From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The The three Despite the qualitatively similar signaling properties of the three
Receptor retention on the lateral subdomain of MDCKII cells likely
involves the third intracellular loop of the Other functional roles have been attributed to the third intracellular
loop of The present studies were undertaken to identify interacting proteins
for the intracellular 3i loops of 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 NEN Life Science
Products. PVDF nylon membranes were from Millipore (Bedford, MA). The
FPLC and DEAE-Sephacel columns were from Amersham Pharmacia Biotech.
Dodecyl- Subcloning and in Vitro Translation of the Gen10
Protein- The residues corresponding to the 3i loops of the
The Gen10-3i loop fusion proteins and 14-3-3 Pig Brain Cortex and MDCK Cell Fractionation
Frozen pig brain cortex (2 g/preparation) was suspended in 20 ml
of ice-cold lysis buffer (20 mM HEPES, 50 mM
KCl, 2 mM MgCl2, 1 mM
CaCl2 with the following protease inhibitors: 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml soybean
trypsin inhibitor, 5 µg/ml leupeptin, 1 mg/ml aprotinin, 1 mg/ml
benzamidine) and homogenized using a Brinkmann Polytron (two 5-s bursts
separated by 30 s on ice). The lysate was filtered through
cheesecloth to remove debris and then centrifuged at 38,000 × g in an SS34 rotor (Sorvall RC 5B centrifuge) for 20 min.
The supernatant of this centrifugation was removed and designated as
the cytosolic fraction. The pellet was resuspended in 4 ml of lysis
buffer, homogenized again, this time using a Teflon/glass homogenizer,
and centrifuged as before. In early experiments, this pellet was
resuspended and an aliquot saved to permit analysis of 3i loop
interacting proteins in membrane protein fractions. To resolve proteins
in the particulate fraction that could be extracted into Triton X-100,
the membrane pellet was re-homogenized into 4 ml of ice-cold
detergent-containing buffer (20 mM HEPES, 150 mM KCl, 2 mM MgCl2, 0.5% Triton
X-100, and the protease inhibitors indicated above). All fractions were stored at Cultured MDCK cells (two 100-mm dishes/preparation) were harvested at
confluence by scraping into 1 ml of lysis buffer (see above) using a
rubber policeman. MDCK cell lysates were disrupted further by 10 up and
down passages through a 25-gauge needle mounted onto a 5-ml syringe.
The supernatant of the 15 min, 4 °C centrifugation (estimated at
30,000 × g in an Eppendorf centrifuge) was saved and
defined as the cytosolic fraction.
Gel Overlay Assay
A gel overlay procedure (31, 32) was used to detect binding of
[35S]Met-labeled Gen10- For gel overlay analysis, PVDF membranes were blocked at least 1 h
in blocking buffer: Tris-HCl/NaCl (50 and 200 mM,
respectively, referred to hereafter as TBS), containing Tween 20 (3%
v/v) and non-fat powdered milk (5% w/v). The PVDF membranes were then
washed for 30 min in rinsing buffer: TBS containing Tween 20 (0.1%
v/v) and non-fat powdered milk (5% w/v). The PVDF membrane strips were then incubated with 300,000 cpm of the appropriate
[35S]Met-labeled Gen10- In experiments where the duration of the incubation or the amount of
radioligand was varied, the times and concentrations evaluated are
indicated in the figure legends. Following incubation with the various
loop structures (or radiolabeled Gen10, as a control), membranes were
washed 3 times with rinsing buffer, twice with cold TBS, and air-dried
before autoradiography. Autoradiography was performed using a Molecular
Dynamics PhosphorImager, and band intensities were calculated using the
manufacturer's software, presented as arbitrary intensity units.
Following quantitation, strips were exposed to x-ray film for 24-72 h.
For Raf competition experiments, phosphorylated and non-phosphorylated
peptides corresponding to a 14-3-3 binding region of Raf-1
(LSQRQRSTS(PO4)TPNVHMV and LSQRQRSTSTPNVHMV, respectively (33)) were incubated with the membranes for 0.5 h prior to the addition of the [35S]Met-labeled Gen10- Purification of the DEAE Chromatography--
For each purification protocol, 15 ml
of a cytosolic protein fraction prepared from 2 g of frozen pig
brain cortex were loaded onto a 2-ml DEAE-Sephacel column equilibrated
overnight with ice-cold column equilibration buffer (20 mM
HEPES (pH 7.0), 50 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, and protease
inhibitors as utilized above). The column pass-through was saved for
evaluation of 3i loop binding activity. The DEAE column was washed with
100 ml of 150 mM KCl-containing lysis buffer. The proteins
were eluted using a gradient of KCl (from 150 to 500 mM
KCl) in lysis buffer, at a rate of 6 ml/h. One-ml fractions were
collected and subsequently evaluated for 3i loop binding activity using
gel overlay analysis.
