c-Src Tyrosine Kinase Binds the
2-Adrenergic Receptor via
Phospho-Tyr-350, Phosphorylates G-protein-linked Receptor Kinase
2, and Mediates Agonist-induced Receptor Desensitization*
Gao-feng
Fan
,
Elena
Shumay
,
Craig C.
Malbon
§, and
Hsien-yu
Wang¶
From the
Department of Molecular Pharmacology,
Diabetes and Metabolic Diseases Research Program, University Medical
Center, State University of New York, Stony Brook,
New York 11794-8651 and the ¶ Department of Physiology and
Biophysics, Diabetes and Metabolic Diseases Research Program,
University Medical Center, State University of New York, Stony Brook,
New York 11794-8661
Received for publication, December 21, 2000, and in revised form, January 16, 2001
 |
ABSTRACT |
The nonreceptor tyrosine kinase Src has
been implicated in the switching of signaling of
2-adrenergic
receptors from adenylylcyclase coupling to the mitogen-activated
protein kinase pathway. In the current work, we demonstrate that Src
plays an active role in the agonist-induced desensitization of
2-adrenergic receptors. Both the expression of dominant-negative Src
and treatment with the
4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine
phosphorylation of the
2-adrenergic receptor and
recruitment and activation of Src. Because phosphorylation of
the Tyr-350 residue of the
2-adrenergic receptor creates a
conditional, canonical SH2-binding site on the receptor, we examined
the effect of the Y350F mutation on Src phosphorylation, Src
recruitment, and desensitization. Mutant
2-adrenergic receptors with
a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced
desensitization, Src recruitment, or Src activation. Downstream of
binding to the receptor, Src phosphorylates and activates
G-protein-linked receptor kinase 2 (GRK2), a response obligate for
agonist-induced desensitization. Constitutively active Src increases
GRK phosphorylation, whereas either expression of dominant-negative Src
or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation
of GRK and desensitization. Thus, in addition to its role in signal
switching to the mitogen-activated protein kinase pathway, Src
recruitment to the
2-adrenergic receptor and activation are obligate
for normal agonist-induced desensitization.
 |
INTRODUCTION |
The nonreceptor tyrosine kinase family of Src functions in a
wide spectrum of cell signaling (1). The recruitment and activation of
Src kinases is obligate for the mediation of Ras activation by
G-protein-linked receptors
(GPLRs)1 (2, 3). GPLRs
display four well known responses to the stimulation by agonists:
namely, activation, desensitization, internalization, and eventual
resensitization (4, 5). The activation of adenylylcyclase in response
to agonist stimulation of
2-adrenergic receptors results in
elevation of intracellular cyclic AMP levels, activation of protein
kinase A, and later phosphorylation of the receptor by protein kinase A
and G-protein-linked receptor kinases (GRKs) (6). Phosphorylation leads
to desensitization and receptor sequestration, dependent upon the
binding of
-arrestin to the phosphorylated receptor as well as to
clathrin (7, 8). Internalization occurs via clathrin-mediated processes
(9), and resensitization/de-phosphorylation follows by the action of the protein phosphatase 2A (10) or 2B (11). These activities are
organized by the protein kinase A-anchoring protein AKAP250 or
gravin (12). Gravin acts as a scaffold for interactions among protein
kinase A, protein kinase C, PP2B, and the
2-adrenergic receptor
(13).
Src has been shown to participate in the formation of
2-adrenergic
receptor-Src complexes, in association with
-arrestin, to switch the
signaling from adenylylcyclase to activation of Ras and its downstream
effector, the mitogen-activated protein kinase (3). Src has been
reported to phosphorylate and activate GRKs (14), observations confined
to systems in which the elements are overexpressed at high levels or
examined in reconstituted systems, leaving unanswered the question
whether or not Src interacts directly with the
2-adrenergic receptor
and functions in desensitization. The proposed model for Src action
defines a temporal sequence in which GRK2 phosphorylates the
2-adrenergic receptor, and an Src-arrestin complex then targets to
the receptor, facilitating internalization and signaling to Ras (3). In
the current work we reveal that Src targets a conditional SH2-binding
site of the
2-adrenergic receptor, is recruited to the
phospho-Tyr-350 receptor, is activated, and then phosphorylates GRK2.
This temporal sequence is shown to be obligate for agonist-induced desensitization.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Human epidermoid carcinoma cells (A431) and
Chinese hamster ovary (CHO) cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
(HyClone, Logan, UT), penicillin (60 µg/ml), and streptomycin (100 µg/ml) and grown in a humidified atmosphere of 5% CO2
and 95% air at 37 °C. Cells were stably transfected with mammalian
expression vectors harboring the constitutively active form (Y527F) or
the dominant-inhibitory form (K295R/Y527F) of c-Src, the hamster
wild-type
2-adrenergic receptor, the hamster Y350F
mutant form of the
2-adrenergic receptor, or a green
fluorescent protein-tagged
2-adrenergic receptor fusion protein (8), as previously described (13).
Suppression via Antisense Oligodeoxynucleotides--
Antisense,
sense, and control missense oligodeoxynucleotides with the same base
composition, but in scrambled order, were synthesized and purified to
cell culture-grade (Operon, Alameda, CA), as described (15). Before
addition to cells, oligodeoxynucleotides were mixed at a ratio of 1:3
(w/w) with
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (Roche Molecular Biochemicals), a cationic
diacylglycerol in liposomal form that serves as a delivery vehicle.
A431 cells were treated with oligodeoxynucleotides (ODNs, 5 µg/ml)
for at least 48-72 h prior to the analysis of the expression of the
target molecule. The sequences employed for the antisense and sense
ODNs for Src were 24 nucleotides in length, terminating with the
initiator codon for Src. Missense was created using a random sequence
with the same base composition as the antisense ODN for Src.
