From the Departments of Pharmacology and of
Molecular Physiology and Biophysics, Vanderbilt University
Medical Center, Nashville, Tennessee 37232-6600
Received for publication, December 26, 2000
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ABSTRACT |
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Previously, we demonstrated that the third
intracellular (3i) loop of the heptahelical
The three Although all three Receptor retention on the lateral subdomain of MDCKII cells likely
involves the third intracellular loop of the Based on our findings that the Materials--
The pGEMEX-2 vector and TnT in
vitro translation kit were from Promega (Madison, WI). The
[35S]methionine (1000 Ci/mmol, at 10 mCi/ml) was
purchased from PerkinElmer Life Sciences (Boston, MA). PVDF nylon
membranes were from Millipore (Bedford, MA). The fast protein liquid
chromatography and DEAE-Sephacel columns were from Amersham Pharmacia
Biotech (Piscataway, NJ). Dodecyl- MDCKII Cell Culture and Polarization--
MDCKII cells were
plated at confluence (~1-2.5 × 105 cells) and
grown on 12-mm Transwell filters (0.4-µm pore size, Costar, Cambridge, MA) in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (Sigma) and 100 units/ml
penicillin and 10 µg/ml streptomycin at 37 °C/5% CO2
as described previously (19) except with daily media changes for 5-7
days. Under these conditions, cells form a monolayer and functionally
polarize with distinct apical and basolateral surfaces separated by
tight junctions. We routinely verify that tight junctions have formed
and that the apical and basolateral compartments are functionally
separated from one another using the nontransportable molecule
[3H]methoxy-inulin (19). For these leak assays, 2 µCi
of [3H]methoxy-inulin is added to the apical
subcompartment and incubated for 1 h at 37 °C/5%
CO2 followed by counting 100 µl of the medium in each of
the apical and basolateral subcompartments. Leaks range from 5-10%,
and we discard from study any culture wells of >10% leak.
Immunofluorescent Labeling and Confocal Microscopy
Antibody Purification--
Rabbit anti-spinophilin antibodies
were generated by injection of purified glutathione
S-transferase (GST) fusion proteins (fused to spinophilin
amino acids 286-390) as described previously by MacMillan et
al. (26). Antibodies were purified from serum by affinity
chromatography. Affinity matrices were generated by mixing 2 ml of
Affi-Gel-15 and 1 ml of Affi-Gel-10 (Bio-Rad) equilibrated in 0.1 M HEPES, pH 7.0, in a 10 ml of a Poly Prep chromatography column (Bio-Rad). Purified GST-Sp286-390 fusion protein (11.7 mg in
6.5 ml of PBS) was loaded onto the column and incubated with inversion
for 4 h at 4 °C. The resin was washed with 1× PBS until free
of unbound GST-Sp286-390, as determined by
A280. Unbound sites on the Affi-Gel matrix were
blocked by incubation with 1 M ethanolamine for 1 h at
4 °C with inversion. The column was equilibrated with 1× PBS
(0.05% NaN3) and stored at 4 °C. A GST "subtraction
column" was prepared in the same manner, except GST alone was coupled
to the Affi-Gel 10/15 mixed matrix.
Serum (2 ml) was added to the GST-Sp286-390 affinity matrix and
incubated with rotation for 2 h at room temperature. The column was washed three times with 1× PBS, once with 333 mM NaCl
in 1× PBS, and then twice more with 1× PBS. Antibody was eluted twice with 2 ml of 100 mM glycine, pH 2.5, and collected into 200 µl of 1 M Tris-HCl, pH 9.0, to neutralize the sample.
Eluted antibody was pooled, concentrated, and exchanged into 1× PBS
using an Amicon Stirred Cell with a YM30 filter (Amicon). To
remove antibody directed against the GST portion of the GST-spinophilin
fusion protein, concentrated antibody was incubated with the GST
subtraction column, prepared as described above, by rotation for
30 min at room temperature. The pass-through from this column was
collected and concentrated using an Amicon Stirred Cell as described
above, and utilized as the anti-Sp286-390 antibody. Antibody
concentration was determined to be 1.44 mg/ml by protein assay
(Bradford). Optimal working concentrations of antibody in Western and
immunolocalization were derived empirically via Western blot analysis
and immunofluorescence staining.
