From the Departments of Medicine and
§ Cell Biology and ¶ Howard Hughes Medical Institute,
Duke University Medical Center, Durham, North Carolina 27710
Received for publication, March 16, 2001
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ABSTRACT |
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Agonist-dependent desensitization of
the Phosphoinositide 3-kinases
(PI3Ks)1 are a family of
enzymes that can be divided into three classes based on their structure and substrate specificity (1). The class I PI3Ks are heterodimeric enzymes consisting of catalytic and regulatory subunits that are divided into the subgroups IA and IB. The class
IA PI3K (consisting of p110 The exposure of a GPCR to agonist produces rapid attenuation of its
signaling ability that involves uncoupling of the receptor from its
cognate heterotrimeric G-protein. The cellular mechanism mediating
agonist-specific or homologous desensitization is a two-step process in
which agonist-occupied receptors are phosphorylated by a
G-protein-coupled receptor kinase and then bind arrestin proteins (4).
Homologous desensitization of agonist-occupied Previous studies have shown that phospholipids are required for
receptor internalization (9). In particular, the deletion of
PtdIns(3,4,5)P3 binding sites from Cell Culture--
Mouse NIH-3T3 and HEK 293 cells were
maintained in either Iscove's modified Dulbecco's medium or minimal
essential medium supplemented with 10% fetal bovine serum and 1:100
penicillin-streptomycin (10,000 units/ml) at 37 °C. Cells were
seeded at a density of ~1-3 × 105 cells/35-mm dish
and, at 70-80% confluence, were transiently transfected using the
transfection reagent FUGENE6 (Roche Molecular Biochemicals) or calcium
phosphate precipitation. Cells were harvested 24 h after
transfection, re-plated in triplicate, allowed to grow overnight, and
serum-starved for 2-4 h before agonist stimulation.
Plasmid Constructs--
The cDNA encoding bovine Cytosolic and Membrane Fractionation--
Cell monolayers were
scraped in 1 ml of buffer containing 25 mM Tris-HCl (pH
7.5), 5 mM EDTA, 5 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml each leupeptin and aprotinin
and disrupted further by using Dounce homogenizer. Intact cells and
nuclei were removed by centrifugation at 1,000 × g for
5 min. The collected supernatant was further subject to a
centrifugation at 38,000 × g for 25 min. The pellet
was resuspended in lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 20 mM Tris-Cl (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium
orthovanadate, and 2 µg/ml each aprotinin and leupeptin) and used as
membrane fraction, and the supernatant was diluted in lysis buffer and used as the cytosolic fraction. Heart samples also underwent similar procedures to obtain membrane and cytosolic fractions. Purity of the
membrane fraction preparation was confirmed by measuring enzyme
activity of the membrane marker enzyme K+-stimulated
p-nitrophenylphosphatase (18) (membrane fraction: 9.4 µmol/mg of protein/min, cytosol: 2.6 µmol/mg of protein/min).
Lipid Kinase Assays--
PI3K assays were carried out as
previously described (19). Briefly, cells were lysed in lysis buffer,
and cytosolic extract was used for immunoprecipitation with either the
C5/1 monoclonal antibody directed against Immunoblotting and Detection--
Immunoblotting and detection
of PI3K, Determination of Confocal Microscopy and In Vivo Pressure Overload Hypertrophy and Isoproterenol
Infusion--
Four-month-old adult C57BL/6 wild type mice of either
sex were used for this study. Microsurgical procedures and hemodynamic evaluation of pressure overload hypertrophy induced through transverse aortic constriction (T) was performed as previously described (19, 22).
After 7 days of aortic constriction, mice were anesthetized, and both
carotid arteries were cannulated to measure the trans-stenotic pressure
gradient (22). Hearts were then rapidly excised, and individual
chambers were separated, weighed, and frozen in liquid N2
for later biochemical analysis. In separate experiments, adult wild
type mice underwent intravenous infusion of 10 µM
isoproterenol for 3 min at 50 µl/min. Hearts were removed and
flash-frozen in liquid N2 for later biochemical analysis.
The animals in this study were handled according to approved protocols
by the animal welfare regulations of Duke University Medical Center.
Statistical Analysis--
Data are expressed as means ± S.E. Statistical comparisons was performed using an unpaired Student's
t test. Results for the Agonist-dependent Agonist-dependent Translocation of PI3K Activity Is Required for the Sequestration of the
Receptor--
Because
Alteration of receptor endocytosis with overexpression of the
There is increasing evidence for a requirement of D-3
phosphoinositides in the process of membrane trafficking and receptor internalization (9-12). Since, PtdIns(3,4,5)P3 is the main product catalyzed by the lipid kinase activity of P13K (3), we investigated whether there is a mechanism that links the
agonist-dependent recruitment of PI3K to GPCR endocytosis.
The data presented here provide evidence for an interaction between
Recent structural studies of PI3K Our data are consistent with a mechanism that allows A previous study shows that the PI3K yeast homologue,
Vps34p, plays an important role in endocytosis (27).
Furthermore, a recent study shows that treatment with wortmannin blocks
agonist-induced Previous studies show that both the clathrin adaptor molecule AP2 (11)
and Although the observed attenuation of To determine the association of PI3K with A recent study using rat cardiomyocytes links the v-Akt
(protein kinase B)-glycogen synthase kinase 3 Based on our study we propose the idea that agonist-stimulated
recruitment of PI3K is linked to -adrenergic receptor requires translocation and activation of
the
-adrenergic receptor kinase1 by liberated G
subunits.
