# Determinants Present in the Receptor Carboxy Tail  Are Responsible for Differences in Subtype-Specific Coupling ofβ-Adrenergic Receptors to Phosphoinositide 3-Kinase

Authors: Julie Simard; Matthieu Boucher; Rachel Massé; Terence E. Hébert; Guy Rousseau
Journal: International Journal of Cell Biology
 (2009)

---

## Abstract

An agonist-occupiedβ2-adrenergic receptor (β2-AR) recruits G protein receptor kinase-2 (GRK2) which is recruited to the membrane. Thus, the physical proximity of activated β2-AR and PI-3K allows the activation of the latter. In contrast, it has been observed that the β1-AR is unable to activate the PI-3K/Akt pathway. We hypothesized that the difference might be due to molecular determinants present in the carboxy termini of the two β-AR subtypes. Using transiently transfected HEK 293 cells expressing either β1- or β2-AR, we also observed that in presence of an agonist, β2-AR, but not β1-AR, is able to activate the PI-3K/Akt pathway. Switching the seventh transmembrane domain and the carboxy tail between the two receptors reverses this phenotype; that is, β1×β2-AR can activate the PI-3K/Akt pathway whereas β2×β1-AR cannot. Pretreatment with pertussis toxin abolished the activation of PI-3K by β2- or β1×β2-AR stimulation. Ligand-mediated internalization  of the β2-AR induced by a 15-minute stimulation with agonist was abolished in the presence of a dominant negative of PI-3K or following pertussis toxin pretreatment. These results indicate that the subtype-specific differences in the coupling to PI-3K/Akt pathway are due to molecular determinants present in the carboxy tail of the receptor and further that β2-AR activates  PI-3K via a pertussis toxin-sensitive mechanism.


---

## Body

## 1. Introduction

β2-AR activation induces antiapoptotic
effects in cardiomyocytes mediated by stimulation of the PI-3K pathway [1]. The proposed mechanism by
which this activation  occurs is dependent on G protein-coupled receptor kinases
(GRKs). Under basal conditions, GRK2 forms a complex with PI-3K in the cytosol [2]. When β2-AR is occupied by an agonist, GRK2
is translocated  to the membrane by a Gβγ subunit-dependent mechanism and subsequently recruits PI-3K from
the cytosol to the membrane [2]. The proximity of the
PI-3K and the stimulated receptor induces the activation of the enzyme. In
contrast, β1-AR has proapoptotic activity in cardiomyocytes due to stimulation
of PKA or CAM KII [3]. Indeed, it has been
observed that stimulation of β1-AR with an agonist induces apoptosis
and this can be prevented in presence of inhibitors of either PKA or CAM-KII [4].Interestingly it has been shown that
stimulatedβ1-AR can
also recruit GRK to the membrane. The interaction of either β-AR subtype with GRKs is mainly via
the intracellular loops and carboxy tail of the receptor [5]. When activated, GRK induces the
phosphorylation of certain serine/threonine residues in the carboxy tail of
both β-AR subtypes resulting in functional
uncoupling of the receptor from their primary signalling pathways. GRK2
phosphorylation also favours subsequent interaction with β-arrestin. This interaction with β-arrestin further desensitizes the receptor and is subsequently involved
in receptor endocytosis [6, 7].As GRK interacts with molecular
determinants in carboxy tail of the receptor, we hypothesized that differences
in the coupling of theβ1- and β2-AR with PI-3K may be due to
determinants located in this portion of the receptor. This study was designed to
determine the respective efficiencies of β1-AR and β2-AR to stimulate the PI-3K pathway
and to test the above hypothesis.


## 2. Material and Methods

### 2.1. DNA Constructions, Cell Transfection, and Culture

Murineβ1-AR and human β2-AR subcloned into pcDNA3 were used
in this study. Two chimeric receptors consisting of the β1-AR with the seventh transmembrane
domain and the carboxyl-terminal tail of the β2-AR and the reciprocal β2-AR with the seventh transmembrane
domain and the carboxyl-terminal tail of the β1-AR were constructed as follows. A
restriction site for Hpa I was created by polymerase chain reaction (PCR) at
position 2070 in the β1-AR. The new restriction site in the β1-AR was created with three primers.
Two primers were used for the hybridation with the receptor sequence. These
primers contained 21 base pairs and had, respectively, CGCCTCAGAAGCCATAGAGCC and TCGTGTGCACAGTGTGGGCCA sequences. The third primer was utilized to introduce
the restriction site. This primer which contained 24 base pairs had the
following sequence: GGTGGAAAGCGTTAACCACGTTGG. The mutated β1-AR and the β2-AR wt were double digested with Hpa
I and Xho I. The result of this digestion is two fragments of 6540 bp and 486 bp
for the mutated β1-AR and two fragments 5998 bp and
1362 bp for the β2-AR wt. The appropriate restriction
fragments (containing the seventh transmembrane domain and the carboxy-terminal
portion of the receptor) were isolated, exchanged for their counterparts, and
religated. Positive clones were selected by enzymatic digestion and confirmed
by sequencing.Theβ-AR wild type (wt), chimeric receptors, dominant-negative PI-3
kinase (p85ΔPI-3K),
and/or carboxy-terminal domain of GRK2 (ct-GRK2) were transiently transfected
in human embryonic kidney (HEK 293) cells using the calcium phosphate
precipitation method. We performed all experiments at 48 hours
posttransfection, that is, at maximal receptor expression determined by ligand
binding. Cells were starved overnight 24 hours before the experiments in a
medium without fetal bovine serum. HEK 293 cells were maintained in DMEM
supplemented with 10% fetal bovine serum, 100 U/mL penicillin and streptomycin,
1 mM glutamine, 0.25 μg/mL fungizone in an atmosphere of 95% air/5%
CO2 at 37°C.
On the day of the experiment, cells were treated with 1 μM isoproterenol for the indicated times and fractionated for the
cytosolic or membrane compartments. In some experiments, cells were pretreated
with pertussis toxin (0.1 μg/mL; Sigma), 18 hours before stimulation with
isoproterenol.