Fixation and Immunolabeling--
Polarized MDCKII cells stably
expressing the individual
After fixation, cells were rinsed two more times in PBS-CM,
permeabilized in 0.2% Triton X-100 added to the cell surface of the
excised Transwell for 20 min, and incubated in blocking buffer (0.1%
Triton X-100 and 2% bovine serum albumin in PBS-CM) for 1 h.
Primary antibody was added to the cell side of excised Transwells and
incubated for either 1 h at room temperature or overnight (~15
h) at 4 °C. Mouse 12CA5 anti-HA antibodies were diluted at 1:250 (4 µg/ml), and rabbit anti-spinophilin 286-390 antibodies were used at
a dilution of 1:100 (~10 µg/ml). MDCKII cells were washed three
times for 15 min in PBS-CM at 22 °C before adding secondary
antibodies. The secondary antibodies were Alexa488- or Cy3-conjugated
anti-rabbit or anti-mouse antibodies, diluted 1:1000 (2 µg/ml) and
were incubated with the cells for 1 h at room temperature. Cells
were again rinsed three times for 15 min in PBS-CM and mounted
cell-side-up onto a glass slide with Aqua-Polymount and sealed under a
glass coverslip. Images were visualized on a Zeiss LSM 410, laser-scanning, confocal microscope in the Vanderbilt Cell Imaging Core
Facility. Images were taken through a 40× oil objective at 1.5× magnification.
Generating [35S]Met-labeled The residues corresponding to the 3i loops of the
The Gen10-3i loop fusion proteins and (Met)4-3i loops were
transcribed, translated, and [35S]Met-labeled using the
Promega transcription and translation-coupled (TnT) rabbit reticulocyte
lysate kit, as follows: 25 µl of TnT reticulocyte lysate was added to
1 µl of amino acid mix (1 mM, minus methionine), 2 µl
of reaction buffer, 1 µl of TnT T7 RNA polymerase, 4 µl of
[35S]methionine (1000 Ci/mmol, at 10 mCi/ml), and 1 µl
of RNasin ribonuclease inhibitor (40 units/µl). Then, 1 µg of the
appropriate plasmid DNA template was added, and the volume was adjusted
to 50 µl with nuclease-free water. The mixture was incubated for 90 min at 30 °C. Following each synthesis, products were analyzed and
quantitated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The band representing each probe was cut out of the dried gel and counted in scintillation mixture. GST-pull-down assays were performed such that each incubation contained an equivalent amount of [35S]Met-labeled 3i
loop as radioligand.
GST-spinophilin Fusion Protein Generation
GST-spinophilin fusion proteins were generated with spinophilin
amino acid regions 151-444 and 169-255 and expressed in DH5 Binding of 3i Loops to GST-spinophilin
Equimolar concentrations of GST-spinophilin fusion protein were
incubated with 300,000 cpm (estimated to represent ~40
pM) [35S]Met-labeled Detergent Extraction and Coimmunoprecipitation of Full-length
HA-tagged CosM6 cells were plated at 1.75 × 106 cells on
10-cm plates and maintained in DMEM supplemented with 10% fetal bovine
serum and 100 units/ml penicillin and 10 µg/ml streptomycin at
37 °C/5% CO2. The following day, cells (at ~60-80%
confluence) were transfected using FuGENE 6 reagent (Roche Molecular
Biochemicals), according to the manufacturer's specifications, with an
empirically optimized ratio of 3 µl FuGENE 6 reagent/1 µg of
plasmid DNA. The Medium was changed 24 h after the FuGENE transfection.
Approximately 48 h after transfection, cells were rinsed in
serum-free DMEM and incubated with 100 µM epinephrine, or
not (control), for 3 min at 37 °C. Regulation of signaling pathways
by Western Blot Analysis
PVDF membranes were blocked for 15 min in Tris-buffered saline
(20 mM Tris, pH 7.6, 137 mM NaCl) with 0.1%
Tween 20 (TBST) and 5% Carnation Instant powdered milk (w/v). The
appropriate primary antibody was then added at a dilution of 1:1000
(Rat anti-HA) or 1:2000 (mouse anti-Myc monoclonal antibody) in
blocking buffer and incubated at room temperature for 1.5-2 h. Blots
were washed three times for 15 min with TBST and exposed to horseradish
peroxidase-conjugated anti-rat or anti-mouse secondary antibodies, as
appropriate, at a 1:2000 dilution in blocking buffer for 45 min at room
temperature. Blots were washed again three times for 15 min in TBST,
incubated with ECL Western blotting detection reagent (Amersham
Pharmacia Biotech, Buckinghamshire, UK) for 1.5 min, and then exposed
to x-ray film for variable times ranging from 5 s to 30 min.