Subsequent internalization of agonist-occupied receptors occurs as a
result of the binding of
-arrestin to the phosphorylated receptor
followed by interaction with the AP2 adaptor and clathrin proteins.
Receptor internalization is known to require D-3 phosphoinositides that
are generated by the action of phosphoinositide 3-kinase.
Phosphoinositide 3-kinases form a family of lipid kinases that couple
signals via receptor tyrosine kinases and G-protein-coupled receptors.
The molecular mechanism by which phosphoinositide 3-kinase acts to
promote
-adrenergic receptor internalization is not well understood.
In the present investigation we demonstrate a novel finding that
-adrenergic receptor kinase 1 and phosphoinositide 3-kinase form a
cytosolic complex, which leads to
-adrenergic receptor kinase
1-mediated translocation of phosphoinositide 3-kinase to the membrane
in an agonist-dependent manner. Furthermore,
agonist-induced translocation of phosphoinositide 3-kinase results in
rapid interaction with the receptor, which is of functional importance,
since inhibition of phosphoinositide 3-kinase activity attenuates
-adrenergic receptor sequestration. Therefore,
agonist-dependent recruitment of phosphoinositide 3-kinase
to the membrane is an important step in the process of receptor
sequestration and links phosphoinositide 3-kinase to G-protein-coupled
receptor activation and sequestration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
catalytic
subunits) associates with the p85 regulatory subunit and is essential
for coupling signals through receptor tyrosine kinases (1). The class
IB PI3K (p110
catalytic subunit) associates with the
p101 adaptor subunit and is activated by
subunits of G-proteins
(2). Stimulation of a variety of G-protein-coupled receptors (GPCRs)
through G
-mediated activation of PI3K
leads to an increase in
the level of 3'-phosphorylated phosphatidylinositol (PtdIns), which in
turn mediates diverse cellular effects including cell proliferation,
cell survival, cytoskeletal rearrangements, and endocytosis (3).
-adrenergic receptors
(
ARs) occurs after translocation of G-protein-coupled receptor
kinase 2 (GRK2;
-adrenergic receptor kinase 1 (
ARK1)) to the
plasma membrane.
ARK1 association with the plasma membrane is
facilitated by binding to liberated G
subunits and the
interaction of its pleckstrin homology domain to the membrane
phospholipids (5). After
ARK1-mediated
AR phosphorylation, the
phosphorylated receptor becomes desensitized by binding
-arrestin
(4, 6) and is targeted to the clathrin-coated pit for endocytosis (6, 7). In addition to functioning as docking proteins linking GPCRs to
components of the endocytic machinery,
-arrestins also bind other
intracellular regulatory proteins such as c-Src (8).
-arrestin results in
inhibition of GPCR endocytosis (10), and binding of
polyphosphoinositides to AP2 (11) is important for targeting the
receptor-arrestin complex to a clathrin-coated pit (12). Since
PtdIns(3,4,5)P3 is the main product catalyzed by the lipid
kinase activity of P13K (3), and the extended PH domain of
ARK1 and
the helical domain of PI3K
allow for a protein-protein interaction
(13, 14), we explored whether
ARK1 and PI3K might interact to
promote
AR internalization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ARK1 and
the carboxyl terminus of
ARK1 (
ARKct) was described previously
(15, 16). Genomic DNA isolated from mouse tail was used for
amplification of
1AR gene by polymerase chain reaction
with Pfu Taq polymerase (Stratagene) using
the 5' primer
(5'-AATTCgCCgCCATGgACTACAAggACgACgATgATAAgggCgCgggggCgCTCgCCCTg-3') containing an EcoRI site for subcloning followed by Kozak
consensus sequence and a FLAG tag and 3' primer
(5'-AAGCTTCTACTTGGACTCCGAGGA-3') containing a consensus stop codon with
a HindIII site for subcloning. Pfu Taq
polymerase-amplified PCR product was subcloned in zero-blunt TOPO
vector (Invitrogen) and was cut with
EcoRI/HindIII and subcloned into pRK5 mammalian
expression vector. The cloned
1AR in pRK5 was then
sequenced to check for its authenticity. FLAG-tagged
2AR
was a generous gift from Dr. Robert J. Lefkowitz. HA-tagged pCMV-PI3Kp110
wild type (PI3K
), HA-tagged pCMV-PI3Kp110
mutant (
PI3K
) (
942-981, deletion in ATP binding site) were generous gifts from Dr. Charles S. Abrams (17). Myc-PI3K
was a generous gift
from Dr. Michael J. Waterfield.
ARK1 (20) or the anti-FLAG
M2 monoclonal antibody (Sigma). Beads were washed and assayed for PI3K
activity. The organic phase was spotted on TLC plates and resolved by
chromatography. No associated background PI3K activity was found with
beads used for immunoprecipitation (data not shown). TLC plates were
subjected to autoradiography, and PIP was quantified by
phosphorimaging. PtdIns(4)P (Sigma) was used as a standard. Lipids were
prepared as previously described (19).
ARK1, HA-PI3K
, HA-
PI3K
, and Myc-PI3K
were
carried out as previously described (19, 20). Immunoprecipitating
antibodies were added to 500 µg of cell lysate in lysis buffer
followed by the addition of 35 µl of 1:1 protein A- or G-agarose.