### 2.2. Preparation of Cytosolic and Membrane Fractions

Cells were washed
three times with 10 mL of phosphate-buffered saline at 4°C and
mechanically detached in 1 mL of ice-cold buffer containing 5 mM Tris-HCl, pH 7.4, 2 mM
EDTA, 5 μg/mL leupeptin, 5 μg/mL soybean trypsin inhibitor, and 10 μg/mL benzamidine. Cells were then lysed with a sonicator (3 bursts
of 10 seconds at max speed), and the lysates were centrifuged at 1000  × g for 5
minutes at 4°C. The supernatant was centrifuged at 45 000  × g for 20 minutes  and  was considered as the cytosolic preparation.
Protein content was assessed using the Bradford method (Bio-Rad). The
pelleted membranes were resuspended in 250 μL of a solubilization buffer (buffer A) containing 50 mM Tris pH
7.5, 20 mM β-glycerophosphate, 20 mM NaF, 5 mM EDTA, 10 mM EGTA, 1 mM Na3VO4, 10 mM
benzamidine, 0.5 mM PMSF, 10 μg/mL leupeptin, 5 mM DTT, 1 μM microcystin LR, and 1% Triton X-100; and solubilized for 2 hours
at 4°C. Then the membranes were centrifuged at 10 000  × g for 15
minutes. The protein content was assessed using the Lowry method
(Bio-Rad).


### 2.3. Radioligand Binding Assay

Radioligand binding
assays were conducted essentially as described previously [8] with ~5 μg of membrane proteins in a total volume of 0.5 mL containing 250 pM
[125I]CYP in the presence or absence of 10 μM alprenolol to define nonspecific binding. The binding reactions
were incubated at room temperature for 90 minutes and terminated by rapid
filtration with ice-cold 25 mM Tris-HCl, pH 7.4, over Whatman GF/C glass fiber
filters preincubed for ≥30 minutes in
a buffer containing 25 mM Tris-HCl, pH 7.4, 0.1% bovine serum albumin, and 0.3%
polyethylenimine.


### 2.4. Western Blotting

Western blotting was conducted as described previously [9]. Briefly, aliquots of 50–75 μg of cytosolic or membrane protein preparations
were subjected to 10% denaturing polyacrylamide gel electrophoresis as
previously described. Transfer was performed with a Trans-Blot SD Semi-dry
transfer cell (Bio-Rad) on Protran nitrocellulose membrane (Mandel, Montréal,
QC, Canada). Protein transfer efficiency was assessed using Ponceau S
staining. Membranes were blocked using 5% nonfat dry milk in TBS-T (10 mM Tris
(pH 7.4), 150 mM NaCl, and 0.05% Tween 20) and membranes were incubated at 4°C overnight with primary antibody (anti-Phospho-Akt (Ser 473) and
anti-Akt from Cell Signaling Technology, (Missisauga, Canada) or anti-PI-3K,
anti-β1- or anti-β2-AR from Santa-Cruz (Calif, USA)
diluted 1:1000 in 5% nonfat dry milk into TBS-T. Subsequently, membranes were
washed and incubated for 45 minutes at room temperature with the secondary
antibody (diluted 1:5000 in 5% nonfat dry milk into TBS-T) conjugated to
horseradish peroxidase. Membranes were washed and exposed to scientific imaging
film (Perkin Elmer Life Sciences, ON) or quantified using a Kodak
ImageStation 440CF using enhanced chemiluminescence reagent (Perkin Elmer Life
Sciences). Band intensities were analyzed using Kodak 1D v.3.5.5 Scientific
Imaging Software.


### 2.5. Immunohistochemistry and Receptor Internalization

Sequestration ofβ-AR
was observed
by immunolocalisation. After agonist treatment, cells were washed with PBS and
fixed with 3% paraformaldehyde for 15 minutes. After these washes, nonspecific
sites were blocked with 0.2% BSA and 0.15% Triton x-100 (blocking solution) for
10 minutes. Primary antibody (β1- and β2-AR from Santa-Cruz), prepared in the
blocking solution (1:200), was added for 30 minutes at room temperature. After
another series of washes, secondary antibody (antirabbit, Santa-Cruz,) also
prepared in the blocking solution (1:500) was added for 30 minutes. After a
final series of washes, slides were mounted and viewed using a Leica epi-illumination microscope.


### 2.6. PI-3K Activity

PI-3K activity was measured as previously described [10]. Briefly, 250–375 μg of cytosolic and membrane proteins were precipitated with
anti-phosphotyrosine antibody conjuged to biotin (1:50, Santa-Cruz, Calif, USA)
overnight at 4°C. The immune complex was pelleted (with streptavidin
beads) and washed three times with lysis buffer and twice with
phosphate-buffered saline buffer containing 0.1 mM Na3VO4.
The immune pellet was then suspended in activation buffer (35 mM ATP, 0.2 mM
adenosine, 30 mM MgCl2, 10 mg/mL L-α-phosphatidylinositol, and 20 μCi [γP32]-ATP; (Amersham Pharmacia
Biotech, Baie-d'Urfé, Canada) and incubated at room temperature for 20 minutes.
The reaction was stopped with the addition of 100 μL HCl 1 M and 200 μL of chloroform:methanol (1:1). The aqueous
phase was then discarded. Eighty μL
of HCl:methanol (1:1) were then added before discarding the aqueous phase.
Twenty μL
of the organic phase containing 32P-phosphatidylinositol were
resolved by thin layer chromatography on K6 Silica Gel plates (Whatman,
Clifton, NJ, USA) in a solvent system containing chloroform:methanol:ammonium
hydroxide (45:35:10). Plates were exposed to film for three to five days
(−80°C).


### 2.7. Statistical Analysis

Results are expressed as mean ± SEM and were evaluated using
analysis of variance adapted for factorial experimental design.
Orthogonalization was performed when necessary [11]. P<.05 was considered significant.