Immunoprecipitation and Immunoblotting by Antibodies against
2-Adrenergic Receptor, c-Src, or GRK2--
For most studies, A431
cells were either untreated or stimulated with 10 µM
isoproterenol for periods up to 60 min. Cells were harvested and lysed
in a lysis buffer (1% Triton X-100, 0.5% Nonidet-P40, 10 mM dithiothreitol, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 100 µg/ml bacitracin, 100 µg/ml benzamidine, 2 mM sodium orthovanadate, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 40 mM sodium pyrophosphate, 50 mM KH2PO4, 10 mM sodium molybdate, and 20 mM Tris-HCl, pH
7.4) at 4 °C for 20 min. After centrifugation of the cell debris at
10,000 × g for 15 min, the lysates were precleared with protein A/G-agarose for 90 min at 4 °C. The mixture was then subjected to immunoprecipitation for 2 h with antibodies specific for the
-adrenergic receptor (CM04), c-Src, or GRK2. The primary antibodies were linked covalently to a protein A/G-agarose matrix. Immune complexes were washed three times with RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM
EDTA, 10 mM dithiothreitol, 1% Triton X-100, pH 8.0) and
separated on 4 to 12% linear gradient SDS-acrylamide Laemmli gels.
Immunoblotting and detection of Src, of GRK2, and of the
2-adrenergic receptor by immunostaining were performed as previously
described (13). The anti-phospho-Src (Y416) antibodies were purchased
from Upstate Biotechnology (Lake Placid, NY).
Epifluorescence Imaging--
Microscopy of live cells stably
transfected to express a green fluorescent protein-tagged
-adrenergic receptor fusion protein was performed on the Eclipse
TE300 (Nikon) inverted microscope equipped with a × 40 objective
and a set of filters (13). Images were acquired using MicroMAX Imaging
System (Princeton Instruments Inc.) and WinView32 software. Fluorescent
dyes were imaged sequentially in frame-interface mode to eliminate
spectral overlapping between the channels (12).
Radioligand Binding Assay--
The number of
2-adrenergic receptors was determined by radioligand
binding. Approximately 106 stably transfected CHO cells
were incubated with 30 pM
[125I]iodocyanopindolol (PerkinElmer Life
Sciences) in the absence or presence of 10 µM
propranolol (for defining the amount of nonspecific binding) at
23 °C for 90 min. The incubation buffer contained 50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, and 150 mM NaCl (16). The cells were collected on GF/C
binder-free glass giber filter membranes at reduced pressure and washed
rapidly. The radioligand bound to the washed cell mass retained by the
filter was quantified by use of a
-counter. Nonspecific binding was
less than 5% of radioligand binding. Each experimental point was
performed in triplicate.
Cyclic AMP Assay--
One day prior to the analysis, A431 cells
and CHO clones stably transfected with
2-adrenergic receptor and
2-adrenergic receptor-GFP-pcDNA3 were seeded in 96-well
plates at the density of 20,000 cells/well. Cells were washed and
challenged with or without 10 µM isoproterenol in 50 µl
of HEM buffer (20 mM Hepes, pH 7.4, 135 mM
NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM NaHCO3, containing Ro-20-1724 (0.1 mM; Calbiochem)) and adenosine deaminase (0.5 unit/ml) for
the times indicated in the figure legends. The first challenge with
agonist was for 0-60 min. For some studies, the cells first challenged
with agonist were washed free of agonist (three times) at the end of
the first incubation and then challenged again for 5 min with agonist.
The incubations were stopped by addition of 100 µl of ice-cold
ethanol. The agonist-stimulated cyclic AMP production was determined as
described elsewhere (15).
 |
RESULTS |
The nonreceptor tyrosine kinase Src has been shown to play a
prominent role in linking G-protein-linked receptors to Ras and downstream signaling to the mitogen-activated protein kinase network (2, 3). For the
2-adrenergic receptor, Src has been shown to act as
a switch from receptor regulation of Gs and adenylylcyclase to
activation of Ras following the normal progression of receptor activation, desensitization, and sequestration. The role of Src in
desensitization is far less clear. Src has been proposed to target to
GPLRs via its interaction with
-arrestin, suggesting in a temporal
sense that Src would be functioning in post-receptor desensitization.
To test the hypothesis that Src may be participating in events prior to
receptor sequestration and Ras activation, we focused on an analysis of
2-adrenergic receptor-mediated activation and desensitization, using
the accumulation of intracellular cyclic AMP as the readout.
Human epidermoid A431 carcinoma cells are a widely used model of
2-adrenergic receptor action (18, 19). Challenging A431 cells with
the
2-adrenergic agonist isoproterenol (10 µM) leads to peak accumulation of cyclic AMP at 5 min, which declines to approximately half-maximal levels within 30-60 min, reflecting ongoing
agonist-induced desensitization (Fig. 1).
The cyclic AMP response of A431 cells to agonist, following a 30 or 60 min first challenge with isoproterenol, a washout of agonist,
and a second challenge with isoproterenol for 5 min, is rectified to
the level of naïve cells that have been challenged with agonist
for 5 min. To explore what role, if any, Src may have in
agonist-induced desensitization of
2-adrenergic receptors, we
performed the same experiments in A431 cells pretreated for 30 min with
the Src family tyrosine kinase inhibitor PP2 (Fig. 1). In the presence
of the Src inhibitor, agonist-induced desensitization was abolished, and intracellular cyclic AMP levels continued to increase over the
period of 60 min of challenge with agonist. These data suggest that Src
activity is important not only for subsequent downstream signaling to
Ras but also for agonist-induced desensitization itself. To test the
observations by an independent approach that targets Src only, A431
cells were treated for 4 days with ODNs antisense to Src to suppress
Src levels. Treatment with ODNs antisense to Src also abolished
agonist-induced desensitization (Fig. 1). Suppression of Src levels by
antisense ODNs yields increased cyclic AMP accumulation in A431 cells
challenged with isoproterenol for up to 60 min. Treatment of cells with
ODNs sense or missense to Src, in contrast, was without effect (data
not shown).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 1.
Inhibition of Src kinase activity or
suppression of Src expression by antisense oligodeoxynucleotides leads
to loss of desensitization. A431 cells were untreated, pretreated
for 30 min with the Src kinase inhibitor PP2 (50 nM), or
pretreated with oligodeoxynucleotides antisense to Src
(+AS-Src) for 4 days and then challenged with 10 µM isoproterenol (ISO) for 0, 5, 30, or 60 min, and intracellular cyclic AMP accumulation was determined. Some
cells were first treated with isoproterenol for 30 or 60 min and then
washed free of agonist and rechallenged with isoproterenol for 5 min.