Samples were rocked overnight at 4 °C then centrifuged at
12,000 × g for 5 min. Immunoprecipitates were washed
twice with lysis buffer, twice with 1× phosphate-buffered saline, and
resuspended in 1× SDS gel loading buffer. Proteins were resolved by
SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene
difluoride membranes (Bio-Rad). Blots were incubated with antibodies
recognizing PI3K, Myc (Santa-Cruz), and HA (Roche Molecular
Biochemicals) at 1:2000 dilution and the
ARK1 monoclonal antibody at
1:10,000 dilution. Blots were subsequently incubated with appropriate
secondary antibody (1:2000 dilution) conjugated to horseradish
peroxidase (Amersham Pharmacia Biotech), and detection was carried out
using enhanced chemiluminescence.
2AR Sequestration in HEK 293 Cells by 125I-Cyanopindolol (CYP)
Binding--
2AR sequestration was performed as
previously described (7). Briefly, HEK 293 cells were plated at a
density of 2.5 × 106 cells/dish and transfected the
following day with plasmids containing either the
2AR
(150-250 ng), PI3K
(4.0 µg), or
PI3K
(4.0 µg) cDNAs.
Twelve hours after transfection, cells were split into six-well Falcon
plates at a density of 750,000 cells/well. The following day the media
was replaced with minimal essential medium containing 1 µM isoproterenol and 100 µM ascorbate for 0 to 30 min. In separate experiments, cells transfected with
2AR were treated with the PI3K inhibitors wortmannin
(100 nM) or LY294002 (100 µM) for 15 min
before isoproterenol stimulation. To determine the amount of
internalized receptor, 100-µl aliquots of whole cells were added to
150 µl of binding buffer (75 mM Tris-HCl, 10 mM MgCl2, 5 mM EDTA (pH 7.5)).
Total binding was determined in the presence of 175 pM
125I-CYP alone, the number of internalized receptors was
determined by using 175 pM 125I-CYP plus 100 nM CGP12177, and nonspecific binding was determined using
175 pM 125I-CYP plus 1 µM
propranolol (7). Sequestration was calculated as the ratio of (specific
receptor binding of 125I-CYP in the presence of
CGP12177)/(specific receptor binding of 125I-CYP in the
absence of CGP12177).
2AR
Phosphorylation--
Confocal microscopy was performed as previously
described (21). In brief, HEK 293 cells were transfected with plasmids
containing the
2AR (2 µg) and
-arrestin-GFP (2 µg) or the
2AR and
-arrestin-GFP along with either
PI3K
(2.5 µg) or
PI3K
(2.5 µg). Cells were split into
35-mm dishes with glass bottoms for observation using a Zeiss LSM-510
confocal microscope. The cells were treated with isoproterenol (1 µM) for the indicated times (0 to 5 min), and the
fluorescence images were exported as Tiff files by the LSM software to
Adobe Photoshop.
2AR phosphorylation was performed in
intact cells as previously described (7). Cells were transfected with
plasmids containing the FLAG
2AR (2 µg) and vector DNA
(2.5 µg), the FLAG
2AR (2 µg) and PI3K
(2.5 µg), or the
2AR (2 µg) and
PI3K
(2.5 µg).
24 h after transfection, cells were washed and metabolically
labeled for 1 h with medium containing 100 µCi of
32P/ml. After stimulation with 10 µM
isoproterenol for 5 min, incubations were terminated by adding 2 ml of
ice-cold phosphate-buffered saline/well, and then cells were
solubilized with the addition of 0.75 ml/well of radioimmune
precipitation buffer. After centrifugation at 38000 × g for 20 min at 4 °C, the supernatants were processed for
immunoprecipitation of the FLAG-
2AR as described above.
Phosphorylated receptors were resolved by 10% SDS-polyacrylamide gel
electrophoresis, and dried gels were subjected to autoradiography.
2AR sequestration by
CYP binding was analyzed using Graphpad Prism.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ARK1 and PI3K Form a Complex in the Cytoplasm--
Since both
ARK1 and PI3K
are activated by
subunits of G-proteins, we
explored whether they interact to form a complex in the cytosol. We
used a mouse NIH-3T3 cell line since it has a relatively low level of
endogenous PI3K activity compared with other cell lines (data not
shown). Cells were transfected with
ARK1 and HA-PI3K
cDNAs,
and
ARK1 was immunoprecipitated from extracts using a monoclonal
antibody directed against the
ARK1 catalytic domain (20). As shown
in Fig. 1, A-C, PI3K activity and protein were found associated with
ARK1. The association was
independent of PI3K activity since co-transfection with a catalytically
inactive PI3K mutant (HA-
PI3K
, deletion in the ATP binding site)
did not prevent the association of the PI3K mutant with
ARK1 (Fig.
1C). Furthermore, the association of PI3K with
ARK1
occurred with either PI3K isoform (Fig. 1D), and the associated PI3K activity was wortmannin-sensitive (Fig. 1E).
Lysates from cell extracts before immunoprecipitation were
immunoblotted for
ARK1, PI3K
, and
PI3K to determine levels of
expression.
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Fig. 1.