Coimmunolocalization of Endogenous Spinophilin with the
The 3i Loops of All Three
As shown in Fig. 2B, GST pull-down assays revealed that the
3i loop of each of the Interaction of the Our studies have demonstrated an interaction between the third
intracellular loops of the A variety of novel interactions are being reported for G
protein-coupled receptors via their C terminus (34-40) or 3i loops (30, 40, 41). In some cases, the interactions are fostered by agonist
occupancy of the receptor such as for interaction of the
Interactions between 2A-adrenergic receptor (
2AAR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their
direct targeting to this surface. Findings that the 3i loops of the
D2 dopamine receptors interact with spinophilin (Smith,
F. D., Oxford, G. S., and Milgram, S. L. (1999)
J. Biol. Chem. 274, 19894-19900) and that spinophilin
is enriched beneath the basolateral surface of polarized MDCK cells
prompted us to assess whether
2AR subtypes might also
interact with spinophilin. [35S]Met-labeled 3i loops of
the
2AAR (Val217-Ala377),
2BAR (Lys210-Trp354), and
2CAR (Arg248-Val363) subtypes
interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169-255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because
2AAR and spinophilin
coimmunoprecipitation from cells is enhanced by prior treatment with
agonist. These findings suggest that spinophilin may contribute not
only to
2AR localization but also to agonist modulation
of
2AR signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor
(
2AR)1
subtypes are members of the type II, biogenic amine-binding, G
protein-coupled receptor family. These receptor subtypes all couple via
the Gi/Go family of GTP-binding proteins to the
inhibition of adenylyl cyclase, inhibition of
voltage-dependent calcium channels, potentiation of
potassium currents via G protein-coupled, inwardly rectifying potassium
channels, activation of phospholipase D, and activation of MAP kinase
in native cells (1-4). In heterologous cell systems, these receptors
also couple to the activation of a variety of signaling molecules,
including Ras (5-7), p70S6 kinase (8), MAP kinase (9, 10),
and phospholipase D (11).
2ARs appear to activate similar
signaling pathways, differences in the cellular trafficking of these
subtypes have been reported, both in naive cells and following agonist activation. Subtype-selective differences in agonist-elicited
2AR redistribution have been noted in several
experimental systems (12-18). The
2BAR subtype is
readily internalized following agonist activation, whereas the
2AAR subtype typically is not (14, 18). The
2CAR subtype has not been explored in as much detail with regard to agonist-elicited redistribution because of its considerable accumulation intracellularly (14). The
2AR
subtypes also manifest different trafficking itineraries in polarized
Madin-Darby canine kidney II (MDCKII) cells, even in the absence of
agonist treatment. The
2AAR subtype is targeted directly
to the basolateral surface (19), whereas the
2BAR
subtype is delivered randomly to both the apical and basolateral
surfaces but is selectively retained on the basolateral surface
(t1/2 = 10-12 h) in contrast to its rapid loss from
the apical surface (t1/2 = 5-15 min) (20). These
findings suggest that there is a molecular mechanism responsible for
the selective retention of the
2BAR on the basolateral
sub-domain of MDCK cells, probably a retention mechanism shared by the
basolaterally targeted
2A- and
2CAR
subtypes (20). Although
2CARs, like
2AARs, are directly targeted to and retained on the
basolateral subdomain, a significant proportion of these receptors is
identifiable in an intracellular pool at steady state (14, 18, 20); the functional relevance of this intracellular
2CAR pool has
yet to be clarified.
