
Biosciences) for 16 h to 5 days.
3 TM3-TM4 loop fused to GST, or GST alone. Proteins were expressed under
control of the Rous sarcoma virus promoter. Cells were lyzed in RIPA
buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS
in phosphate-buffered saline, pH 7.4) containing 1 mM
dithiothreitol (Sigma), 25 units/ml Benzonase, and a protease inhibitor
mixture (Roche Molecular Biochemicals) and sedimented for 30 min at
13,200 × g at 4 °C. The supernatant was diluted 1:4
in phosphate-buffered saline supplemented with 1 mM
dithiothreitol and mixed with glutathione-Sepharose beads
(pre-incubated as above) for 5 h at 4 °C. Subsequent washing, elution, and Western blot detection of interacting proteins was performed as described above.
3 TM3-TM4 loop, or GST and His tags as negative controls
for 5 h under slow agitation. Bound proteins were washed, eluted
and visualized as described above. All protein preparation steps were
carried out on ice or at 4 °C.
(1:10.000, Sigma), mouse anti-Kv
2 (1:500, Upstate
Biotechnology, Lake Placid, NY), goat anti-PICK1 (1:500, N-18, Santa
Cruz Biotechnology, Santa Cruz, CA), and goat anti-PP1
(1:2000,
C-19, Santa Cruz Biotechnology).
1,
2, and
3 subunits of the rat GABAC receptor (19), a mouse monoclonal antibody against
PKC-
(1:200; Dunn Labortechnik, Asbach, Germany)-labeling rod
bipolar cells, and a rabbit polyclonal antiserum against PKC-
(1:10.000; Sigma). In double-labeling immunocytochemical experiments,
co-expression of PKC-
and PKC-
and of PKC-
and
GABAC receptors was examined. The binding sites of the
primary antibodies were revealed by the secondary antibodies
AlexaTM 594 (red fluorescence) and AlexaTM 488 (green fluorescence) goat anti-mouse or goat anti-rabbit IgG (H + L)
conjugates, respectively (1:500; Molecular Probes, Eugene, Oregon).
Double-labeled sections were examined and analyzed with a confocal
laser-scanning microscope (LSM 5 Pascal, Zeiss, Oberkochen, Germany),
and resulting images were adjusted in brightness and contrast using
Adobe PhotoShop 5.5 (Adobe Systems Inc., San Jose, CA).
reverse transcriptase
(Invitrogen). Incubation times were 15 min at room temperature followed
by 2 h at 42 °C. PCR amplification was performed with 250 ng of
reverse transcribed RNA in 50 µl of PCR-buffer (in mM: 20 Tris-HCl, 50 KCl, 1.5 MgCl2, 0.2 dNTPs, and 0.5 µM each ZIP3 primer (sense 5'-CAGCAAGCTCATCTTTCCcaac-3', nt 480-501; antisense 5'-CTACTTATGACACTTAAAgcca-3', nt 684-705), 5 units Taq-polymerase (Invitrogen), pH 8.0) in a thermocycler (Applied Biosystems) using the following parameters: 94 °C for 2 min
followed by 30 cycles at 94 °C for 45 s, 60 °C for 60 s, 72 °C for 30 s and a final incubation at 72 °C for 10 min. To compare amounts of RNA isolated from each tissue as well as the efficiency of reverse transcription between RNA samples, a PCR with
oligonucleotides recognizing
-actin (sense:
5'-tgagaccttcaacaccccag-3', nt 372-391; antisense:
5'-gtagacgaccttccacctgt-3', nt1065-1046) was performed for 25 cycles
with the parameters described above. Five microliters of each PCR
product were separated on a 1.5% agarose gel and stained with ethidium
bromide. Controls were treated as above without adding template and/or
reverse transcriptase and showed no PCR products. To identify the PCR
products, a Southern blot was performed using standard techniques (47).
Oligonucleotides for ZIP3 (5'-catgggcactttggctggc-3', nt 559-577), and
-actin (5'-cggtcaggtcatcactatc-3', nt 732-750) were tailed with
DIG-ddUTP, hybridized and detected following the protocol of the
"DIG-oligonucleotide Tailing Kit" and the "DIG Luminescent
Detection Kit" (Roche Molecular Biochemicals) as described in the
user manuals.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-interacting Protein Family--
To identify proteins that bind
to the GABAC receptor
3 subunit, a rat brain cDNA
library was screened against the intracellular TM3-TM4 loop of this
subunit. Of ~1.9 × 107 transformed yeast cells, 100 yeast colonies were isolated on selection plates and analyzed further.