ARK1 and PI3K interact to form
a cytosolic complex. NIH-3T3 cells were transfected with the
cDNAs represented in the boxes on the top of the
panel. The control (C) represents transfection
with an equivalent quantity of vector DNA. A, 48 h
after transfection, 500 µg of cytosolic extract was
immunoprecipitated using
ARK1 monoclonal antibody directed against
its catalytic domain, and the associated lipid kinase activity was
measured. Shown is a representative autoradiograph of a TLC plate where
PIP and phosphatidylinositol bisphosphate (PIP2) are
visualized. Ori, origin of resolution. B,
ARK1-associated PI3K activity quantified by phosphorimaging of the
TLC plates from four independent experiments. Results are expressed as
fold over control. *, p < 0.05 versus
control. IP, immunoprecipitation. C,
immunoprecipitations (IP) were performed from cytosolic
extracts with an anti-HA or anti-
ARK1 monoclonal antibody and
immunoblotted (IB) with a
ARK1 monoclonal (upper
panel) or anti-HA monoclonal antibody (lower panel).
ARK1- and HA-PI3K
-transfected cells were used as positive
controls (last lane). D, 500 µg of cytosolic
extract was immunoprecipitated with a
ARK1 monoclonal antibody, and
the associated lipid kinase activity was measured. Shown is a
representative autoradiograph where PIP is visualized. E,
500 µg of cytosolic extract was immunoprecipitated with a
ARK1
monoclonal antibody, and the associated PI3K activity was measured from
cells with and without treatment with 100 nM wortmannin
(Wort) for 15 min before lysis. Lysates from cytosolic
extracts before immunoprecipitation were used to monitor the levels of
ARK1 and HA-PI3K
expression.
ARK1-mediated Translocation of
PI3K
--
Since
ARK1 associates with PI3K, we tested whether
ARK1 could also promote translocation of PI3K to the cell membrane
in response to agonist stimulation. Experiments were performed in NIH-3T3 cells co-transfected with
ARK1 and PI3K
cDNAs, and
the endogenous
AR receptors were stimulated with 10 µM
isoproterenol for 2 min. Cytosolic and membrane fractions were prepared
and analyzed for
ARK1-associated PI3K activity. Little change in the
ARK1-associated PI3K activity was noted in the cytosolic fraction
after isoproterenol stimulation (Fig.
2A). In contrast, the membrane
fraction showed a significant increase in the
ARK1-associated PI3K
activity (Fig. 2, A and D) at 2 min after agonist
stimulation. The
ARK1-mediated recruitment of PI3K
to the
membrane was analyzed by immunoprecipitating
ARK1 from both
fractions and blotting for HA-PI3K. As shown in Fig. 2B,
treatment with isoproterenol resulted in a greater level of
ARK1-associated PI3K
protein in the membrane. Taken together
these data show that
ARK1 and PI3K
interact to form a complex in
the cytosol, and
ARK1 recruits PI3K
to the membrane in an
agonist-dependent manner.
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Fig. 2.
ARK1 and
PI3K
form a complex in the cytosol and
translocate to the membrane on agonist stimulation in a
G
-dependent
manner. A, NIH-3T3 cells were transfected with
ARK1
and HA-PI3K
and stimulated with isoproterenol (ISO, 10 µM) for a period of 2 min. 250 µg of protein from the
membrane and cytosolic fraction was used to immunoprecipitate
(IP)
ARK1 using
ARK1 monoclonal antibody and assayed
for associated PI3K activity. Shown is a representative autoradiograph
of a TLC plate where PIP is visualized (samples in duplicate except for
control (C), which was transfected with vector DNA and
treated with isoproterenol).
ISO, no treatment with
isoproterenol; +ISO, treatment with isoproterenol.
B,
ARK1 was immunoprecipitated from the membrane and
cytosolic fractions with a
ARK1 monoclonal antibody and
immunoblotted (IB) with an anti-HA monoclonal antibody.
Cells transfected with HA-PI3K
cDNA were used as a positive
control. MEM, membrane fraction; CYTO, cytosolic
fraction. C, cells were co-transfected with
ARK1 and
PI3K
cDNAs along with or without the
ARKct cDNA (the
carboxyl terminus of
ARK1 that attenuates the
G
-dependent signaling). 250 µg of protein from the
membrane and cytosolic fraction was used to immunoprecipitate
(IP)
ARK1 with a
ARK1 monoclonal antibody and assayed
for associated PI3K activity after isoproterenol stimulation (2 min).
Shown is a representative autoradiograph of the TLC plate where PIP and
phosphatidylinositol bis-phosphate (PIP2) are visualized.
D,
ARK1-associated PI3K activity was quantified by
phosphorimaging the TLC plates from four independent experiments.
Results are expressed as fold over basal (no isoproterenol treatment).
*, p < 0.05 versus membrane fraction
without isoproterenol treatment. E, NIH-3T3 cells were
transfected with the cDNAs represented in the boxes on
the top of the panel. Cytosolic extracts were used for
immunoprecipitation (IP) with a monoclonal HA-PI3K
or
Myc-PI3K
antibody and immunoblotted (IB) with a
polyclonal antibody against the carboxyl terminus of
ARK1 that
recognizes both full-length
ARK1 and the
ARKct.
Control (C), cells transfected with vector DNA. Lysates from
cytosolic extracts before immunoprecipitation were used to monitor the
level of expression of PI3K,
ARK1, and
ARKct. Endogenous
AR
density of NIH-3T3 cells was determined by a
125I-cyanopindolol binding study and found to contain
20.7 ± 3.2 fmols/mg of whole cell protein, which is sufficient to
activate and translocate
ARK1 after agonist stimulation (34,
35).
