From § Biofrontera Pharmaceuticals AG, Hemmelratherweg
201, 51377 Leverkusen, Germany, the ** Department of Animal
Physiology, Ruhr-University of Bochum, 44780 Bochum, Germany,
CNRS UPR 9023, Centre CNRS-INSERM de Pharmacologie
et Endocrinologie, 141 Rue de la Cardonille, 34094 Montpellier
Cedex 05, France, and the ¶ Department of Molecular Cell Biology,
Weizmann Institute of Science, 76100 Rehovot, Israel
Received for publication, September 5, 2000, and in revised form, December 13, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By using the yeast two-hybrid system, we
previously isolated a cDNA clone encoding a novel member of the
multivalent PDZ protein family called MUPP1 containing 13 PDZ domains.
Here we report that the C terminus of the 5-hydroxytryptamine
type 2C (5-HT2C) receptor selectively interacts with
the 10th PDZ domain of MUPP1. Mutations in the extreme C-terminal SSV
sequence of the 5-HT2C receptor confirmed that the
SXV motif is critical for the interaction. Co-immunoprecipitations of MUPP1 and 5-HT2C receptors from
transfected COS-7 cells and from rat choroid plexus verified this
interaction in vivo. Immunocytochemistry revealed an
SXV motif-dependent co-clustering of both
proteins in transfected COS-7 cells as well as a colocalization in rat
choroid plexus. A 5-HT2C receptor-dependent
unmasking of a C-terminal vesicular stomatitis virus epitope of MUPP1
suggests that the interaction triggers a conformational change within
the MUPP1 protein. Moreover, 5-HT2A and 5-HT2B,
sharing the C-terminal EX(V/I)SXV sequence with
5-HT2C receptors, also bind MUPP1 PDZ domains in
vitro. The highest MUPP1 mRNA levels were
found in all cerebral cortical layers, the hippocampus, the granular
layer of the dentate gyrus, as well as the choroid plexus, where
5-HT2C receptors are highly enriched. We propose that MUPP1
may serve as a multivalent scaffold protein that selectively assembles
and targets signaling complexes.
There is ample evidence suggesting that the function of a receptor
is dependent on its specific subcellular localization. Sequence-specific interactions between proteins provide the basis for
the structural and functional organization of receptors within cells.
For a few members of the G-protein-coupled receptor family, these
interactions have been described to be mediated by C-terminal interactions with PDZ (PSD-95/discs
large/ZO-1) domain-containing proteins. The best
investigated example is the C-terminal interactions of G-protein-coupled receptors with PDZ
domain-containing proteins extends to a member of the somatostatin receptor family (sst). The subtype sst2 interacts selectively with a highly homologous PDZ domain contained within the protein CortBP1/ProSAP1/Shank2 (5) and the recently cloned synaptic protein
SSTRIP (somatostatin
receptor-interacting
protein)/Spank1/synamon/Shank1 (6). Both proteins belong to
a common family recently termed Shanks (7, 8) or ProSAP (9), sharing
essentially identical domain structures such as ankyrin repeats, an SH3
domain, the PDZ domain, a sterile 5-HT2C receptors are broadly expressed in the central
nervous system and in the choroid plexus (11, 12) and are involved in a
diversity of physiological functions such as the control of
nociception, motor behavior, endocrine secretion, thermoregulation, modulation of appetite, and the control of exchanges between the central nervous system and the cerebrospinal fluid (13-17). These receptors contain a C-terminal sequence, SSV* (where the asterisk indicates a carboxyl group), corresponding to the
T/SXV* motif. This motif is potentially implicated in
protein-protein interactions with PDZ domains as originally described
for the C termini of the
N-methyl-D-aspartate receptor and
K+ channel subunits (18, 19). The T/SXV* motif
is also present in C termini of various other G-protein-coupled
receptors, including the 5-HT2A (18) and human
5-HT2B receptors. By virtue of its interaction with the C
terminus of the 5-HT2C receptor, we recently isolated a
novel cDNA encoding MUPP1, a protein with 13 PDZ domains (20).
MUPP1 belongs to the family of multi-PDZ proteins comprising CIPP
(channel-interacting PDZ domain
protein) (21, 22), INADL (INAD-like
protein) (23), and a putative Caenorhabditis elegans polypeptide referred to as C52A11.4 (20), containing 4, 7, or 10 PDZ
domains, respectively, and no other obvious catalytic domain. PDZ
domains highly similar to those of MUPP1 are arrayed in the same order
in all four proteins, implying the requirement of a precise arrangement
for the assembly into a functional macromolecular complex (20). The
present work provides biochemical and immunohistochemical evidence that
the 5-HT2C receptor interaction with MUPP1 takes place
in vitro and in vivo. Among the 13 PDZ domains of
MUPP1, 5-HT2C receptors exclusively interact with the 10th
PDZ domain, emphasizing the high selectivity of PDZ domain
interactions. Moreover, the interaction induces a conformational change
in the MUPP1 molecule and a co-clustering that might trigger a
downstream signal transduction pathway.
Two-hybrid Screening--
Yeast two-hybrid screening was
performed using the CG1945 strain (24) harboring the HIS3
and Domain Analysis of the Interaction--
To assemble the
full-length clone pBSKSII-rMUPP1, a PCR fragment covering region
Mutagenesis--
The 90-amino acid C-terminal fragment of the
human 5-HT2C receptor was mutagenized by PCR using reverse
primers harboring nucleotide exchanges and by using pAS2-1/hu2C as
template. The amplified DNA was directionally subcloned into the
BamHI and EcoRI sites of pBluescript KSII
(Stratagene) and sequenced. The inserts were transferred into either
pAS2-1 (BamHI and SalI) or pGEX-3X (BamHI and EcoRI; Amersham Pharmacia Biotech).
The mutant plasmid pRK5/h5-HT2C-SSA was obtained by PCR
using a reverse primer encoding the V458A mutation by a GCG codon using
pRK5/h5-HT2C as template.
In Vitro Protein-Protein Interaction--
The partial cDNA
of human MUPP1 was amplified by PCR from pACT-2/huMUPP1 (clone 1) (20)
and inserted directionally into the HindIII and
EagI sites of pBAT (27). In vitro translation of
[35S]methionine-labeled MUPP1 was performed using T3 RNA
polymerase with the TNT coupled reticulolysate lysate system (Promega,
Mannheim, Germany) according to the manufacturer's instructions. The
C-terminal tails of the 5-HT2A (residues 380-471),
5-HT2B (residues 414-481), and 5-HT2C
(residues 368-458) receptors were subcloned into pGEX-3X, giving rise
to plasmids pGEX2A92, pGEX2B67 and pGEX2C90, respectively. Synthesis of
recombinant proteins (GST-2A92 and GST-2C90) in BL21 cells (Amersham
Pharmacia Biotech) was induced by 0.25 mM
isopropyl- Expression Plasmids--
pBSKSII-rMUPP1 was cut with
EcoRI and XhoI and ligated with a PCR product
containing a C-terminal VSV tag flanked by two XhoI sites
into pXMD1 (28), generating pXMD1/rMUPP-VSV. pBSKSII-rMUPP1 was cut
with NdeI and SalI and ligated into pCI-neo
(Promega) EcoRI and SalI sites using
adapter oligonucleotides containing the VSV tag (YTDIEMNRLGK) DNA
sequence (TACACCGATATCGAGATGAACAGGCTGGGAAAGTGA). The resulting plasmid,
pCI-neo/VSV- Cell Culture and Transfection--
Expression plasmids were
introduced into COS-7 cells by electroporation as described (31).
