From the Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, December 4, 2002 , and in revised form, April 23, 2003.
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ABSTRACT |
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INTRODUCTION |
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Despite similarities in signaling, there are several striking physiological
differences between the 1AR and
2AR subtypes
(5,
6). For example,
1AR and
2AR subtypes play opposing roles in
regulating cardiac myocyte apoptosis: stimulation of the
1AR
increases apoptosis, whereas stimulation of the
2AR inhibits
apoptosis (7,
8).
1AR-induced apoptosis is implicated in the transition from
cardiac hypertrophy to heart failure
(9,
10). The differential
regulation of cardiac cell survival by these
AR subtypes has been
partially explained by the ability of the
2AR to couple to
the pertussis toxin (PTX)-sensitive G protein, Gi
(11).
2ARs in
human and mouse cardiomyocytes have been shown to activate both Gs
and Gi, whereas
1AR stimulation has, thus far,
only been shown to activate Gs
(12). Activation of the
2AR, stimulates a Gi-mediated PI3K-Akt-dependent
cell-survival signaling pathway and prevents cardiomyocytes from undergoing
Gs-mediated apoptosis
(11). Recent studies suggest
that Gs to Gi switching by the
2AR is
the consequence of PKA-mediated receptor phosphorylation
(13,
14).
2AR/Gi coupling has also been implicated in
receptor-meditated ERK activation
(13,
15).
2AR-stimulated ERK1/2 activation has been reported in
cultured HEK-293 and COS-7 cells, and in isolated cardiac myocytes
(16,
17). This
2AR-stimulated ERK1/2 activation is mediated by
subunits of PTX-sensitive G proteins (Gi) through a pathway
involving the non-receptor tyrosine kinase c-Src, small G protein Ras and
Raf-1 kinase (15,
18). The mechanism(s) of
1AR-stimulated ERK activation is somewhat more controversial.
A number of groups have reported that the
1AR is unable to
stimulate ERK activation, a conclusion based on the perceived inability of the
1AR to couple to Gi
(19,
20). However, recent data from
cardiac myocytes suggest that the
1AR can activate ERK and
p38 in a Gi-dependent manner; even though the
1AR
is less potent in stimulating ERK activation than the
2AR
(11,
21). The mechanisms behind the
observed differential activation of ERK are still unknown.
Recent data suggest that ARs may transduce signals that are both
dependent and independent of heterotrimeric G proteins
(22,
23). Well known
receptor-interacting proteins, such as
-arrestins and G protein-coupled
receptor kinases (GRKs), which were thought previously only to regulate
receptor desensitization, have been implicated in signaling pathways that may
be G protein-independent
(2426).
Another G protein-independent mechanism underlying
AR-mediated cell
signaling has recently been demonstrated. Both
1ARs and
2ARs have carboxyl-terminal PDZ binding motifs reported to
bind to PDZ domain-containing proteins
(27,
28). The
2AR
has been shown to interact with Na+/H+ exchanger
regulatory factors (NHERF) in an agonist-dependent manner via this PDZ binding
domain (27); while the
1AR interacts with the postsynaptic density-enriched
proteins, PSD-95 and MAGI-2
(28,
29). Association with PSD-95
regulates agonist stimulated
1AR internalization and may also
provide a molecular mechanism by which
1ARs are localized to
the synapse, regulating synaptic plasticity
(28). Interactions between the
1AR and PDZ domain-containing proteins can be regulated by
GRK5 (30). Furthermore,
studies in cardiac myocytes have recently demonstrated that the PDZ binding
motif of the
1AR modulates receptor trafficking and signaling
(31).
In order to identify proteins that interact with the PDZ binding domain of
the 1AR, we performed a yeast two-hybrid screen on a human
heart cDNA library, using the
1AR carboxyl-terminal tail as
bait. From this screen we identified GIPC (GAIP-interacting protein, carboxyl
(C) terminus), a PDZ-containing protein, as a novel binding partner of the
1AR. Here we characterize this interaction and its functional
consequences.