ARK1-associated
PI3K Activity Is G
-dependent--
The
agonist-dependent translocation of
ARK1 to the membrane
requires the presence of G
subunits (5). We tested whether G
subunits were required for the translocation of the
ARK1·PI3K
complex to the membrane by overexpressing the
carboxyl-terminal portion of
ARK1, the
ARKct, which is known to
inhibit the ability of
ARK1 to bind G
(15). Cells were
co-transfected with the
ARK1 and PI3K
plasmids along with or
without the
ARKct cDNA. Robust PI3K activity was found
associated with immunoprecipitated
ARK1 in the absence of
ARKct
(Fig. 2C). In contrast, the agonist-dependent translocation of
ARK1-associated PI3K activity was abolished in the
presence of the
ARKct (Fig. 2, C and D). These
data suggest that the sequestration of G
by
ARKct can
interrupt the process of
ARK1-mediated translocation of PI3K
to
the membrane (Fig. 2D). Interestingly, in the presence of
ARKct, there was also loss of
ARK1-associated PI3K activity in
the cytosolic fraction (Fig. 2, C and D). Since
the
ARKct contains the same PH domain as
ARK1, it appears that
ARKct competitively inhibits the
ARK1/PI3K interaction. We
tested this using cells expressing
ARK1,
ARKct, and either
PI3K
or PI3K
followed by immunoprecipitation with antibodies
directed against either HA-PI3K
or Myc-PI3K
. As shown in Fig.
2E, both
ARK1 and
ARKct were found to interact with either of the PI3K isoforms. To exclude the possibility that
overexpression of the
ARKct altered the level of expression of
either
ARK1 or PI3K
, lysates prepared from the co-transfected
cells were immunoblotted for
ARK1,
ARKct, and PI3K
. No
significant difference in the level of expression levels for either
ARK1 or PI3K
was seen in presence of the
ARKct (data not shown).
ARK1 Translocates PI3K to
AR--
The ability of
ARK1 to
translocate PI3K to the cell membrane suggests a mechanism for
co-localization of PI3K with
ARs. To test this, we used HEK 293 cells transfected with either FLAG epitope-tagged
1AR or
2AR plasmids and monitored the association of PI3K to
the receptor after agonist stimulation. HEK 293 cells are known to
contain adequate levels of
ARK1 to support agonist-induced receptor
phosphorylation (8). Transfected cells were split into separate dishes
and stimulated with 10 µM isoproterenol from 0 to 10 min.
The FLAG epitope was immunoprecipitated from cell extracts, and PI3K
activity was measured. As shown in Fig.
3, A-D, FLAG
1AR- and FLAG
2AR-associated PI3K
activity was observed by 2 min after agonist stimulation, with gradual
decline by 10 min. Moreover, the PI3K activity associated with the FLAG
1AR and
2AR was wortmannin-sensitive
(Fig. 3E). In addition, we could also detect the
agonist-dependent association of endogenous PI3K
protein
with the transfected FLAG
2AR at 2-10 min after
isoproterenol treatment (data not shown).
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Fig. 3.
ARK1 translocates
PI3K
to the
-adrenergic receptor. A, HEK 293 cells were transfected with FLAG
1AR and split into
separate dishes that were then individually treated with 10 µM isoproterenol for the indicated times. Control
(C), transfected with vector DNA. At indicated times points,
cell monolayers were scraped, and 500 µg of protein from the
cytosolic extract was used for immunoprecipitation (IP) with
an anti-FLAG monoclonal antibody, and the associated PI3K activity was
measured. Shown is a representative autoradiograph of the TLC plate
where PIP is visualized. B, FLAG
1AR-associated PI3K activity was quantified by
phosphorimaging of the TLC plates from five independent experiments.
Results are expressed as fold over basal (no isoproterenol treatment).
*, p < 0.05 versus 0 min. C, as
for panel A, except cells were transfected with FLAG
2AR cDNA. D, as for panel B,
except the levels of FLAG
2AR-associated PI3K activity
was quantified. *, p < 0.05 versus 0 min.
E, HEK 293 cells were transfected with FLAG
1AR (upper panel) and FLAG
2AR
(lower panel). A set of transfected cells was treated with
100 nM wortmannin 15 min before agonist stimulation. Both
wortmannin-treated and untreated cells were then stimulated with 10 µM isoproterenol for the indicated times. Cell extracts
were used to immunoprecipitate
1AR and
2AR using the anti-FLAG antibody, and PI3K activity was
measured in the immunoprecipitates. Shown are the autoradiograph of TLC
plates where PIP was visualized.
2AR endocytosis is regulated by
ARK1, and
ARK1-mediated PI3K translocation provides a mechanism
for the interaction of PI3K with receptor, we determined whether the
PI3K inhibitors, wortmannin and LY294002, could attenuate
AR
internalization. Agonist-dependent sequestration was
studied in HEK 293 cells transfected with the FLAG
2AR
plasmid. As shown in Fig. 4A,
a significant attenuation in the rate of
2AR
sequestration was observed in wortmannin-treated cells for up to 20-30
min. Similarly, a 50% reduction in sequestration was also observed
with LY294002-treated cells (data not shown). To directly address
whether PI3K is required for the process of
AR sequestration, a time
course of agonist-dependent sequestration was studied in
HEK 293 cells transfected either with the FLAG
2AR, FLAG
2AR and PI3K
, or FLAG
2AR and
PI3K
plasmids. Similar to the pattern of inhibition by
wortmannin,
PI3K
-transfected cells showed a significant
attenuation in the rate of receptor sequestration (Fig. 4B).