Briefly, cells were trypsinized, centrifuged, and resuspended in
electroporation buffer (50 mM K2HPO4, 20 mM
CH3CO2K, 20 mM KOH, and 26.7 mM MgSO4 (pH 7.4)) with 1 µg of
pRK5/h5-HT2C and 1.5 µg of pXMD1/rMUPP-VSV. The total amount of DNA was kept constant at 15 µg by filling up with pRK5 DNA.
After 15 min at room temperature, 107 cells were
transferred to a 0.4-cm electroporation cuvette (Bio-Rad) and pulsed
using a Gene Pulser apparatus (setting at 1000 microfarads and 280 V).
Cells were resuspended in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) containing 10% dialyzed fetal bovine serum (Life
Technologies, Inc.) and plated on 10-cm Falcon Petri dishes or into
12-well clusters.
Antibodies--
The production, characterization, and
purification of the rabbit polyclonal 522 antibody raised against the
mouse 5-HT2C receptor have been described (32). The mouse
monoclonal anti-c-Myc antibody was a gift from B. Mouillac. Rabbit
polyclonal anti-MUPP1 antiserum was raised against GST fusion protein
containing the third PDZ domain of rat MUPP1 (residues 318-451)
(antibody 2324) or amino acids 780-1063 of human MUPP1, containing the
region between the fourth and fifth PDZ domains (antibody
2526).2 The mouse monoclonal
anti-VSV antibody was purchased from Sigma. The secondary antibodies
used were Oregon green-conjugated goat anti-rabbit or anti-mouse
antibody and Texas Red-conjugated goat anti-mouse or anti-rabbit IgG
antibody (all from Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA).
Membrane Preparations and Immunoprecipitation--
Rat choroid
plexus or COS-7 cells were briefly centrifuged (3 min at 200 × g), and pellets were resuspended in lysis buffer (50 mM Tris-HCl, 1 mM EDTA, and protease inhibitor
mixture (2.5 µg/ml each leupeptin, aprotinin, and antipain and 0.5 mM benzamidine (pH 7.4))), homogenized 20 times with a
glass-Teflon homogenizer at 4 °C, and centrifuged at 100,000 × g for 1 h. Each membrane pellet was resuspended in
CHAPS extraction buffer (50 mM Tris-HCl (pH 7.4) containing
0.05 mM EDTA, 10 mM CHAPS, and protease
inhibitor mixture (see above)) for 2 h in rotation at 4 °C.
After centrifugation (1 h at 100,000 × g),
CHAPS-soluble proteins were incubated overnight at 4 °C with 2 µl
of anti-5-HT2C receptor 522 antibody, anti-VSV antibody, or
anti-MUPP1 antiserum for the immunoprecipitation. 50 µl of protein
A-Sepharose beads (Sigma) was added to the supernatant, and the mixture
was then rotated at 4 °C for 1 h. After five washes in
CHAPS-free buffer, the immunoprecipitates and co-immunoprecipitates were dissociated in Laemmli sample buffer. The samples were
centrifuged, and the supernatants were fractionated by
SDS-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel,
electrotransferred to nitrocellulose membrane (Hybond C extra, Amersham
Pharmacia Biotech), probed with rabbit anti-5-HT2C receptor
antibody or anti-MUPP1 antiserum (1:500), and then detected by enhanced
the chemiluminescence method (Renaissance Plus, PerkinElmer Life Sciences).
Determination of Inositol Phosphate Accumulation--
6 h after
transfection, cells were incubated overnight in serum-free Dulbecco's
modified Eagle's medium with 1 µCi/ml
myo-[3H]inositol (17 Ci/mol; PerkinElmer Life
Sciences). Total inositol phosphate accumulation in response to
serotonin in the presence of 10 mM LiCl for 10 min at
37 °C was determined as described (33). The amount of
[3H]inositol phosphate formed was separated according to
the ion exchange method (34). Concentration-response curves,
EC50 values, and Scatchard analysis were calculated with
Synergy software.
Membrane Preparations and Radioligand Binding
Assay--
Membranes were prepared from transiently transfected COS-7
cells. 6 h after transfection, cells were incubated overnight in serum-free Dulbecco's modified Eagle's medium. Cells were washed twice in PBS, scraped with a rubber policeman, harvested in PBS, and
centrifuged at 4 °C for 4 min at 200 × g. The
pellet was resuspended in 10 mM HEPES (pH 7.4), 5 mM EGTA, 1 mM EDTA, and 0.32 M
sucrose and homogenized 10 times with a glass-Teflon homogenizer
at 4 °C. The homogenate was centrifuged at 100,000 × g for 20 min, and the membrane pellet was resuspended in 50 mM HEPES (pH 7.4) and stored at Immunocytochemistry--
Cells were grown on 35-mm dishes, fixed
24 h after transfection in 4% paraformaldehyde and PBS (pH 7.4)
for 20 min at room temperature, washed three times in 0.1 M
glycine buffer (pH 7.4), and permeabilized with 0.05% Triton X-100 for
5 min. Cells were washed in 0.2% gelatin and PBS and incubated
overnight at 4 °C with the primary antibody diluted 1:500 in 0.2%
gelatin and PBS. Cells were washed and incubated for 1 h at room
temperature with the secondary antibody diluted 1:1000 in 0.2% gelatin
and PBS. Cultures were washed, mounted on glass slides using Gel Mount (Biomeda Corp., Foster City, CA), and viewed on a Zeiss Axioplan 2 microscope (Zeiss, Göttingen, Germany).
Enzyme-linked Immunosorbent Assay--
Cells were seeded at a
density of 8 × 105 cells/well on a 12-well plate,
fixed 24 h after transfection in 4% paraformaldehyde and PBS (pH
7.4) for 20 min at 4 °C, and washed three times in 0.1 M
glycine buffer (pH 7.4). Cells were incubated for 5 min in 3%
H2O2 and PBS to minimize endogenous peroxidase
activity. The anti-c-Myc antibody (diluted 1:500 in PBS and 0.2%
bovine serum albumin) was applied for 1.5 h at 37 °C. Plates
were rinsed five times with PBS and incubated for 1 h at 37 °C
with horseradish peroxidase-conjugated anti-mouse antibody diluted
1:250 in PBS and 0.2% bovine serum albumin. Plates were rinsed seven
times with PBS, and the reaction was developed by adding the substrate ABTS. Absorbance was measured at 410 nm using an enzyme-linked immunosorbent assay reader. Control plates without cells were included
to determine background activity, which was subtracted from the
A410 readings. Each experiment was performed in quadruplicates.
Immunohistochemistry--
Rats were deeply anesthetized with
pentobarbital and transcardially perfused with a fixative
solution containing 4% paraformaldehyde and 0.1 M PBS (pH
7.4). Brains were removed, post-fixed at 4 °C for 2 h in the
same fixative solution, and stored overnight at 4 °C in PBS
containing 30% sucrose for cryoprotection. Sections of 10 µm were
cut with a cryostat (Microm HM500), collected on slides, and then
stored at In Situ Hybridization--
Rats (10-week-old Wistar male adult)
were killed by decapitation, and their brains were removed and frozen
immediately on dry ice. Microtome cryostat sections (20 µm) were
thaw-mounted onto gelatin-coated slides, air-dried, and kept at
Characterization of the PDZ Domain Interaction--
It is evident
that PDZ domains display sufficient variability to allow distinct
protein-protein interactions (36, 37). Since rat MUPP1 has 13 PDZ
domains, we analyzed the interaction of all parts of MUPP1 with the C
terminus of the 5-HT2C receptor. A library of fusion
proteins with the Gal4 activation domain was constructed through a
random generation of ~500-base pair DNA fragments by sonication of
pBSKSII-rMUPP1. This tagged fragment library was cotransfected into
yeast with the vector encoding the C terminus of the 5-HT2C
receptor attached to the Gal4 DNA-binding domain, and resultant yeast
colonies were selected by histidine starvation. DNAs from 13 selected
colonies were amplified by PCR with plasmid-specific primers, and the
amplified DNAs were sequenced. All sequences shared the entire MUPP1
PDZ10 coding region (Fig. 1), indicating
a selective interaction of the 5-HT2C receptor C terminus
with PDZ10. The complete PDZ10 domain seems to be required since PDZ10
was complete in all selected clones.