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EXPERIMENTAL PROCEDURES |
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Polyclonal (A-14) and monoclonal (9E10) anti-Myc antibodies, and polyclonal anti-ERK2 antibody were from Santa Cruz Biotechnologies. Anti-FLAG M2 and Anti-FLAG M2 affinity gel were from Sigma. Polyclonal anti-phospho-ERK1/2 antibody was from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were from Amersham Biosciences.
Yeast Two-hybrid ScreeningPlasmid
pAS21/1AR-CT was used as bait to screen a human heart
cDNA library (in pACT2) (Clontech). Bait plasmid and library cDNAs were
co-transformed into the yeast strain PJ694A using a standard yeast
transformation protocol. Yeast were plated on selective medium
(S.D.-Leu/Trp/His, +10 mM 3AT) and allowed to grow for 46
days at 30 °C. Positive colonies were then restreaked on selective medium
(S.D.-Leu/Trp/Ade or S.D.-Leu/Trp/His) plates. Plasmid DNA was rescued from
positive colonies that grew on both -His and -Ade plates and subject to
further sequence analysis. To further confirm positive interactions, isolated
library cDNAs were co-transformed back into yeast together with a bait
plasmid, either pAS21/
1AR-CT, empty vector
pAS21, or other test plasmids as indicated in the text. Yeast was then
subjected to growth tests on selective plates.
Cell Culture and TransfectionAll tissue culture media and related reagents were purchased from Invitrogen. COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a 37 °C incubator under 5% CO2. Cells in 100-mm dishes were transfected with LipofectAMINE (at 4:1 ratio with DNA) according to the manufacturer's protocol. HEK-293 cells were maintained under the same conditions as COS-7 cells except minimal essential medium (MEM) was used. HEK-293 cells were transfected with FuGENE 6 (at 2.5:1 ratio with DNA) according to the manufacturer's protocol (Roche Applied Science). After transfection, cells were grown 3648 h before agonist stimulation.
Cellular Immunoprecipitations and ImmunoblottingTransfected COS-7 cells in 100-mm plates were incubated in serum-free medium for 60 min before agonist stimulation. The cells were then incubated in the presence or absence of 10 µM isoproterenol (ISO) for 10 min at 37 °C. The cells were rinsed with cold Dulbecco's phosphate-buffered saline (DPBS) and replaced with 1 ml of cross-linking buffer (DPBS containing 10 mM HEPES, pH 7.4, and 2.5 mM of the cell permeable cross-linking reagent dithiobis(succinimidyl propionate) (DSP, Pierce)). For the stimulated cells, 10 µM ISO was present in the cross-linking buffer. The cells were incubated for 30 min at room temperature with continuous slow rocking. The cross-linking reaction was terminated by quickly removing the cross-linking buffer and replacing it with 1 ml of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, and protease inhibitors). The cells were lysed by incubation on ice for 30 min and then clarified by centrifugation at 21,000 x g for 12 min at 4 °C. The clarified supernatants were then used in cellular coimmunoprecipitation experiments. 50 µl of each supernatant was diluted into an equal amount of 3x SDS-PAGE sample buffer to serve as whole cell extract controls.
1 ml of clarified cell extract was then incubated with 25 µl of anti-FLAG M2 affinity gel slurry at 4 °C with gentle rotation for 4 h to overnight. Beads were washed four to five times with ice-cold lysis buffer, and the bound proteins were eluted with 50 µl of 3x SDS-PAGE sample buffer. Equivalent amounts of sample in 3x sample buffer were resolved on precast 420% polyacrylamide gels (Invitrogen) and then transferred to nitrocellulose membranes by semi-dry blotting. Nitrocellulose membranes were blocked with 5% fat-free milk in TBST (20 mM Tris, pH 7.4, 500 mM NaCl, and 0.1% Tween 20) and incubated with the appropriate primary antibody at room temperature for 1 h. After washing with TBST, membranes were incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or rabbit IgG secondary antibody (1:2000). Protein bands were visualized via SuperSignal Chemiluminescence Substrate (Pierce). Quantitation of band density was performed with Bio-Rad Fluor-S MultiImager.