Gel Filtration--
Preparative gel filtration using fast
protein liquid chromatography (FPLC) was performed as follows: a 120-ml
Superdex 200 column was equilibrated for 2 h with ice-cold buffer
(20 M HEPES (pH 7.0), 150 mM KCl, 2 mM MgCl2, 1 mM CaCl2)
at a rate of 2 ml/min. A 2-ml sample, corresponding to peak fractions
from the DEAE-Sephacel column, was injected. Eluate fractions (2 ml/fraction) were collected at a rate of 2 ml/min for 2 h. The 3i
loop binding activity in individual fractions was determined by
assaying aliquots using sequential SDS-PAGE and gel overlay analysis;
in some studies, the proteins were concentrated and desalted using
Centricon-10 concentrators before assaying 3i loop binding activity.
Two-dimensional Gel Electrophoresis--
To be confident that
the bands on SDS-PAGE manifesting 3i loop binding activity were not
"contaminated" by underlying bands, two-dimensional gel
electrophoresis was performed using protocols and the tube gel adapter
kit provided by Hoefer Scientific instruments.
Microsequencing--
Microsequencing was performed at the
Harvard Microsequencing Laboratory facility (Dr. William Lane,
Director) by Edman degradation of tryptic digests of the ~30-kDa
bands hydrolyzed in polyacrylamide gels, resolved by high pressure
liquid chromatography, and assessed by mass spectrometry.
Detergent Extraction of Functional MDCKII cells, parental or stably transfected with the
Because the 3i loops of the 2-adrenergic receptors
(
2ARs) are localized to and function on the
basolateral surface in polarized renal epithelial cells via a mechanism
involving the third cytoplasmic loop. To identify proteins that may
contribute to this retention, [35S]Met-labeled Gen10
fusion proteins with the 3i loops of the
2AAR (Val217-Ala377),
2BAR
(Lys210-Trp354), and
2CAR
(Arg248-Val363) were used as ligands in gel
overlay assays. A protein doublet of ~30 kDa in Madin-Darby canine
kidney cells or pig brain cytosol (
2B
2C
2A) was identified. The
interacting protein was purified by sequential DEAE and size exclusion
chromatography, and subsequent microsequencing revealed that they are
the
isoform of 14-3-3 proteins. [35S]Met-14-3-3
binds to all three native
2AR subtypes, assessed using a
solid phase binding assay (
2A
2B>
2C), and this binding depends on the presence of the 3i
loops. Attenuation of the
2AR-14-3-3 interactions in the
presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251-266), but not by its
non-phosphorylated counterpart, provides evidence for the functional
specificity of these interactions and suggests one potential interface
for the
2AR and 14-3-3 interactions. These studies
represent the first evidence for G protein-coupled receptor
interactions with 14-3-3 proteins and may provide a mechanism for
receptor localization and/or coordination of signal transduction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor
(
2AR)1
subtypes, encoded by distinct genes (1), all couple via the
Gi/Go family of GTP-binding proteins to
inhibition of adenylyl cyclase, suppression of voltage-sensitive calcium channels, and activation of receptor-operated potassium channels (2). These receptors also couple to activation of Ras (3, 4),
the mitogen-activated protein kinase cascade (3, 5-7), and to
activation of phospholipase D (8, 9).
2AR subtypes, differences in trafficking of these
receptors have been reported. For example, subtype-selective
differences in agonist-elicited
2AR redistribution occur
(10-15). In addition, selective itineraries for the
2AR
subtypes are observed in polarized Madin-Darby canine kidney (MDCKII)
renal epithelial cells. Thus, the
2AAR subtype is
targeted directly to the basolateral surface (16), whereas the
2BAR subtype is delivered randomly to both the apical
and basolateral surfaces but is rapidly lost from the apical
(t1/2 = 5-15 min) and selectively retained on the
basolateral (t1/2 = 10-12 h) surface (17). These
findings suggest that there is a molecular mechanism responsible for
the selective retention of the
2BAR on the basolateral
domain of MDCK cells that may be shared by all three
2AR
subtypes, as they manifest comparable half-lives on that surface
(17).
2AAR, since deletion of this loop, creating the mutant
2A
3iAR,
results in accelerated basolateral 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) (18). The accelerated turnover of
the
2A
3iAR when compared with the wild-type
2AAR structure suggests that the third intracellular
loop interacts with proteins that either tether
2AAR to
a particular surface domain or, alternatively, mask the
2AAR from interacting with endocytosis machinery.