Characterization of the 5-HT2C Receptor C-terminal
Interaction--
Ion channels such as the
N-methyl-D-aspartate and Shaker-type
K+ channels share a C-terminal T/SXV* motif
known to interact with PDZ domains (18, 19). The 5-HT2C
receptor has a similar C-terminal SSV* sequence that may define a part
of the critical motif for interaction. To determine whether the 3 C-terminal amino acid residues in the 5-HT2C receptor are
essential for binding to the PDZ10 domain of MUPP1, mutational analysis
was performed in which the residue at each position was replaced with
an alanine, and interactions with MUPP1 were tested in the yeast
two-hybrid assay. A partial human MUPP1 clone (clone 1) (see Ref. 20)
encoding the C-terminal 454 amino acids including PDZ10-13 served as
bait. Substitution of Ser456 and Val458 with
Ala abolished the interaction with MUPP1 (Table
I). In contrast, the conservative
mutation of Ser456 to Thr and the mutation of
Ser457 to Ala were tolerated, although the resultant yeast
clones displayed reduced growth on histidine-lacking medium. To
independently demonstrate the interaction of MUPP1 with the
5-HT2C receptor C terminus, fusion proteins of either the
wild-type form or various mutants of C-terminal 5-HT2C
receptor sequences (90 amino acids) fused to GST were bound to
glutathione-Sepharose (Fig.
2A) and incubated with
in vitro translated 35S-labeled MUPP1 (clone 1, PDZ10-13). MUPP1 bound to the GST-5-HT2C fusion proteins
was resolved on an SDS-polyacrylamide gel and visualized by
autoradiography. Only the S457A mutant (Fig. 2A, fifth
lane) retained MUPP1-binding activity, as observed in the yeast
two-hybrid assay (Table I), which confirmed the SXV motif as
a critical determinant for the PDZ domain interaction.
Interaction of MUPP1 with 5-HT2A and 5-HT2B
Receptors--
Multiple sequence alignment of the C-terminal ends from
all members of the 5-HT2 receptor family (Fig.
2B) revealed a common C-terminal amino acid sequence motif,
EX(V/I)SXV*. We therefore analyzed whether the
5-HT2A or 5-HT2B receptor C terminus would also
interact with MUPP1. GST fusion proteins of the C-terminal 92 amino
acids of the 5-HT2A receptor (GST-2A92) and the C-terminal 67 amino acids of the 5-HT2B receptor (GST-2B67) were bound
to glutathione-coupled Sepharose beads and incubated with in
vitro translated 35S-labeled MUPP1 (clone 1, PDZ10-13). MUPP1 bound to both the GST-2A92 (Fig. 2C,
second lane) and GST-2B67 (fourth lane) fusion
proteins, but not to GST alone (first lane). Specificity of
the interaction with MUPP1 PDZ domains was demonstrated by
co-incubation with a 9-amino acid synthetic peptide mimicking the
5-HT2C receptor C terminus, which prevented MUPP1 binding
to all three GST-5-HT2 receptor fusion proteins (Fig.
2C, third, fifth, and seventh
lanes). A control peptide harboring the S456A and V458A mutations
did not compete with MUPP1 binding (data not shown), confirming the requirement of Ser (position Interaction of Heterologously Expressed 5-HT2C
Receptors with MUPP1--
To determine whether MUPP1 forms a protein
complex with the 5-HT2C receptor in living cells, COS-7
cells were transfected with cDNA encoding the human
5-HT2C receptor in the presence or absence of the
C-terminally VSV-tagged rat MUPP1 protein (MUPP1-VSV). In a
CHAPS-soluble cell extract, the anti-5-HT2C receptor 522 antibody (11) revealed two bands between 60 and 50 kDa, corresponding to the glycosylated and unglycosylated 5-HT2C receptor,
respectively (Fig. 3 first and
fifth lanes). In fact, treatment with
N-glycosidase F to remove N-linked sugars caused
a shift of the upper band to the level of the lower band (data not
shown). CHAPS-soluble extracts were immunoprecipitated by an anti-VSV
antibody and then immunoblotted with the anti-5-HT2C
receptor 522 antibody. 5-HT2C receptors were co-immunoprecipitated by MUPP1-VSV from cells cotransfected with 5-HT2C receptor and MUPP1 expression plasmids (Fig. 3,
second lane). This indicates that the MUPP1 protein and the
5-HT2C receptor are able to interact when expressed in a
heterologous system. In contrast, when cells were transfected with the
5-HT2C receptor alone, the receptor was not revealed in the
immunoprecipitates (Fig. 3, sixth lane), indicating the
specificity of the co-immunoprecipitation. The mutant
5-HT2C receptor construct in which the C-terminal Val of
the SXV PDZ-binding motif was replaced with Ala (V458A,
5-HT2C-SSA) was extracted in amounts similar to those of
the native 5-HT2C receptor, but to a higher extent
in the glycosylated form (Fig. 3, third lane). When
cells expressed MUPP1-VSV and the 5-HT2C-SSA receptor, the
mutant receptor could not be immunoprecipitated with the anti-VSV
antibody (Fig. 3, fourth lane). This is in accordance with
the in vitro experiments, confirming that the SXV
motif at the extreme C terminus of the 5-HT2C receptor is
the critical determinant for the interaction with MUPP1.
MUPP1 Induces 5-HT2C Receptor Clustering in COS-7
Cells--
Because the antibodies to both MUPP1 and 5-HT2C
receptors were derived from rabbits, a c-Myc epitope-tagged version of
the human 5-HT2C receptor was constructed.
Immunofluorescence was performed with COS-7 cells transiently
expressing the N-terminally c-Myc-tagged 5-HT2C
receptor. Staining with an anti-c-Myc antibody revealed a random
distribution of 5-HT2C receptors on membrane-type structures including intracellular membranes similar to the
distribution described previously (11) and in neurons (12) (Fig.
4A). In contrast, the
MUPP1-VSV protein, which was stained with an antibody raised against
the PDZ3 domain of MUPP1, was homogeneously distributed throughout
transiently transfected COS-7 cells (Fig. 4B). The specificity of the anti-MUPP1 antibody was confirmed using the following criteria: the preimmune serum did not show any labeling (data
not shown); preincubation with an excess (80 µg/ml) of a peptide
(GST-PDZ3) corresponding to the PDZ3 domain suppressed the
immunofluorescent staining (data not shown); and a single protein with
the expected molecular mass of 230 kDa was revealed on Western blots
prepared from total extracts of MUPP1-expressing COS-7 cells (data not
shown) or from rat choroid plexus (see Fig. 8). Coexpression of the
c-Myc-tagged 5-HT2C receptor together with MUPP1-VSV
revealed a distribution that was distinct from those observed for each
of them. Both proteins were colocalized and formed many clusters (Fig.
4C). A similar pattern of immunoreactive receptors was
obtained when the c-Myc-tagged 5-HT2C receptor was coexpressed with a truncated form of the rat MUPP1 protein
(VSV-
Confocal microscopy revealed that the clustered complex of MUPP1 and
5-HT2C receptors was localized on intracellular membranes, but also on the cell surface (Fig. 4E). To further
investigate cell-surface localization of the clustered complex,
selective permeabilization was applied. In non-permeabilized cells,
cell-surface expression of the N-terminally c-Myc-tagged
5-HT2C receptor could be immunostained by the anti-c-Myc
antibody. When tagged 5-HT2C receptors were expressed
alone, no significant labeling could be detected (data not shown).