ERK PhosphorylationTwenty-four hours after transfection, cells were split into 6-well dishes and then incubated in serum-free media (DMEM for COS-7 and MEM for HEK-293, 0.1% bovine serum albumin, 10 mM HEPES, pH 7.4) overnight before agonist stimulation. Agonist stimulation was performed at 37 °C in serum-free media for the times indicated in the figure legends. The medium was removed, and cells were then solubilized in 3x SDS-PAGE sample buffer. The whole cell lysates were sonicated and resolved by SDS-PAGE. ERK phosphorylation was detected by Western blot using anti-phospho-ERK1/2 antibody as described above. The total ERK2 in the cell lysates was detected using anti-ERK2 polyclonal antibody. Protein bands were visualized via SuperSignal Chemiluminescence substrate and quantitated with Bio-Rad Fluor-S MultiImager.
Receptor Internalization and Cyclic AMP Accumulation
AssaysFor receptor internalization assays, HEK-293 cells in 100-mm
dishes were transiently transfected with pcDNA3/FLAG-1AR in
the presence and absence of Myc-GIPC. One day after transfection, cells were
split into poly-D-lysine-coated 6-well plates (Biocoat) and grown
overnight at 37 °C. Cells were serum-starved for 1 h before stimulation
with 10 µM ISO for 30 min at 37 °C. Cells were placed on ice
and cell surface FLAG-tagged receptors were detected with anti-FLAG M2
antibody followed by FITC-conjugated anti-mouse IgG as described previously
(35). Receptor internalization
was defined as the percentage of cell surface receptors lost after agonist
stimulation, measured by cell flow cytometry.
For cyclic AMP accumulation assays, HEK-293 cells in 100-mm dishes were
transiently transfected with pcDNA3/FLAG-1AR in the presence
and absence of transfected Myc-GIPC. Twenty-four hours after transfection,
cells were split into 12-well collagen-coated plates and then labeled with
modified essential medium supplemented with 5% fetal bovine serum and 2
µCi/ml [3H]adenine for 4 h to overnight. Cells were
serum-starved for 30 min and then stimulated with
1AR
selective agonist dobutamine or 10 µM forskolin for 10 min.
Cyclic AMP accumulation was quantitated by chromatography and expressed as a
percentage of 3H incorporated into cyclic AMP as described
previously (35).
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RESULTS |
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The specificity of the interaction between GIPC and the
1AR-CT was confirmed by further yeast two-hybrid analysis.
GIPC clones were transformed back into yeast strain PJ69-4A together with
baits (either
1AR-CT or
2AR-CT). Transformed
yeast containing both bait and GIPC were then subjected to growth tests on
selective medium. As shown in Fig.
1, only yeast hosting both GIPC and the
1AR-CT
were able to grow on both -His and -Ade plates. The interaction between the
1AR and GIPC is specific, since the
2AR-CT
did not support yeast growth under the same conditions when co-transformed
with GIPC.
The 1-Adrenergic Receptor Interacts with GIPC Through
Its Carboxyl-terminal PDZ Binding MotifIn order to determine
whether the full-length
1AR could associate with GIPC in the
cell, COS-7 cells were co-transfected with FLAG-
1AR and
Myc-GIPC. Immunoprecipitation of the
1AR followed by Western
blotting for Myc-GIPC revealed robust co-immunoprecipitation of a
GIPC/
1AR complex (Fig.
2). Treatment of the cells with the
-adrenergic receptor
agonist ISO had no significant effect on the GIPC/
1AR
association (data not shown). Consistent with the yeast two-hybrid results,
GIPC did not co-immunoprecipitate with the full-length
FLAG-
2AR when the two proteins were co-expressed in COS-7
cells.