Assays of cyclic AMP accumulation were performed in triplicate. The
data shown are mean values ± S.E. from three to five separate
experiments performed on separate occasions.
|
|
To further test this newly discovered role for Src in agonist-induced
desensitization of
2-adrenergic receptors, we tested the effects of
stable transfection of A431 cells with an expression vector harboring
either the constitutively active form (Y527F) or the dominant-negative
form (K295R/Y527F) of c-Src (Fig. 2). Stable expression of the dominant-inhibitory form of Src abolishes agonist-induced desensitization of
2-adrenergic receptor with respect to cyclic AMP accumulation. The accumulation of intracellular cyclic AMP increased throughout the time course of
-adrenergic stimulation, for up to 60 min, in the clones expressing the
dominant-negative form of Src. This observation confirms the results
obtained with cells treated with the Src family inhibitor PP2 and with
ODNs antisense to suppress Src levels (Fig. 1). Thus, inhibition of Src
activity, suppression of Src expression, and expression of a
dominant-inhibitory form of Src all lead to a loss of agonist-induced desensitization of
2-adrenergic receptor-mediated activation of
adenylylcyclase. The cyclic AMP responses to isoproterenol in the cells
with Src suppression or inhibition are the greatest cyclic AMP
responses that we have observed with these cells to date. Expression of
the constitutively active form (Y527F) of Src, in contrast, had no
demonstrable effect on agonist-induced desensitization (Fig. 2).
Although we expected that the expression of the constitutively active
Src would further amplify the desensitization response, no such
alteration was noted. This observation suggests that endogenous Src may
be fully activated with respect to desensitization only when the
2-adrenergic receptors first have been activated. Under these
circumstances, the introduction of constitutively active Src without
proper targeting to
2-adrenergic receptor, as in the
basal state, may have little effect on desensitization.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2.
Expression of a dominant-negative, but not
the constitutively active, mutant form of Src abolishes agonist-induced
desensitization of the cyclic AMP response to stimulation by a
-adrenergic agonist. A431 cells (control) and
clones stably transfected to express either the Y527F constitutively
active mutant of Src (CA-Src (Y527F)) or the
dominant-negative mutant of Src (DN-Src (K295R/Y527F)) were
used to study cyclic AMP accumulation in response to isoproterenol over
time. The cells were challenged with 10 µM isoproterenol
(ISO) for 0, 5, 30, or 60 min, and intracellular cyclic AMP
accumulation was determined. Some cells were first treated with
isoproterenol for 30 or 60 min and then washed free of agonist and
rechallenged with isoproterenol for 5 min. Assays of cyclic AMP
accumulation were performed in triplicate. The data shown are mean
values ± S.E. from three to five separate experiments performed
on separate occasions.
|
|
We tested the hypothesis that agonist treatment of A431 cells activated
endogenous Src activity in A431 cells (Fig.
3). Cells were challenged with
isoproterenol (10 µM) for periods up to 60 min. At the
conclusion of the activation, whole-cell extracts were prepared, and
the extracts were subjected to an immune precipitation "pull down"
assay using antibodies against the
2-adrenergic receptor (antibody
CM04). The immune precipitations were subjected to SDS-PAGE analysis,
and the resultant immunoblots were stained with antibodies to c-Src.
Some Src was found in association with the
2-adrenergic receptor
under basal, unstimulated cell culture conditions, probably reflecting
the observation that growth factors such as insulin catalyze the
phosphorylation of the
2-adrenergic receptor on residue Tyr-350,
which then constitutes a canonical SH2-binding site (20-22). In
response to challenge with isoproterenol, the level of Src association
increased by more than 3.0-fold within 30 min and was maintained until
at least 60 min. To determine whether the Src associated with the
2-adrenergic receptor was activated, we performed immunoblotting of
the Src associated with the
2-adrenergic receptor using an antibody
that detects only Src phosphorylated at the Tyr-416 position
(anti-phospho-Src (Tyr-416)). The results of the blotting for activated
Src revealed that the Src associated with the
2-adrenergic receptor
following the challenge with isoproterenol was phosphorylated at
Tyr-416 and therefore activated. Thus, not only were Src expression and
activity necessary for agonist-induced desensitization of
2-adrenergic receptors, but association and activation by this
G-protein-linked receptor were also increased sharply in response to
agonist.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 3.
Stimulation of cells with isoproterenol
provokes recruitment of Src to the -adrenergic
receptor and tyrosine phosphorylation (activation) of Src. A431
cells were challenged with 10 µM isoproterenol
(ISO) for 0, 5, 30, or 60 min and then used to harvest
whole-cell extracts. The extracts were subjected to immune
precipitation (IP) pull down assays with antibodies
against the -adrenergic receptor (CM04),and the immune complexes
were subjected to SDS-PAGE. The resultant immunoblots
(IB) were stained with antibodies specific for the
phospho-Tyr-417 activated version of Src, an antibody against
Src itself, or a second antibody against the -adrenergic receptor
(CM02) to establish equivalent loading of each lane. Shown is a blot
representative of three such experiments with essentially identical
results. 2AR, 2-adrenergic receptor.
|
|
The increase in the amount of Src associated with the
2-adrenergic
receptor, in tandem with the increase in Src activation, prompted the
question what the basis was for this increased association and activity
of Src with the receptor. The simplest hypothesis to explain the
agonist-induced increase in Src association and activation was that the
2-adrenergic receptor might display increased phosphorylation of
Tyr-350, a conditional canonical SH2-binding site. To test this
possibility, we challenged A431 cells with isoproterenol (10 µM) for periods up to 60 min and performed immune precipitation pull-downs of
2-adrenergic receptors from the
whole-cell extracts (Fig. 4). The immune
precipitates were subjected to SDS-PAGE, and the resultant immunoblots
were stained with anti-phosphotyrosine antibody (PY99). These data show
conclusively and unexpectedly that the phosphotyrosine content of the
2-adrenergic receptor increased upon challenge of the cells with
isoproterenol. Within 5 min of challenge with isoproterenol, the
content of phosphotyrosine more than doubled, increasing to 2.5- and
then 3.0-fold by 30 and 60 min, respectively. We confirmed these data
using metabolic labeling of an independent hamster smooth muscle cell
line (DDT1-MF2 vas deferens) with
[32P]Pi overnight and stimulation with 10 µM isoproterenol for 5 or 15 min (data not shown).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Stimulation of cells with
-adrenergic agonist leads to increased
phosphotyrosine content of the 2-adrenergic
receptor. A431 cells were challenged with isoproterenol
(ISO) (10 µM) for 0, 5, 30, and 60 min and
then used to prepare whole-cell extracts. The cell extracts were
subjected to immune precipitation (IP) pull-down
assays with antibody (CM04) against the 2-adrenergic receptor,
separation of the immune precipitate on SDS-PAGE, and immunoblotting.