However, when cells coexpressed tagged receptors and MUPP1, few
clusters were observed at the cell surface of non-permeabilized cells
(Fig. 4F, left panel). This difference could be
due to a better visualization of clustered versus dispersed
receptors. Subsequently, the same cells were permeabilized and
incubated with the anti-MUPP1 antibody, which stained numerous clusters
(Fig. 4F, center panel). As expected, only
those clusters localized on cell membranes were colocalized with
5-HT2C receptors (Fig. 4F, right
panel). To investigate whether MUPP1 modulates cell-surface
expression of 5-HT2C receptors, a cell-surface
enzyme-linked immunosorbent assay was performed using non-permeabilized
cells. Immunolabeled c-Myc-tagged 5-HT2C receptors were
quantified by a secondary horseradish peroxidase-conjugated anti-mouse
antibody. When c-Myc-tagged 5-HT2C receptors were
transfected alone, the enzymatic activity was 223.45 ± 2.31% of the control values (mock-transfected cells), whereas the
values were 212.95 ± 5.45% of the control values when
cotransfected with MUPP1. This suggests that MUPP1 does not alter
cell-surface expression of 5-HT2C receptors. In conclusion,
these results indicate that MUPP1 induces clustering of a few
5-HT2C receptors at the cell surface, but the total number
of cell-surface receptors remains unchanged. To test if this clustering
is mediated by the C-terminal interaction of the 5-HT2C
receptor with MUPP1, the mutant 5-HT2C-SSA receptor was
coexpressed with MUPP1. Whereas the mutant receptor displayed a
distribution similar to that of the wild-type 5-HT2C receptor in transfected COS-7 cells (data not shown), coexpression failed to form clusters (Fig. 4G).
5-HT2C Receptors Induce a Conformational Change in
MUPP1--
When MUPP1-VSV was transiently expressed in COS-7 cells,
the anti-VSV antibody was unable to detect the MUPP1-VSV protein (Fig.
5A). However, when MUPP1-VSV
was coexpressed with the 5-HT2C receptor, the same anti-VSV
antibody succeeded in staining the MUPP1 protein (Fig. 5B).
This clearly demonstrates that the VSV tag fused to the C terminus of
the MUPP1 molecule is unmasked upon interaction of MUPP1 with the
5-HT2C receptor C terminus. This interaction may induce a
conformational change and render the VSV tag accessible to the anti-VSV
antibody.
Spatial Distribution of MUPP1 Transcripts in Rat Brain and
Colocalization of MUPP1-5-HT2C Receptor Proteins in the
Choroid Plexus--
To verify that the MUPP1-5-HT2C
receptor interaction does exist within a tissue that endogenously
expresses both proteins, we first studied the spatial distribution of
MUPP1 transcripts in brain, where the distribution of
5-HT2C receptors is well known (11, 12). In situ
hybridization studies were carried out using an
[
To demonstrate that 5-HT2C receptors and MUPP1 also
interact in vivo, immunoprecipitation was performed with
CHAPS-soluble extracts of rat choroid plexus. Western blot analysis
revealed the presence of the MUPP1 protein in the choroid plexus (Fig. 8, first lane), which could
also be immunoprecipitated with the anti-MUPP1 antiserum directed
against the PDZ3 domain (second lane). The
preimmune serum failed to immunoprecipitate the MUPP1 protein (Fig. 8,
fourth lane). The anti-5-HT2C receptor antiserum was able to co-immunoprecipitate the MUPP1 protein (Fig. 8, third lane), confirming that MUPP1 and the 5-HT2C receptor
interact in rat choroid plexus tissues.
Phosphoinositide Hydrolysis Assays--
To test possible
functional implications of the MUPP1-5-HT2C receptor
interaction, serotonin-mediated phosphoinositide hydrolysis was
determined in transfected COS-7 cells expressing 5-HT2C
receptors in the absence or presence of MUPP1. In the both cases,
dose-dependent stimulation of 5-HT2C receptors
demonstrated identical EC50 values (0.9 ± 0.1 and
0.6 ± 0.1 nM, respectively), with an approximate 4-5-fold increase in basal inositol phosphate accumulation (Fig. 9A). The 5-HT2C
receptor density as determined by Scatchard analysis of
radioligand-saturation binding experiments was on the same order
for transfected COS-7 cells in absence or presence of MUPP1 (Fig.
9B). These data indicate that the interaction of
heterologously expressed 5-HT2C receptors with MUPP1 does
not influence receptor expression levels or the second messenger
activity of phosphoinositide-mediated phospholipase C
activation.
The results presented here demonstrate that MUPP1 selectively
interacts with the C-terminal SXV motif of the
5-HT2C receptor via PDZ10 in vitro and in
vivo. Using a random tagged fragment library of MUPP1, we showed
in yeast that the C terminus of the 5-HT2C receptor selects
exclusively PDZ10 for MUPP1 interaction. This interaction requires the
complete PDZ10 domain. In agreement with the literature, the
5-HT2C receptor terminating with a serine at position Immunoprecipitation of 5-HT2C receptors with CHAPS-soluble
extracts of transfected COS-7 cells revealed glycosylated and
unglycosylated receptors, which could be co-immunoprecipitated with
anti-MUPP1 antibodies. This suggests that MUPP1 interacts with the
glycosylated membrane-bound receptor as well as with the unglycosylated
intracellular receptor reserve. Immunofluorescence in transfected COS-7
cells revealed a different localization of MUPP1 and 5-HT2C
receptors when both were transfected separately. MUPP1 was
homogeneously distributed, whereas 5-HT2C receptors
localized within membrane-type structures, including intracellular
membranes. Coexpression induced the formation of MUPP1 clusters that
were strikingly different from the distribution observed with MUPP1
only. This suggests that the clustering within membranous structures
was mediated by the presence of 5-HT2C receptors.
Concerning the expression at the cell surface, MUPP1 appears to induce
clustering of a few 5-HT2C receptors, but the total number
of cell-surface receptors remains unchanged. The direct physical
interaction of MUPP1 with the PDZ-binding motif of 5-HT2C
receptors is manifested by the conformational change triggered within
the MUPP1 molecule. The functional importance of the 5-HT2C
receptor interaction with MUPP1 remains to be answered since MUPP1 did
not influence receptor expression levels or the activation of
phospholipase C. However, these experiments were performed in COS-7
cells, which might resemble an artificial environment. Since MUPP1
contains 13 different PDZ domains (20), a maximum of 13 different
players have to be taken into account that could potentially link the
MUPP1 molecule to specialized submembranous sites, thereby selectively
assembling unique signaling complexes. These proteins are unlikely to
be completely present in COS-7 cells. Therefore, to investigate the functional relevance of the MUPP1-5-HT2C receptor
interaction, tissues that endogenously express both proteins have to be
analyzed. Indeed, using confocal microscopy, we demonstrated that MUPP1 and 5-HT2C receptors colocalize exclusively at the apical
surface of epithelial cells from choroid plexus tissues. The apical
localization is in agreement with an early report suggesting an
activation of 5-HT2C receptors by cerebrospinal
fluid-borne serotonin (41).