2AAR. The N- and C-terminal 10-15 residues of
the 3i loop, predicted to form amphipathic helices, are involved in
coupling to G proteins (19-22). The C-terminal third of the 3i loop of
the
2AAR subtype is implicated in the interaction with
-arrestin, a protein that preferentially associates with G
protein-coupled receptor kinase-phosphorylated receptors sustaining agonist-elicited homologous desensitization (23). For the
2AR subtypes, G protein-coupled receptor kinase
phosphorylation sites are in the N-terminal region of the
2AAR 3i loop (24, 25), widely distributed throughout the
2BAR 3i loop, and presumed to be absent in the
2CAR 3i loop (13, 26).
2AR subtypes. In vitro translation of Gen10-
2AR 3i loop
fusion proteins (gen10-
23i) served as a means
to create [35S]methionine-radiolabeled 3i loops as
ligands for identifying interacting proteins via a gel overlay
strategy. Our findings reveal that these loops, in a subtype-selective
fashion, interact with the
isoform of 14-3-3 proteins (14-3-3
).
Using a solid phase binding assay with [35S]Met-14-3-3
as a probe and solubilized
2AR as the target indicates that 14-3-3 proteins can bind to native
2AR subtypes in
a way that relies on the 3i loop in the receptor structure. Further evidence for the functional relevance of these interactions is the
ability of a Raf peptide, corresponding to a 14-3-3-interacting domain,
to block Gen10-
23i loop interactions with 14-3-3
in its phosphorylated, but not in its non-phosphorylated, state.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-maltoside and cholesterol hemisuccinate were purchased from
Calbiochem and Sigma, respectively. Staph A immunoprecipitin was
obtained from Life Technologies, Inc. 12CA5 monoclonal antibody against
the hemagglutinin epitope engineered into the
2AR
structures was obtained from Babco; the M2 monoclonal antibody against
the FLAG epitope engineered into the N terminus of 14-3-3
was from
Eastman Kodak Co., and the rabbit anti-14-3-3
(or pan) and anti-
isoform antibodies were from Santa Cruz Laboratories (Santa Cruz, CA).
Protein A beads were from Vector (Burlingame, CA). Centricon-10
concentrating filters were purchased from Amicon (Beverly, MA). The
tube gel adapter kit was from Hoefer Scientific instruments (San
Francisco, CA).
2AR 3i Loop Fusion Proteins
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
pGEMEX-2 vector. The residues utilized are shown schematically in Fig.
2B. These 3i loop sequences were inserted in frame within the polylinker located downstream of the sequence encoding the Gen10
protein, a methionine-rich phage structural protein. The sequence
encoding an epitope of the c-Myc protein was inserted 3' to the
sequence of the
2AR 3i loop sequences.
were produced and
[35S]Met-labeled using an in vitro T7 RNA
polymerase-coupled translation system in reticulocyte lysates as
follows: 25 µl of TNT lysate were added to 1 µl of amino acid mix
(1 mM, minus methionine, TNT kit), 2 µl of TNT reaction
buffer, 1 µl of TNT T7 RNA polymerase, 4 µl of
[35S]methionine (1000 Ci/mmol, at 10 mCi/ml), 1 µl of
RNasin ribonuclease inhibitor (40 units/µl). Then, 1 µg of the
appropriate DNA template (presented as the circular plasmid DNA) was
added, and the volume was adjusted to 50 µl with nuclease-free water.
The mixture was incubated for 90 min at 30 °C. Products were
analyzed and quantitated following each synthesis by 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
autoradiography, and the band representing each probe was cut out of
the dried gel and counted in scintillation mixture. If smaller
molecular weight species were generated during the translation
reaction, they were eliminated by P30 size exclusion chromatography
before use as probes. These 35S-labeled
Gen10-
23i loop fusion proteins were used as radioactive ligands in subsequent gel overlay assays as described previously (30)
and detailed below.
70 °C. The protein concentration in each fraction was estimated using the Bradford assay.
23i loops to
fractionated cellular proteins resolved via SDS-PAGE. Protein aliquots
(2.5-3.0 mg/sample) were separated by SDS-PAGE using 7.5-20%
polyacrylamide gradients on 16-cm long and 1.5-mm thick gels.
Prestained molecular weight markers also were run to permit estimation
of the approximate molecular weights of the proteins identified by gel
overlay analysis. The resolved proteins were transferred at 4 °C to
PVDF nylon membranes (Millipore) by electrophoresis overnight at 30 V
in Tris/glycine buffer (25 and 192 mM, respectively). The
membranes were then cut in 2-4-mm strips for gel overlay and Western
blot analysis.
