From the Howard Hughes Medical Institute and the
Departments of § Pharmacology and Cancer Biology,
¶ Medicine, and ** Surgery, Duke University Medical Center,
Durham, North Carolina 27710
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ABSTRACT |
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Some forms of G protein-coupled receptor
signaling, such as activation of mitogen-activated protein kinase
cascade as well as resensitization of receptors after hormone-induced
desensitization, require receptor internalization via
dynamin-dependent clathrin-coated pit mechanisms. Here we
demonstrate that activation of Agonist stimulation of cell surface receptors results in rapid
attenuation of receptor responsiveness, a process termed
desensitization. In the case of G protein-coupled receptors
(GPCRs),1 this process is
initiated by phosphorylation of agonist-occupied receptors by the G
protein-coupled receptor kinase family (1) and the subsequent formation
of high affinity complexes with cytosolic proteins termed The process of cell surface receptor internalization (also termed
endocytosis) is dependent on the invagination and fission of
clathrin-coated vesicles from the plasma membrane into the cytosol.
Endocytosis of many GPCRs and receptor tyrosine kinases (RTKs) requires
the GTPase activity of dynamin. Receptor stimulation leads to
recruitment of cytosolic dynamin to coated pits where it induces
constriction of the pits and fission of vesicles (7, 8). In
vitro, dynamin is activated by a variety of mechanisms including
binding to microtubules (9), phospholipids (10), and several proteins
containing SH3 domains (11). Collectively, these molecules are thought
to induce a conformational change in dynamin, inducing
homo-oligomerization and subsequent increase in GTPase activity (12).
Recently, dynamin was shown to contain a GTPase effector domain that
interacts with its N-terminal GTPase domain to stimulate GTP hydrolysis
(13). GTPase defective dynamin mutants (e.g. K44A)
specifically block endocytic coated vesicle formation and
agonist-mediated internalization of GPCRs and RTKs (14, 15).
Accumulating evidence suggests that tyrosine protein phosphorylation is
critical for the internalization of cell surface receptors. Exposure of
cells to tyrosine kinase inhibitors profoundly attenuates cross-linking-induced internalization of B cell receptors (16) and
hormone-induced internalization of tyrosine kinase growth factor
receptors (17). Recently, overexpression of the nonreceptor tyrosine
kinase c-Src was found to cause an increase in the internalization rate
constant of the epidermal growth factor (EGF) receptor following EGF
treatment (18). Although these results suggest involvement of tyrosine
kinases in the process of receptor internalization, their role(s) in
this process is not well defined. Therefore, we tested the hypothesis
that tyrosine kinase activity might regulate endocytosis by acting on
accessory molecules important for receptor internalization such as
dynamin. We find not only that Src-mediated tyrosine phosphorylation of
dynamin is required for Cell Culture and Transfection--
HEK293 cells were maintained
and transfected precisely as described (5, 6). All assays were
performed 40 h after transfection. HEK293 cells stably
overexpressing dynamin were generated as described (19).
Sequestration of Plasmid Construction and Site-specific Mutagenesis--
To
generate single tyrosine mutants (Y231F and Y597F), tyrosine 231 or
tyrosine 597 of rat dynamin I was mutated to phenylalanine by
overlapping polymerase chain reaction (UAC(Y) GST-Grb2 Fusion Proteins--
Isolation of total cellular
dynamin for analysis of tyrosine phosphorylation was performed by
affinity purification with full-length GST-Grb2 fusion protein
according to the method of Gout et al. (11). The cDNA
encoding GST-Grb2 fusion protein was generously provided by A. R. Saltiel. GST fusion proteins were prepared as glutathione-agarose
conjugates as described previously (6, 21). Complexed proteins were
dissociated from agarose beads with SDS-polyacrylamide gel
electrophoresis sample buffer.
Protein Cross-linking--
For the detection of transient,
agonist-promoted association of dynamin and c-Src, covalent protein
cross-linking with Dithiobis(succinimidylpropionate) (DSP, Pierce) was
employed. Cells were stimulated in 4.6 ml of phosphate-buffered saline
containing 10 mM Hepes, pH 7.4. Stimulation was terminated
by the addition of 0.4 ml of 2.5 mM DSP in
Me2SO, and plates were rocked for 30 min at room
temperature. Reactions were quenched by the addition of 0.1 ml of 1 M Tris, pH 7.5, followed by two washes with ice-cold
phosphate-buffered saline/Hepes to remove unreacted DSP. Cell pellets
were lysed in RIPA buffer (5, 6) prior to immunoprecipitation of
c-Src-dynamin complexes.