Human 5-HT2A and 5-HT2B receptors share the
C-terminal EX(V/I)SXV sequence with
5-HT2C receptors even though all three 5-HT2 receptors differ greatly in the overall identity of their intracellular C-terminal amino acid sequences. In accordance with the striking conservation of a common PDZ-binding motif at their extreme C terminus,
all three receptors bind to MUPP1 PDZ domains in vitro. Since we demonstrated the in vivo interaction of MUPP1 with
the 5-HT2C receptor, interactions with 5-HT2A
or 5-HT2B receptors may occur at least in tissues
displaying overlapping expression with MUPP1. In contrast to
5-HT2C receptors, which appear to be exclusively expressed
in neuronal tissues (42), 5-HT2A and 5-HT2B receptors are also present in peripheral organs. 5-HT2A
receptor mRNA has been shown to be present in various human smooth
muscle cells (26), and 5-HT2B receptor mRNA in human
heart, placenta, liver, kidney, and pancreas (43, 44), tissues that are
known to express MUPP1 (20). In rat brain, MUPP1 expression coincides with 5-HT2A transcripts in the cerebral cortex, the
olfactory system including the mitral cell layer and the piriform
cortex, the CA3 pyramidal cell layer, and the pontine nuclei (45).
5-HT2B receptors are coexpressed with MUPP1
transcripts in the medial amygdala, dorsal hypothalamus, frontal
cortex, and granular layer of the cerebellum (46, 47).
This is the first report of a protein interacting with a serotonin
receptor. The presence of a PDZ-binding motif at the C termini of the
5-HT2A, 5-HT2B, and 5-HT2C
receptors has been documented (18, 20, 48). To date, there are two
reports about possible functions of the C-terminal PDZ-binding motifs
of the mouse 5-HT2B (48) and rat 5-HT2C (49)
receptors. The serine at position In a murine cell line expressing 5-HT2B receptors, it was
demonstrated that stimulation of 5-HT2B receptors triggers
an increase in intracellular cGMP through dual activation of
constitutive and inducible nitric-oxide synthase. This activity is
dependent on the C-terminal PDZ-binding motif (48). 5-HT2C
receptors have also been shown to be involved in nitric oxide signaling
such as inhibition of the
N-methyl-D-aspartate/nitric oxide/cGMP pathway in the rat cerebellum (51) and stimulation of cGMP formation in the
choroid plexus (52), which in turn inhibits phosphoinositide turnover
in the choroid plexus (53). PDZ interactions with MUPP1 could provide
the basis for 5-HT2C receptor-mediated cross-talks between
second messenger pathways such as phosphoinositide hydrolysis and
nitric oxide signaling. By searching for the proteins that interact
with the remaining 12 PDZ domains of MUPP1, novel intracellular targets
that might contribute to our understanding of 5-HT2C
receptor trafficking and/or signaling will be identified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor, which
interacts with the Na+/H+ exchanger regulatory
factor (NHERF/EBP50)1 (1).
The interaction of NHERF with the
2-adrenergic receptor is mediated via binding of the first PDZ domain of NHERF to the extreme
C terminus of the
2-adrenergic receptor in an
agonist-dependent manner, thereby regulating
Na+/H+ exchange (2). NHERF has also been
described to link proteins with the actin cytoskeleton through
association with ERM
(ezrin-radixin-moesin) proteins
(3). In fact, the PDZ-mediated interaction of the
2-adrenergic receptor with NHERF family proteins has
been shown to control recycling of internalized
2-adrenergic receptors. Disrupting the
2-adrenergic receptor-NHERF interaction perturbs the
endocytic sorting of the
2-adrenergic receptor,
resulting in lysosomal degradation (4).
motif domain, and a
proline-rich region that links Shanks to cortactin, a constituent of
the actin cytoskeleton (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase reporter genes under the control of upstream
GAL4-binding sites (CLONTECH). The yeast
culture was transformed using the polyethylene glycol/LiAc method (25).
Interactions between bait and prey were monitored by
-galactosidase
activity in colonies transferred onto Hybond N filters (Amersham
Pharmacia Biotech, Freiburg, Germany).
183 to 2000 with a silent mutation of C1979 to T to
delete a BamHI site was generated by reverse
transcription-PCR from rat femoral muscle cDNA that was prepared as
described (26) using recombinant Pfu polymerase (Stratagene,
La Jolla, CA). The resulting 5'-end was ligated into the remaining
BamHI site of the partial rat MUPP1 cDNA
(pXMD1/rMUPP1) (20). pBSKSII-rMUPP1 was sonicated to an average size of
500 base pairs, and blunt-ended DNA fragments were cloned into the
SrfI site of pCR-Script (Stratagene). The inserts of 20,000 recombinant plasmids retrieved by NotI and EcoRI
digestion were directionally cloned into the XhoI and
EcoRI sites of pACT-2 using NotI/XhoI
adapter oligonucleotides. The resulting library was transformed into
yeast strain CG1945 carrying pAS2-1/hu2C. pAS2-1/hu2C was described
previously (20). The resulting plasmid fuses amino acids 369-458 of
human 5-HT2C receptors to the Gal4 DNA-binding
domain (amino acids 1-147) of yeast Gal4 in the yeast expression
vector pAS2-1 (CLONTECH). Transformants selected
for HIS3 expression were picked after 5 days. Plasmid DNA
was extracted, and inserts were retrieved by PCR with plasmid-specific primers, sequenced, and aligned with the rat MUPP1 cDNA sequence.
-D-thiogalactopyranoside for 3 h at
30 °C. GST-2B67 was induced by 0.1 mM
isopropyl-
-D-thiogalactopyranoside for 3 h at
25 °C. Cells were sonicated in buffer S (20 mM HEPES (pH
7.9), 100 mM KCl, 0.5 mM EDTA, 1 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) and
pelleted in buffer S, 1% Triton, and 10% glycerol. GST proteins were
purified on bulk glutathione-Sepharose 4B (Amersham Pharmacia Biotech)
according to the manufacturer's instructions, with the exception of
using buffer S instead of PBS. GST proteins were eluted with buffer S,
10% glycerol, and 10 mM glutathione and dialyzed against
buffer S and 10% glycerol (2 × 3 h at 4 °C).
35S-Labeled MUPP1 was incubated in buffer S, 10% glycerol,
and 1% Nonidet P-40 with glutathione-Sepharose 4B beads saturated with 10 µg of GST fusion protein or GST for 3.5 h at 4 °C in the
presence or absence of 1.5 mM synthetic peptide VVSERISSV
or control peptide VVSERIASA (B&G Biotech GmbH, Freiburg). Beads were
washed four times for 10 min in buffer S, 10% glycerol, and 1%
Nonidet P-40. Bound proteins were resolved by SDS-polyacrylamide gel
electrophoresis and visualized by autoradiography.
MUPP1, fuses the N-terminal VSV tag and amino acids
1337-2055 of the rat MUPP1 protein. pRK5/h5-HT2C was
directionally subcloned from pXMD1/h5-HT2C (29) by
releasing the cDNA insert with the restriction digest enzymes
EcoRI and XbaI. Using PCR, the human
5-HT2C receptor cDNA was N-terminally tagged with a
stretch of nucleotides (ATGGAACAAAAGCTTATTTCTGAAGAAGACTTG) encoding a 10-amino acid epitope (EQKLISEEDL) of the human c-Myc protein (30). The amplified DNA was directionally subcloned into the
EcoRI and XbaI sites of pRK5, yielding
pRK5/c-Myc-hu5-HT2C.
80 °C until used.
5-HT2C receptor densities were estimated using the specific
radioligand
[N6-methyl-3H]mesulergine
at a saturating concentration of 4 nM. Mianserin (1 µM) was used to determine nonspecific binding. Protein
concentrations were determined using the Bradford protein assay
(Bio-Rad).