The immunoblots (IB) were stained with
anti-phosphotyrosine antibody (PY99) as well as with another antibody
(CM02) against the 2-adrenergic receptor to establish equivalent
loading for each lane. Shown is a blot representative of three such
experiments with essentially identical results. 2AR,
2-adrenergic receptor.
|
|
Earlier studies demonstrated both in vivo and in
vitro that phosphorylation of the
2-adrenergic receptor at
Tyr-350 generates an SH2-binding site that Grb2,
1-phosphatidylinositol 3-kinase, or dynamin can bind via SH2 domains
(23, 24). We tested whether Tyr-to-Phe substitution of the Tyr-350
residue of the
2-adrenergic receptor would alter association of the
receptor with Src (Fig. 5). These studies
were performed in CHO-K cells that express very low levels of
endogenous
2-adrenergic receptors. Stably transfected CHO clones
were created that express either the wild-type
2-adrenergic receptor
or the Y350F mutant form of the receptor. Clones were selected that
express comparable levels of expression of
2-adrenergic receptors,
0.25 pmol of receptor/mg of membrane protein, as measured using
the high affinity, radiolabeled
2-adrenergic antagonist ligand
iodocyanopindolol. CHO clones were challenged with isoproterenol (10 µM) for periods up to 60 min and then harvested for
whole-cell extracts. The extracts were subjected to immune
precipitation pull-down assays with antibodies to the
2-adrenergic
receptor (CM04). The immune precipitates were subjected to SDS-PAGE,
and the resultant blots were stained with antibodies against Src as well as with a second antibody (CM02) to ensure equivalent loading of
samples. As noted above with pull-down assays from whole-cell extracts
prepared from A431 cells (Fig. 3), the amount of Src associated with
the wild-type
2-adrenergic receptor increased in response to
challenge of the cells with isoproterenol (Fig. 5). In sharp
contrast to the situation observed for pull-down assays in cells
expressing the wild-type receptor, the pull-down assays performed with
whole-cell extracts of CHO clones expressing the Y350F mutant form of
the
2-adrenergic receptor do not reveal an increase in Src
association in response to agonist challenge. In fact, contrary to the
increase shown in Src-
2-adrenergic receptor association for the
wild-type receptor, Src association with the Y350F mutant form of
2-adrenergic receptor actually declines in response to challenge
with isoproterenol. Thus, the likely basis for the increase in
Src-
2-adrenergic receptor association and Src activation in response
to agonist challenge is Src association with the phosphorylated Tyr-350
residue of the receptor that creates an SH2-binding site.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5.
The Y350F mutation of the
2-adrenergic receptor
( 2AR) abolishes recruitment of Src to a
receptor-Src complex in response to stimulation by
-adrenergic agonist. CHO clones expressing
either wild-type (WT) 2-adrenergic receptors or the Y350F
mutant form of the 2-adrenergic receptor were challenged with
isoproterenol (ISO) (10 µM) for 0, 5, 30, and
60 min and then used to prepare whole-cell extracts. The clones
expressed equivalent levels of 2-adrenergic receptors, as measured
by radioligand binding studies with iodocyanopindolol. The cell
extracts were subjected to immune precipitation (IP)
pull-down assays with antibody (CM04) against the 2-adrenergic
receptor, separation of the immune precipitate on SDS-PAGE, and
immunoblotting. The immunoblots (IB) were stained
with anti-Src antibody as well as with another antibody (CM02) against
the 2-adrenergic receptor to establish equivalent loading for each
lane. Shown is a blot representative of three such experiments with
essentially identical results.
|
|
To test whether or not the agonist-induced increase in phosphotyrosine
content of the
2-adrenergic receptor is indeed a reflection of the
phosphorylation of the Tyr-350 residue, we challenged CHO clones stably
expressing either the wild-type receptor or the Y350F mutant form of
the receptor with isoproterenol for periods up to 60 min. Whole-cell
extracts were prepared from both clones and then employed for pull-down
assays for the
2-adrenergic receptor using the CM04 anti-receptor
antibody that recognizes an exofacial epitope of the receptor not
influenced by the Tyr-350 residue localized to the cytoplasmic,
C-terminal tail of the
2-adrenergic receptor. The immune
precipitates were subjected to SDS-PAGE and immunoblotting and stained
with either anti-phosphotyrosine PY99 antibody or anti-receptor CM02
antibody to establish equivalent loading of samples. For the clones
expressing wild-type receptor, the phosphotyrosine content increased
(Fig. 6), as observed above. In the cells
expressing the Y350F mutant form of the
2-adrenergic receptor,
however, phosphotyrosine content at 30 and 60 min post-challenge with
isoproterenol was reduced in comparison with that obtained for
wild-type receptors. An increase in phosphotyrosine content was
observed at 5 min in the clones expressing the Y350F mutant form of the
receptor, which presumably reflects increased phosphorylation of either
Tyr-132 and Tyr-141 in the second intracellular loop or Tyr-354 and/or
Tyr-364 in the cytoplasmic, C-terminal tail of the hamster
2-adrenergic receptor. Each of these tyrosine residues has been
characterized earlier with respect to phosphorylation, but only the
Tyr-350 site creates an SH2-binding site upon phosphorylation (20).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 6.
The Y350F mutation of the
2-adrenergic receptor
( 2AR) attenuates
-adrenergic agonist-induced tyrosine
phosphorylation of the receptor. CHO clones expressing either
wild-type (WT) 2-adrenergic receptors or the Y350F mutant
form of the 2-adrenergic receptor were challenged with isoproterenol
(ISO) (10 µM) for 0, 5, 30, and 60 min and
then used to prepare whole-cell extracts. The clones expressed
equivalent levels of 2-adrenergic receptors, as measured by
radioligand binding studies with iodocyanopindolol. The cell
extracts were subjected to immune precipitation (IP)
pull-down assays with antibody (CM04) against the 2-adrenergic
receptor, separation of the immune precipitate on SDS-PAGE, and
immunoblotting. The immunoblots (IB) were stained
with anti-phosphotyrosine antibody (PY99) as well as with another
antibody (CM02) against the 2-adrenergic receptor to establish
equivalent loading for each lane. Shown is a blot representative of
three such experiments with essentially identical results.
|
|
Using the cyclic AMP response to isoproterenol stimulation as a
readout, we examined in parallel the effects of the Y350F mutation of
the
2-adrenergic receptor on agonist-induced desensitization in
these CHO clones (Fig. 7). Clones stably
expressing either the wild-type or the Y350F mutant forms of the
receptor were challenged with isoproterenol for up to 60 min. In some
cases (30 and 60 min) the clones were then washed free of the agonist
and then challenged a second time for 5 min with isoproterenol, and
intracellular cyclic AMP accumulation was measured. Agonist-induced
desensitization in the CHO clones expressing the wild-type
2-adrenergic receptor was essentially complete within 30 min.