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To examine the structural determinants of the GIPC/1AR
interaction, we used several
1AR mutants, in which three out
of the last five residues were replaced with Ala individually
(28). Similar to that observed
with the
1AR/PSD-95 interaction, when the Ser residue at the
-2 position of the
1AR was mutated to either Ala (S475A) or
Asp (S475D), the association of GIPC with the
1AR could no
longer be detected. When GIPC was co-expressed with the
1AR
mutant S473A, co-immunoprecipitation of GIPC was reduced only slightly
relative to the wild-type
1AR. However, the
1AR mutant V477A, which is unable to bind with PSD-95, showed
a very strong interaction with GIPC. These studies demonstrate by both yeast
two-hybrid screening and cellular co-immunoprecipitation experiments that the
GIPC specifically interacts with the
1AR through the PDZ
domain. Unlike the association of the
1AR with PSD-95, the
Val in the last position (Val-477) of the receptor is not required for the
interaction with GIPC.
GIPC Has No Effect on Receptor-stimulated Cyclic AMP
AccumulationNext we examined the effect of GIPC on
1AR-mediated whole cell cAMP accumulation. HEK-293 cells were
transiently transfected with wild-type
1AR, and either
Myc-tagged GIPC or vector alone. Dose-response curves of cAMP accumulation
induced by the selective
1AR agonist dobutamine are shown in
Fig. 3. Similar to previous
results with PSD-95, GIPC expression had no effect on
1AR-mediated whole cell cAMP accumulation, changing neither
the maximal level of cAMP accumulation nor the EC50. Because PDZ
domain-containing proteins have previously been shown to regulate receptor
internalization (28,
29), we also examined the
effect of GIPC on
1AR internalization in HEK-293 cells. GIPC
expression had no effect on agonist stimulated
1AR
internalization (data not shown).
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1AR-stimulated ERK ActivationIn
order to examine
1AR-mediated ERK activation, we used a
plasmid encoding an ERK2-GFP fusion protein. This allowed for GFP-ERK2 to be
easily separated from endogenous ERK1/2 by SDS-PAGE. In COS-7 cells
transiently transfected with pcDNA3/FLAG-
1AR and GFP-ERK2,
ISO stimulation induced robust ERK phosphorylation/activation compared with
non-stimulated cells (Fig.
4A). In control cells transfected with GFP-ERK2 alone,
ISO-stimulated ERK phosphorylation was not detected using phospho-ERK
antibody.
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Similar to previous studies of the 2AR,
1AR-stimulated ERK activation was a relatively fast process,
peaking at around 5 min. At maximum, ISO-stimulated ERK activation was
68 fold greater than that of non-stimulated control cells
(Fig. 4B). After
peaking at 5 min,
1AR-stimulated ERK activation steadily
decreased to
50% of maximum at 15 min.
1AR-stimulated
ERK activation was observed over a range of agonist concentrations. The dose
response curve for ISO-stimulated ERK activation is shown in
Fig. 4C.
Next we examined the effect of different agonists on
1AR-mediated ERK activation in COS-7 cells. The
1AR has a high affinity for both norepinephrine and
epinephrine (2). As predicted,
norepinephrine (NE) stimulation dramatically increased ERK activation
(Fig. 4D). The
1AR specific agonist dobutamine (Dobu) also stimulated ERK
activation, but was much less active than either NE or ISO. As a control, PMA,
the direct activator of PKC, demonstrated robust ERK activation, whereas the
direct activator of adenylate cyclase, forskolin (Forsk), showed no
demonstrable ERK activation. As expected, ISO-stimulated ERK activation was
completely blocked by the
-adrenergic receptor antagonists propranolol
(PRO) and alprenolol (ALP) (Fig.
4E).