80 °C. Prior to the experiment, sections were rinsed
serially for 5 min once with PBS containing 20% sucrose, 10%
sucrose, and without sucrose, respectively. Preincubation was performed
in 0.25% Triton X-100, 20% horse serum, and PBS (pH 7.4) for 1 h
at room temperature. Sections were incubated with either the rabbit
polyclonal anti-5-HT2C receptor 522 antibody or
affinity-purified anti-MUPP1 2324 or 2526 antibody diluted 1:500 in 1%
horse serum and PBS overnight at 4 °C. After three washes in PBS,
sections were incubated with donkey Cy3-conjugated anti-rabbit IgG
antibody (1:2000; Jackson ImmunoResearch Laboratories, Inc.) in 1%
horse serum and PBS at 4 °C for 3 h. Sections were rinsed with
PBS and mounted on slides, which were coverslipped with Mowiol (Calbiochem).
20 °C until used. Hybridization was performed at 60 °C
essentially as described (35) using radioactive
[
-33P]UTP-labeled riboprobes that were prepared as
follows. The plasmid pXMD1/rMUPP1 (20) was cut with SmaI to
release a fragment of 2669 base pairs covering the region from 4653 to
the 3'-end of the rat MUPP1 cDNA, which was ligated into
the SrfI site of pCR-Script. For in vitro
transcription, both sense and antisense constructs were linearized with
XhoI and transcribed using T3 RNA polymerase. RNA synthesis
was performed in a 20-µl reaction with 1 µg of linearized plasmid
DNA, 10 mM dithiothreitol, 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.1 mM UTP, 70 µCi of [
-33P]UTP, 40 units of RNasin (Promega), and
20 units of RNA polymerase (Roche Molecular Biochemicals, Mannheim) and
incubated for 1 h at 37 °C. Synthesis was continued with the
addition of 20 units of RNasin and 20 units of RNA polymerase for
1 h. DNA was digested with 10 units of RQ1 RNase-free DNase
(Promega) for 20 min. The reaction was stopped with 5 µl of EDTA (0.5 M) and applied to a Sephadex G-50 spin column (Roche
Molecular Biochemicals). RNA was hydrolyzed in 20 mM
NaHCO3 and 30 mM NaCO3 for 20 min
and neutralized in 3.3 mM HCl.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
The C-terminal domain of the
5-HT2C receptor interacts with PDZ10 of MUPP1.
Randomly generated MUPP1 fragments interacting in the yeast two-hybrid
system with the C-terminal sequence of the 5-HT2C receptor
are displayed relative to the domain map and are identified by amino
acid numbers. All selected clones share the PDZ10 coding sequence
(shaded).
Sequence requirements in the 5-HT2C receptor carboxyl terminus
mediating interaction with MUPP1
, no
significant growth.
-Galactosidase (
-Gal) activity was determined
from the time taken for colonies to turn blue in the
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal)
filter lift assay: ++, <3 h; +, 3-6 h;
, no significant
-galactosidase activity.
View larger version (25K):
[in a new window]
Fig. 2.
In vitro binding of C-terminal
receptor sequences to MUPP1. DNA encoding C-terminal receptor
sequences in-frame with the GST moiety were bacterially expressed and
purified on glutathione-Sepharose. GST pull-down reactions were
performed with in vitro translated 35S-labeled
MUPP1 (clone 1, PDZ10-13), and the adsorbed proteins were separated by
SDS-polyacrylamide gel electrophoresis. MUPP1 that was copurified with
the fusion protein was identified by autoradiography. A,
analysis of GST fusion proteins that contained 90-amino acid C-terminal
5-HT2C receptor sequences from either the wild-type form
(GST-2C90-SSV) or mutants in which 1 of the last 3 amino acids was
replaced by alanine as indicated. B, alignment of the
C-terminal 30 amino acids of human 5-HT2A,
5-HT2B, and 5-HT2C receptors. All sequences
share the C-terminal EX(V/I)SXV* motif, where the
asterisk indicates a carboxyl group. C, analysis of the GST
fusion protein with the C-terminal 92 amino acids of the
5-HT2A receptor sequence (GST-2A92) or the C-terminal 67 amino acids of the 5-HT2B receptor (GST-2B67) or GST-2C90.
Where indicated, an 8.5-fold excess of the
EX(V/I)SXV sequence-containing peptide was
present during incubation.
2) and Val (position 0) for PDZ domain interaction.
View larger version (24K):
[in a new window]
Fig. 3.
Co-immunoprecipitation of MUPP1 and
5-HT2C receptors from transfected COS-7 cells. Cells
were transfected with MUPP1-VSV and the 5-HT2C receptor
(5-HT2C R) (first and second
lanes), MUPP1-VSV and the mutant 5-HT2C-SSA receptor
(third and fourth lanes), or the
5-HT2C receptor alone (fifth and sixth lanes).
Membrane fractions were solubilized with CHAPS and then
immunoprecipitated (IP) using the anti-VSV antibody
(second, fourth, and sixth lanes). The
immunoprecipitates were analyzed by Western blotting using the
anti-5-HT2C receptor antiserum.
MUPP1) containing only the last six of the 13 PDZ domains
(PDZ8-13) (Fig. 4D). When cells coexpressing MUPP1 and
5-HT2C receptors were treated with 5-hydroxytryptamine (30 min, 1 h, and overnight), no changes in the distribution of
5-HT2C receptors and MUPP1 were observed (data not
shown).
View larger version (23K):
[in a new window]
Fig. 4.
MUPP1 induces 5-HT2C receptor
clustering in transiently transfected COS-7 cells.
A, cells expressing c-Myc-tagged 5-HT2C
receptors (5-HT2C R) were immunostained using the anti-c-Myc
antibody. B, cells expressing MUPP1-VSV were immunostained
using the anti-MUPP1 antiserum. C, in cells expressing
c-Myc-tagged 5-HT2C receptors and MUPP1-VSV, both proteins
were contained within the same clusters, as indicated by
arrows. D, in cells expressing 5-HT2C
receptors and VSV- MUPP1, the same clusters formed as well.
E, shown is a confocal section of a cell expressing
c-Myc-tagged 5-HT2C receptors and MUPP1-VSV. Most clusters
were formed intracellularly. Arrows indicate clusters at or
near the cell-surface membrane. F, cells expressing
c-Myc-tagged 5-HT2C receptors and MUPP1-VSV were fixed and
stained with the anti-N-terminal c-Myc antibody to label surface
5-HT2C receptors (left panel). The same cells
was then permeabilized and immunostained using the anti-MUPP1 antiserum
(center panel). Arrows indicate clusters at the
cell-surface membrane. G, cells coexpressing the mutant
5-HT2C-SSA receptor and MUPP1-VSV failed to form
clusters.
View larger version (29K):
[in a new window]
Fig. 5.
5-HT2C receptors unmask the VSV
tag of MUPP1-VSV. A, COS-7 cells were transiently
transfected with MUPP1-VSV. MUPP1 was detected with the anti-MUPP1
antiserum (right panel), but not with the anti-VSV antibody
(left panel). B, in cells expressing
5-HT2C receptors (5-HT2C R) and MUPP1-VSV,
5-HT2C receptors were immunostained using the
anti-5-HT2C receptor antiserum (left panel), and
MUPP1 was immunostained using the anti-VSV antibody (right
panel). 5-HT2C receptors and MUPP1 were contained
within the same clusters, indicated by arrows.
-33P]UTP-labeled riboprobe (Fig.
6). MUPP1 transcripts are
abundant in all cerebral cortical layers, especially the piriform
cortex, the pyramidal cells of the CA1-CA3 subfields of the
hippocampus, as well as the granular layer of the dentate gyrus.