23i loop structure
in a 1-ml incubation for 4 h (to overnight) at 4 °C with
constant rocking in rinsing buffer. Based on the concentration of
methionine contributed to the [35S]Met-labeling reaction
by the rabbit reticulocyte lysate (5 µM) and the specific
activity of the [35S]Met radiolabel, we estimated that
this 300,000 cpm of Gen10-
23i loop represents 5-10 pmol
of probe.
23i
loop probes, and the incubation was continued for 90 min followed by
washing and detection, as described above.
2AR 3i Loop Interacting
Proteins
2AR Subtypes
2A,
2B or
2CAR subtype,
were grown to confluence on 150-mm plates, serum-starved overnight,
harvested in lysis buffer (15 mM HEPES, 5 mM
EGTA, and 5 mM EDTA (pH 7.6), containing 10 units/ml
aprotinin and 100 µM phenylmethylsulfonyl fluoride),
disrupted using a Teflon/glass homogenizer, split into 2 aliquots, and
centrifuged at 30,000 × g. One aliquot was extracted
with detergent, and the other was used to monitor receptor available
for extraction. For detergent extraction, one pellet was resuspended in
2.25 ml/150-mm plate D
M/CHS extraction buffer (4 mg/ml
dodecyl-
-D-maltoside (D
M), 0.8 mg/ml cholesterol
hemisuccinate (CHS), 25 mM glycylglycine, 20 mM
HEPES, 100 mM NaCl, 5 mM EGTA, 1 µg/ml
soybean trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml aprotinin,
and 100 µM phenylmethylsulfonyl fluoride), homogenized
using a 27-gauge needle, and centrifuged at 100,000 × g at 4 °C for 1 h. The resulting supernatant was defined as the detergent-solubilized receptor. To assess the
2AR binding capacity of these preparations,
[3H]rauwolscine was used as a radioligand, and Sephacel
G-50 chromatography was used to separate bound from free ligand, as
described previously (34). To assess the relative efficiency of the
detergent to extract receptor from membranes, the results of the G-50
chromatography binding assays were compared with radioligand binding
assays performed on the membrane pellet, derived from a fraction of the
original preparation, with [3H]rauwolscine. Based on
these determinations we estimate that we extract >50% of the
2AR subtypes using this protocol. Equal concentrations
of detergent-solubilized receptor were incubated with mouse
anti-hemagglutinin antibodies for 1 h and then with 100 µl of
protein A-agarose (1:1 slurry with D
M/CHS wash buffer) for a 2nd h.
The protein A-agarose was rinsed twice with D
M/CHS wash buffer (1 mg/ml D
M, 0.2 mg/ml CHS, 25 mM glycylglycine, 20 mM HEPES, 100 mM Na, 5 mM EGTA, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml
aprotinin and 100 µM phenylmethylsulfonyl fluoride) and
incubated for 16 h with [35S]Met-14-3-3
rotating
end over end at 4 °C. To terminate the incubation, the protein A
resin was pelleted at 18,500 × g and rinsed twice with
D
M/CHS wash buffer before resuspension in Laemmli buffer and
fractionated by 12% SDS-PAGE. The gels were dried prior to
autoradiography; the amount of [35S]Met-14-3-3
bound
to receptor was quantitated by cutting the bands and counting in NEF
963 scintillation fluor. To assess the extent of
non-receptor-dependent [35S]Met-14-3-3
binding, detergent extracts of parental MDCKII cells expressing no
2ARs were prepared, and volumes of this extract equal to
the largest volume receptor-containing preparations was adsorbed to
protein A resin and served as the control for these studies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AAR have been
implicated in stabilization of these receptors on the basolateral
surface of polarized renal epithelial cells, we sought to identify
proteins that interact with these intracellular domains. We created
fusion proteins of the 3i loops with the methionine-rich Gen10 protein.
In vitro translation of these fusion proteins in the
presence of [35S]methionine generated radiolabeled 3i
loops that served as ligands for the identification of interacting
proteins via gel overlay analysis. As can be seen in Fig.
1, the 3i loops of the
2BAR and
2CAR subtypes readily identified
a doublet of apparent molecular mass of 30 kDa in cytosolic fractions
of MDCKII cell lysate that was not detected by Gen10 protein or by the
3i loop of the
2AAR under these incubation conditions
(see Fig. 2, later, for delayed binding
by
2AAR 3i loop). The ~30-kDa doublet identified by
the
2B3i and
2C3i loops is enriched in
the cytosolic fraction and is barely detected in the membrane fractions
of MDCKII cells. Similar binding profiles were seen in fractions from
porcine brain cortex, albeit with greater membrane-associated binding
activity, and from lysates of MDCKII cells that had been grown in
Transwell® culture to foster polarization (data not shown), consistent
with the published experience that confluent MDCKII cells grown in regular culture dishes manifest many of the properties characteristic of the polarized cellular phenotype (16, 35).