Immunoprecipitation and Immunoblotting--
Cell lysates in RIPA
buffer were incubated with the specified antibodies and protein
A/G-Sepharose beads for 2 h at 4 °C. Rabbit polyclonal anti-Src
antibody (Src-2, Santa Cruz Biotech. Inc.) was used for c-Src
immunoprecipitation after protein cross-linking. Dynamin proteins were
immunoprecipitated without cross-linking using either mouse monoclonal
anti-dynamin antibody (Hudy-1; Upstate Biotech. Inc.) or rabbit
polyclonal anti-dynamin I-specific antibody (kind gift of S. L. Schmid). Immunoprecipitates or GST-Grb2 fusion complexed proteins were
resolved on acrylamide gels, transferred to nitrocellulose filters,
immunoblotted with anti-dynamin or anti-phosphotyrosine antibodies
(RC20H or PY20H, Transduction Laboratories), and visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Metabolic Labeling and Microsequencing--
HEK293 cells stably
expressing dynamin I were metabolically labeled with
[32P]orthophosphate as described (19). Dynamin was
purified using GST-Grb2 binding as described above. Partially purified
dynamin was resolved by SDS-polyacrylamide gel electrophoresis, and the dynamin band was excised from the gel and subsequently used for microsequencing after tryptic digestion (22).
MAP Kinase Assay--
HA-Erk2 was immunoprecipitated from HEK293
lysates using 6 µg of 12CA5 antibody. Immunoprecipitates were washed
three times with RIPA buffer and twice with kinase buffer (23).
In vitro kinase reactions using myelin basic protein (MBP)
as substrate were performed as described (23). Labeled MBP was
quantified using a Storm PhosphorImager (Molecular Dynamics).
Spatial distribution and trafficking of receptors control their
function. For example, mitogenic signaling emanating from certain GPCRs
and RTKs is dependent on receptor internalization (5, 6, 24-26). To
determine whether tyrosine kinase activity is required for G
protein-coupled receptor endocytosis, isoproterenol-induced internalization of the 2-adrenergic receptors (
2-ARs) leads to c-Src-mediated tyrosine
phosphorylation of dynamin, which is required for receptor
internalization. Two tyrosine residues, Tyr231 and
Tyr597, are identified as the major phosphorylation sites.
Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following
2-AR
stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits
2-AR internalization and the isoproterenol-stimulated
mitogen-activated protein kinase activation. Thus, agonist-induced,
c-Src-mediated tyrosine phosphorylation of dynamin is essential for its
function in clathrin mediated G protein-coupled receptor endocytosis.
INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References
-arrestins
(2).
-Arrestin-bound, desensitized GPCRs then undergo
-arrestin-mediated targeting to clathrin-coated pits (3, 4), wherein
they are sequestered, internalized, and ultimately either recycled to
the cell surface or targeted for degradation. Interestingly, the
process of GPCR internalization also plays a critical, albeit poorly
understood, role in some aspects of GPCR signal transduction,
e.g. MAP kinase activation (5, 6).
2-AR internalization but that
this phosphorylation event itself is regulated by receptor stimulation.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
2-ARs--
The
2-AR sequestration was quantified as loss of cell
surface receptors determined by immunofluorescence flow cytometry as described (5).
UUC(F)) (20). The
double tyrosine mutant (Y231F/Y597F) dynamin was constructed by
recombination of the two single tyrosine mutant constructs.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
2-AR was examined in HEK293 cells
pretreated with tyrosine kinase inhibitors. Fig.
1A shows that pretreatment of
cells with the generic tyrosine kinase inhibitor herbimycin A reduced
isoproterenol-mediated internalization of the
2-AR by
60%. More dramatically, acute exposure of cells to the specific c-Src
kinase inhibitor PP1 reduced the isoproterenol-induced
2-AR sequestration by approximately 80% (Fig.
1A). As shown in Fig. 1B, expressing either a
c-Src kinase inhibitor (Csk) or a kinase defective, dominant inhibitory
form of c-Src (K298M) impaired the isoproterenol-mediated
internalization of the
2-AR. Conversely, expression of
an activated form of Src (v-Src) increased
agonist-dependent sequestration of the
2-AR
(Fig. 1B). These results suggest that Src tyrosine kinase
activity is required for agonist-dependent internalization
of the
2-AR.
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Fig. 1.
Effect of tyrosine kinase inhibitors on
agonist-induced 2-AR sequestration. HEK293 cells
transiently expressing Flag-tagged
2-AR were exposed to
10 µM isoproterenol for 30 min at 37 °C and analyzed for their
plasma membrane content of the
2-AR by flow cytometry.