Following the sharp increase in cyclic AMP accumulation observed in
response to isoproterenol at 5 min post-challenge, the clones displayed
desensitization. The cyclic AMP levels of CHO cells expressing the
wild-type
2-adrenergic receptors declined in the continued presence
of agonist. By 30 or 60 min of challenge with agonist, cyclic AMP
levels of these clones declined to that of unstimulated CHO cells.
Washout of the agonist, followed by a second 5-min challenge with
agonist, enabled resensitization, the restimulation provoking a sharp
rise in intracellular cyclic AMP accumulation, albeit a somewhat
smaller response than that observed with the naïve cells.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
The Y350F mutation of the
2-adrenergic receptor
( 2AR) abolishes desensitization of receptor
signaling to adenylcyclase in response to stimulation by
-adrenergic agonist. CHO clones expressing
either wild-type (WT) 2-adrenergic receptors or the Y350F
mutant form of the 2-adrenergic receptor were challenged with 10 mM isoproterenol (ISO) for 0, 5, 30, or 60 min,
and intracellular cyclic AMP accumulation was determined. Some cells
were first treated with isoproterenol for 30 or 60 min and then washed
free of agonist and rechallenged with isoproterenol for 5 min. Assays
of cyclic AMP accumulation were performed in triplicate. The data shown
are mean values ± S.E. from three to five separate experiments
performed on separate occasions.
|
|
Expression of the Y350F mutant form of the
2-adrenergic receptor
does not alter the ability of the receptor to activate adenylylcyclase, because the cyclic AMP response of the mutant receptor is essentially equal to that observed for the clones expressing the wild-type receptor
(Fig. 7). What is striking, however, is that the clones expressing the
Y350F mutant form of the
2-adrenergic receptor display a
desensitization response that is substantially reduced in magnitude
from that observed in clones expressing the wild-type receptor. At both
30 and 60 min post-challenge with isoproterenol, clones expressing the
wild-type receptor display complete desensitization, whereas the CHO
clones expressing the Y350F mutant retain ~50% of the cyclic AMP
response observed following a single 5-min challenge with agonist.
Although the fundamental observations obtained from the studies of the
A431 cells and CHO clones stably expressing wild-type receptors are the
same, the cellular context does play a role in the magnitude of the
biological readout of cyclic AMP accumulation.
Because phosphorylation of the
2-adrenergic receptor by protein
kinase A and GRK2 precedes association of the phospho-receptor with
-arrestin and because
-arrestin acts as an adaptor for clathrin-mediated internalization of the
2-adrenergic receptor, we
explored the phosphorylation state of GRK2 in A431 cells in the basal
and isoproterenol-stimulated states (Fig.
8). Src has been shown to be capable of
phosphorylating and increasing the activity of GRK2 (14). A proposed
model of Src activity, however, suggests that Src is targeted to the
receptor, not directly, as suggested by the prominent role of Tyr-350
and the SH2-binding site of the
2-adrenergic receptor created upon
phosphorylation, but rather via
-arrestin, following the
phosphorylation of the
2-adrenergic receptor by GRK2. As an
alternative, we hypothesize that Src associates directly with, and is
activated by, the phospho-Tyr-350
2-adrenergic receptor. Src then is
activated through association with the
2-adrenergic receptor and
phosphorylates GRK2, driving the desensitization response. The
2-adrenergic receptor phosphorylated by Src-activated GRK2 then
favors association of
-arrestin and clathrin with the receptor
complex. This hypothesis was tested by measuring the amount of
phosphotyrosine content (phosphorylation) in GRK2 under the same
conditions that were employed to test agonist-induced desensitization
and sequestration.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 8.
Inhibition of Src kinase activity or
expression of dominant-negative Src blocks the stimulation of increased
phosphotyrosine content (activation) of GRK2 in response to
-adrenergic agonist. A431 cells with or
without pretreatment for 30 min with the Src kinase inhibitor PP2 or
clones stably transfected to express the constitutively active
(CA-Src) or the dominant-negative (DN-Src) form
of Src were challenged with isoproterenol (Iso) (10 mM) for 0, 5, 30, and 60 min and then used to prepare
whole-cell extracts. The cell extracts were subjected to immune
precipitation (IP) pull-down assays with antibody
against GRK2, separation of the immune precipitate on SDS-PAGE, and
immunoblotting. The immunoblots (IB) were stained
with anti-phosphotyrosine antibody (PY99) as well as with anti-GRK2
antibody to establish equivalent loading for each lane. Shown is a blot
representative of three such experiments with essentially identical
results.
|
|
A431 wild-type and stably transfected clones were either untreated or
challenged with isoproterenol (10 µM) for periods of up
to 60 min, and the whole-cell extracts were prepared for immune precipitation pull-down assays using antibodies specific for GRK2 (Fig.
8). The immune precipitates were subjected to SDS-PAGE, and the
resultant immunoblots were stained with either anti-phosphotyrosine antibodies (PY99) or antibodies against GRK2 to establish equivalent loading. For the wild-type cells, GRK2 appears to have some
phosphotyrosine content in the basal state. Stimulation with
2-adrenergic agonist leads to a sharp increase in phosphotyrosine
content that peaks at 5 min (>5.0-fold) and then declines by 30 to 60 min post-challenge at a level about 3-fold greater than basal.