2AR
subtypes. For example, deletion of this loop in the
2AAR
subtype (
3i
2AAR) results in accelerated basolateral
receptor turnover (t1/2
4.5 h) when
compared with that for the wild-type receptor or with
2AAR structures that have been mutated in the N terminus or the C-terminal tail (all possessing a t1/2 of
10-12 h) (21). Similarly, the
3i
2BAR is not
enriched at the basolateral surface of MDCKII cells at steady state
(22).
2BAR is rapidly removed
from the apical surface following random delivery and that removal of
the 3i loops of the
2A- and
2
AR
subtypes accelerates surface turnover of these receptors, we
hypothesize that
2ARs interact, via their 3i loops, with
protein(s) enriched beneath the basolateral surface of MDCKII cells to
stabilize their steady-state localization. Consequently, we were
particularly intrigued by recent findings that the 3i loop of another
Gi/Go-coupled G protein-coupled receptor, the
D2 dopamine receptor, interacts with spinophilin (23-25),
and that this protein is enriched beneath the basolateral surface of
polarized MDCK cells (24). In addition, the multiple protein-interacting domains within spinophilin (24) suggest that its
interaction with the receptor may facilitate the formation of a
signaling complex to modulate signaling or recruitment of other
proteins to a functional microdomain. The present studies were
undertaken to identify whether spinophilin interacts with the 3i loops
of the
2AR subtypes and, if so, if these interactions are regulated by agents that modify
2AR function.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-maltoside and
cholesteryl-hemisuccinate were purchased from Calbiochem (San Diego,
CA) and Sigma Chemical Co. (St. Louis, MO), respectively. Antibodies
against the HA epitope engineered into the
2AR
structures was obtained from BABCo (mouse) or from Roche Molecular
Biochemicals (rat and mouse). Mouse anti-Myc antibodies were purchased
from CLONTECH (Palo Alto, CA). Protein A-agarose was from Vector (Burlingame, CA). Centricon-10 concentrating filters were purchased from Amicon (Beverly, MA). Horseradish
peroxidase-labeled anti-mouse and anti-rat antibodies were from
Amersham Pharmacia Biotech. Horseradish peroxidase substrate for
Western detection was Enhanced Chemiluminescence (ECL, Amersham
Pharmacia Biotech). Cy3 and Alexa488 secondary antibodies were from
Molecular Probes (Eugene, OR).
2AR subtypes were grown on
Transwells, as described above, and then rinsed once with PBS-CM
(phosphate-buffered saline with 1 mM MgCl2 and
0.5 mM CaCl2) and fixed for 15 min with either
100% methanol (MeOH) at
20 °C or with 4% paraformaldehyde at
room temperature (~22 °C) followed by quenching with two
sequential 7.5-min incubations with 50 mM NH4Cl
in PBS-CM. Spinophilin immunolocalization was best observed after MeOH
fixation, whereas the
2AR localization ("signal-to-background" ratio) was best visualized following
paraformaldehyde fixation and quenching. For colocalization studies, we
used MeOH for fixation of the polarized MDCKII cells.
2AR 3i
Loops as Ligands
2AAR (amino acids 217-377 (27)), the
2BAR (amino acids 210-354 (28)), and the
2CAR (amino acids 248-363 (29)) were subcloned into the
pGEMEX2 vector in-frame within the polylinker located downstream of the
sequence encoding the methionine-rich viral coat protein Gene 10 (30).
Alternatively, constructs were generated in which four methionines were
inserted via polymerase chain reaction into the N-terminal region of
the
2AAR 3i loop
((Met)4-
2A3i) and subcloned into the pGEMEX2
vector. All DNA constructs were verified by sequencing.
. Bacteria were grown at 37 °C to an A600 of
0.6. GST or GST fusion protein expression was initiated with the
addition of 1 mM
isopropyl-
-D-thiogalactopyranoside and allowed to
proceed for 2-6 h at 37 °C. Bacteria were collected by
centrifugation at 10,000 × g and then lysed in 50 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 1 mg/ml lysozyme,
200 mM NaCl, 100 µM PMSF, 1 µg/ml soybean
trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml aprotinin
(TT+ buffer) by one freeze-thaw cycle followed by probe
sonication for three 30-s bursts on ice. GSH-agarose (1 ml of a 1:1
slurry equilibrated in TT+ buffer) was added to the
supernatant of a 13,000 × g centrifugation and
incubated for 1 h at 4 °C with inversion. This solution was transferred to a 0.8- × 4-cm Poly-Prep column (Bio-Rad) and washed with 12 ml of TT+ buffer, 3 ml of 333 mM NaCl
in TT+ buffer, and then with 6 ml of TT+
buffer. GST or GST fusion protein was eluted from the GSH-agarose by
adding 3 ml of 10 mM free acid GSH in TT+, pH
7.5. Eluted protein was concentrated and exchanged into PBS buffer
using an Amicon Stirred Cell.