View larger version (87K):
[in a new window]
Fig. 1.
Subcellular distribution of the
2AR 3i loop interacting proteins in
MDCKII cells and pig brain cortex. Pig brain cortex or MDCK cells
were fractionated, separated by SDS-PAGE, transferred to PVDF
membranes, and probed with [35S]Met-labeled
Gen10-
23i loop fusion proteins as described under
"Experimental Procedures." Incubations contained 300,000 dpm of
each of the probes in 1 ml of buffer (corresponding to 5-10 pmol of
[35S]Met-labeled 3i loop or Gen10 control) and were
performed at 4 °C; the incubations were terminated by washing three
times in rinse buffer ("Experimental Procedures") and examined by
autoradiography. The data shown are from a single experiment
representative of at least six other experiments.
View larger version (29K):
[in a new window]
Fig. 2.
Time course of the interaction of
[35S]Met-labeled
Gen10- 23i loop fusion proteins
with the cytosolic fraction of porcine brain cortex. A,
the [35S]Met-labeled Gen10-
23i loop fusion
proteins for each of the
2AR receptor subtypes, or
labeled Gen10 alone (control), were incubated with PVDF membranes, as
described under "Experimental Procedures" and the legend to Fig. 1.
The autoradiogram was used to identify the radioactive regions on the
PVDF filter; these regions were cut and counted in NEN 965 scintillation fluor to obtain the cpm/gel overlay shown on the
y axis. B, schematic representation of the
2AR with the portions of the 3i loop of each subtype
(shown with bolder line and amino acid (a.a.) numbers) used
as probes in the gel overlay assays.
Fig. 2A demonstrates the time course for interaction of the
2AR 3i loops with the 30-kDa doublet in porcine brain
cytosolic fractions. The 3i loops of the
2BAR and
2CAR interacted more readily and to a significantly
greater extent than the 3i loop of the
2AAR subtype,
whose binding to the 30-kDa doublet was detectable above background
labeling only after longer (>4 h) incubations. When the ability of a
10× molar excess of unlabeled 3i loops to compete for binding of the
35S-labeled 3i loops for each subtype was evaluated after a
2- or 4-h incubation, it was evident that competition for the binding of the
2AAR 3i loop was more facile than for the binding
of the
2BAR or the
2CAR loop (data not
shown), consistent with the apparent lower affinity of the
2AAR 3i loop for the 30-kDa interacting proteins in the
gel overlay assay (Fig. 2). The Gen10 fusion protein (control probe)
did not compete for any of the 3i loop-specific binding nor did a Gen10
fusion protein encoding 58 amino acids of the distal C-terminal tail of
the
1 adrenergic receptor (data not shown), a region
previously implicated in
1AR stabilization on the cell
surface (36, 37).
To reveal the molecular identity of the 30-kDa doublet, we undertook
its purification from cytosolic fractions of porcine brain cortex. As
shown in Fig. 3A, the
interacting proteins were quantitatively adsorbed to DEAE-Sephacel and
eluted, using a 50-500 mM KCl gradient, at approximately
250-300 mM KCl (fractions 82-95). Binding to these peak
fractions showed a specificity of 3i loop binding characteristic of the
unfractionated cytosol, as shown in Fig. 3B. The peak
fractions were pooled and purified further using size exclusion FPLC,
which removed most of the proteins migrating on SDS-PAGE at >50
kDa and <20 kDa (Fig. 4). The
elution position of the 3i loop interacting proteins on FPLC
corresponded to an Mr of 50,000-80,000 (data
not shown), suggesting that the ~30-kDa proteins on SDS-PAGE may
exist as a dimer, in a complex with other proteins, or both.
|
|
Material that had been purified by sequential chromatography on
DEAE-Sepharose, FPLC, and concentrated by a second application to
DEAE-Sepharose was subjected to two-dimensional isoelectric focusing
and SDS-PAGE. As shown in Fig.
