From the Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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The classical paradigm for G protein-coupled
receptor (GPCR) signal transduction involves the
agonist-dependent interaction of GPCRs with heterotrimeric G
proteins at the plasma membrane and the subsequent generation, by
membrane-localized effectors, of soluble second messengers or ion
currents. Termination of GPCR signals follows G protein-coupled
receptor kinase (GRK)- and -arrestin-mediated receptor uncoupling
and internalization. Here we show that these paradigms are inadequate
to account for GPCR-mediated, Ras-dependent activation of
the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293
cells expressing dominant suppressor mutants of
-arrestin or
dynamin,
2-adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma
membrane-delimited steps in the GPCR-mediated activation of the MAP
kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase
activation by Ras, are unaffected by inhibitors of receptor
internalization. Thus, GRKs and
-arrestins, which uncouple GPCRs and
target them for internalization, function as essential elements in the
GPCR-mediated MAP kinase signaling cascade.
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INTRODUCTION |
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Stimulation of G protein-coupled receptors
(GPCRs)1 facilitates the
exchange of bound GDP for GTP on heterotrimeric G proteins, resulting
in dissociation of the G protein into active G-GTP and G
subunits. The interaction of G
-GTP and G
subunits with effectors initiates and accounts for the known signaling events mediated by GPCRs. Exposure of GPCRs to an agonist often results in
rapid attenuation of receptor responsiveness, a process termed desensitization. Signal termination is initiated by phosphorylation of
agonist-occupied receptors, mediated by the G protein-coupled receptor
kinase (GRK) family (1-3). The GRK-mediated phosphorylation of
activated GPCRs promotes binding of members of a family of cytosolic
proteins,
-arrestins, to the receptor (4, 5). Binding of
-arrestins to phosphorylated receptors serves two functions. First,
it uncouples the receptor from its cognate G protein and thus leads to
diminished receptor signaling (4, 5). Second, it initiates the process
of receptor internalization (also termed sequestration) by targeting
the receptor to clathrin-coated pits (6, 7).
G protein-coupled receptors and receptor tyrosine kinases (RTKs) stimulate mitogenesis in part via mitogen-activated protein (MAP) kinase cascades. The mechanism of activation of MAP kinase signaling pathways by GPCRs is poorly understood, although it is becoming evident that signal transduction by certain GPCRs utilizes many of the same intermediates as those activated by RTKs (see Reaction 1).
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Transfection--
Human embryonic kidney (HEK)
293 cells, maintained in minimum essential medium supplemented with
10% fetal bovine serum and 50 µg/ml gentamicin were transiently
transfected using calcium phosphate coprecipitation (14). Cells were
starved overnight in medium containing 10 mM Hepes (pH 7.4)
and 0.1% (v/v) bovine serum albumin prior to agonist stimulation. All
assays were performed 48 h after transfection. Transient
expression of -arrestin1,
-arrestin1 V53D, dynamin, and dynamin
K44A transfected plasmids were verified by immunoblotting of whole cell
lysates using commercially available antibodies.
Sequestration Assay--
The 2-AR sequestration
was determined by immunofluorescence flow cytometry (15). Cells
expressing epitope-tagged
2-AR at 300-400 fmol/mg of
whole cell protein were exposed to 10 µM isoproterenol
for 30 min at 37 °C prior to addition of antibodies. Sequestration
is defined as the fraction of total cell surface receptors which are
removed from the plasma membrane (and thus are not accessible to
antibodies added to the cells) following agonist treatment.
cAMP Production-- Cells were metabolically labeled with 1 µCi of [3H]adenine/ml, washed in PBS, and incubated with 1 mM isobutylmethylxanthine for 25 min at 37 °C. Agonist was added for 5 min followed by the addition of 1 ml of stop solution (0.1 mM cAMP, 4 nCi of [14C]cAMP/ml, 2.5% perchloric acid). Cell lysates were neutralized with KOH, and total [3H]cAMP was assayed by anion exchange chromatography (14).