2A,
2B, or
2C 3i loop ligand (see above).
GSH-agarose (1:1 slurry equilibrated with TT+ buffer) was
then added to this incubation, rotated for 2 h at 4 °C, and the
resin collected by centrifugation. The resin was then exposed to
four 1-ml TT+ washes. Interaction with GST-spinophilin
versus GST (controls) was determined by elution of the 3i
loop into 1× Laemmli buffer (400 mM Tris, pH 6.8, 700 mM
-mercaptoethanol, 1% SDS, 10% glycerol) and
separation of the eluates by 12% SDS-PAGE. The degree of interaction was quantitated by cutting and counting the bands corresponding to 3i
loop (determined via autoradiography) in scintillation mixture.
2AR Subtypes with Full-length Myc-tagged
Spinophilin
2A,
2B, and
2CARs (GenBankTM accession numbers A38316,
X74400, and X57659, respectively) were encoded in pCMV4 and tagged at
their 5'-end after the start ATG codon with the sequence corresponding to the hemagglutinin tag (HA; YPYDVPDYA), as described previously (19,
20). Full-length spinophilin (GenBankTM accession AF016252) was
expressed in pCMV4, and epitope-tagged with a Myc sequence inserted 5'
after the start ATG codon (Myc; QKLISEEDLLRKR).
2ARs (e.g. inhibition of adenylyl cyclase
or stimulation of MAP kinase) typically is maximal following incubation
with 100 µM epinephrine for 2-3 min (9). Cells were then
rinsed twice in cold (4 °C) PBS-CM and extracted into lysis
buffer (15 mM HEPES, 5 mM EDTA, 5 mM EGTA, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml
leupeptin, 10 units/ml aprotinin, and 100 µM PMSF).
Membranes were collected by centrifugation at 12,000 × g at 4 °C for 30 min and solubilized in
n-dodecyl-
-D-maltoside (D
M)/cholesteryl
hemi-succinate (CHS) extraction buffer (4 mg/ml D
M, 0.8 mg/ml CHS,
20% glycerol, 25 mM glycylglycine, pH 7.6, 20 mM HEPES, pH 7.6, 5 mM EGTA, 1 µg/ml soybean
trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml aprotinin, and 100 µM PMSF) by five passes through a 25-gauge needle
followed by ten passes in a glass/Teflon homogenizer. The supernatant
of a 100,000 × g centrifugation for 1 h at
4 °C was defined as the D
M/CHS-solubilized preparation. A 0.75-ml
aliquot of this preparation was "precleared" by a 15-min incubation
with 30 µl of protein A-agarose equilibrated with D
M/CHS buffer.