Upon sequencing, clones 56 and 72 were of specific interest since they
represented two independent clones coding for an unknown C-terminal
splice variant of the PKC-
-interacting proteins ZIP1 and ZIP2 (38).
Therefore, the protein was termed ZIP3. The N-terminal part of ZIP3 was
identical to ZIP1 and ZIP2 and contained a recently characterized
acidic putative protein-binding motif described as cdc-homology domain (37, 39), a ZZ-zinc finger domain that also has been associated with
protein-protein interactions (48), and two consensus sequences for
phosphorylation by PKC (Fig.
1A). However, due to the new C
terminus, ZIP3 did not contain the PEST and ubiquitin-associated domains present in ZIP1 and ZIP2 (39). Interestingly, the ZIP3-specific C terminus started at the same splice site that is used to generate the
ZIP1-specific cassette missing in ZIP2 (Fig. 1A; Refs. 39, 49).
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Fig. 1.
Cloning and tissue distribution of ZIP3, a
new C-terminal splice variant of the protein kinase
C- interacting protein family.
A, schematic representation of ZIP protein family members
drawn to scale. Functional domains are represented by boxes, consensus
sequences for PKC are shown by black circles, and the splice
site is indicated by an arrow. The splice cassette of ZIP1,
absent in ZIP2, is indicated by a dotted line, and the amino
acid sequence of the ZIP3 C terminus is given in the single letter
code. B, Southern blots of PCR products for ZIP3, obtained
after reverse transcription of total RNA isolated from the indicated
tissues. Amplification of fragments for
-actin indicated similar
cDNA concentrations in all samples. The calculated size of the PCR
products is shown on the left.
-actin indicated that approximately equal amounts of
cDNA from each tissue were used.
Subunits--
The highest sequence diversity between
GABAC receptor subunits is found in their TM3-TM4 loops.
Thus we analyzed whether besides the
3 subunit additional subunits
of the GABAC or the structurally related GABAA
receptor were able to interact with ZIP3. PCR products representing
TM3-TM4 loops were cloned in the bait vector of the yeast two-hybrid
system and expressed fusion proteins were analyzed for their ability to
interact with ZIP3. Protein-protein interaction was monitored by the
ability of transformed yeast cells to grow on selective media,
containing 3 mM or 3-AT. To our surprise, TM3-TM4 loops of
all three
subunits bound ZIP3, with the
3 loop showing the
highest binding affinity (Fig.
2A, left panel). In
contrast to GABAC receptor
subunits, no interaction was
observed between ZIP3 and subunits of the GABAA receptor
(Fig. 2A, middle panel).
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Fig. 2.
Domain mapping in ZIP3 and
GABAC receptor subunits. A, the TM3-TM4 loops of
GABAA/C receptor subunits were tested in binary yeast
two-hybrid experiments for their ability to bind ZIP3. Encoded amino
acids are represented by numbers in parentheses. Activation
of HIS3, ADE2, and
-galactosidase reporter genes was monitored by
the ability of transformants to grow on selective media with the
addition of 3 mM (+) or 10 mM 3-AT (+++),
indicating low or high binding strength, respectively. (
) indicates
no growth of yeast colonies. B, upper half:
alignment of the TM3-TM4 loops of the rat
1-3 subunits. (/)
replaces a region not shown because of low homology. Lower
half: the complete
3 TM3-TM4 loop and deletion constructs were
tested for binding to ZIP3 as described in A. The identified
minimal interacting region identical between the
subunits is
highlighted in gray. Relative binding intensities of
interacting constructs were quantified to 1.88 ± 0.41 (
3),
1.87 ± 0.09 (
3-D1), 1.40 ± 0.15 (
3-D2), 1.25 ± 0.51 (
3-D3), 1.0 ± 0.12 (
3-D6), 1.66 ± 0.42 (
3-D7), and 1.88 ± 0.38 (
3-D8) arbitrary
-galactosidase
units. Each bar represents the mean of three yeast clones. Error
bars are ± S.E. As a reference for high binding affinity,
the strength of the interaction between the tumor suppressor protein
p53 and the SV40 large T-antigen, encoded by the two positive control
vectors pGBKT7-53 and pGADT7-T of the Matchmaker Gal4 Two-Hybrid
System 3 (Clontech), was calculated to 2.42 ± 0.29 arbitrary
-galactosidase units (not shown). C, N-
and C-terminal deletion constructs of ZIP3 were tested for binding to
the TM3-TM4 loop of the
3 subunit as described in A and
relative affinities were calculated to 1.37 ± 0.14 (ZIP3-D2),
2.15 ± 0.15 (ZIP3-D3), 1.86 ± 0.47 (ZIP3-D4), 2.13 ± 0.9 (ZIP3-D5), 1.58 ± 0.04 (ZIP3-D6), 1.12 ± 0.21 (ZIP3-D7), and 1.29 ± 0.08 (ZIP3-D9) arbitrary
-galactosidase
units as in B. The mapped binding region of ZIP3 is marked
by a gray background.