5A, the material migrating in
the 30-kDa region on one-dimensional SDS-PAGE was resolved into three
distinct spots upon two-dimensional gel analysis, as revealed by Zoion
Coomassie staining. Gel overlay analysis indicated that two of the
three spots, migrating at isoelectric points of 5 and 5.6, represented
the 2AR 3i loop interacting proteins (Fig. 5B). In fact, the spot migrating at a pI of 5.0 has greater
35S-Gen10-3i loop binding relative to its Coomassie
labeling intensity than the protein migrating with a pI
5.6. Since
the C-terminal tail of both the
2AAR and the
2AR interacts with a ~30-kDa protein that corresponds
to eIF2B
(38), as revealed in yeast two-hybrid screens, we examined
whether or not eIF2B
represented any of the three spots detected on
the two-dimensional gel. Indeed, the upper of the three spots on the
two-dimensional gel analysis of the highly purified 3i loop interacting
proteins, migrating at a pI of 4.8, corresponds to eIF2B
, based on
Western analysis with a polyclonal antibody directed against this
protein (Fig. 5C).
|
Microsequencing of the 2AR 3i loop interacting proteins
revealed the spots corresponding to the proteins of pI 5 and 5.6 on
two-dimensional gel analysis both represented the
isoform of 14-3-3 proteins. As shown in Fig. 6, the 14-3-3 proteins represent a family of closely related proteins containing
several highly homologous domains, as well as sequences unique to each
of the isoforms. The name 14-3-3 derives from the particular migration pattern of these proteins following systematic analysis by starch gel
electrophoresis and DEAE chromatography (39). The highlighted sequences
shown in Fig. 6 correspond to those we obtained by microsequence analysis of the purified 3i loop interacting proteins. Since two of
these four sequences are uniquely represented in the
isoform, our
interpretation is that the upper interacting band of the 30-kDa doublet, migrating on two-dimensional electrophoresis with a pI of 5.0, represents the phosphorylated form of 14-3-3
(sometimes referred to
as 14-3-3
), and the lower band, which migrates with a pI of 5.6, represents non-phosphorylated 14-3-3
. Since the pI 5.0 species
appears to bind more extensively to the 3i loop relative to its protein
content than its pI 5.6 counterpart, it is reasonable to postulate that
the 3i loops of
2BAR and
2CARs preferentially interact with 14-3-3
in its phosphorylated form.
|
Solid phase binding assays revealed that intact, native
2ARs are also able to interact specifically interact
with 14-3-3
(see Fig. 7A).
2-Adrenergic receptor subtypes stably expressed in
MDCKII cells were extracted with dodecyl-
-maltoside/cholesterol hemisuccinate, a detergent mixture that extracts the receptor in a form
that is functional in terms of ligand binding properties. A solid phase
matrix was formed by incubating the epitope-tagged
2AR-subtype extracts with 12CA5 anti-hemagglutinin
antibodies and protein A-Sepharose, as described under "Experimental
Procedures." After washing to remove unbound protein, the
resin-
2AR subtype matrix was incubated with
[35S]Met-labeled 14-3-3
. Retained
[35S]Met-14-3-3
, resistant to washing, was identified
after separation by 12% SDS-PAGE and autoradiography and quantitated
by cutting and counting the corresponding bands in scintillation fluor.
Non-transfected (parental) MDCKII cells served as the negative control
in these experiments. [35S]Met-14-3-3
was consistently
retained on the solid phase pellet containing each of the
2AR subtypes. As in gel overlays, the
2BAR subtype shows a prominent interaction with
14-3-3
. Interactions with the
2AAR subtype are more
readily detectable for the native receptor in the solid phase binding
assays than for the third intracellular loop alone in gel overlay
assays. The precise reasons for these quantitative differences in
receptor subtype-14-3-3
interactions in gel overlay
versus native receptor solid phase binding assays are not
known. This difference likely relates to the different state (native
versus partially denatured) of 14-3-3
in the two settings
as well as that of the third intracellular loop versus the
whole receptor in a native form. Nonetheless both methods demonstrate a
reliably detectable interaction, especially for the
2BAR
subtype. Furthermore, receptor interaction with 14-3-3
essentially
was not detectable upon deletion of the third intracellular loop of
either the
2AAR or the
2BAR
(
2A
3i (deletion of amino acids 217-377) or
2B
3i (deletion of amino acids 214-357); Fig.
7B), further verifying that it is the third cytoplasmic loop of these receptors that promotes the interaction with 14-3-3
.
|
In order to characterize the functional specificity of the
2AR-14-3-3
interaction, competition experiments were
performed using Raf and Raf-PO4 peptides in gel overlay
assays. These peptides represent a Raf-14-3-3
binding motif when the
peptide is phosphorylated on serine 259 (see "Experimental
Procedures" and Refs. 33 and 40). As can be seen in Fig.