HA-tagged
2AAR or Myc-tagged spinophilin was then
immunoprecipitated following the addition of rat anti-HA monoclonal
antibody or mouse anti-Myc monoclonal antibody, respectively, each at a
1:100 dilution, and incubation at 4 °C for 1 h. The immune
complex was isolated by centrifugation following a 1-h adsorption to
protein A-agarose; nonspecifically adsorbed proteins were removed by
washing the protein A resin three times in D
M/CHS wash buffer (1 mg/ml D
M, 0.2 mg/ml CHS, 20% glycerol, 25 mM
glycylglycine, pH 7.6, 20 mM HEPES, pH 7.6, 100 mM NaCl, 5 mM EGTA, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml aprotinin, and 100 µM PMSF) and centrifugation at 4 °C. Proteins were
eluted with the addition of 1× Laemmli buffer and heating to 70 °C
for 5 min. Eluates were separated via 10% SDS-PAGE, transferred to an
Immobilon P membrane (PVDF; Millipore) with a constant current of 1 amp for 72 min in CAPS transfer buffer (1 M
cyclohexylamino-1-propane sulfonic acid (CAPS), pH 11, 10% methanol),
and subjected to Western blot analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR Subtypes in MDCKII Cells--
Spinophilin is a
ubiquitously expressed multidomain protein (25) composed of an F-actin
binding domain (amino acids 1-153), a PP1 binding/regulatory region
(amino acids 427-470 (26, 31, 32)), a single PDZ binding
domain, and a C terminus that possesses a series of coiled-coil domains
(see schematic in Fig. 2A). Satoh et al. (24)
showed that spinophilin was localized to the lateral sub-domain in
polarized MDCK cells. As shown previously, the
2A-adrenergic receptor also is enriched on the lateral
sub-domain of these cells (19, 20) and is revealed here using a Cy3
(red signal)-conjugated secondary antibody directed against the 12CA5
antibody that recognizes the N-terminal HA epitope in the
2AAR (Fig. 1). A rabbit
polyclonal antibody was raised against amino acids 286-390 in
spinophilin (26), a region that has virtually no sequence similarity to spinophilin's structural homolog, neurabin I (Fig.
2A). As shown in Fig. 1, the
affinity-purified polyclonal antibody against spinophilin, visualized
here via Alexa488 (green signal)-conjugated secondary antibody, reveals considerable enrichment of endogenous spinophilin at
the lateral surface of these polarized cells, corroborating initial
reports of Satoh et al. (24). The overlap of expression of
the
2AAR and spinophilin in the lateral domain of MDCKII
cells is demonstrated by the considerable amount of yellow
signal present in the red/green overlay. Similar
results were observed upon colocalization of the
2BAR
subtype and spinophilin (data not shown). It should be noted, however,
that some spinophilin also is detected intracellularly, including in a
sub-apical compartment.
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Fig. 1.
Laser-scanning confocal microscopy reveals
colocalization of 2AARs with
endogenous spinophilin in polarized MDCKII epithelial cells.
MDCKII cells stably expressing HA-tagged
2AARs
were grown and polarized on 12-mm Transwells for 6-8 days. The
immunolocalization of the receptor and spinophilin was performed as
described under "Experimental Procedures." Secondary antibodies
were Cy3-conjugated donkey anti-mouse (red) and
Alexa488-conjugated goat anti-rabbit (green). The presence
of yellow in the overlay image indicates colocalization of
the fluorescent signal. Images were taken through a 40× oil objective
(NA = 1.4) via a Leica-TCS laser-scanning confocal microscope at
1.5× magnification in both the XY (top panels) and Z planes
(lower panels corresponding to the blue line), as
shown in the schematic diagram to the left of the confocal
images.
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Fig. 2.
The 3i loops of all three of the
2AR subtypes interact with amino acids
169-255 in spinophilin. A, schematic diagram of the
predicted spinophilin domain structure and of GST fusion proteins that
were used in pull-down assays. Neurabin is a closely related protein to
spinophilin (51% identical and 74% functionally similar at the amino
acid level) with its region of least homology being within the region
from amino acids 151-444 of spinophilin, especially 286-390 (the
region against which the anti-spinophilin antibody used in these
studies was developed (25)). B, GST fusion protein pull-down
assays were performed as described under "Experimental Procedures."
The amount of 35S-labeled Gen10-3i loop fusion protein
retained in the GSH-agarose-GST fusion protein pellet was visualized by
autoradiography and quantitated via scintillation counting.
C, the
2AAR-binding domain of spinophilin can
be further refined to Sp169-255; virtually no interaction with
Neurabin 146-453 can be observed. For these assays, the radiolabeled
probe was [35S]Met-(Met)4-
2A
3i loop GST-Nb146-453. The "input" lane reflects 1/15
of the amount of
[35S]Met-(Met)4-
2A 3i loop
added to each of the GST fusion protein binding incubations.
2AR Subtypes Interact with
Spinophilin--
Smith et al. (23) have demonstrated, via
yeast two-hybrid screens and gel overlay strategies, that the 3i loops
of the D2 dopamine receptor (short and long forms) interact
with spinophilin in the region between the F-actin binding and PP1
domains (Fig. 2A). Consequently, we created GST fusion
proteins of spinophilin bounded between amino acids 151 and 444 (GST-Sp151-444).