subunits of the
GABAC receptor allowed the identification of amino acids
important for the binding. An alignment of the TM3-TM4 loops of the rat
1-3 subunits identified two regions of high similarity, located at
the very N- and C-terminal ends of the loops (Fig. 2B,
upper half). To determine the involvement of these regions
in ZIP3 binding, subsequent N- and C-terminal deletions were generated
in the
3 TM3-TM4 loop, and the resulting protein fragments were
tested directly for their ability to interact with ZIP3 in yeast cells (Fig. 2B, lower half) as described above. While
amino acid regions located at the C-terminal part of the
3 loop
showed no binding to ZIP3, 10 amino acids at the very N-terminal
part of the loop were sufficient for the interaction (gray
background in Fig. 2B). To analyze if the deletions
changed the
3 binding affinity for ZIP3, the relative binding
strength was estimated using a semi-quantitative
-galactosidase
assay. Compared with the binding strength of the complete
3 TM3-TM4
loop, the relative ZIP3 binding affinities of the
3 constructs
decreased slightly with subsequent deletions. Thus, construct
3-D3
contained a minimal ZIP3 binding region; however, additional amino
acids present in constructs
3-D1 and
3-D2, which are absent in
the TM3-TM4 loops of
1 and
2, support the interaction. This
result is consistent with our finding that all three
subunits did
interact with ZIP3 and that
3 showed a higher binding strength than
1 and
2 (Fig. 2A, left panel).
3-D3 construct, we generated three
additional deletions of the
3 TM3-TM4 loop. Upon deletion of the
first 10 amino acids (construct
3-D6), the binding affinity for ZIP3
was reduced by about 50%, indicating that this domain is an important
but not the only mediator for the interaction between
3 and ZIP3.
When the first 10 amino acids were used in combination with more
C-terminal domains of the
3 TM3-TM4 loop (constructs
3-D7 and
3-D8), the binding strength increased to values comparable with the
wild-type sequence.
3 TM3-TM4 loop, again using a strategy of subsequent deletions. The
ZIP3/
3 interaction was mediated by the ZZ-zinc finger and a
C-terminal adjacent protein region (amino acids 119-221, gray background in Fig. 2C), while the
ZIP3-specific C terminus and the cdc-homology region were not involved
in the binding. Dividing the interacting protein region in two parts
resulted in a reduction in binding affinities (constructs ZIP3-D2 and
ZIP3-D7). However, interaction with
3 was still present, indicating
that amino acids in both protein regions contribute to the binding site
in a synergistic manner. Indeed, the ZZ-zinc finger domain alone
(construct ZIP3-D9) was able to bind the
3 TM3-TM4 loop at a binding
intensity similar to those of constructs ZIP3-D2 and ZIP3-D7.