## 2.1. DNA Constructions, Cell Transfection, and Culture

Murineβ1-AR and human β2-AR subcloned into pcDNA3 were used
in this study. Two chimeric receptors consisting of the β1-AR with the seventh transmembrane
domain and the carboxyl-terminal tail of the β2-AR and the reciprocal β2-AR with the seventh transmembrane
domain and the carboxyl-terminal tail of the β1-AR were constructed as follows. A
restriction site for Hpa I was created by polymerase chain reaction (PCR) at
position 2070 in the β1-AR. The new restriction site in the β1-AR was created with three primers.
Two primers were used for the hybridation with the receptor sequence. These
primers contained 21 base pairs and had, respectively, CGCCTCAGAAGCCATAGAGCC and TCGTGTGCACAGTGTGGGCCA sequences. The third primer was utilized to introduce
the restriction site. This primer which contained 24 base pairs had the
following sequence: GGTGGAAAGCGTTAACCACGTTGG. The mutated β1-AR and the β2-AR wt were double digested with Hpa
I and Xho I. The result of this digestion is two fragments of 6540 bp and 486 bp
for the mutated β1-AR and two fragments 5998 bp and
1362 bp for the β2-AR wt. The appropriate restriction
fragments (containing the seventh transmembrane domain and the carboxy-terminal
portion of the receptor) were isolated, exchanged for their counterparts, and
religated. Positive clones were selected by enzymatic digestion and confirmed
by sequencing.Theβ-AR wild type (wt), chimeric receptors, dominant-negative PI-3
kinase (p85ΔPI-3K),
and/or carboxy-terminal domain of GRK2 (ct-GRK2) were transiently transfected
in human embryonic kidney (HEK 293) cells using the calcium phosphate
precipitation method. We performed all experiments at 48 hours
posttransfection, that is, at maximal receptor expression determined by ligand
binding. Cells were starved overnight 24 hours before the experiments in a
medium without fetal bovine serum. HEK 293 cells were maintained in DMEM
supplemented with 10% fetal bovine serum, 100 U/mL penicillin and streptomycin,
1 mM glutamine, 0.25 μg/mL fungizone in an atmosphere of 95% air/5%
CO2 at 37°C.
On the day of the experiment, cells were treated with 1 μM isoproterenol for the indicated times and fractionated for the
cytosolic or membrane compartments. In some experiments, cells were pretreated
with pertussis toxin (0.1 μg/mL; Sigma), 18 hours before stimulation with
isoproterenol.


## 2.2. Preparation of Cytosolic and Membrane Fractions

Cells were washed
three times with 10 mL of phosphate-buffered saline at 4°C and
mechanically detached in 1 mL of ice-cold buffer containing 5 mM Tris-HCl, pH 7.4, 2 mM
EDTA, 5 μg/mL leupeptin, 5 μg/mL soybean trypsin inhibitor, and 10 μg/mL benzamidine. Cells were then lysed with a sonicator (3 bursts
of 10 seconds at max speed), and the lysates were centrifuged at 1000  × g for 5
minutes at 4°C. The supernatant was centrifuged at 45 000  × g for 20 minutes  and  was considered as the cytosolic preparation.
Protein content was assessed using the Bradford method (Bio-Rad). The
pelleted membranes were resuspended in 250 μL of a solubilization buffer (buffer A) containing 50 mM Tris pH
7.5, 20 mM β-glycerophosphate, 20 mM NaF, 5 mM EDTA, 10 mM EGTA, 1 mM Na3VO4, 10 mM
benzamidine, 0.5 mM PMSF, 10 μg/mL leupeptin, 5 mM DTT, 1 μM microcystin LR, and 1% Triton X-100; and solubilized for 2 hours
at 4°C. Then the membranes were centrifuged at 10 000  × g for 15
minutes. The protein content was assessed using the Lowry method
(Bio-Rad).


## 2.3. Radioligand Binding Assay

Radioligand binding
assays were conducted essentially as described previously [8] with ~5 μg of membrane proteins in a total volume of 0.5 mL containing 250 pM
[125I]CYP in the presence or absence of 10 μM alprenolol to define nonspecific binding. The binding reactions
were incubated at room temperature for 90 minutes and terminated by rapid
filtration with ice-cold 25 mM Tris-HCl, pH 7.4, over Whatman GF/C glass fiber
filters preincubed for ≥30 minutes in
a buffer containing 25 mM Tris-HCl, pH 7.4, 0.1% bovine serum albumin, and 0.3%
polyethylenimine.


## 2.4. Western Blotting

Western blotting was conducted as described previously [9]. Briefly, aliquots of 50–75 μg of cytosolic or membrane protein preparations
were subjected to 10% denaturing polyacrylamide gel electrophoresis as
previously described. Transfer was performed with a Trans-Blot SD Semi-dry
transfer cell (Bio-Rad) on Protran nitrocellulose membrane (Mandel, Montréal,
QC, Canada). Protein transfer efficiency was assessed using Ponceau S
staining. Membranes were blocked using 5% nonfat dry milk in TBS-T (10 mM Tris
(pH 7.4), 150 mM NaCl, and 0.05% Tween 20) and membranes were incubated at 4°C overnight with primary antibody (anti-Phospho-Akt (Ser 473) and
anti-Akt from Cell Signaling Technology, (Missisauga, Canada) or anti-PI-3K,
anti-β1- or anti-β2-AR from Santa-Cruz (Calif, USA)
diluted 1:1000 in 5% nonfat dry milk into TBS-T. Subsequently, membranes were
washed and incubated for 45 minutes at room temperature with the secondary
antibody (diluted 1:5000 in 5% nonfat dry milk into TBS-T) conjugated to
horseradish peroxidase. Membranes were washed and exposed to scientific imaging
film (Perkin Elmer Life Sciences, ON) or quantified using a Kodak
ImageStation 440CF using enhanced chemiluminescence reagent (Perkin Elmer Life
Sciences). Band intensities were analyzed using Kodak 1D v.3.5.5 Scientific
Imaging Software.


## 2.5. Immunohistochemistry and Receptor Internalization

Sequestration ofβ-AR
was observed
by immunolocalisation. After agonist treatment, cells were washed with PBS and
fixed with 3% paraformaldehyde for 15 minutes. After these washes, nonspecific
sites were blocked with 0.2% BSA and 0.15% Triton x-100 (blocking solution) for
10 minutes. Primary antibody (β1- and β2-AR from Santa-Cruz), prepared in the
blocking solution (1:200), was added for 30 minutes at room temperature. After
another series of washes, secondary antibody (antirabbit, Santa-Cruz,) also
prepared in the blocking solution (1:500) was added for 30 minutes. After a
final series of washes, slides were mounted and viewed using a Leica epi-illumination microscope.