2AR subtypes is able to interact
with GST-Sp151-444. Radiolabeled [35S]Met- Gen10
2AR 3i loops specifically interacted with GST-Sp151-444 but not with GST alone. To further refine the interacting regions of
spinophilin, the 3i loop of the
2AAR was incubated with
a fusion protein of GST-spinophilin amino acids 169-255 (Fig.
2C). This region was selected because it represents the
region of least homology with the spinophilin-related protein, neurabin
I, a brain-specific protein (see Fig. 2A (33)). However,
because there is a stretch of 14 amino acids identical between these
protein family members within this region, it was of considerable
importance to evaluate the ability of the corresponding region of
neurabin I (Nb146-453) to interact with the
2AAR 3i
loop. As shown in Fig. 2C, little or no interaction was
detected between the
2AAR 3i loop and GST-neurabin 146-453 when compared with GST-spinophilin 151-444. Furthermore, binding to the 3i loop is demonstrated by the more restricted region of
Sp169-255; in fact, binding of the
2A-3i loop to this region is more readily detected to this region, than to
GST-Sp151-444.
2AAR and Spinophilin within the
Cell Is Regulated by Agonist--
It was of interest to determine
whether or not these
2AAR-3i loop-spinophilin
interactions, detected in vitro via GST fusion protein
assays, could be detected in the context of a living cell. For these
studies, we transiently coexpressed cDNAs encoding full-length HA-tagged
2AAR and Myc-tagged spinophilin in CosM6
cells. On the day of analysis, cells were incubated with or without 100 µM epinephrine prior to extraction of the cell membranes
and solubilization with D
M/CHS, a detergent that extracts receptor
in a functional conformation (30). As shown in Fig.
3, immunoisolation of Myc-tagged spinophilin leads to the coisolation of the HA-
2AAR in
cells expressing both receptor and spinophilin (lanes 2 and
3; lane 1 is an extract from CosM6 cells
expressing only the HA-tagged
2AAR). HA-tagged
2AAR also has been coimmunoprecipitated from cells
transfected with only HA-
2AAR using anti-spinophilin
286-390 antibodies, indicating that it does not require the
overexpression of Myc-spinophilin to detect an interaction with
2AARs (data not shown). Of particular interest, however,
is the ability of the
2AAR agonist, epinephrine, to
increase the amount of
2AAR seen in association with
spinophilin, suggesting that regulated interactions with spinophilin
could contribute both to receptor localization and coordination of
signal transduction events mediated by
2ARs.
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Fig. 3.
The HA-tagged
2AAR coimmunoprecipitates with
Myc-tagged spinophilin in an agonist-regulated fashion.
A, CosM6 cells were transiently cotransfected with the
HA-
2AAR and Myc-spinophilin (lanes 2 and
3) or with the HA-
2AAR alone (lane
1). Cells were treated with vehicle (
) or 100 µM
Epi (+) as indicated. Lysates were prepared and precleared as described
under "Experimental Procedures." Top, mouse anti-Myc
antibody adsorbed to protein A-agarose was used to immunoprecipitate
Myc-spinophilin. Resulting precipitates were separated via SDS-PAGE,
transferred to a PVDF membrane, and blotted with rat anti-HA antibody.
Bottom, the blot was stripped and reprobed with the rabbit
anti-spinophilin amino acids 286-390 antibody. B, histogram
representing the -fold increase over basal levels of
coimmunoprecipitated receptor detected via Western blot following
epinephrine treatment. *, two-tailed p < 0.005 determined using a paired t test comparing differences in
band densities before and after epinephrine treatment
(n = 4 independent experiments with single or duplicate
immunoprecipitates).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR subtypes and spinophilin
and significantly extend previous findings of in vitro
studies demonstrating that the 3i loops of the D2 dopamine
receptor short and long isoforms interact with spinophilin fusion
proteins (23). Furthermore, our studies have refined this interaction
to amino acids 169-255 of spinophilin (neurabin II), a region that
shares little homology with what is otherwise a very homologous
protein, neurabin I. Finally, our studies are the first to document G
protein-coupled receptor-spinophilin interactions in the context of a
native cell and to demonstrate that these interactions are fostered by
agonist binding to the receptor.