A, cells in serum-free media were exposed to 1 µM
herbimycin A (Herb A) for 12-14 h or to 5 µM PP1 for 30 min prior to agonist stimulation. CN, control cells.
B, Flag-tagged
2-AR was transfected together
with plasmids expressing the following: empty pRK5 vector
(CN), C-terminal c-Src kinase (CSK),
catalytically inactive c-Src (c-Src K298M), or
constitutively activated Src (v-Src). Values shown are
expressed as the percentage of loss of agonist-induced cell surface
receptor over unstimulated cells. Data represent means ± S.E.
from five (A) and three (B) independent
experiments done in triplicate.
A direct interaction between c-Src and dynamin in neuronal cells has
been described (27). Because c-Src kinase activity was required for the
agonist-mediated internalization of 2-AR, we examined
whether dynamin is a phosphotyrosyl protein and a substrate for c-Src.
Cellular dynamin was isolated using a GST-Grb2 affinity purification
following agonist exposure. As shown in Fig.
2A, isoproterenol stimulation
resulted in 2-2.5-fold increase in tyrosine phosphorylation of dynamin
(upper immunoblot), which was maximal within 2 min of
agonist exposure. These results demonstrate that
2-AR
activation induces the rapid tyrosine phosphorylation of dynamin.
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Next, we examined whether c-Src is the kinase responsible for the
tyrosine phosphorylation of dynamin following activation of the
2-AR. We expressed the c-Src inhibitors, Csk and K298M c-Src, or the constitutively active v-Src and examined their effect on
isoproterenol-induced tyrosine phosphorylation of dynamin in HEK293
cells stably expressing dynamin I. Fig. 2B shows that
expression of Csk or K298M c-Src inhibited the agonist-induced tyrosine
phosphorylation of dynamin, whereas expression of activated Src (v-Src)
further enhanced the level of tyrosine phosphorylation of dynamin
(upper immunoblot). Thus, agonist stimulation resulted in a
net increase in the phosphotyrosine content of the cellular dynamin
pool, which could be reversed by inhibition of Src activity. These data
mirror the effects of these reagents on
2-AR
sequestration (Fig. 1B), suggesting that c-Src-regulated
internalization of the
2-AR may be mediated by the
c-Src-induced tyrosine phosphorylation of dynamin.
Foster-Barber and Bishop (27) have reported that the purified SH3
domain of c-Src binds dynamin in vitro and that dynamin co-precipitates with c-Src from PC12 cell lysates. These authors did
not describe tyrosine phosphorylation of dynamin or modulation of
c-Src-dynamin complex formation following receptor activation. To test
the hypothesis that the agonist-regulated association of dynamin and
c-Src might account for the agonist-promoted increase in dynamin
tyrosine phosphorylation, we examined the ability of c-Src to form a
complex with dynamin following 2-AR activation. Protein
cross-linking was used to stabilize potentially transient, agonist-mediated, dynamin-c-Src interactions. HEK293 cells stably expressing dynamin I were treated with or without isoproterenol for 2 min followed by covalent protein cross-linking with DSP. As shown in
Fig. 2C, treatment of cells with isoproterenol increased the
amount of tyrosine phosphorylated dynamin present in c-Src immunoprecipitates.
Reverse phase high pressure liquid chromatography analysis of trypsin-digested phosphodynamin isolated from isoproterenol-stimulated cells revealed the presence of two major phosphopeptides. Microsequencing and amino acid sequence analyses (19, 22) of one peptide revealed that Tyr597 is phosphorylated (Fig. 3A). Sequence analysis of the second phosphopeptide revealed sequences just N-terminal to a GYIG motif (Tyr231) that displays high sequence homology to consensus c-Src phosphorylation sites (28).
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To determine whether tyrosine phosphorylation of dynamin affected its function, each of these tyrosine residues (at positions 231 and 597) was mutated to phenylalanine singly and in combination. As shown in Fig. 3 (B and C), whereas isoproterenol treatment increased the tyrosine phosphorylation content of wild-type dynamin, the single tyrosine mutants (Y231F and Y597F), and the double tyrosine mutant (Y231F/Y597F) of dynamin exhibited diminished tyrosine phosphorylation in both isoproterenol-stimulated and nonstimulated cells. The dramatic decrease in tyrosine phosphorylation of Y231F/Y597F mutant strongly suggests that these residues represent the phosphorylated sites in vivo.
In agreement with previous results (5, 15), overexpression of wild-type
dynamin did not enhance agonist-promoted sequestration of the
2-AR (Fig. 4A).
However, expression of Y231F or Y597F dynamin resulted in a modest
reduction of agonist-induced
2-AR internalization.