## 2.6. PI-3K Activity

PI-3K activity was measured as previously described [10]. Briefly, 250–375 μg of cytosolic and membrane proteins were precipitated with
anti-phosphotyrosine antibody conjuged to biotin (1:50, Santa-Cruz, Calif, USA)
overnight at 4°C. The immune complex was pelleted (with streptavidin
beads) and washed three times with lysis buffer and twice with
phosphate-buffered saline buffer containing 0.1 mM Na3VO4.
The immune pellet was then suspended in activation buffer (35 mM ATP, 0.2 mM
adenosine, 30 mM MgCl2, 10 mg/mL L-α-phosphatidylinositol, and 20 μCi [γP32]-ATP; (Amersham Pharmacia
Biotech, Baie-d'Urfé, Canada) and incubated at room temperature for 20 minutes.
The reaction was stopped with the addition of 100 μL HCl 1 M and 200 μL of chloroform:methanol (1:1). The aqueous
phase was then discarded. Eighty μL
of HCl:methanol (1:1) were then added before discarding the aqueous phase.
Twenty μL
of the organic phase containing 32P-phosphatidylinositol were
resolved by thin layer chromatography on K6 Silica Gel plates (Whatman,
Clifton, NJ, USA) in a solvent system containing chloroform:methanol:ammonium
hydroxide (45:35:10). Plates were exposed to film for three to five days
(−80°C).


## 2.7. Statistical Analysis

Results are expressed as mean ± SEM and were evaluated using
analysis of variance adapted for factorial experimental design.
Orthogonalization was performed when necessary [11]. P<.05 was considered significant.


## 3. Results

### 3.1. Expression ofβ-AR Subtypes and P85ΔPI-3K in HEK 293 Cells

HEK 293 cells were transiently transfected with cDNAs encoding forβ1-AR Wt, β2-AR Wt,
β1×β2-AR, or
β2×β1-AR and in some case, p85ΔPI-3K.
Forty-eight hours after transfection β-AR expression levels were approximately 500 fmol/mg of proteins
(compared to 10–20 fmol/mg in
untransfected cells). Expression of p85ΔPI-3K
determined by western blot was increased 3.24 times as compared to wild-type
cells (data not shown, n=3).


### 3.2. Stimulation ofβ2-AR but not β1-AR Induces Activation of PI-3A/Akt Pathway

PI-3K activation by theβ1- or β2-AR was measured by in vitro phosphorylation of L-α-phosphatidylinositol. Transfected cells were stimulated with
isoproterenol 1 μM for 0, 5, or 15 minutes at 37°C. Stimulation of β2-AR- for 5 or 15 minutes induced a
significant augmentation in PI-3K activity compared to basal conditions
(nonstimulated; Figure 1). In contrast, β1-AR stimulation had no effect on
PI-3K activity. Thus, the stimulation of β2-AR but not β1-AR by isoproterenol induces
activation of PI-3K in our model. We suspect that the apparent high basal level
of PI-3K activation observed in our cell line may be due to the phosphotyrosine
antibody used to immunoprecipitate the activated PI-3K. Using this antibody, we
immunoprecipitate other PI-3K subtypes as well as phosphotyrosine proteins that
although not activated by β-AR may still contribute to the basal level of activation.Figure 1
PI-3K activity withβ-AR stimulation. HEK 293 cells were
transfected with β1-AR or β2-AR. Forty-eight hours after
transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. PI-3K activity was determined by the level of [P32]-PI
produced by the stimulation of β1-AR (□) or β2-AR (■) expressing cells. Top panel
is a representative autoradiograph of TLC separation (n=4−5). Data from
these experiments is quantitated in the bottom panel as described in Section
2
.
P*<.05 versus
0 minute.To confirm differences between stimulation of the twoβ-AR subtypes on PI-3K activity, we used a measure of Akt (a
downstream PI-3K effector) activation. Akt activation, as determined by the phosphorylation
status of Serine 473, was significantly increased after β2-AR-stimulation for 5 and 15 minutes
as compared to the basal state (Figure 2). Stimulation of β1-AR did not modify the
phosphorylation status of Akt as compared to control, confirming that β1-AR activation cannot stimulate
PI3-kinase/Akt pathway activation. Stimulation of
untransfected HEK293 with isoproterenol did not result in any significant
activation of Akt (data not shown).Figure 2
Akt activation followingβ-AR stimulation. HEK 293 cells were
transfected with β1-AR or β2-AR. Forty-eight hours after the
transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. Akt activation status was determined by immunoblot for the
phosphorylated form of Akt compared to the total amount of Akt after
stimulation of β1-AR (□) or β2-AR (■) expressing cells. Top panel is a
representative immunoblot of the experiments (n=4−5). Data from
these experiments is quantitated in the bottom panel as described in Section
2
.
P*<.05 versus
0 minute.


### 3.3. Stimulation of Eitherβ1-or β2-AR Induces PI-3K Recruitment to the Plasma Membrane

To determine whetherβ1-AR can recruit PI-3K to the plasma
membrane, immunolocalisation of PI-3K was performed using Western blotting with
anti-P110γ antibody. Transiently
transfected cells with β1- or β2-AR were stimulated by isoproterenol
1 μM for 0, 5, or 15
minutes at 37°C. Both β1- and β2-AR stimulation results in recruitment of PI-3K to the
particulate fraction (Figure 3). Compared with control (i.e., nonstimulated
cells), the presence of PI-3K was significantly increased (P<.05) in
membrane fractions by agonist stimulation of either β1-AR or β2-AR for 5 or 15 minutes. No
significant difference was detected between 5 and 15 minutes of stimulation.
Thus, either subtype of β-AR can recruit PI-3K to the membrane after agonist stimulation but
only the β2-AR results in PI-3K activation.Figure 3
PI-3K Partitioning of PI-3K to the membrane. Forty-eight hours after transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. Membranes were isolated, solubilized, and approximately 100 μg of
membrane proteins were separated on a 10% SDS-PAGE gel. Membrane-associated
PI-3K levels increased with cells expressing β1-AR (□) or β2-AR (■). Top panel is a representative immunoblot of
the experiments (n=3−4). Data from
these experiments is quantitated in the bottom panel as described in Section
2
.
P*<.05 versus 0 minute.