Pretreatment (30 min) with the Src kinase family inhibitor PP2 (Fig. 8,
+PP2) was associated with a complete loss of the
2-adrenergic agonist-induced increase in the phosphotyrosine content
of GRK2. In clones expressing the constitutively activated Src mutant
(Fig. 8, CA-Src), the phosphotyrosine content of GRK2 was
sharply elevated in the basal state and equivalent to that observed in
response to stimulation with agonist. In contrast, clones expressing
the dominant-negative form of Src (Fig. 8, DN-Src) showed very little phosphotyrosine content in GRK2, either in the basal
state or in response to stimulation with
2-adrenergic agonist.
Having established that Src plays an important role in
agonist-induced desensitization of
2-adrenergic receptors, we also sought to explore whether Src participated in agonist-induced sequestration. A431 cells were stably transfected with a
GFP-
2-adrenergic receptor fusion protein, so that receptor
localization could be monitored in live cells by epifluorescence
microscopy. The clones stably expressing the GFP-tagged
2-adrenergic
receptor were stimulated for 30 min with isoproterenol (10 µM) and examined for localization of the epifluorescence
signal (Fig. 9). In the unstimulated,
control situation, the GFP-tagged
2-adrenergic receptor is localized predominantly to the plasma membrane (Fig. 9a). The cell
membranes are well defined with GFP-tagged receptor (see
arrows, Fig. 9a). After 30 min of stimulation
with
-adrenergic agonist, the receptor distribution is largely
intracellular, although some plasma membrane-associated receptor is
still obvious (Fig. 9b). When the clones are treated with
the Src family tyrosine kinase inhibitor PP2 for 30 min (Fig. 9c), two changes in the localization of the receptors are
noted. First, there appears a greater level of receptor localized to the cytoplasmic, perinuclear regions of the cell. Second, and more
striking, is the marked thickening of the band of GFP-tagged receptors
in the plasma membrane region (see arrows, Fig.
9c). Challenging the PP2-treated cells with agonist only
increases the thickening of the band of GFP-tagged receptor localized
about the plasma membrane. Washout of the PP2 in the continued presence of isoproterenol leads eventually to a normal pattern of perinuclear receptor localization (data not shown). These data suggest that Src
activity is important not only to desensitization but also to the
subsequent phase of receptor biology in which the desensitized receptors are trafficked to the cytoplasmic, perinuclear regions of the
cells for resensitization and eventual transit back to the plasma
membrane. Inhibition of Src activity stalls the agonist-induced internalization at some point early in vesicular trafficking from the
plasma membrane.

View larger version (110K):
[in this window]
[in a new window]
|
Fig. 9.
Inhibition of Src kinase activity
stalls agonist-induced internalization of
2-adrenergic receptors in response to stimulation
by -adrenergic agonist. A431 clones
stably expressing the GFP-tagged human 2-adrenergic receptor were
analyzed by epifluorescence microscopy. Clones were pretreated without
(a and b) and with (c and
d) the Src kinase inhibitor PP2 (50 nM) for 30 min prior to challenge without (a and c) or with
(b and d) stimulation with the -adrenergic
agonist isoproterenol (Iso) (10 µM) for 30 min. The microscopy was performed on living cells using a Nikon
inverted epifluorescence microscope. The images shown are
representative of those acquired in at least five separate experiments,
sampling several dozen images per experiment. Note that treatment with
agonist leads to a marked thickening of the GFP-tagged receptors at the
proximity of the plasma membrane when the cells are pretreated with the
Src kinase inhibitor PP2 (see arrows).
|
|
 |
DISCUSSION |
The Src family of nonreceptor tyrosine kinases have been
shown to play important roles in the biology of G-protein-linked receptors. In response to agonist binding, GPLRs typically demonstrate several discrete, temporally linked responses, the first of these being
activation of the receptor, transducing activation of the cognate
heterotrimeric G-protein(s) to which the receptor is coupled. Following
activation, virtually all GPLRs display agonist-induced desensitization, sequestration/internalization, and a resensitization phase. Recently, the
2-adrenergic receptor was shown to display signaling "switching" in which the desensitization of its well known cyclic AMP response was accompanied by a second wave of responses
involving a new cast of G-protein partners and signaling to the
mitogen-activated protein kinase pathway via Ras (3, 4). The latter
response to mitogen-activated protein kinase has been shown to involve
internalization in some cell lines (26) and a scaffold role of the
2-adrenergic receptor for mitogen-activated protein kinase signaling
elements in other cell lines (25). Thus, much of what we know about the
role of Src in GPLR generally and
2-adrenergic receptors
specifically involves agonist-induced responses that occur temporally
after desensitization.
Herein we explored the possible role of Src in events that precede
rather than follow desensitization. The results demonstrate an
important role of Src in the ability of the
2-adrenergic receptor to
undergo agonist-induced desensitization. The primary results on which
this claim is made are as follows. Treating cells with the
cell-permeable PP2 inhibitor of Src kinases impairs desensitization; suppression of the expression of Src specifically leads to a loss in
agonist-induced desensitization for the
2-adrenergic receptor; and
expression of a dominant-negative mutant (K295R/Y527F) of Src nullifies
agonist-induced desensitization and enables an impressive cyclic AMP
response for periods up to 60 min post-challenge with agonist. These
data implicate Src itself, rather than other Src family members, in the
desensitization of
2-adrenergic receptors in response to agonist
challenge. Other Src kinase family members, however, may play analogous
roles for GPLRs other than
2-adrenergic receptors.
How Src is recruited to the receptor complex is not understood.
Based upon pull-down assays, it has been suggested that Src associates
with at least one well known member of the desensitization complex,
-arrestin. The interaction between
-arrestin and Src appears to
be either a weak SH3 motif found in the N terminus of
-arrestin
and/or a binding site that interacts with the catalytic domain of Src.
Because the current hypothesis for agonist-induced desensitization
suggests that GRKs first are recruited to the activated GPLR, followed
by Ser/Thr phosphorylation of the receptor and eventual recruitment of
-arrestin to the phosphorylated GPLR, the proposed association of
Src with
-arrestin as a targeting mechanism seems untenable. In
addition, it has been shown that Src both phosphorylates and activates
GRK2 itself (demonstrated in vivo in the current work),
arguing that the proper sequence of Src association with the receptor
would be in advance of (and not following) GRK-catalyzed
phosphorylation of the GPLR (Fig. 10).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 10.