MUPP1 mRNA was detected in the internal granular layer
and the mitral cell layer of the olfactory bulb; in the medial
habenular nucleus; and in amygdaloid, thalamic, hypothalamic, and
pontine nuclei. In the cerebellum, high levels of transcripts
were found in the granular layer. Transcripts were detected in the
lateral ventricle, which was due to staining of the epithelial
ependymal cells as well as the choroid plexus. These results indicate
that MUPP1 mRNA colocalizes with 5-HT2C
receptor expression in all regions of the rat brain (11), including the
choroid plexus, where 5-HT2C receptors are highly enriched
(38, 39). Immunocytochemistry and confocal microscopy were applied to
investigate the colocalization of MUPP1 and 5-HT2C receptor
proteins in choroid plexus tissues. Both proteins were expressed on the
apical membrane of epithelial choroid plexus cells (Fig.
7, A and B).
View larger version (120K):
[in a new window]
Fig. 6.
In situ hybridization analysis of
MUPP1 transcripts in rat brain. Tissue sections
were hybridized to 33P-labeled antisense probes. Shown are
representative autoradiograms of coronal sections sliced at bregma 6.7, 0.4, and 3.14 mm, respectively. A, sagittal section sliced
laterally at 1.9 mm; B, a horizontal section at bregma 4.28 nm; C, according to the atlas of Ref. 54. Only very weak
hybridization signals were observed in the granular layer of the
cerebellum with the radiolabeled sense probe (data not shown).
AN, amygdaloid nuclei; Cx, cortex; fields
CA1-CA3 of hippocampus; DG,
dentate gyrus; GL, granular layer; IGr, internal
granular layer of the olfactory bulb; LV, lateral ventricle;
Mi, mitral cell layer of the olfactory bulb; MHb,
medial habenular nucleus; MnPO, median preoptic nucleus;
Pir, piriform cortex; Pn, pontine nuclei;
TN, thalamic nuclei.
View larger version (99K):
[in a new window]
Fig. 7.
Immunohistochemistry of MUPP1 and the
5-HT2C receptor in the choroid plexus. A,
immunolabeling of a choroid plexus slice (10 µm) with the
anti-5-HT2C receptor (5-HT2C
R) antibody; B, immunolabeling with the
anti-MUPP1 antiserum; C, immunolabeling with the preimmune
antiserum. 4V, fourth ventricle; ECP, epithelial
choroid plexus cells.
View larger version (32K):
[in a new window]
Fig. 8.
Co-immunoprecipitation of MUPP1 and
5-HT2C receptors in CHAPS-soluble extracts of the choroid
plexus. Membrane fractions of rat choroid plexus were solubilized
with CHAPS (first lane) and immunoprecipitated
(IP) with the anti-5-HT2C receptor antiserum
(second lane ) or with the anti-MUPP1 antiserum (third
lane). The immunoprecipitates were analyzed by Western blotting
using the anti-MUPP1 antiserum. MUPP1 was not immunoprecipitated by the
preimmune serum (fourth lane).
View larger version (11K):
[in a new window]
Fig. 9.
Inositol phosphate accumulation and receptor
density in the absence and presence of MUPP1. A,
stimulation of inositol phosphate (IP) accumulation by
increasing concentrations of 5-hydroxytryptamine; B,
Scatchard analysis of saturation experiments of
[3H]mesulergine binding to membranes in COS-7 cells
transfected with 5-HT2C receptors (5-HT2C R) and
MUPP1 or with 5-HT2C receptors alone. The results are
representative of three experiments performed in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
selects the PDZ10 domain, which displays a histidine at the
B1
position (20). His
B1 has been shown to
coordinate the hydroxyl group of the serine at position
2 of the
PDZ-binding motif (40).
2 was shown to enhance
resensitization of the 5-HT2C receptor responses (49). This
critical Ser
2 is part of two possible
phosphorylation sites contained within the 5-HT2C receptor
PDZ-binding motif. The presence of these phosphorylation sites implies
that phosphorylation might regulate the capacity of the binding of the
PDZ recognition sequence to the MUPP1 PDZ10 domain. Phosphorylation
sites for Ser
2 within PDZ-binding
motifs were also observed for the C terminus of the
2-adrenergic receptor (50). It has been suggested that G-protein-coupled-receptor kinase-5-mediated phosphorylation may disrupt the interaction with NHERF and thereby regulate the sorting of
internalized
2-adrenergic receptors (4). A similar role for the PDZ-binding motif of the 5-HT2C receptor can be anticipated.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to M. Staufenbiel and D. Abramowski (Novartis Pharma AG, Basel, Switzerland) for the 5-HT2C receptor 522 antiserum, M. Lerner-Natoli for assistance with immunohistochemistry experiments, M. Sebben for assistance with the binding experiments, and A. Turner-Madeuf for help in language revision.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from CNRS.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.
Incumbent of the Madeleine Haas Russell Career Development Chair.
To whom correspondence should be addressed. Tel.:
49-214-8763235; Fax: 49-214-8763290; E-mail:
ullmer@biofrontera.de.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M008089200
2 S. Poliak and E. Peles, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NHERF, Na+/H+ exchanger regulatory factor; 5-HT2C, 5-hydroxytryptamine type 2C; PCR, polymerase chain reaction; GST, glutathione S-transferase; PBS, phosphate-buffered saline; VSV, vesicular stomatitis virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hall, R. A., Premont, R. T., Chow, C. W., Blitzer, J. T., Pitcher, J. A., Claing, A., Stoffel, R. H., Barak, L. S., Shenolikar, S., Weinman, E. J., Grinstein, S., and Lefkowitz, R. J. (1998) Nature 392, 626-630[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Hall, R. A.,
Ostedgaard, L. S.,
Premont, R. T.,
Blitzer, J. T.,
Rahman, N.,
Welsh, M. J.,
and Lefkowitz, R. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8496-8501 |
3. |
Reczek, D.,
Berryman, M.,
and Bretscher, A.
(1997)
J. Cell Biol.
139,
169-179 |
4. | Cao, T. T., Deacon, H. W., Reczek, D., Bretscher, A., and von Zastrow, M. (1999) Nature 401, 286-290[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Zitzer, H.,
Richter, D.,
and Kreienkamp, H. J.
(1999)
J. Biol. Chem.
274,
18153-18156 |
6. |
Zitzer, H.,
Honck, H. H.,
Bachner, D.,
Richter, D.,
and Kreienkamp, H. J.
(1999)
J. Biol. Chem.
274,
32997-33001 |
7. | Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, C., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569-582[Medline] [Order article via Infotrieve] |
8. |
Lim, S.,
Naisbitt, S.,
Yoon, J.,
Hwang, J. I.,
Suh, P. G.,
Sheng, M.,
and Kim, E.
(1999)
J. Biol. Chem.
274,
29510-29518 |
9. | Boeckers, T. M., Winter, C., Smalla, K. H., Kreutz, M. R., Bockmann, J., Seidenbecher, C., Garner, C. C., and Gundelfinger, E. D. (1999) Biochem. Biophys. Res. Commun. 264, 247-252[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Du, Y.,
Weed, S. A.,
Xiong, W. C.,
Marshall, T. D.,
and Parsons, J. T.
(1998)
Mol. Cell. Biol.