### 3.4. Recruitment of PI-3K to the Plasma Membrane Is Gβγ Subunit-Dependent

It has been shown that PI-3K and GRK2 form a cytosolic complex [2]. To determine whether
the mechanism of PI-3K recruitment to the plasma membrane was Gβγ subunit-dependent, β1- or β2-AR was transiently cotransfected with the
carboxyl-terminal portion (ct) of GRK2 (a sequestering agent for Gβγ) in HEK 293 cells. Cells cotransfected with β1- or β2-AR and ct-GRK2 were stimulated with
1 μM isoproterenol for 5
minutes. ct-GRK2 when transfected alone had no effect on PI-3K activity.
Stimulation of the β2-AR for 5 minutes with isoproterenol
in cells cotransfected with ct-GRK2 resulted in a significantly decreased (P<.001) PI-3K recruitment to the particulate fraction (Figure 4) as
compared to stimulation of the β2-AR expressed alone. Similar results
were obtained when cells expressing the β1-AR were stimulated for 5 minutes
with the agonist, that is, the presence of ct-GRK2 significantly decreased (P<.001) PI-3K recruitment to membranes (Figure 4). Thus, PI-3K
recruitment to the plasma membrane by either β1- or β2-AR stimulation is Gβγ subunit-dependent although only β2-AR-dependent recruitment results in
subsequent PI-3K activation.Figure 4
GRK2 carboxy-terminal domain reduced PI-3K recruitment to membrane fractions
in response toβ-AR stimulation. HEK 293 cells were
cotransfected with ct-GRK-2 and β-AR subtypes. Forty-eight hours after the transfection, cells were
stimulated with 1 μM isoproterenol for 5 minutes. PI3-kinase recruitment to
particulate fractions decreased with cells expressing either β1-AR (□) or β2-AR (■) in presence of cotransfected
ct-GRK2. Top panel is a representative immunoblot of the experiments (n=4–6). Data from
these experiments is quantitated in the bottom panel as described in Section
2.
P*<.05
versus 0 minute.


### 3.5. PI-3K/Akt Pathway Activation Following Stimulation of Chimericβ1×β2-AR and β2×β1-AR

To determine the importance of receptor-specific determinants in theβ-AR carboxy tail for
PI-3K activation, we constructed two chimeric receptors which consisted of the β1-AR with the proximal seventh
transmembrane domain and carboxy-terminal tail of the β2-AR (β1×β2-AR) as well as the reciprocal
receptor which consisted of β2-AR with the carboxy-terminal tail of
the β1-AR (β2×β1-AR). When the
β1×β2-AR was stimulated by 1 μM isoproterenol for 5 or 15 minutes, PI3-kinase activity was
significantly increased (Figure 5; P<.05). On the other hand,
agonist stimulation of the
β2×β1-AR for 5 or 15 minutes did not
result in increased PI3-kinase activity. These results suggest that the
carboxy-terminal tail of the β2-AR contains molecular determinants
which are required for PI3-kinase activation.Figure 5
PI-3K activity with chimericβ-AR stimulation. HEK 293 cells were
transfected with
β1×β2-AR or
β2×β1-AR. Forty-eight hours after
transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. PI-3K activity was determined by the level of [P32]-PI
produced by the stimulation of
β1×β2-AR (□) or
β2×β1-AR (■) expressing cells. Top panel is a
representative autoradiograph of the TLC separation (n=4−5). Data from
these experiments is quantitated in the bottom panel as described in Section
2.
P*<.05 versus 0 minute.To confirm results obtained for the PI-3K activation mediated by the
two chimericβ-ARs, we again
determined the activation status of Akt. Chimeric receptors were stimulated
with 1 μM isoproterenol for 0,
5, or 15 minutes at 37°C.  Akt
phosphorylation was significantly increased (P<.05) in HEK 293 cells
expressing
β1×β2-AR (Figure 6). Stimulation of
β2×β1-AR by isoproterenol had no effect on
Akt phosphorylation as compared to basal conditions. Thus chimeric
β1×β2-AR increased Akt phosphorylation and
confirmed that the
carboxy-terminal tail of the β2-AR contained molecular determinants
necessary and sufficient for PI-3K activation.Figure 6
Akt activity with chimericβ-AR stimulation. HEK 293 cells were
transfected with
β1×β2-AR or
β2×β1-AR. Forty-eight hours after the
transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. Akt activity was determined by measuring levels of phosphorylated Akt
compared with total Akt produced by the stimulation of
β1×β2-AR (□) or
β2×β1-AR (■) expressing cells. Upper panel is a
representative immunoblot of the experiments (n=6−7). Data from
these experiments is quantitated in the bottom panel as described in Section
2.
P*<.05 versus 0 minute.


### 3.6. Effect of Pertussis Toxin Treatment onβ-AR Stimulation of PI3-K Activity

It has been proposed that stimulation of PI-3K byβ2-AR is pertussis toxin sensitive [1, 12]. To determine the effect of pertussis
toxin on β-AR induced stimulation
of PI-3K, we incubated the cells with PTX for 18 hours followed by a
stimulation with 1 μM isoproterenol for 15 minutes. Our results indicate that,
in presence of PTX, no activation of PI-3K was observed with β2-AR stimulation (Figure 7(b)). In
presence of the chimeric receptors, PTX treatment abolished the activation of
PI-3K observed with the chimeric
β1×β2-AR (Figure 7(d)). Consistent with
our earlier data, neither the β1-AR (Figure 7(a)) nor the
β2×β1-AR (Figure 7(c)) was sensitive to PTX
pretreatment. These results indicate that stimulation of PI-3K by β2-AR is pertussis toxin sensitive.Pertussis toxin effects onβ-AR-mediated PI-3K activation. Twenty-six
hours after transfection, cells were incubated with 0.1 μg/mL pertussis toxin
for 18 hours. Cells were stimulated for 5 minutes with 1 μM isoproterenol prior
to processing for TLC as described in Figure 1 and Section
2. PI-3K
activity was determined by the level of [P32]-PI produced by the stimulation of β1-AR (a), β2-AR (b),
β2×β1-AR (c), and
β1×β2-AR expressing cells. (n=3−4; P*<.05 versus basal and PTX-Iso conditions).
(a)(b)(c)(d)