Model of
2-adrenergic receptor-mediated recruitment and
activation of Src, which activates GRK2 and leads both to
desensitization and to -arrestin-mediated
targeting of the receptor-Src complex with clathrin. The
phosphorylation of Tyr-350 of the 2-adrenergic receptor creates an
SH2-binding site to which Src is recruited upon activation of receptor
by -adrenergic agonist. Src association with the receptor
SH2-binding site activates Src and catalyzes its
phosphorylation/activation of GRK2, a Ser/Thr kinase that
phosphorylates the agonist-activated receptor. The Ser/Thr-directed
phosphorylation of the 2-adrenergic receptor recruits -arrestin
to the complex to complete agonist-induced desensitization and commence
clathrin-mediated internalization. Inhibition of Src kinase with PP2,
by expression of a dominant-negative mutant of Src or the Y350F
mutation of the 2-adrenergic receptor, abolishes the ability of Src
to be recruited/activated in response to -adrenergic agonist and
thereby precludes both desensitization and eventually full
internalization of the receptor-Src complex. Not shown for the sake of
simplicity is the scaffold molecule gravin that nucleates the complex
of the receptor with several additional kinases and phosphatases.
AR or bAR, -adrenergic receptor;
TK, tyrosine kinase.
|
|
What other mechanism might be proposed and tested to explain the
association of Src with the
2-adrenergic receptor in advance of GRK
association? The
2-adrenergic receptor harbors a putative, canonical
SH2-binding domain flanking the Tyr-350 residue found in the
cytoplasmic, C-terminal tail of the receptor. Upon phosphorylation, the
Tyr-350 residue constitutes a functional SH2-binding domain that can
recruit SH2 domain-containing partners to the
2-adrenergic receptor,
including the adaptor molecule Grb2, the GTPase involved in trafficking
dynamin, and 1-phosphatidylinositol 3'-kinase. The interaction of the
Tyr-350-phosphorylated
2-adrenergic receptor with Grb2 has been
shown to be dependent upon the integrity of the SH2, but not SH3,
domains of Grb2 (24). The presence of a potential SH2-binding site in
the cytoplasmic, C-terminal region of the
2-adrenergic receptor
provoked us to test the hypothesis that this site may become
phosphorylated in response to challenge with agonist. This working
hypothesis is displayed in Fig. 10. The results displayed in Fig. 6
clearly demonstrate that agonist stimulates the phosphorylation of the
Tyr-350 residue and that a Tyr-to-Phe substitution at this residue
largely attenuates the increase in phosphotyrosine content found in the
2-adrenergic receptor upon agonist treatment (Fig. 6). Earlier it
was shown that the
2-adrenergic receptor is a substrate for tyrosine
phosphorylation in response to activation of several growth factors,
including insulin and insulin-like growth factor 1 (21-23). Insulin
treatment in vivo leads to the phosphorylation of Tyr-350,
as well as Tyr-354 and Tyr-364. Phosphorylation of the Tyr-350 residue
of the
2-adrenergic receptor leads to recruitment of Grb2, dynamin,
and PI3 kinase to the receptor (17, 20, 23, 24). Although the nature of
the tyrosine kinase catalyzing the phosphorylation of Tyr-350 in
response to
2-adrenergic agonist remains to be established, the
Tyr-350 site is shown in the current work to be critical for agonist-induced desensitization.
Cells expressing the Y350F mutant form of the
2-adrenergic receptor
fail to display normal agonist-induced desensitization (Fig. 7). The
phosphotyrosine content of the
2-adrenergic receptor in response to
stimulation by agonist is sharply attenuated when the Y350F mutant
receptor is expressed (Fig. 6). Furthermore, the ability of the
2-adrenergic receptor to recruit Src in response to agonist
stimulation is abolished by the Tyr-to-Phe substitution of the Tyr-350
residue of the receptor (Fig. 5). These data provide a compelling case
for the role of the Tyr-350 residue as a docking site for Src binding
and for Src activation upon phosphorylation. A working model that
captures these features of Src association with the
2-adrenergic
receptor in the process of agonist-induced desensitization is displayed
(Fig. 10). According to this model, it is the phosphorylation of
Tyr-350 by some tyrosine kinase in response to agonist that initiates
the process of desensitization. Upon phosphorylation, the Tyr-350
residue recruits Src to the plasma membrane-localized receptor, both
docking and activating Src. Activated Src, in turn, phosphorylates and
activates GRK2. GRK2 then catalyzes the further phosphorylation of the
2-adrenergic receptor on Ser/Thr residues, which in turn creates the
docking site for
-arrestin, largely completing the desensitization
phase of the receptor biology. Acting as an adaptor between the
phosphorylated receptor and clathrin,
-arrestin then initiates the
sequestration/internalization of the
2-adrenergic receptor, a
process essential for resensitization and relocalization to the plasma
membrane to occur.
Based upon the model (Fig. 10), one can ask, if the Tyr-350
phosphorylated receptor is necessary to recruit Src in advance of GRK2,
what the outcome is of inhibiting Src on the subsequent activation of
GRK. The answers to this question were quite clear. Inhibition of Src
with PP2, expression of a dominant-negative version of Src, or
suppression of Src levels by treatment with antisense
oligodeoxynucleotides all blocked agonist-induced desensitization as
well as the agonist-induced increase in the phosphotyrosine content
(i.e. activation) of GRK2. Similarly, inhibiting Src
activity with PP2 was shown to block agonist-induced changes in
receptor biology, including desensitization and receptor
trafficking. Using autofluorescent protein-tagged
2-adrenergic
receptors and epifluorescence, we demonstrated a marked thickening of
the band of receptor localized in close proximity to the plasma
membrane in response to agonist in the PP2-treated cells. This
observation made in live cells is consistent with the notion that Src
activity is obligate both for desensitization and for proper
trafficking of the
2-adrenergic receptor in response to agonist.