Subunits, Kv
2, and PKC-
--
Several proteins have
been described in the literature to physically interact with ZIP1 or
ZIP2, including PKC-
, shaker-type potassium channel
subunits, or
the ZIP proteins themselves (37, 39). Therefore, we analyzed the
binding characteristics of the newly identified member of the ZIP
protein family, ZIP3. Immobilized GST fusion proteins were incubated
with E. coli protein extracts and bound proteins were
analyzed in Western blots. Unspecific interactions were excluded using
immobilized GST. ZIP3 bound specifically to ZIP1, ZIP2, and ZIP3 and to
the TM3-TM4 loops of the
1,
2, and
3 subunits (Fig.
3A), consistent with the
results obtained from the yeast two-hybrid experiments. Furthermore,
ZIP3 interacted with Kv
2 (Fig. 3A) and with PKC-
(Fig.
3B). Since the N-terminal regions of ZIP1, ZIP2, and ZIP3
are identical, our data indicate that the ZIP dimerization domain and
the binding regions for PKC-
and Kv
2 are located in the N
termini. Indeed, the region important for dimerization and binding of
ZIP1 to PKC-
was mapped to the cdc-homology region (see Fig. 1;
Refs. 37, 39, 41, 50).
View larger version (24K):
[in a new window]
Fig. 3.
Recombinant and native proteins binding to
ZIP3. To analyze protein interactions of ZIP3 in vitro,
GST and GST fusion proteins were immobilized on glutathione-Sepharose
beads and incubated with T7-tagged ZIP3 (A) or PKC-
(B) purified from E. coli as indicated. Bound
proteins were detected on Western blots (upper panels) using
a monoclonal anti-T7 immunserum. The concentration of GST and GST
fusion proteins bound to Sepharose beads is shown on Coomassie-stained
SDS-PAGE (arrowheads in lower panels).
C, to analyze the binding of native proteins to ZIP3,
cytosolic (S1) or membrane (P2) protein preparations of rat brain were
incubated with GST or GST-ZIP3 immobilized on glutathione-Sepharose.
Bound proteins were detected on Western blots using specific immunsera
as indicated on the right. Antibodies against PP1C and PICK1
served as negative controls to ensure specificity of the assay. The
concentration of GST and GST fusion proteins bound to Sepharose beads
is shown on Coomassie-stained SDS-PAGE (arrowheads in the
lowest two panels). D, to verify the interaction
between ZIP3 and the GABAC receptor
3 subunit in
mammalian cells, HEK-293 cells were co-transfected with ZIP3 and GST or
ZIP3 and GST fused to the TM3-TM4 loop of the
3 subunit. Cell
lysates were incubated with glutathione-Sepharose beads, and bound
proteins were analyzed using a monoclonal anti-T7 antibody.
E, to verify the interaction between ZIP3 and the
full-length
3 subunit,
3 was synthesized in vitro
using radioactive methionine in the presence of microsomal membranes.
The lysate was incubated with immobilized GST-ZIP3, and the bound
3
subunit was detected radiographically. In all panels, the Benchmark
prestained protein ladder (Invitrogen) or calculated sizes of
interacting proteins are indicated in kDa.
and Kv
2 (Fig. 3C). Nonspecific interactions were excluded using antibodies against protein phosphatase 1C (PP1C; 51) and against a protein interacting
with C-kinase (PICK1; Ref. 52) as negative
controls for cytosolic (S1) and membrane associated (P2) protein
preparations, respectively. So far, no report described a physical
interaction between PP1C and PICK1 with ZIP proteins, and indeed, no
binding was detected under our experimental conditions, demonstrating
the specificity of the assay (Fig. 3C).
3 subunit on Western blots do not exist, which prevented us from analyzing the interaction between these two proteins in native tissue. To circumvent this fact, we analyzed whether the interaction between ZIP3 and the GABAC receptor
3 subunit occurred
when the binding partners were synthesized in mammalian cells. For this purpose, HEK-293 cells were co-transfected with T7-tagged ZIP3, and the
3 subunit TM3-TM4 loop fused to GST or T7-ZIP3 and GST as a control.
Proteins interacting with the
3 loop were precipitated using
glutathione-Sepharose beads and analyzed on a Western blot using an
anti-T7 immunserum. In agreement with our previous data, ZIP3 was able
to bind specifically to the
3 subunit, while no interaction could be
observed with GST (Fig. 3D) or in cells co-transfected with
the green fluorescent protein instead of ZIP3 (data not shown).