These data demonstrate a role for PI3K in the process of
AR
internalization possibly due to the local production of
PtdIns(3,4,5)P3.
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Fig. 4.
PI3K activity is required for
AR sequestration. A, agonist
promoted (1 µM isoproterenol)
2AR
sequestration in transfected HEK 293 cells studied by
125I-cyanopindolol binding over a time course of 0-30 min
in untreated (
) and wortmannin (100 nM)-treated (
)
cells. Receptor sequestration at each time point has been normalized to
the value of internalized
2AR at 30 min in the absence
of wortmannin. The maximal absolute level of sequestration for the
2AR in the untreated cells at 30 min was 22.3 ± 3.9%, n = 5. Receptor expression (fmol/mg of
whole-cell protein) was 378 ± 123; *, p < 0.05 versus untreated. Inset, upper panel,
inhibition of endogenous lipid kinase activity with wortmannin.
2AR and + represents
2AR-transfected
wortmannin-treated cells;
2AR and
represents
2AR-transfected untreated cells. The first
lane represents endogenous PI3K activity in the
untransfected cells. Lower panel, the level of endogenous
PI3K
expression. B, agonist-promoted
2AR
sequestration in cells co-transfected with
2AR along
with either empty vector (
) or PI3K
(
) or
PI3K
(
)
cDNAs in HEK 293 cells. Receptor expression (fmol/mg of whole-cell
protein) was:
2AR + vector, 316 ± 75;
2AR + PI3K
, 367 ± 81;
2AR +
PI3K
, 267 ± 157. n = 5, *,
p < 0.05 versus
2AR.
Inset upper panel, immunoblot showing the levels of
expression of HA-PI3K
, HA-
PI3K
, and PI3K
in the transfected
cells. Cells were transfected with
2AR (
),
2AR and wild type PI3K
(WT) or
2AR and
PI3K
(
) cDNAs. Inset lower
panel, lipid kinase activity in the HEK 293 cells transfected with
the PI3K
(WT) and
PI3K
(
) cDNAs. The
receptor sequestration at each time point has been normalized to the
value of internalized
2AR at 30 min. The maximal
absolute level for the
2AR in the untreated cells at 30 min was 25.0 ± 2.1%, n = 5. C,
-arrestin 2-GFP recruitment to the membrane on isoproterenol
stimulation (1 µM) was monitored by confocal microscopy
in HEK 293 cells transfected with
2AR and
-arrestin
2-GFP cDNAs along with either empty vector pRK5, PI3K
, or
PI3K
at the indicated times. Marked redistribution of
-arrestin 2-GFP to the membrane occurs within 2.5 min.
Arrows on the 5-min panel highlight regions of
translocated
-arrestin 2-GFP. The upper panel is an
immunoblot for HA from extracts of the same cells showing equal levels
of HA-PI3K
and HA-
PI3K
expression. D, agonist
(isoproterenol (ISO), 5 min) promoted
2AR
phosphorylation in transfected cells after 32Pi
metabolic labeling for 1 h before stimulation (upper
panel). Lower panel, immunoblot for HA showing equal
levels of expression of HA-PI3K
and HA-
PI3K
. + represents
HA-PI3K
control. IB, immunoblot; IP,
immunoprecipitate.
PI3K
mutant could result if
PI3K
inhibited either
recruitment of
-arrestin to the phosphorylated receptor or directly
inhibited receptor phosphorylation. To exclude these possibilities,
cells were co-transfected with plasmids containing FLAG
2AR,
-arrestin 2-GFP, and either PI3K
or
PI3K
cDNAs and were monitored for the recruitment of
-arrestin 2-GFP using confocal microscopy. In cells transfected with
PI3K
, there was prompt recruitment of
-arrestin 2-GFP to the
receptor after exposure to agonist (Fig. 4C). To directly
test the effect of
PI3K
expression on receptor phosphorylation,
32Pi metabolic labeling was performed in HEK
293 cells transfected with plasmids containing FLAG
2AR
and either the PI3K
or
PI3K
cDNAs. As shown in Fig.
4D, the level of agonist-induced receptor phosphorylation
was not affected by the expression of
PI3K
. Thus, the
effect of overexpression of
PI3K
on
2AR
sequestration is not related to processes that are involved with
2AR phosphorylation or
-arrestin 2 recruitment.
ARK1 and PI3K Form a Complex in the Heart--
Since normal
AR function is critical for maintenance of cardiac function,
particularly during periods of increased workload (23), we wanted to
determine whether PI3K and
ARK1 interact in an organ of in
vivo relevance such as the heart. The monoclonal
ARK1 antibody
was used to immunoprecipitate
ARK1 from myocardial extracts prepared
from normal mouse hearts. The immunoprecipitate was tested for the
presence of associated PI3K protein by immunoblotting. As a positive
control, the PI3K polyclonal antibody was used to immunoprecipitate
total PI3K from separate extracts prepared from the same heart. As
shown in Fig. 5A, PI3K was
co-immunoprecipitated along with
ARK1 from the myocardial extract.