Expression of the Y231F/Y597F dynamin inhibited approximately 70% of
the agonist-induced internalization of the
2-AR, which
was equivalent to the degree of inhibition observed with
GTPase-deficient dynamin K44A. These data suggest that tyrosine phosphorylation of dynamin plays an important role in its ability to
support endocytosis.
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Recent data suggest that agonist-promoted endocytosis plays a dual
regulatory role in signaling pathways emanating from GPCRs or RTKs.
Activation of the Erk1/2 MAP kinase pathway by GPCRs, such as those for
isoproterenol, lysophosphatidic acid, thrombin, and bombesin (5, 6), as
well as RTKs, such as those for EGF (25), nerve growth factor (29, 30),
and insulin-like growth factor-1 (26), is dependent on receptor
internalization. In the case of the 2-AR, protein kinase
A-mediated phosphorylation of the receptor confers
receptor-Gi coupling, with subsequent Ras-dependent Erk1/2 activation mediated by
G
-subunits derived from pertussis toxin-sensitive G proteins
(31). In addition, expression of dominant negative mutants of
-arrestin 1 or dynamin, which attenuate agonist-mediated
endocytosis, blocks
2-AR-mediated Erk1/2 activation (5).
Considering the role of dynamin in endocytosis, we investigated the
effects of expressing wild-type or mutant dynamin on the
2-AR-mediated activation of the Erk2 MAP kinase. Expression of the Y231F or Y597F mutated forms of dynamin reduced the
isoproterenol-mediated activation of Erk2 approximately 60% (Fig. 4,
B and C). However, expression of the Y231F/Y597F
mutant reduced the agonist-induced Erk2 activation by 80-90%, similar to the effects observed using the K44A dynamin (Fig. 4, B
and C). These data suggest that c-Src-mediated tyrosine
phosphorylation of dynamin is required for Ras-mediated activation of
MAP kinase by
2-AR by virtue of its involvement in
receptor sequestration.
Our results establish that agonist-induced tyrosine phosphorylation of
dynamin is required for 2-AR internalization and
internalization-dependent signaling to MAP kinase.
Clathrin-coated vesicle-mediated receptor internalization is regulated
by the enzymatic (GTPase) activity of dynamin. As shown in Fig.
3A, Tyr231 and Tyr597 reside in the
GTPase and pleckstrin homology (PH) domains of dynamin, respectively.
Intermolecular interaction between GTPase and GED domains of adjacent
dynamins has been reported to regulate the GTPase activity of the
enzyme (13). Thus, phosphorylation of Tyr231 might regulate
the GTPase activity of dynamin by controlling these intermolecular
interactions. Another mutation that disables the GTPase activity of
dynamin (K44A) also ablates the ability of dynamin to function in
internalization of receptors (14, 15). PH domains in proteins such as
phospholipase C-
1 (32) have been shown to mediate
interactions with acidic phospholipids. Although there is as yet no
direct evidence for dynamin PH domain-phospholipid interaction, acidic
phospholipids such as phosphatidylinositol 4,5-bisphosphate have been
shown to stimulate GTPase activity of dynamin in vitro (10,
33). Additionally, G protein
subunits, which interact with PH
domains of many proteins (34), have been shown to control the GTPase
activity of dynamin (33). Tyr597 is located in one of three
variable loops of the dynamin PH domain that possess positive
electrostatic potential (35). Phosphorylation of Tyr597 may
alter the ability of dynamin to interact with effector molecules such
as phosphatidylinositol 4,5-bisphosphate and G protein
subunits,
resulting in modulation of dynamin GTPase activity. Although the
molecular mechanism(s) whereby tyrosine phosphorylation of dynamin
regulates its function remain unclear, the present results establish
that the c-Src-mediated tyrosine phosphorylation of dynamin provides
one mechanism by which GPCRs regulate their own internalization and MAP
kinase signaling.
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ACKNOWLEDGEMENTS |
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We thank Drs. S. L. Schmid and A. R. Saltiel for reagents and Mary Holben and Donna Addison for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL16037 (to R. J. L.).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.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Dept. of Medicine, Box 3821, Duke
University Medical Center, Durham, NC 27710. Tel.: 919-684-2974; Fax:
919-684-8875; E-mail: lefko001{at}receptor-biol.duke.edu.
The abbreviations used are:
GPCR, G
protein-coupled receptor; MAP, mitogen-activated protein; RTK, receptor
tyrosine kinase; EGF, epidermal growth factor; 2-AR,
2-adrenergic receptor; GST, glutathione
S-transferase; DSP, dithiobis(succinimidylpropionate); MBP, myelin basic protein; PH, pleckstrin homology.
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REFERENCES |
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