### 3.7. Involvement of PI-3K Activation onβ-AR Sequestration

Cells expressingβ1-AR were treated with isoproterenol
for 15 minutes. After stimulation, very little β1-AR was observed in the interior
compartments of treated cells and no significant difference was observed in
presence of either cotransfected p85ΔPI-3K
or PTX pretreatment (Figures
8(a) to 8(d)). In contrast, we observed a significant internalization of β2-AR after a similar period of agonist
stimulation as shown by clustered distribution of the receptor (Figure 8(f);
outline of the cell is poorly defined in this particular condition). β2-AR internalization was inhibited in
the presence of either cotransfected p85ΔPI-3K or PTX pretreatment (Figures
8(g) and 8(h)).
These results confimed that functional PI-3K is important for the sequestration
of β2-AR and attests to the effectiveness of our p85ΔPI-3K cotransfection or PTX pretreatment.p85ΔPI-3K
reduced agonist-inducedβ2-AR sequestration. HEK 293 cells were
transfected with p85ΔPI-3K
and the two β-AR subtypes. Cells
were stimulated with 1 μM isoproterenol for 15 minutes (b) and (f). Stimulation
of β2-AR-transfected cells induced a cluster distribution which is
characteristic of receptor internalization (f). This sequestration was
abolished in presence of either cotransfected p85ΔPI-3K (g) or PTX pretreatment (h). No
significant β1-AR sequestration was observed under the different conditions used
in these experiments (a)–(d). Representative
images of several experiments (n=8–15) are shown.


## 3.1. Expression ofβ-AR Subtypes and P85ΔPI-3K in HEK 293 Cells

HEK 293 cells were transiently transfected with cDNAs encoding forβ1-AR Wt, β2-AR Wt,
β1×β2-AR, or
β2×β1-AR and in some case, p85ΔPI-3K.
Forty-eight hours after transfection β-AR expression levels were approximately 500 fmol/mg of proteins
(compared to 10–20 fmol/mg in
untransfected cells). Expression of p85ΔPI-3K
determined by western blot was increased 3.24 times as compared to wild-type
cells (data not shown, n=3).


## 3.2. Stimulation ofβ2-AR but not β1-AR Induces Activation of PI-3A/Akt Pathway

PI-3K activation by theβ1- or β2-AR was measured by in vitro phosphorylation of L-α-phosphatidylinositol. Transfected cells were stimulated with
isoproterenol 1 μM for 0, 5, or 15 minutes at 37°C. Stimulation of β2-AR- for 5 or 15 minutes induced a
significant augmentation in PI-3K activity compared to basal conditions
(nonstimulated; Figure 1). In contrast, β1-AR stimulation had no effect on
PI-3K activity. Thus, the stimulation of β2-AR but not β1-AR by isoproterenol induces
activation of PI-3K in our model. We suspect that the apparent high basal level
of PI-3K activation observed in our cell line may be due to the phosphotyrosine
antibody used to immunoprecipitate the activated PI-3K. Using this antibody, we
immunoprecipitate other PI-3K subtypes as well as phosphotyrosine proteins that
although not activated by β-AR may still contribute to the basal level of activation.Figure 1
PI-3K activity withβ-AR stimulation. HEK 293 cells were
transfected with β1-AR or β2-AR. Forty-eight hours after
transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. PI-3K activity was determined by the level of [P32]-PI
produced by the stimulation of β1-AR (□) or β2-AR (■) expressing cells. Top panel
is a representative autoradiograph of TLC separation (n=4−5). Data from
these experiments is quantitated in the bottom panel as described in Section
2
.
P*<.05 versus
0 minute.To confirm differences between stimulation of the twoβ-AR subtypes on PI-3K activity, we used a measure of Akt (a
downstream PI-3K effector) activation. Akt activation, as determined by the phosphorylation
status of Serine 473, was significantly increased after β2-AR-stimulation for 5 and 15 minutes
as compared to the basal state (Figure 2). Stimulation of β1-AR did not modify the
phosphorylation status of Akt as compared to control, confirming that β1-AR activation cannot stimulate
PI3-kinase/Akt pathway activation. Stimulation of
untransfected HEK293 with isoproterenol did not result in any significant
activation of Akt (data not shown).Figure 2
Akt activation followingβ-AR stimulation. HEK 293 cells were
transfected with β1-AR or β2-AR. Forty-eight hours after the
transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. Akt activation status was determined by immunoblot for the
phosphorylated form of Akt compared to the total amount of Akt after
stimulation of β1-AR (□) or β2-AR (■) expressing cells. Top panel is a
representative immunoblot of the experiments (n=4−5). Data from
these experiments is quantitated in the bottom panel as described in Section
2
.
P*<.05 versus
0 minute.


## 3.3. Stimulation of Eitherβ1-or β2-AR Induces PI-3K Recruitment to the Plasma Membrane

To determine whetherβ1-AR can recruit PI-3K to the plasma
membrane, immunolocalisation of PI-3K was performed using Western blotting with
anti-P110γ antibody. Transiently
transfected cells with β1- or β2-AR were stimulated by isoproterenol
1 μM for 0, 5, or 15
minutes at 37°C. Both β1- and β2-AR stimulation results in recruitment of PI-3K to the
particulate fraction (Figure 3). Compared with control (i.e., nonstimulated
cells), the presence of PI-3K was significantly increased (P<.05) in
membrane fractions by agonist stimulation of either β1-AR or β2-AR for 5 or 15 minutes. No
significant difference was detected between 5 and 15 minutes of stimulation.
Thus, either subtype of β-AR can recruit PI-3K to the membrane after agonist stimulation but
only the β2-AR results in PI-3K activation.Figure 3
PI-3K Partitioning of PI-3K to the membrane. Forty-eight hours after transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. Membranes were isolated, solubilized, and approximately 100 μg of
membrane proteins were separated on a 10% SDS-PAGE gel. Membrane-associated
PI-3K levels increased with cells expressing β1-AR (□) or β2-AR (■). Top panel is a representative immunoblot of
the experiments (n=3−4). Data from
these experiments is quantitated in the bottom panel as described in Section
2
.
P*<.05 versus 0 minute.