8, co-incubation of membrane strips with
Raf-PO4 attenuates the interaction of
2B3i
[35S]Met-labeled probe with pig brain cortex cytosolic
fraction in gel overlay assays (45% of the intensity in the presence
of probe alone), whereas the non-14-3-3
interacting,
non-phosphorylated Raf peptide does not. Similarly, Raf-PO4
peptides attenuate the interaction of
2C3i
[35S]Met-labeled probe but to a lesser extent (80% of
the intensity in the presence of probe alone), perhaps the reflection
of a higher affinity of the
2C3i loop for 14-3-3
(data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The third cytoplasmic loop of the 2AAR has been
implicated in the retention or stabilization of the receptors on the
surface of cells (18, 41). Consequently we looked for proteins able to
interact with the 3i loop of
2ARs which might implicate
their involvement in receptor stabilization on the surface or perhaps receptor microcompartmentalization and signaling specificity. By using
a gel overlay strategy, we demonstrated that proteins with an apparent
mass of 30 kDa in brain and in MDCKII cell lysates were able to
interact with the 3i loops of the
2AR subtypes. Purification and microsequencing revealed that these interacting proteins were the
isoform of 14-3-3 proteins. Subsequent analyses using [35S]Met-14-3-3
as the ligand demonstrate that
this protein indeed interacts with the native
2AR
subtypes in manner that depends on the
2AR 3i loop (Fig.
7). In addition, these interactions are selectively blocked by a
phosphorylated Raf peptide but not by its non-phosphorylated
counterpart (Fig. 8).
The 14-3-3 proteins are expressed ubiquitously and have been shown to interact with a large number of signaling proteins (42), including Raf kinases (43-45), phosphatases (46, 47), and phosphatidylinositol 3-kinase (48). They have been demonstrated to exist as dimers in the context of the cell and in crystal structures (49, 50). The N-terminal domains of the 14-3-3 monomers interact to form a dimer, whereas surface residues from helices 3, 5, 7, and 9 define a conserved amphipathic groove (42, 46). Both charged and hydrophobic residues in this groove are involved in the interaction with several proteins, as revealed by genetic (51, 52) and biochemical (45, 49) analysis. For example, the Saccharomyces cerevisiae homologs of 14-3-3, BMH1 and BMH2, are essential for the Ras/mitogen-activated protein kinase cascade during pseudohyphal development in the yeast; signaling defects due to mutations in BMH1 are not accompanied by decrements in Ras or other members of the signaling pathway but to the failure of these signaling components to interact with one another, presumably due to changes in the cleft of the amphipathic substrate binding pocket of the BMH1 mutant allele (52). The role of 14-3-3 in increasing the efficiency of Ras-mediated signaling has been similarly demonstrated via genetic analysis of Drosophila eye development (51). It has been postulated that interaction of signaling molecules with 14-3-3 homo- or heterodimers serves as a scaffolding mechanism to facilitate interactions among molecular components of signaling cascades (42, 53).
The present experiments provide the first evidence for an interaction
between 14-3-3 proteins and G protein-coupled receptors and lay the
groundwork for establishing the molecular bases for the coordination of
2AR containing, multicomponent signaling pathways. Since
2ARs are capable of activating the Ras/Raf cascade (3)
via the
subunits of G proteins (54, 55), the interactions revealed in the present study suggest the possibility that a
14-3-3-based scaffold fosters coincident activation of these signaling
components. One speculation that derives from our findings that
phosphorylated Raf peptides compete for receptor-14-3-3
interactions
is that 14-3-3 proteins could interact with inactive receptors and
poise them for immediate coupling to the Ras/Raf cascade upon
agonist-elicited conformational changes in the receptor which
facilitate receptor interaction with G proteins. This would lead to the
liberation of
subunits and simultaneously liberate the 14-3-3 dimer to coordinate activated Ras interactions with Raf in the receptor microenvironment.