18,
5838-5851 |
11. | Abramowski, D., Rigo, M., Hoyer, D., and Staufenbiel, M. (1995) Neuropharmacology 34, 1635-1645[CrossRef][Medline] [Order article via Infotrieve] |
12. | Clemett, D. A., Punhani, T., Duxon, M. S., Blackburn, T. P., and Fone, K. C. (2000) Neuropharmacology 39, 123-132[CrossRef][Medline] [Order article via Infotrieve] |
13. | Lucki, U., Ward, H. R., and Frazer, A. (1989) J. Pharmacol. Exp. Ther. 249, 155-164[Abstract] |
14. | Murphy, D. L., Lesch, K. P., Aulakh, C. S., and Pigott, T. A. (1991) Pharmacol. Rev. 43, 527-552[Medline] [Order article via Infotrieve] |
15. | Tecott, L. H., Sun, L. M., Akana, S. F., Strack, A. M., Lowenstein, D. H., Dallman, M. F., and Julius, D. (1995) Nature 374, 542-546[CrossRef][Medline] [Order article via Infotrieve] |
16. | Millan, M. J., Girardon, S., and Bervoets, K. (1997) Neuropharmacology 36, 743-745[CrossRef][Medline] [Order article via Infotrieve] |
17. | Fone, K. C. F., Austin, R. H., Topham, I. A., Kennett, G. A., and Punhani, T. (1998) Br. J. Pharmacol. 123, 1707-1715[Abstract] |
18. | Kornau, H. C., Schenker, L. T., Kennedy, M. B., and Seeburg, P. H. (1995) Science 269, 1737-1740[Medline] [Order article via Infotrieve] |
19. | Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N., and Sheng, M. (1995) Nature 378, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
20. | Ullmer, C., Schmuck, K., Figge, A., and Lübbert, H. (1998) FEBS Lett. 424, 63-68[CrossRef][Medline] [Order article via Infotrieve] |
21. | Kurschner, C., Mermelstein, P. G., Holden, W. T., and Surmeier, D. J. (1998) Mol. Cell. Neurosci. 11, 161-172[CrossRef][Medline] [Order article via Infotrieve] |
22. | Simpson, E. H., Suffolk, R., and Jackson, I. J. (1999) Genomics 59, 102-104[CrossRef][Medline] [Order article via Infotrieve] |
23. | Philipp, S., and Flockerzi, V. (1997) FEBS Lett. 413, 243-248[CrossRef][Medline] [Order article via Infotrieve] |
24. | Feilotter, H. E., Hannon, G. J., Ruddel, C. J., and Beach, D. (1994) Nucleic Acids Res. 22, 1502-1503[Medline] [Order article via Infotrieve] |
25. | Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425[Medline] [Order article via Infotrieve] |
26. | Ullmer, C., Schmuck, K., Kalkman, H. O., and Lübbert, H. (1995) FEBS Lett. 370, 215-221[CrossRef][Medline] [Order article via Infotrieve] |
27. | Annweiler, A., Hipskind, R. A., and Nordheim, A. (1991) Nucleic Acids Res. 19, 3750[Medline] [Order article via Infotrieve] |
28. | Kluxen, F. W., Bruns, C., and Lübbert, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4618-4622[Abstract] |
29. | Schmuck, K., Ullmer, C., Probst, A., Kalkman, H. O., and Lübbert, H. (1996) Eur. J. Neurosci. 8, 959-967[Medline] [Order article via Infotrieve] |
30. | Ramsay, G., Evan, G. I., and Bishop, J. M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7742-7746[Abstract] |
31. | Claeysen, S., Sebben, M., Journot, L., Bockaert, J., and Dumuis, A. (1996) FEBS Lett. 398, 19-25[CrossRef][Medline] [Order article via Infotrieve] |
32. | Abramowski, D., and Staufenbiel, M. (1995) J. Neurochem. 65, 782-790[Medline] [Order article via Infotrieve] |
33. | Berg, K. A., Maayani, S., and Clarke, W. P. (1996) Mol. Pharmacol. 50, 1017-1023[Abstract] |
34. | Berridge, M. J., Dawson, C. P., Downes, C. P., Heslop, J. P., and Irvine, R. F. (1983) Biochem. J. 212, 473-482[Medline] [Order article via Infotrieve] |
35. | Bartsch, S., Bartsch, U., Döries, U., Faissner, A., Weller, A., Ekblom, P., and Schachner, M. (1992) J. Neurosci. 12, 736-749[Abstract] |
36. |
Songyang, Z.,
Fanning, A. S.,
Fu, C.,
Xu, J.,
Marfatia, S. M.,
Chishti, A. H.,
Crompton, A.,
Chan, A. C.,
Anderson, J. M.,
and Cantley, L. C.
(1997)
Science
275,
73-77 |
37. | Schultz, J., Hoffmüller, U., Krause, G., Ashurst, J., Marcias, M. J., Schmieder, P., Schneider-Mergener, J., and Oschkinat, H. (1998) Nat. Struct. Biol. 5, 19-24[Medline] [Order article via Infotrieve] |
38. | Pazos, A., Hoyer, D., and Palacios, J. (1984) Eur. J. Pharmacol. 106, 539-546[CrossRef][Medline] [Order article via Infotrieve] |
39. | Yagaloff, K., and Hartig, P. R. (1985) J. Neurosci. 5, 3178-3183[Abstract] |
40. | Doyle, D. A., Lee, A., Lewis, J., Kim, E., Sheng, M., and MacKinnon, R. (1996) Cell 85, 1067-1076[Medline] [Order article via Infotrieve] |
41. | Hartig, P. R. (1989) in Serotonin: Actions, Receptors, Pathophysiology (Mylecharone, E. J. , Angus, J. A. , De la Londe, I. S. , and Humphrey, P. P. A., eds) , pp. 180-187, Macmillan Press Ltd., London |
42. | Julius, D., MacDermott, A. B., Axel, R., and Jessell, T. M. (1988) Science 241, 558-564[Medline] [Order article via Infotrieve] |
43. | Schmuck, K., Ullmer, C., Engels, P., and Lübbert, H. (1994) FEBS Lett. 342, 85-90[CrossRef][Medline] [Order article via Infotrieve] |
44. | Bonhaus, D. W., Bach, C., DeSouza, A., Salazar, F. H., Matsuoka, B. D., Zuppan, P., Chan, H. W., and Eglen, R. M. (1995) Br. J. Pharmacol. 115, 622-628[Abstract] |
45. | Pompeiano, M., Palacios, J. M., and Mengod, G. (1994) Mol. Brain Res. 23, 163-178[Medline] [Order article via Infotrieve] |
46. | Choi, D., and Maroteau, L. (1996) FEBS Lett. 391, 45-51[CrossRef][Medline] [Order article via Infotrieve] |
47. | Duxon, M. S., Flanigan, T. P., Reavley, A. C., Baxter, G. S., Blackburn, T. P., and Fone, K. C. F. (1996) Neuroscience 76, 323-329[CrossRef] |
48. |
Manivet, P.,
Mouillet-Richard, S.,
Callebert, J.,
Nebigil, C. G.,
Maroteaux, L.,
Hosoda, S.,
Kellermann, O.,
and Launay, J. M.
(2000)
J. Biol. Chem.
275,
9324-9331 |
49. |
Backstrom, J. R.,
Price, R. D.,
Reasoner, D. T.,
and Sanders-Bush, E.
(2000)
J. Biol. Chem.
275,
23620-23626 |
50. |
Fredericks, Z. L.,
Pitcher, J. A.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
13796-13803 |
51. |
Marcoli, M.,
Maura, G.,
Tortarolo, M.,
and Raiteri, M.
(1998)
J. Pharmacol. Exp. Ther.
285,
983-986 |
52. | Kaufman, M. J., Hartig, P. R., and Hoffman, B. J. (1995) J. Neurochem. 64, 199-205[Medline] [Order article via Infotrieve] |
53. | Kaufman, M. J., and Hirata, F. (1996) Neurosci. Lett. 206, 153-156[CrossRef][Medline] [Order article via Infotrieve] |
54. | Paxinos, G., and Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates , Academic Press, Inc., San Diego, CA |