## 3.4. Recruitment of PI-3K to the Plasma Membrane Is Gβγ Subunit-Dependent

It has been shown that PI-3K and GRK2 form a cytosolic complex [2]. To determine whether
the mechanism of PI-3K recruitment to the plasma membrane was Gβγ subunit-dependent, β1- or β2-AR was transiently cotransfected with the
carboxyl-terminal portion (ct) of GRK2 (a sequestering agent for Gβγ) in HEK 293 cells. Cells cotransfected with β1- or β2-AR and ct-GRK2 were stimulated with
1 μM isoproterenol for 5
minutes. ct-GRK2 when transfected alone had no effect on PI-3K activity.
Stimulation of the β2-AR for 5 minutes with isoproterenol
in cells cotransfected with ct-GRK2 resulted in a significantly decreased (P<.001) PI-3K recruitment to the particulate fraction (Figure 4) as
compared to stimulation of the β2-AR expressed alone. Similar results
were obtained when cells expressing the β1-AR were stimulated for 5 minutes
with the agonist, that is, the presence of ct-GRK2 significantly decreased (P<.001) PI-3K recruitment to membranes (Figure 4). Thus, PI-3K
recruitment to the plasma membrane by either β1- or β2-AR stimulation is Gβγ subunit-dependent although only β2-AR-dependent recruitment results in
subsequent PI-3K activation.Figure 4
GRK2 carboxy-terminal domain reduced PI-3K recruitment to membrane fractions
in response toβ-AR stimulation. HEK 293 cells were
cotransfected with ct-GRK-2 and β-AR subtypes. Forty-eight hours after the transfection, cells were
stimulated with 1 μM isoproterenol for 5 minutes. PI3-kinase recruitment to
particulate fractions decreased with cells expressing either β1-AR (□) or β2-AR (■) in presence of cotransfected
ct-GRK2. Top panel is a representative immunoblot of the experiments (n=4–6). Data from
these experiments is quantitated in the bottom panel as described in Section
2.
P*<.05
versus 0 minute.


## 3.5. PI-3K/Akt Pathway Activation Following Stimulation of Chimericβ1×β2-AR and β2×β1-AR

To determine the importance of receptor-specific determinants in theβ-AR carboxy tail for
PI-3K activation, we constructed two chimeric receptors which consisted of the β1-AR with the proximal seventh
transmembrane domain and carboxy-terminal tail of the β2-AR (β1×β2-AR) as well as the reciprocal
receptor which consisted of β2-AR with the carboxy-terminal tail of
the β1-AR (β2×β1-AR). When the
β1×β2-AR was stimulated by 1 μM isoproterenol for 5 or 15 minutes, PI3-kinase activity was
significantly increased (Figure 5; P<.05). On the other hand,
agonist stimulation of the
β2×β1-AR for 5 or 15 minutes did not
result in increased PI3-kinase activity. These results suggest that the
carboxy-terminal tail of the β2-AR contains molecular determinants
which are required for PI3-kinase activation.Figure 5
PI-3K activity with chimericβ-AR stimulation. HEK 293 cells were
transfected with
β1×β2-AR or
β2×β1-AR. Forty-eight hours after
transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. PI-3K activity was determined by the level of [P32]-PI
produced by the stimulation of
β1×β2-AR (□) or
β2×β1-AR (■) expressing cells. Top panel is a
representative autoradiograph of the TLC separation (n=4−5). Data from
these experiments is quantitated in the bottom panel as described in Section
2.
P*<.05 versus 0 minute.To confirm results obtained for the PI-3K activation mediated by the
two chimericβ-ARs, we again
determined the activation status of Akt. Chimeric receptors were stimulated
with 1 μM isoproterenol for 0,
5, or 15 minutes at 37°C.  Akt
phosphorylation was significantly increased (P<.05) in HEK 293 cells
expressing
β1×β2-AR (Figure 6). Stimulation of
β2×β1-AR by isoproterenol had no effect on
Akt phosphorylation as compared to basal conditions. Thus chimeric
β1×β2-AR increased Akt phosphorylation and
confirmed that the
carboxy-terminal tail of the β2-AR contained molecular determinants
necessary and sufficient for PI-3K activation.Figure 6
Akt activity with chimericβ-AR stimulation. HEK 293 cells were
transfected with
β1×β2-AR or
β2×β1-AR. Forty-eight hours after the
transfection, cells were stimulated with 1 μM isoproterenol for the indicated
times. Akt activity was determined by measuring levels of phosphorylated Akt
compared with total Akt produced by the stimulation of
β1×β2-AR (□) or
β2×β1-AR (■) expressing cells. Upper panel is a
representative immunoblot of the experiments (n=6−7). Data from
these experiments is quantitated in the bottom panel as described in Section
2.
P*<.05 versus 0 minute.


## 3.6. Effect of Pertussis Toxin Treatment onβ-AR Stimulation of PI3-K Activity

It has been proposed that stimulation of PI-3K byβ2-AR is pertussis toxin sensitive [1, 12]. To determine the effect of pertussis
toxin on β-AR induced stimulation
of PI-3K, we incubated the cells with PTX for 18 hours followed by a
stimulation with 1 μM isoproterenol for 15 minutes. Our results indicate that,
in presence of PTX, no activation of PI-3K was observed with β2-AR stimulation (Figure 7(b)). In
presence of the chimeric receptors, PTX treatment abolished the activation of
PI-3K observed with the chimeric
β1×β2-AR (Figure 7(d)). Consistent with
our earlier data, neither the β1-AR (Figure 7(a)) nor the
β2×β1-AR (Figure 7(c)) was sensitive to PTX
pretreatment. These results indicate that stimulation of PI-3K by β2-AR is pertussis toxin sensitive.Pertussis toxin effects onβ-AR-mediated PI-3K activation. Twenty-six
hours after transfection, cells were incubated with 0.1 μg/mL pertussis toxin
for 18 hours. Cells were stimulated for 5 minutes with 1 μM isoproterenol prior
to processing for TLC as described in Figure 1 and Section
2. PI-3K
activity was determined by the level of [P32]-PI produced by the stimulation of β1-AR (a), β2-AR (b),
β2×β1-AR (c), and
β1×β2-AR expressing cells. (n=3−4; P*<.05 versus basal and PTX-Iso conditions).
(a)(b)(c)(d)


## 3.7. Involvement of PI-3K Activation onβ-AR Sequestration

Cells expressingβ1-AR were treated with isoproterenol
for 15 minutes. After stimulation, very little β1-AR was observed in the interior
compartments of treated cells and no significant difference was observed in
presence of either cotransfected p85ΔPI-3K
or PTX pretreatment (Figures
8(a) to 8(d)). In contrast, we observed a significant internalization of β2-AR after a similar period of agonist
stimulation as shown by clustered distribution of the receptor (Figure 8(f);
outline of the cell is poorly defined in this particular condition). β2-AR internalization was inhibited in
the presence of either cotransfected p85ΔPI-3K or PTX pretreatment (Figures
8(g) and 8(h)).
These results confimed that functional PI-3K is important for the sequestration
of β2-AR and attests to the effectiveness of our p85ΔPI-3K cotransfection or PTX pretreatment.p85ΔPI-3K
reduced agonist-inducedβ2-AR sequestration. HEK 293 cells were
transfected with p85ΔPI-3K
and the two β-AR subtypes. Cells
were stimulated with 1 μM isoproterenol for 15 minutes (b) and (f). Stimulation
of β2-AR-transfected cells induced a cluster distribution which is
characteristic of receptor internalization (f). This sequestration was
abolished in presence of either cotransfected p85ΔPI-3K (g) or PTX pretreatment (h). No
significant β1-AR sequestration was observed under the different conditions used
in these experiments (a)–(d). Representative
images of several experiments (n=8–15) are shown.