Taken together, these results reveal a novel, important role for
Src in the regulation of
2-adrenergic receptors that may well extend
to many other members of the GPLR superfamily. Src is critical for
agonist-induced desensitization. For the
2-adrenergic receptor,
recruitment of Src requires phosphorylation of a tyrosine residue in
the cytoplasmic, C-terminal tail of the receptor, which creates a
conditional, canonical SH2-binding site for Src. Through this early
receptor-Src association and Src activation, Src is positioned to
phosphorylate incoming GRK2 and to facilitate the subsequent
phosphorylation of the receptor on Ser/Thr residues of the cytoplasmic,
C-terminal tail that recruit
-arrestin and ultimately clathrin. The
interaction of Src with
-arrestin, observed when both signaling
molecules are overexpressed (3), may reflect a later role of Src in
receptor internalization or signaling to Ras. These studies are the
first to illuminate the role of Src in agonist-induced desensitization.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Joan Brugge (Department of Cell
Biology, Harvard Medical School) for providing us with the expression
vectors harboring the constitutively activated and dominant-negative
forms of Src, and we thank Dr. Jeffrey Benovic (Department of
Pharmacology, Thomas Jefferson Medical School) for providing us with
the expression vector harboring the GFP-tagged
2-adrenergic receptor and antibodies against GRK2.
 |
FOOTNOTES |
*
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.
§
To whom correspondence should be addressed: Pharmacology-HSC,
SUNY/Stony Brook, Stony Brook, NY 11794-8651. Tel.: 516-444-7873; Fax:
516-444-7696; E-mail: craig@pharm.som.sunysb.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011578200
 |
ABBREVIATIONS |
The abbreviations used are:
GPLR, G-protein-linked receptor;
GRK, G-protein-linked receptor kinase;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;
CHO, Chinese hamster ovary;
ODN, oligodeoxynucleotide;
GFP, green
fluorescent protein;
SDS-PAGE, SDS-polyacrylamide gel
electrophoresis.
 |
REFERENCES |
1.
|
Thomas, S. M.,
and Brugge, J. S.
(1997)
Annu. Rev. Cell Dev. Biol.
13,
513-609[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Ahn, S.,
Maudsley, S.,
Luttrell, L. M.,
Lefkowitz, R. J.,
and Daaka, Y.
(1999)
J. Biol. Chem.
274,
1185-1188[Abstract/Free Full Text]
|
3.
|
Luttrell, L. M.,
Ferguson, S. S.,
Daaka, Y.,
Miller, W. E.,
Maudsley, S.,
Della Rocca, G. J.,
Lin, F.,
Kawakatsu, H.,
Owada, K.,
Luttrell, D. K.,
Caron, M. G.,
and Lefkowitz, R. J.
(1999)
Science
283,
655-661[Abstract/Free Full Text]
|
4.
|
Lefkowitz, R. J.
(1998)
J. Biol. Chem.
273,
18677-18680[Free Full Text]
|
5.
|
Morris, A. J.,
and Malbon, C. C.
(1999)
Physiol. Rev.
79,
1373-1430[Abstract/Free Full Text]
|
6.
|
Carman, C. V.,
and Benovic, J. L.
(1998)
Curr. Opin. Neurobiol.
8,
335-344[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Benovic, J. L.,
Kuhn, H.,
Weyand, I.,
Codina, J.,
Caron, M. G.,
and Lefkowitz, R. J.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
8879-8882[Abstract]
|
8.
|
Gagnon, A. W.,
Kallal, L.,
and Benovic, J. L.
(1998)
J. Biol. Chem.
273,
6976-6981[Abstract/Free Full Text]
|
9.
|
Goodman, O. B. J.,
Krupnick, J. G.,
Santini, F.,
Gurevich, V. V.,
Penn, R. B.,
Gagnon, A. W.,
Keen, J. H.,
and Benovic, J. L.
(1996)
Nature
383,
447-450[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Pitcher, J. A.,
Payne, E. S.,
Csortos, C.,
DePaoli-Roach, A. A.,
and Lefkowitz, R. J.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
8343-8347[Abstract]
|
11.
|
Shih, M.,
and Malbon, C. C.
(1996)
J. Biol. Chem.
271,
21478-21483[Abstract/Free Full Text]
|
12.
|
Shih, M.,
Lin, F.,
Scott, J. D.,
Wang, H. Y.,
and Malbon, C. C.
(1999)
J. Biol. Chem.
274,
1588-1595[Abstract/Free Full Text]
|
13.
|
Lin, F.,
Wang, H.,
and Malbon, C. C.
(2000)
J. Biol. Chem.
275,
19025-19034[Abstract/Free Full Text]
|
14.
|
Sarnago, S.,
Elorza, A.,
and Mayor, F., Jr.
(1999)
J. Biol. Chem.
274,
34411-34416[Abstract/Free Full Text]
|
15.
|
Shih, M.,
and Malbon, C. C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12193-12197[Abstract/Free Full Text]
|
16.
|
Malbon, C. C.
(1980)
J. Biol. Chem.
255,
8692-8699[Free Full Text]
|
17.
|
Karoor, V.,
Shih, M.,
Tholanikunnel, B.,
and Malbon, C. C.
(1996)
Prog. Neurobiol. (Oxf.)
48,
555-568[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Kashles, O.,
and Levitzki, A.
(1987)
Biochem. Pharmacol.
36,
1531-1538[Medline]
[Order article via Infotrieve]
|
19.
|
Wang, H. Y.,
Berrios, M.,
Hadcock, J. R.,
and Malbon, C. C.
(1991)
Int. J. Biochem.
23,
7-20[Medline]
[Order article via Infotrieve]
|
20.
|
Baltensperger, K.,
Karoor, V.,
Paul, H.,
Ruoho, A.,
Czech, M. P.,
and Malbon, C. C.
(1996)
J. Biol. Chem.
271,
1061-1064[Abstract/Free Full Text]
|
21.
|
Karoor, V.,
Baltensperger, K.,
Paul, H.,
Czech, M. P.,
and Malbon, C. C.
(1995)
J. Biol. Chem.
270,
25305-25308[Abstract/Free Full Text]
|
22.
|
Karoor, V.,
and Malbon, C. C.
(1998)
Adv. Pharmacol.
42,
425-428[Medline]
[Order article via Infotrieve]
|
23.
|
Karoor, V.,
Wang, L.,
Wang, H. Y.,
and Malbon, C. C.
(1998)
J. Biol. Chem.
273,
33035-33041[Abstract/Free Full Text]
|
24.
|
Shih, M.,
and Malbon, C. C.
(1998)
Cell. Signal.
10,
575-582[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Wang, H.,
Doronin, S.,
and Malbon, C. C.
(2000)
J. Biol. Chem.
275,
36086-36093[Abstract/Free Full Text]
|
26.
|
Luttrell, L. M.,
Daaka, Y.,
and Lefkowitz, R. J.
(1999)
Curr. Opin. Cell Biol.
11,
177-183[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.