Treatment of cells with 10 µM PTX overnight decreased
1AR-mediated ERK activation considerably. To further
demonstrate that PTX inhibited the activation of ERK by inhibiting the
i subunit of the G protein, we overexpressed a G protein
i1 subunit (C351S), which cannot be ADP-ribosylated and is
therefore unaffected by PTX treatment. As shown in
Fig. 4F,
overexpression of Gi (C351S), while having little effect on the
overall ISO-stimulated ERK activity, leads to a marked increase in
ISO-stimulated ERK activation observed in the presence of PTX. The residual
effect of PTX is presumably due to inhibition of endogenous Gi.
These data further support the idea that the
1AR activates
ERK through a Gi-mediated pathway, similar to that reported for the
2AR (13). In
contrast to findings with the
2AR
(13), the PKA inhibitor H89
had no significant effect on
1AR-mediated ERK activation.
GIPC Regulates 1AR-mediated ERK
ActivationGIPC is a PDZ domain-containing protein that interacts
with a Gi-specific RGS protein called GAIP (G
-interacting
protein) (34); therefore, GIPC
could be involved in the regulation of Gi-mediated signaling. Here
we tested the effect of GIPC on
1AR-stimulated ERK
activation, which may be mediated by the PTX-sensitive G protein,
Gi. As shown in Fig.
5A, expression of GIPC substantially decreased
ISO-stimulated
1AR-mediated ERK activation. ISO-stimulated
ERK activation in the presence of GIPC was decreased by 45% compared with
control cells lacking GIPC (Fig. 5,
A and D). Under similar conditions, expression
of GIPC had little effect on
2AR-mediated ERK activation
(Fig. 5B). The
inhibitory effect of GIPC appeared to be specific for the
1AR. GIPC had no significant effect on the ability of the
1AR mutant (S475A), which is unable to interact with GIPC, to
activate ERK (Fig.
5C). These data suggest that the ability of GIPC to
regulate
1AR-mediated ERK activation is dependent on its
ability to interact directly with the
1AR via its PDZ
domain.
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DISCUSSION |
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Here we report a specific interaction between the 1AR and
GIPC. Despite the fact that both the
1AR and
2AR contain similar PDZ binding motifs, GIPC interacts only
with the
1AR, as demonstrated by both yeast two-hybrid
screening and co-immunoprecipitation experiments. The GIPC PDZ domain
facilitates the interaction with the
1AR. Mutation of Ser-475
to either Ala (S475A) or Asp (S475D) completely eliminated the interaction.
However, unlike that reported for PSD-95, changing the last residue Val-477 of
the
1AR to Ala had no effect on the
1AR/GIPC
association. TAX, a protein containing a similar carboxyl-terminal ETEA-COOH
motif has also been shown to interact with GIPC
(43). GIPC expression has no
significant effect on
1AR-induced cAMP accumulation.
Therefore, similar to PSD-95, the association of GIPC with the
1AR had no effect on
1AR coupling to
Gs. In contrast to PSD-95, which inhibits receptor internalization
in HEK-293 cells (31), GIPC
had little to no effect on agonist-stimulated receptor internalization.
Recently, many GPCRs have been reported to activate MAP kinase cascades
regulating cell growth and/or proliferation
(18). In COS-7 cells, it has
been clearly demonstrated that stimulation of the 2AR
activates the MAP kinase ERK through a Gi-mediated pathway
(13,
44). In cardiac myocytes,
2AR stimulation has been shown to activate the MAP kinases
ERK, JNK, and p38 (21,
45). However,
1AR-mediated ERK activation or coupling to Gi is
still quite controversial. Here, we demonstrate
1AR
stimulated, PTX-sensitive, ERK activation; strongly suggesting that the
1AR, like the
2AR, can signal through both
Gs and Gi.