## 4. Discussion

The results of the current study demonstrate that stimulation ofβ2-AR, but not β1-AR, induces PI-3K activation by a
pertussis toxin-sensitive mechanism. This subtype-dependent activation has been
reinforced by the fact that Akt, a downstream PI-3K effector, was also
selectively activated by β2-AR stimulation. However, both
receptor subtypes can recruit the PI-3K to the plasma membrane via a Gβγ subunit-dependent mechanism. Naga Prasad et al. [2] had previously
demonstrated that PI-3K and GRK2 formed a cytosolic complex and the recruitment
of the enzyme to the plasma membrane was facilitated by Gβγ subunits in an agonist-dependent
manner. Our results confirmed that PI-3K is recruited to the plasma membrane
via a Gβγ subunit-dependent mechanism. GRK-2 is important for PI-3K
recruitment to the plasma membrane, thus we thought this might suggest that the
carboxyl-terminal domain of the receptor is important for the PI-3K activation
because the carboxyl-terminal domain of the β-AR contains sites phosphorylated by GRK2. Furthermore, Shiina et
al. [13] had demonstrated that
the carboxyl-terminal domain and the third cytosolic loop are the regions
mostly responsible for the difference in internalization behavior between both β-AR subtypes.To determine the potential contribution of the carboxyl-terminal
domains of theβ1- and β2-AR in the PI-3K activation, chimeric
β1- and β2-AR in which the seventh
transmembrane domain and the carboxyl-terminal domain have been exchanged were
constructed. We observed that the chimeric
β1×β2-AR could activate PI-3K in
contrast to the wild type β1-AR. Reciprocally, β2-AR lost its ability to activate PI-3K when its
carboxy-terminal domain was exchanged for that of the β1-AR. This result demonstrated that
the β2-AR carboxyl-terminal domain contains important molecular
determinants for PI-3K activation.The present study confirms PI-3K activation byβ2-AR stimulation as observed in other
studies [2, 12]. However the kinetics of
PI-3K activation previously reported for β2-AR stimulation was different to that
observed in the present study. PI-3K was rapidly activated following β2-AR stimulation and returned to basal
levels after 10 minutes. In our study, we observed a significant PI-3K
activation with a 5-minute β2-AR stimulation that was maintained
after 15 minutes. The difference in activation patterns may be due to the
agonist concentration used. In the previous study [2], 10 μM isoproterenol was used which is 10
times higher than the concentration used here. Higher
concentration of agonist may induce more rapid PI-3K recruitment to the
membrane and thus activation of the enzyme but may also more rapidly stimulate
mechanisms which terminate these signals as well.Pretreatment with pertussis toxin results in a loss of PI-3K
activation byβ2-AR stimulation confirming results obtained in other studies [1, 12]. The new information afforded by the present study is that the
carboxy-terminal portion of the receptor is important for the interaction
between the receptor, Gi heterotrimers (as stimulation depends on both the Gα and Gβγ subunits), and PI-3K.
Indeed, the chimeric receptor
β1×β2-AR can activate the
PI-3K by a pertussis toxin sensitive mechanism whereas the chimeric receptor
β2×β1-AR is unable to activate
PI-3K. However, other regions of the receptor may also be important for full
activation or regulation of the response by the desensitization machinery.Our results demonstrate that isoproterenol-stimulatedβ1-AR (for up to 15 minutes) cannot
activate PI-3K. These results contrast with those reported in a
previous study in which activation  of PI-3K was observed after stimulation of β1-AR transfected into HEK 293 cells [2]. Several possibilities
can explain the discrepancy between both studies. First, 1 μM isoproterenol
although sufficient to induce submaximal adenylyl cyclase activity [8] cannot induce PI-3K
activation. Secondly, in our study we use transiently transfected cells that
express approximately 500 fmol receptor/mg of membrane proteins, which is
closer to physiological levels than seen with the study of Naga Prasad et al. [2]. Also, it is clear that
the interplay between the signalling and internalization machinery is different
for the two receptors even though they can interact with many of the same
proteins. This study provides another such example that both β1-AR and β2-AR lead to PI-3K and GRK2
recruitment; both
the signalling and desensitization outcomes for these events are markedly
different. Some studies have also shown that β1-AR, in contrast to the β2-AR, cannot activate
Gi
[14] while others suggest that it can [15]. It is possible that other cell- and tissue-specific factors might
regulate these events as well.In this study, we observed thatβ2-AR internalization is inhibited in
the presence of a dominant negative PI-3K, p85ΔPI-3K, results that are similar to previous
studies [2, 16]. We also observed that β2-AR sequestration can be abolished
with pertussis toxin pretreatment. In contrast to β2-AR, we observed that the β1-AR sequestration is minimal with 15
minutes of stimulation. This is similar
to the 10% sequestration observed in β1-AR expressing cells obtained by
Suzuki et al. [17]. Since phosphorylated
lipids generated by PI-3K are critical for the receptor internalization
dynamics [18, 19], it is plausible that the weak sequestration observed for the β1-AR might be due to the weak PI-3K
activation induced by the stimulation of this receptor subtype. Results
obtained with the pertussis pretreatment and p85ΔPI-3K also suggest that activated PI-3K is
important for receptor sequestration.In conclusion, we observed that PI-3K activation isβ-AR subtype specific and the difference is due to molecular determinants
present in the carboxy-terminal tail of the receptors. PI-3K activation is
pertussis toxin sensitive and is necessary for the sequestration of the β2-AR.



---

*Source: 959168-2009-02-05.xml*