To determine whether the association of
ARK1 with PI3K in the heart
would also result in the
ARK1-mediated translocation of PI3K to the
membrane, anesthetized mice were stimulated with isoproterenol (10 µM) by infusion through a cannulated jugular vein for 3 min. Membrane and cytosolic fractions were prepared from the treated
hearts, and PI3K activity was measured after immunoprecipitation with
the
ARK1 monoclonal antibody. As show in Fig. 5B, a
significant increase in
ARK1-associated PI3K activity was found in
the membrane fraction after isoproterenol treatment (fold induction
over untreated, 5.60 ± 1.10, p < 0.05, n = 3). No difference in
ARK1-associated PI3K
activity was found in the cytosolic fraction after isoproterenol
treatment (fold induction over untreated, 1.19 ± 0.47, n = 3). We previously documented an induction of both
ARK1 and PI3K
in the pressure overloaded heart (19, 20) and,
therefore, tested whether the interaction of
ARK1 with PI3K would
also be enhanced in the hypertrophied heart. As shown in Fig. 5,
C and D, greater wortmannin-sensitive PI3K
activity and protein were found complexed with
ARK1 in hypertrophied hearts compared with sham-treated hearts.
View larger version (39K):
[in a new window]
Fig. 5.
ARK1 and PI3K form a complex in
heart. A, 4 mg of myocardial extract from a mouse heart
was used to immunoprecipitate (IP)
ARK1 (left
lane), and 2 mg of the extract was used to immunoprecipitate PI3K
as positive control (right lane). IB, immunoblot.
B, myocardial membrane fractions were prepared, and
ARK1
was immunoprecipitated using
ARK1 monoclonal antibody, assayed for
the associated PI3K activity.
ISO, hearts without
isoproterenol treatment; +ISO, hearts treated with
isoproterenol. C,
ARK1-associated PI3K activity was
measured in the myocardial extracts from the sham (S) and
transverse aortic constricted (T) hearts (19, 20). 4 mg of
the myocardial extracts was used for immunoprecipitation with
ARK1
monoclonal antibody and then assayed for the PI3K activity (fold
induction in T over S 2.1 ± 0.13; p, < 0.05, n = 3).
Wort and +Wort,
reactions performed in the absence or presence of wortmannin.
D, myocardial extracts from sham (S) and T hearts were used
to immunoprecipitate
ARK1 and PI3K
with a monoclonal antibody
directed against
ARK1 (4 mg of extract) and a polyclonal antibody
for PI3K
(2 mg of extract), respectively, as a positive control
(C). The protein bands were visualized using ECL
chemiluminescence.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ARK1 and PI3K in the cytosol. Then, in response to agonist, PI3K
undergoes
ARK1-mediated translocation to the membrane. Furthermore,
the
ARK1-dependent translocation of PI3K allows for
colocalization of PI3K with the receptor, which is of functional
importance since catalytically inactive PI3K or inhibitors of PI3K
activity attenuate
AR sequestration. These studies thus demonstrate
that the agonist-dependent recruitment of PI3K to the
membrane is an important step in the process of receptor sequestration
and links class I PI3Ks to GPCR signaling and endocytosis.
show that it contains many modular
regions including a helical domain (also called a HEAT sequence motif)
(14) that could be involved in protein-protein interactions (24, 25).
Since the HEAT sequence is common to PI3K
, -
, and -
isoforms,
it is possible that this is the common interacting domain for all
PI3Ks. Indeed, in this study we show that
ARK1 can interact with
both PI3K
and -
isoforms but cannot rule out the possibility of
ARK1 interacting with PI3K
, since it was not tested. In this
regard there is increasing interest in the role of the PI3K
isoform
in cell signaling as shown by a recent study demonstrating activation
of downstream signal molecules like v-Akt (protein kinase
B) and protein kinase C
by PI3K
after GPCR stimulation (26).
Thus, the HEAT domain of PI3K could provide the necessary structure for
the interaction of PI3Ks with
ARK1. Depending on the cellular
signaling pathway that is activated (i.e. growth factor
stimulation versus GPCR stimulation), the PI3K isoform that
interacts with
ARK1 might change.
ARK1 to mediate
agonist-dependent translocation of PI3K
to the membrane. Several studies have shown activation of PI3K
in the presence of
G
but have not documented PI3K
recruitment to the membrane after agonist stimulation. Our data suggest that a PI3K molecule interacting with
ARK1 is recruited to the plasma membrane by
ARK1, and this recruitment is G
-dependent, since
expression of the
ARKct (a G
-sequestering peptide) interrupted
the
ARK1-mediated translocation of PI3K to the membrane. It is
interesting that there was a loss of
ARK1-associated PI3K activity
in cytosolic fractions with overexpression of
ARKct despite the
equal expression of
ARK1 or PI3K
. These data indicate that the
ARKct can compete with
ARK1 for interaction with PI3K
and
further points to the carboxyl-terminal PH domain of
ARK1 as the
interacting domain, since it is common within both
ARKct and
ARK1. This conclusion is also supported by our data showing that
both the
ARKct and
ARK1 can be co-immunoprecipitated along with
either of the PI3K isoforms. Based on these experiments, it appears
that overexpression of the
ARKct acts to inhibit the recruitment of
ARK1 to the membrane by sequestering G
subunits (16) and may
also directly interrupt the
ARK1/PI3K
interaction.
2AR endocytosis (28). Our data clearly
show that in transfected HEK 293 cells, the presence of agonist
promotes the association of wortmannin-sensitive PI3K
activity with
both
1- and
2-adrenergic receptors.