Phosphoinositide Hydrolysis-- Cells were metabolically labeled for 16-18 h with 2 µCi of [3H]inositol/ml and washed in PBS containing 20 mM LiCl alone (basal) or with agonist for 5 min at 37 °C. Reactions were terminated by the addition of an equal volume of 0.8 M perchloric acid, and total inositol phosphates were assayed by anion exchange chromatography (14).
MAP Kinase Assay-- Agonist-treated cells were lysed by direct addition of Laemmli sample buffer. Aliquots were resolved by SDS-PAGE, and phosphorylated MAP kinases on nitrocellulose filters were detected using a phosphospecific MAP kinase IgG (Promega). Bands corresponding to MAP kinase were visualized with enzyme-linked chemiluminescence (ECL; Amersham Corp.) and quantitated by scanning laser densitometry.
Shc Phosphorylation-- Agonist-treated HEK293 cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate, 1% Nonidet P-40, 0.5% deoxycholate) and clarified by centrifugation. Shc proteins were immunoprecipitated using rabbit polyclonal anti-Shc antibodies (Transduction Laboratories) and resolved by SDS-PAGE. Phosphorylated Shc proteins on nitrocellulose were detected using anti-phosphotyrosine antibodies (RC20H, Transduction Laboratories), visualized with ECL, and quantitated by scanning laser densitometry.
Raf Kinase Assay-- Lysates were prepared in RIPA buffer from cells treated with agonists for 5 min at 37 °C. Raf-1 was immunoprecipitated with 0.5 µg of anti-Raf-1 polyclonal antibody (C-12, Santa Cruz Biotechnology). Immunocomplexes were washed with cold RIPA, wash buffer (137 mM NaCl, 20 mM Tris, pH 7.4, 2 mM EDTA, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM Na3VO4, 10% glycerol, 1% Nonidet P-40), and kinase buffer (75 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EDTA, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM Na3VO4, 30 µM ATP). Raf-1 kinase activity was determined by incubating the resuspended immune complexes in kinase buffer containing 10 µCi of [32P]ATP and 0.5 µg of MEK at ambient temperature for 15 min. Reactions were terminated by the addition of SDS sample buffer, and the phosphorylated substrate bands were resolved by SDS-PAGE and quantitated by phosphorimaging.
Subcellular Fractionation-- Cells stimulated with or without 10 µM isoproterenol were washed with ice-cold PBS, scraped into 5% (w/v) sucrose in buffer A (10 mM Tris, pH 7.4, 1 mM EDTA), and disrupted by Dounce homogenization (16). Nuclei and cell debris were removed by centrifugation at 500 × g for 10 min. The supernatant was loaded on a sucrose cushion (4 ml of 35% sucrose in buffer A) and centrifuged at 150,000 × g for 90 min at 4 °C. The 35% (vesicle) sucrose interface fractions were collected, diluted with buffer A, and pelleted. The pellets were resuspended in SDS sample buffer; 25 µg for each protein sample was analyzed. The presence of clathrin and Raf was detected by protein immunoblotting. Clathrin was detected using a 1:500 dilution of a monoclonal anti-clathrin IgM (ICN), and Raf-1 was detected using a 1:1000 dilution of a rabbit polyclonal anti-Raf-1 IgG (Santa Cruz Biotechnology). Immune complexes on nitrocellulose were detected using the appropriate horseradish peroxidase-conjugated secondary antibody and visualized by ECL. Binding assays were performed exactly as described (17).
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RESULTS AND DISCUSSION |
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To determine the role of GPCR internalization in signal
transduction we employed dominant suppressor mutants of -arrestin1 and dynamin. Dominant suppressor
-arrestin1 (V53D) prevents GPCR targeting to clathrin-coated pits, while the dominant suppressor form
of dynamin (K44A) inhibits fission of the budding vesicle from the
plasma membrane (18). Fig. 1A
depicts the effects of wild type and mutant
-arrestin1 and dynamin
expression on
2-AR internalization. Whereas
overexpression of the wild type
-arrestin1 protein increases
isoproterenol-mediated internalization of the
2-adrenergic receptor (
2-AR) modestly,
overexpression of wild type dynamin has no effect. In cells
overexpressing the dominant suppressor forms of these proteins
(
-arrestin1 V53D or dynamin K44A) a dramatic decrease in
agonist-mediated internalization of the
2-AR is
observed, in agreement with previous results (6, 19). The
-arrestin1
V53D mutant inhibits sequestration of the receptor by 50%, and the
dynamin K44A mutant inhibits sequestration by 70%.