3 subunit would be
able to bind to ZIP3. The
3 subunit was synthesized in vitro using a rabbit reticulocyte lysate in the presence of canine pancreatic microsomal membranes and subsequently incubated with immobilized GST-ZIP3 fusion proteins. Consistent with our data, we
observed an interaction between the full-length
3 subunit and ZIP3,
but not between
3 and GST (Fig. 3E).
/ZIP3/GABAC
Receptor-containing Macromolecular Complex--
Our findings
demonstrated that ZIP3 was able to dimerize with ZIP family members and
to interact with GABAC receptor
subunits, PKC-
and
Kv
2, in vitro. We mapped the ZIP3 binding site for
subunits to a region different from the cdc-homology domain, that has
been shown to mediate the interaction with PKC-
(see Fig.
2C; Ref. 37). Therefore, ZIP3 could bind to
subunits and
PKC-
at the same time, acting as a scaffold, physically linking PKC-
to GABAC receptors. To analyze whether the
3
subunit and PKC-
were indeed able to bind simultaneously to ZIP3,
competition experiments were performed. Glutathione-Sepharose beads
coated with the TM3-TM4 loop of the
3 subunit were first incubated
with 500 µl (equivalent to 100% of the total volume) of ZIP3
containing E. coli protein extract, washed extensively, and
subsequently mixed with increasing amounts of PKC-
containing
protein extract. PKC-
was not able to displace ZIP3 from binding to
the
3 subunit (Fig. 4A),
indicating that PKC-
and the
3 subunit interacted with different
regions of ZIP3, consistent with our previous data (Fig.
2C). Furthermore, this experiment directly demonstrated that
ZIP3, PKC-
, and the
3 subunit were able to form a ternary complex
in vitro, suggesting the possibility that ZIP3 could
serve as a scaffold protein at synapses expressing GABAC
receptors.
View larger version (26K):
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Fig. 4.
ZIP3, PKC- , and the
GABAC receptor
3
subunit form a ternary complex in vitro.
A, to analyze if PKC-
and the
3 subunit would interact
simultaneously with ZIP3, competition experiments were performed. Equal
amounts of GST/
3TM3-TM4 immobilized on Sepharose beads
(arrowhead in lowest panel) were first incubated
with constant quantities of ZIP3-containing E. coli protein
extracts, washed, and subsequently mixed with increasing concentrations
of PKC-
extracts (12-100% of the total volume). Different volumes
of PKC-
protein extracts were adjusted to 500 µl (defined as 100%
of the total volume) by adding protein extracts of non-transfected
E. coli cells. Bound proteins were detected on Western blots
as described in Fig. 3A. B, to test for a direct
interaction between PKC-
and the
3 subunit, cytosolic protein
preparations of rat brain were incubated with the His-tagged TM3-TM4
loop of the
3 subunit immobilized on Ni-NTA beads. Bound proteins
were detected using a PKC-
specific immunserum. In all panels, the
Benchmark prestained protein ladder (Invitrogen) or calculated sizes of
interacting proteins are indicated in kDa.
in close vicinity of
3-containing GABAC receptors. The
3 TM3-TM4 loop
contains a consensus sequence for phosphorylation by PKC; however, a
direct binding of PKC-
to the
3 subunit would be needed for its
phosphorylation. Indeed, we could show a direct interaction between
PKC-
and the
3 TM3-TM4 loop in native protein preparations (Fig.
4B). Nonspecific interactions between
3 and the protein
extract were excluded using antibodies against PP1C (51), that has not
been reported to bind GABAC receptors. In addition to the
native proteins, PKC-
and the
3 loop did also interact when
recombinant proteins synthesized in E. coli were used (data
not shown).
and GABAC Receptor
Subunits Are
Co-expressed in the Same Cellular Compartments in the Mammalian
Retina--
A prerequisite for any protein-protein interaction is the
co-expression of the binding partners in the same cellular
compartments. As said before, ZIP3-specific immunsera needed to detect
the protein in native tissues were not available. Therefore, we
analyzed whether proteins that physically interact with ZIP3, namely
PKC-
and GABAC receptor
subunits, were co-expressed
in the rat retina.
subunits was detected in the retina,
where they are clustered at synaptic terminals of bipolar cells
(22-25). Vertical cryostat sections of adult rat retinas were
double-labeled with antibodies recognizing GABAC receptor
subunits/PKC
and PKC
/PKC-
. Stained sections were analyzed
using confocal laser-scanning microscopy. Co-expression of PKC-
and
the
subunits of the GABAC receptor had to be shown
indirectly as the antisera are generated in the same species (rabbit).