GIPC, similar to many PDZ domain-containing proteins, has been shown to
play an important role in organizing signaling cascades
(46), anchoring proteins in
specific subcellular compartments
(40), and regulating cell
signaling (39). GIPC regulates
the expression of the TGF- receptor at the cell surface and enhances the
cellular response to TGF-
(39). GIPC has also been
linked to G protein signaling by its ability to interact with the RGS protein
GAIP (46). Overexpression of
GIPC in PC12 cells decreases NGF induced MAP kinase (ERK1/2) activation
(38). Interestingly, a
GIPC-like protein Kermit, which interacts with the Frizzed receptor, has been
reported to regulate frizzed 3 signaling in neural crest development
(47). Here we provided further
evidence that GIPC regulates G protein signaling by directly interacting with
the
1AR in a PDZ-dependent manner. Expression of GIPC
decreased the ability of the
1AR to stimulate ERK activation,
but had no effect on
1AR-mediated cAMP accumulation. This
regulatory effect of GIPC on ERK activation is specific for the
1AR and depends on a PDZ-mediated interaction, since
expression of GIPC has no effect on the ERK activation stimulated by either
the
2AR or the
1AR mutant (S475A). These
data suggest that the GIPC/
1AR association affects receptor
Gi- but not Gs-mediated signaling.
In mouse cardiac myocytes, stimulation of the 1AR leads to
a PKA-dependent increase in the rate of contraction
(48).
2AR
stimulation shows a biphasic effect on the rate of cardiac myocyte
contraction, with an initial PKA-dependent increase followed by a
PTX-sensitive decrease, mediated by Gi
(48). Disruption of the PDZ
binding motif in the
1AR changed the signaling profile of the
1AR in cardiac myocytes to more resemble that of the
2AR (31).
These data suggest that the
1AR is capable of coupling to
Gi, but that association with PDZ-containing proteins such as
PSD-95 could prevent this interaction from occurring. GIPC interacts with the
1AR through its PDZ domain and thereby specifically inhibits
1AR-stimulated Gi-mediated signaling. Thus, the
GIPC effect on
1AR-mediated ERK activation might explain the
differential effects of
1AR and
2AR
stimulation on the rate of cardiac myocyte contraction.
The effect of GIPC on 1AR/Gi-mediated signaling
could also explain the differential effects of
1AR and
2AR stimulation on cardiac myocyte apoptosis.
1AR stimulation leads to cardiac myocyte apoptosis, whereas
stimulation of the
2AR concurrently activates pro-apoptotic
and anti-apoptotic signals, the net effect being increased cell survival
(8). Differential coupling to
Gs and Gi has been used to explain this differential
effect of
1AR versus
2AR
stimulation (10). Here we
demonstrate that the
1AR can couple to both Gs and
Gi, similar to a number of other GPCRs including the histamine,
serotonin, and glucagon receptors
(20). The continued study of
receptor-interacting proteins, such as PDZ-containing proteins, will further
our understanding of differential signaling by receptors.
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FOOTNOTES |
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Recipient of a fellowship from the American Heart Association.
To whom correspondence should be addressed: Howard Hughes Medical Institute,
Box 3821, Duke University Medical Center, Durham, NC, 27710. Tel.:
919-684-2974; Fax: 919-684-8875; E-mail:
lefko001{at}receptor-biol.duke.edu.
1 The abbreviations used are: AR,
-adrenergic receptor;
1AR,
1-adrenergic receptor;
2AR,
2-adrenergic receptor; CT, carboxyl
terminus; ERK, extracellular signal-regulated kinase; GAIP,
G
-interacting protein, GIPC, GAIP-interacting protein,
carboxyl-terminal; GPCR, G protein-coupled receptor; HEK, human embryonic
kidney; GRK, G protein-coupled receptor kinase; MAP kinase, mitogen-activated
protein kinase; NHERF, Na+/H+-exchanger regulatory
factor; PDZ, PSD-95/Dlg/ZO-1 homology domain; PTX, pertussis toxin; ISO,
isoproterenol.
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ACKNOWLEDGMENTS |
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REFERENCES |
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