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As shown in Fig. 1B, cells expressing -arrestin1 V53D or
dynamin K44A exhibit normal
2-AR coupling efficiency to
Gs, as measured by the accumulation of intracellular cAMP.
Similarly, coupling of the LPA receptor to Gi, as measured
by the accumulation of intracellular inositol phosphates, was
unaffected by expression of
-arrestin1 V53D or dynamin K44A proteins
(Fig. 1C). Thus, neither inhibitor of receptor sequestration
significantly impaired classical receptor-G protein-effector-mediated
generation of soluble second messengers.
Stimulation of the endogenous receptors for lysophosphatidic acid (LPA)
and isoproterenol (ISO; 2-AR) in HEK293 cells induces a
6- to 8-fold increase in phosphorylated MAP kinase (Erk1/2) levels
(Fig. 2A). Activation of MAP
kinases by the
2-AR in HEK293 cells, like in Cos-7 cells
(20), is c-Src- and Ras-dependent (21). Phosphorylation of
MAP kinase reflects the enzymatic activation of MEK (13). Expression of
-arrestin1 V53D and dynamin K44A mutants impaired the ability of
these receptors to activate MAP kinase (Fig. 2A).
-Arrestin1 V53D inhibited LPA- and ISO-mediated phosphorylation of
MAP kinase by 56% and 63%, respectively. Similarly, the dynamin K44A
protein inhibited LPA- and ISO-stimulated phosphorylation of MAP kinase
by 60% and 55%, respectively. Other known inhibitors of GPCR
sequestration, concanavalin A, monodansylcadavarine, and low
temperature (22-24), all inhibited LPA- and isoproterenol-stimulated MAP kinase phosphorylation by approximately 70% (data not shown). In
contrast, as shown in Fig. 2B, phorbol ester
(PMA)-stimulated MAP kinase phosphorylation in HEK293 cells, which is
not receptor-mediated, is not affected by the presence of
-arrestin1
V53D or dynamin K44A proteins. Together, these data demonstrate that
inhibition of receptor sequestration attenuates activation of MAP
kinase without affecting early, plasma membrane-delimited, signaling events such as receptor coupling to G proteins.
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Signal transduction between receptors on the plasma membrane and MAP
kinase in the cytosol requires a series of events leading first to the
assembly of an activated Ras-Raf complex on the membrane, followed by
initiation of the cytosolic MEK-MAP kinase cascade. To determine the
point in the signaling cascade at which receptor internalization is
required, we assayed the effects of -arrestin1 V53D and dynamin K44A
on two intermediate steps in the pathway: tyrosine phosphorylation of
the Shc adaptor protein and activation of the Raf kinase. Activation of
protein-tyrosine kinases by GPCRs and RTKs is obligatory for signal
transduction to MAP kinases (8-13; see Reaction 1). Coincident with
the increase in protein-tyrosine kinase activity is the tyrosine
phosphorylation of the adaptor protein Shc, thought to play an
important role in the nucleation and assembly of the multiprotein, Ras
activation complex (11, 12). In HEK293 cells, stimulation with LPA or
isoproterenol results in an increase of 4.2- or 3-fold, respectively,
in tyrosine phosphorylation of Shc relative to unstimulated cells (Fig.
3A). Expression of neither
-arrestin1 V53D nor dynamin K44A proteins had any effect on LPA- or
ISO-stimulated Shc phosphorylation (Fig. 3A). Stimulation of
the endogenous receptors for LPA or isoproterenol increases Raf-1
enzymatic activity 2-fold relative to unstimulated cells, in agreement
with results reported recently in rat hepatocytes (25). In cells
expressing
-arrestin1 V53D or dynamin K44A mutant proteins Raf-1
activity was similar to that in wild type cells following stimulation
of these endogenous receptors (Fig. 3B). Thus, the initial
steps in the GPCR-mediated MAP kinase cascade, including tyrosine
phosphorylation of adaptors and activation of Raf, are unaffected by
inhibitors of receptor sequestration.