Fig. 5A shows that at the rod
bipolar cell terminals that stratify deep in the inner plexiform layer and are stained with the antibody against PKC
, GABAC
receptor
subunits are clustered. Importantly, in the terminals of
the PKC
-labeled rod bipolar cells, PKC-
is co-expressed (Fig.
5B). Co-expression can be clearly seen in the merge of the
stainings, showing the terminals of the rod bipolar cells in a higher
power view (broken lines mark the region of the inner plexiform layer shown). The results of the staining experiments suggest, although indirectly, the co-expression of GABAC receptor
subunits and PKC-
in rod bipolar cell terminals.
View larger version (37K):
[in a new window]
Fig. 5.
Co-expression of
GABAC receptors, PKC
and PKC-
, in the rat retina.
Confocal micrographs of vertical sections through adult rat retinas
double-immunolabeled for PKC-
and GABAC receptor
subunits (A) and for PKC-
and PKC-
(B).
A, rod bipolar cells are immunoreactive for PKC-
(green) and their axon terminals are decorated with
GABAC receptor immunoreactive puncta (red). This
can be clearly seen in the merge of the two stainings, showing a higher
power view of rod bipolar cell terminals (broken lines mark
the region of the IPL shown). GABAC receptors present on
PKC-
labeled terminals appear orange-yellow.
B, PKC-
immunoreactive axon terminals of rod bipolar
cells (green) also express PKC-
(red), shown
as a yellowish color in the merge of the two stainings
(broken lines mark the region of the IPL shown)
(OPL, outer plexiform layer; INL, inner nuclear
layer; IPL, inner plexiform layer). Scale bars,
10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to target proteins, such as
potassium channels (39) or the death-domain kinase RIP, a protein
involved in the activation of the transcription factor NF-
B (40,
41). In this study, we identified ZIP3 as a new member of the ZIP
protein family, which is abundantly expressed in the brain and retina.
ZIP3 was able to form homo- and heterodimers and bound to PKC-
and
Kv
2. Furthermore, ZIP3 interacted with GABAC receptor
subunits using a different binding site, allowing simultaneous
binding of
subunits and PKC-
in vitro. These results suggest a possible formation of a PKC-
/ZIP3/GABAC
receptor containing macromolecular complex.
1-3 subunits. Compared with the complete
3
TM3-TM4 loop, subsequent C-terminal deletions of the
3 loop resulted in a slight decrease in ZIP3 binding affinity, indicating that the 10 amino acids represent a minimal binding motif, while additional
3
sequences support the interaction. Indeed, when these 10 amino acids
were deleted, the binding affinity for ZIP3 was reduced by nearly 50%,
while deletion of internal regions of the loop did not significantly
alter the binding strength compared with the wild-type. Therefore, we
propose at least two binding domains in the
3 TM3-TM4 loop
contacting ZIP3, one present in the first 10 amino acids that acts in
combination with a more C-terminally located motif. TM3-TM4 loops of
GABAA receptor subunits were not able to bind ZIP3. This is
consistent with the fact that the identified 10 amino acids of the
subunits are not conserved in GABAA receptor subunits.
3 TM3-TM4
loop. This protein region is identical between ZIP1, ZIP2, and ZIP3,
and indeed all ZIP proteins bound to
3 in yeast cells (data not
shown). ZIP1 and ZIP2 had been described to bind PKC-
and to form
homo- as well as heterodimers using the cdc-homology domain (37,
39, 41, 50). This domain is identical in ZIP3, and accordingly, ZIP3
was able to dimerize with all ZIP proteins and to bind to PKC-
. The
cdc-homology region is located N-terminal of the domain mediating the
ZIP3/
subunit interaction, indicating that the binding of ZIP3 to
GABAC receptors might be independent from its binding to
PKC-
or other ZIP proteins. Therefore, ZIP3 could physically link
PKC-
to the TM3-TM4 loops of GABAC receptor subunits.