2AR with NHERF/EBP50 (35) or the somatostatin receptor with cortactin binding protein 1 (38). In some cases, interactions appear to affect receptor signaling (37, 39, 42), whereas in others,
the interactions may be critical for receptor trafficking (43).
2AARs and spinophilin in the cell
should be considered in the context of interactions between the 3i loop
of
2ARs and other protein partners. Regions of the third intracellular loop of the
2AAR have been shown to
interact with 14-3-3
(30),
arrestin (41) and heterotrimeric G
proteins (44). The
2B- and
2CAR 3i loops
also have been demonstrated to interact with 14-3-3
(30). These
interactions with 14-3-3
are competed for by a phosphorylated
peptide of raf that blocks raf-14-3-3 interactions (45), suggesting
that receptor activation of downstream signaling pathways, such as the
raf-ras cascade, might disrupt pre-existing
2AR-14-3-3 interactions, or vice versa. Interactions
between
2AAR and
-arrestin are expected to occur following agonist-evoked G protein-coupled receptor
kinase-mediated
2AR phosphorylation (10). The 3i
loop sequence employed for the
2AAR in these studies
includes regions that have been proposed to contribute to interactions
with heterotrimeric G proteins (44), but these amphipathic helical
sequences are not present in the amino acids encoded by the
2BAR and
2CAR 3i loop ligands. The ability of all three 3i loop ligands to interact with spinophilin comparably (e.g. Fig. 2) suggests that the
2AR-spinophilin interactions can occur independent of
interactions with G proteins. It also is probable that
2AR-spinophilin interactions do not prevent interactions
with G proteins, because agonist occupancy of the
2AAR
increases the amount of
2AAR that coimmunoprecipitates with spinophilin. Because agonist occupancy of
2AAR also
favors receptor interactions with G proteins (46), it is likely that the
2AAR can interact simultaneously with spinophilin
and its cognate Gi protein. What remains to be established
is whether this agonist-modulated interaction with spinophilin
regulates acute or tonic receptor-mediated signaling, by analogy with
findings for the D1 dopamine receptor (39, 47, 48) or
mediates retention at the basolateral surface of polarized epithelial
cells (Fig. 1), previously demonstrated to require the 3i loop of the
AR subtypes
2A (21) and
2B (22).
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ACKNOWLEDGEMENTS |
---|
We are grateful to David M. Lovinger for his critical input and to Carol Ann Bonner for her superb technical support. We also are grateful for the exchange of reagents and data with Donelson Smith and Sharon Milgram, University of South Carolina and Chapel Hill, especially in early phases of these studies. We appreciate the shared enthusiasm of the other members of the Limbird laboratory.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK43879 (to L. E. L.) and NS37508 (to R. J. C.).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.
§ Funded by Postdoctoral Training Grant T32DK07563 during part of these studies.
¶ Funded by an advanced pre-doctoral Fellowship in Pharmacology and Toxicology from the Pharmaceutical Research and Manufacturers Assn. Foundation. Confocal microscopy was performed in the Vanderbilt University Medical Center Cell Imaging Core Resource Facility (supported by NIH Grants CA68485 and DK20593).
** To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, Rm. 464, Nashville, TN 37232-6600. Tel.: 615-343-3538; Fax: 615-343-1084; E-mail: Lee. Limbird@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M011679200
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ABBREVIATIONS |
---|
The abbreviations used are:
2AR,
2 adrenergic receptor;
GST, glutathione
S-transferase;
MDCK, Madin-Darby canine kidney;
PAGE, polyacrylamide gel electrophoresis;
PP1, protein-phosphatase 1;
Sp, spinophilin (neurabin II);
3i loop, third intracellular loop;
MAP, mitogen-activated protein;
PVDF, polyvinylidene difluoride;
HA, hemagglutinin;
DMEM, Dulbecco's modified Eagle's medium;
PBS, phosphate-buffered saline;
PMSF, phenylmethylsulfonyl fluoride;
GSH, reduced glutathione;
D
M, n-dodecyl-
-D-maltoside;
CHS, cholesteryl
hemi-succinate;
CAPS, cyclohexylamino-1-propane sulfonic acid.
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