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These data demonstrate that the process of vesicle-mediated endocytosis is required for mitogenic signaling initiated by G protein-coupled receptors. Inhibition of endocytosis blocks phosphorylation of MAP kinase, but does not affect plasma membrane-delimited processes such as receptor coupling to G proteins, tyrosine phosphorylation of Shc, or Raf activation. Rather, inhibition of endocytosis impairs signal transduction between activated Ras-bound Raf and the cytosolic MEK kinase. Regulation of Raf kinase activity has been shown to be complex; dependent, in part, upon its translocation to the plasma membrane and binding to Ras and 14-3-3 proteins, serine and tyrosine phosphorylation, and oligomerization (26, 27). Our data suggest an additional mechanism for regulating the biological actions of Raf; targeting the activated form of the enzyme to an intracellular compartment wherein its substrate, MEK, resides.
Since both -arrestin V53D and dynamin K44A exert their inhibitory
effects downstream of Raf, why does inhibition of receptor internalization (by
-arrestin V53D) impair the mitogenic signaling cascade initiated by the GPCRs? A likely possibility is that the agonist-occupied,
-arrestin-bound receptor actually comprises part
of a multicomponent signaling complex, assembled at the plasma membrane, which includes not only the receptor, but also various intermediates in the pathway up to and including Raf. Stimulation of
HEK293 cells with isoproterenol results in the internalization of
25-35% of the receptor into a vesicular compartment (17). In
addition, agonist treatment induces recruitment of soluble clathrin
onto the plasma membrane where it forms cage-like structures that
enclose a membrane vesicle (18). Following fission of the vesicle from
the plasma membrane, clathrin rapidly dissociates from the internalized
vesicle into the cytosolic compartment. Fig.
4 shows the agonist-mediated
translocation of
2-AR (4.2-fold), clathrin (3.7-fold),
and Raf (2.4-fold) proteins to the internalized vesicles. These data
support the notion that agonist stimulates the assembly of a
multiprotein signaling complex, including Raf, which is internalized by
the clathrin-coated vesicle pathway. These results, however, do not
exclude the possibility that Raf internalization, independent of
receptor, is required for signal transduction.
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A high degree of parallelism exists among GPCR- and RTK-mediated
mitogenic signal transduction pathways to MAP kinases. Both receptor
types utilize many of the same intermediates in this signaling cascade
(13). Recently, inhibition of TrkA (nerve growth factor receptor)
internalization was found to block the nerve growth factor-mediated
phosphorylation of the transcription factor CREB (28). Likewise, in
endocytosis-defective HeLa cells, epidermal growth factor
receptor-mediated activation of phospholipase C is unaffected, but
phosphorylation of MAP kinase is impaired (29). Taken together, these
data demonstrate yet another emerging analogy between RTKs and GPCRs in
mitogenic signal transduction, a requirement for receptor
internalization via clathrin-coated pits.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Sudhof for the cDNA encoding dynamin and Dr. P. Dent for purified (His)6-MEK. We thank Dr. D. Ginty for sharing data prior to publication, Dr. A. Howe for help with the Raf assay, and Drs. J. Pitcher, W. Wetzel, and D. Luttrell for helpful discussion and critical reading of the manuscript. We also thank M. Holben and D. Addison for excellent 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.
Recipient of a National Institutes of Health Clinical Investigator
Development Award.
§ Supported by National Institutes of Health MSTP Grant T32GM-07171.
¶ To whom correspondence should be addressed. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: lefko001{at}mc.duke.edu.
1
The abbreviations used are: GPCR, G
protein-coupled receptor; GRK, G protein-coupled receptor kinase; MAP,
mitogen-activated protein 2-AR.
2-adrenergic receptor; RTK, receptor tyrosine kinase;
LPA, lysophosphatidic acid; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; ISO, isoproterenol; PMA, phorbol
12-myristate 13-acetate.
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REFERENCES |
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