3
subunit to ZIP3 with increasing amounts of PKC-
and could demonstrate the in vitro formation of a ternary complex
composed of ZIP3, PKC-
, and the GABAC receptor
3
subunit. The binding sites for PKC-
and for the
3 subunit were
mapped to N-terminal domains of ZIP proteins, which are identical
between ZIP1, ZIP2, and ZIP3. Therefore, in principle all ZIP proteins
might be able to associate with PKC-
and GABAC receptor
subunits into a ternary protein complex. However, ZIP3 lacks the
PEST and ubiquitin domains present in ZIP1 and ZIP2. Since these
domains are associated with protein degradation, ZIP3-containing
macromolecular complexes might be more stable within cells compared
with ZIP1- and ZIP2-containing complexes.
, and GABAC receptors is the
expression of the proteins in the same cellular compartments. Since
ZIP3 specific immunsera are not available, we compared the expression patterns of the proteins that bind to ZIP3, namely PKC-
and
GABAC receptors. Within the central nervous system, the
highest concentration of GABAC receptor
subunits is
observed in the retina (22, 24, 25), where also ZIP3 is abundantly
expressed (this study). Therefore, we analyzed the distribution of
GABAC receptors and of PKC-
in rat retina with
immunocytochemistry and confocal microscopy. The results of the
experiments suggest the co-expression of the GABAC receptor
subunits and of PKC-
in the terminals of rod bipolar cells.
showed contradictory results. While one study found that PKC-
co-localized with PKC-
in bipolar cells (55), another study showed
PKC-
expression exclusively in the inner segments of photoreceptors (56). Here we confirm the co-localization of PKC-
and PKC-
in rod
bipolar cells. Cerebellar Purkinje cells also express GABAC receptors (23) and, similar to the results reported in this study,
co-express ZIP proteins and PKC-
(39).
1-3
subunits, and GABAC receptor currents are modulated by PKC
(27-29). This suggests a functional link between GABAC
receptors and PKC-
. Indeed, we could show a direct interaction
between PKC-
and the
3 TM3-TM4 loop in recombinant and native
protein preparations, which would be needed for a protein
phosphorylation. However, mutation of PKC consensus sequences of the
1 subunit did not prevent its modulation by PKC (30, 31, 57),
indicating that residues other than those of the consensus sequences
are phosphorylated (58). On the other hand, proteins other than the
1 subunit might be the target of the PKC activity. So far, no
similar studies have been performed for the
3 subunit, and therefore
it remains elusive whether the PKC consensus sequences of this protein
are used. Alternatively, two consensus sequences for PKC present in
ZIP3 might represent new players in the modulation of GABAC
receptors. Furthermore, the synaptic clustering of GABAC receptors might be influenced by its interaction with ZIP3 and PKC-
since PKC-
interacts with tubulin and the actin cytoskeleton (60-61).
-interacting
protein family that it highly expressed in the mammalian retina and
demonstrated in vitro its interaction with PKC-
,
GABAC receptor
subunits and Kv
2. Therefore we
suggest the formation of a postsynaptic macromolecular protein complex
at GABAC receptor-containing synapses using ZIP3 as a scaffold.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Min Li (The Johns Hopkins University, Baltimore, MD) for providing ZIP-specific PCR primers, Erika Jung-Körner, Ines Walter, Anja Hildebrand, and Petra Wenzeler for excellent technical assistance, Adaling Ogilvie and Cord-Michael Becker for support, and Stefan Stamm for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant of the Deutsche Forschungsgemeinschaft EN349 (to R. E.) and by a Heisenberg Fellowship (to J. H. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF439403.
¶ To whom correspondence should be addressed. Tel.: 49-9131-852-6205; Fax: 49-9131-852-2485; E-mail: ralf.enz@biochem.uni-erlangen.de.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M205162200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GABA, -aminobutyric acid;
PP, protein phosphatase;
ZIP, protein kinase
C-
-interacting protein;
PKC, protein kinase C;
TM, transmembrane
region;
MAP, microtubule-associated protein;
3-AT, 3-amino-1,2,4-triazole;
GST, glutathione-S-transferase;
PB, phosphate buffer;
Ni-NTA, nickel-nitrilotriacetic acid.
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