It is of interest that the highly purified preparation of
2AR3i loop interacting proteins contained both a 14-3-3 protein doublet as well as eIF2B
(cf. Fig. 5). Recent
studies have demonstrated that the C-terminal tail of the
2- and
2-adrenergic receptors interact
with eIF2B
in two-hybrid screens (38). Unexpectedly, overexpression
of eIF2B
in HEK293 cells leads to an enrichment of this molecule in
blebs on the surface membrane and co-localization of
2AR
in those eIF2B
-enriched blebs (38). If the co-purification of 14-3-3 proteins and eIF2B
represents a molecular interaction that exists in
the context of the cell and persists during the protein isolation
steps, then it could be postulated that the cytoplasmic domains of
these adrenergic receptors provide a surface for interaction of this
14-3-3-eIF2B
complex, with the C-terminal tail interacting with the
eIF2B
member of the complex and the third intracellular loops
interacting with the 14-3-3 dimer component of the complex. One
potential strategy to address this possibility would be via
co-immunoprecipitation studies; however, we have been unable to detect
reliably the co-precipitation of the
2AR with 14-3-3, perhaps because the interaction is of too low affinity to persist
during immunoisolation procedures. This interpretation is consistent
with the poor efficiency of
2AR-eIF2B
co-immunoprecipitation noted by Von Zastrow and co-workers (38), only
detectable in transient expression systems where both the receptor and
eIF2B
are simultaneously overexpressed. However, it is also possible that the detection of 14-3-3 and eIF2B
in the highly purified preparations of
2AR 3i loop interacting proteins simply
reflects the similar fractionation properties of these proteins on DEAE and size exclusion chromatography. Future studies will be necessary to
distinguish between these possibilities.
A number of recent studies have suggested that the intracellular
domains of G protein-coupled receptors represent a surface for
association with proteins that may coordinate cellular signaling, beyond the well characterized interactions of these receptors with
heterotrimeric G proteins. For example, phosphoinositide-linked metabotropic glutamate receptors have been shown to interact with a PDZ
domain-containing protein, dubbed Homer, via interactions with their
C-terminal tails (56); the functional consequence of these interactions
for receptor-mediated signaling may play a role in long term
potentiation (57). The interaction of the C-terminal tail of G
protein-coupled receptors with PDZ domain-containing proteins may
represent a recurrent theme. For example, the
2-adrenergic receptor C-terminal tail recently has been
shown to interact with the PDZ domain-containing
Na+/H+ exchanger regulatory factor, resulting
in sequestration of this regulatory protein and attenuation of its
phosphorylation-dependent inhibition of ion translocation
(58).
These studies represent the first report that 2AR
subtypes can interact with 14-3-3 proteins in a manner mediated by the third intracellular loop. This interaction is suppressed by a phosphorylated Raf peptide but not by its non-phosphorylated
counterpart. Genetic strategies have provided strong evidence that
14-3-3 proteins, via homo- and hetero-dimeric complexes, serve as
scaffolds that facilitate interactions among molecular components of
signaling cascades (43, 51, 53). The present findings suggest that 14-3-3 proteins may play a similar role in linking G protein-coupled receptors to diverse signaling pathways by coordinating these interactions in the receptor microenvironment.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. John Scott (HHMI,
Vollum Institute) for critical input and discussions regarding these
studies, particularly in their early phase. We thank Dr. Christine
Saunders (Vanderbilt University) for input to the project on an
intellectual level as well as for the generation of the
2B
3i construct and stable cell line. We also thank
Dr. Bih-Hwa Shieh (Vanderbilt University) for detailed advice regarding
in vitro translation and gel overlay analysis. L. P. thanks Suzanne Kloeker and Dr. Brian Wadzinski (Vanderbilt University)
for instruction regarding two-dimensional gel electrophoresis and FPLC
purification and for access to their instrumentation for the studies
described. We are grateful to Dr. William Lane and colleagues at the
Harvard Microsequencing Facility for their sequencing of the 3i loop
interacting proteins evaluated in this study. The Western analyses
shown in Fig. 5C were performed by Dr. Scott R. Kimball and
Dr. Leonard S. Jefferson at Penn State University College of Medicine.
The Raf peptides were provided by Dr. Haian Fu (Emory University) to
whom we are also grateful for the creative and critical input into
these studies. We are grateful to Carol Ann Bonner for superb technical
support and to the other members of the Limbird laboratory for their
shared enthusiasm.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK 43185 (to L. E. L.), DK13499, and DK15658 (to L. S. J.).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 during part of these studies by INSERM.
§ These two authors made equivalent contributions to this manuscript.
¶ Funded by National Institutes of Health Postdoctoral Fellowship T32DK07563 during part of these studies.
To whom correspondence should be addressed: Dept. of
Pharmacology, Vanderbilt University Medical Center, Rm. 468, Nashville, TN 37232-6600. Tel.: 615-343-3534; Fax: 615-343-1084; E-mail: Lee.Limbird{at}mcmail.vanderbilt.edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
2AR,
2 adrenergic receptor;
GPCR, G protein-coupled receptor(s);
MDCK, Madin-Darby canine kidney;
PVDF, polyvinylidene
difluoride;
PAGE, polyacrylamide gel electrophoresis;
eIF2B
, eukaryotic initiation factor 2B
;
FPLC, fast pressure liquid
chromatography;
D
M, dodecyl-
-D-maltoside;
CHS, cholesterol hemisuccinate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|