Moreover, treatment with PI3K inhibitors or overexpression of
catalytically inactive PI3K attenuates
2AR sequestration.
-arrestins (10) bind PtdIns(3,4,5)P3 with high
affinity and PtdIns (4,5)P2 at a 1000-fold lower affinity. Therefore, in our experiments with overexpression of
PI3K,
generation of PtdIns(3,4,5)P3 would be inhibited, resulting
in an increase in the concentration of PtdIns(4,5)P2, which
would be much less efficient in promoting receptor endocytosis. Studies
in macrophages have shown inhibition of phagocytosis in the presence of
the PI3K inhibitor wortmannin, and this inhibition prevented
recruitment of dynamin to endocytic vesicles (29). This suggests the
possibility that generation of phosphoinositides by PI3K within the
receptor complex may regulate internalization of
2ARs
through recruitment of dynamin (30). Importantly, whereas
phosphoinositides have been implicated in receptor internalization
after platelet-derived growth factor (31) and insulin receptor (9)
stimulation, we provide evidence here for the involvement of
phosphoinositides after
AR stimulation. Taken together these data
demonstrate an important role of PI3K
in the process of
AR
internalization, possibly due to the local production of
PtdIns(3,4,5)P3.
AR sequestration by
PI3K suggests a critical role for generation of phosphoinositides in
AR sequestration, it is also possible that
PI3K interrupts either
-arrestin recruitment to the phosphorylated receptor or directly
inhibits
ARK1-mediated
AR phosphorylation by sequestering G
. To exclude these possibilities we showed that overexpression of
PI3K inhibits neither the
-arrestin 2-GFP recruitment to the
receptor nor
ARK1-mediated
2AR phosphorylation. These
data suggest that inhibition of
AR internalization is not linked to events that are involved with phosphorylation and desensitization of
the receptor.
ARK1 in a tissue of
in vivo relevance, we studied the heart under several
conditions. In the unstimulated heart,
ARK1 and PI3K form a complex
in myocardial extracts. Importantly, after isoproterenol stimulation
there was increased
ARK1-associated PI3K activity in myocardial
membranes corroborating the cell culture studies. We have shown in
earlier experiments an increase in both
ARK1 and PI3K
activity in
the pathophysiological state of in vivo pressure overload
hypertrophy (19, 20). Our present results show that in the
hypertrophied heart there is an increase in
ARK1-associated PI3K
activity and protein that may be important for the regulation of
adrenergic receptors during this pathologic state. For instance, under
conditions of in vivo pressure overload hypertrophy, there
would be a higher level of
ARK1-mediated PI3K recruitment to the
membrane. This would lead to both diminished receptor number (due to
more efficient receptor sequestration) and impaired receptor function
(enhanced phosphorylation from increased
ARK1 activity). Consistent
with this hypothesis are the characteristic findings of diminished
AR number and reduced
AR coupling to G-proteins in failing human hearts and in experimental models of heart failure (23, 32). Our
studies suggest that in pathophysiological states such as hypertrophy
and heart failure, alterations in PI3K activation and recruitment may
contribute to abnormalities in
AR function.
pathway to activation of atrial natriuretic factor (ANF) transcription (26), which is
widely considered a marker for cardiac hypertrophy and heart failure.
Furthermore, studies using adenoviral-mediated expression system in
neonatal rat cardiomyocytes show that activation of glycogen synthase
kinase 3
, a negative regulator of cardiomyocyte hypertrophy, is
mediated by a PI3K pathway (33). These studies are consistent with our
earlier findings of activation of PI3K in hearts with pressure overload
hypertrophy (19) and, combined with our present study, support our view
for an important role of phosphoinositides and PI3K in cardiac
hypertrophy and failure.
AR internalization. PI3K that
colocalizes with the receptor may serve to increase the local membrane
concentration of PtdIns(3,4,5)P3, thus enhancing the functional interaction of determinants of the endocytic process such as
-arrestin, AP-2, clathrin, and dynamin. Whether the process leads to
a more efficient recruitment of GPCRs to preformed clathrin-coated pits
or the newly formed pits are interesting possibilities to explore further.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL56687 (to H. A. R.), NS19576 (to M. G. C.), and HL61365 (to L. S. B.) and the Burroughs Wellcome Fund (to H. A. R.).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.
An Investigator of the Howard Hughes Medical Institute.
** A recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. To whom correspondence should be addressed: Dept. of Medicine and Cell Biology, Duke University Medical Center, CARL Bldg., Rm. 226, DUMC 3104, Durham, NC 27710. Tel.: 919-668-2520; Fax: 919-668-2524; E-mail: h.rockman@duke.edu.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M102376200
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ABBREVIATIONS |
---|
The abbreviations used are:
PI3K, phosphoinositide 3-kinase;
GPCR, G-protein-coupled receptor;
AR,
-adrenergic receptor;
ARK1,
-adrenergic receptor kinase 1;
ARKct, carboxyl-terminal peptide of
-adrenergic receptor kinase;
PtdIns, phosphatidylinositol;
PtdIns(3, 4,5)P3,
phosphatidylinositol 3,4,5-triphosphate;
PIP, phosphatidylinositol
monophosphate;
T, transverse aortic constriction;
GFP, green
fluorescent protein;
HEK cells, human embryonic kidney cells;
PCR, polymerase chain reaction;
HA, hemagglutinin;
CYP, cyanopindolol.
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