Epidermal Growth Factor (EGF) Receptor-dependent ERK Activation by G Protein-coupled Receptors

A CO-CULTURE SYSTEM FOR IDENTIFYING INTERMEDIATES UPSTREAM AND DOWNSTREAM OF HEPARIN-BINDING EGF SHEDDING*

Kristen L. PierceDagger, Akira TohgoDagger, Seungkirl Ahn, Michael E. Field, Louis M. Luttrell§, and Robert J. Lefkowitz

From the Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Box 3821, Duke University Medical Center, Durham, North Carolina 27710 and the § Geriatrics Research, Education and Clinical Center, Durham Veterans Affairs Medical Center, Durham, North Carolina 27705

Received for publication, February 9, 2001, and in revised form, April 3, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha 2A-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, alpha 2AAR expressing "donor" cells are co-cultured with "acceptor" cells lacking the alpha 2AAR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha 2AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the alpha 2AAR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha 2AAR stimulation requires Gbeta gamma subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G protein-coupled receptors (GPCRs)1 can employ multiple distinct pathways to activate the ERK/MAPK cascade. At least one of these pathways, demonstrated for both Gi- and Gq-coupled receptors, involves transactivation of classical receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) (1-3). A characteristic feature of transactivation-dependent ERK activation is that stimulation of the GPCR leads to activation of the intrinsic tyrosine kinase activity of the RTK. Whereas the early steps leading from GPCR activation to tyrosine phosphorylation of the RTK have remained largely unknown, once transactivation of the RTK has occurred, RTK- and GPCR-mediated ERK activation are indistinguishable. Thus, tyrosine phosphorylation of the EGFR leads to the rapid tyrosine phosphorylation of adapter proteins such as SHC and Gab1 and the recruitment Grb2-Sos1 complexes to the activated RTK. The Ras guanine nucleotide exchange factor, Sos1, catalyzes the exchange of GDP for GTP on the low molecular weight G protein, Ras. Ras activation, in turn, initiates the phosphorylation cascade consisting of Raf, MEK, and ERK.

Recently, Prenzel et al.(4) have demonstrated that GPCR-stimulated tyrosine phosphorylation of the EGFR involves release of a soluble EGFR ligand, heparin-binding EGF (HB-EGF). HB-EGF is a single transmembrane-spanning protein that is proteolytically cleaved at a juxtamembrane site leading to the "shedding" of a soluble EGFR ligand that activates the EGFR in an autocrine/paracrine manner (5). GPCR-mediated HB-EGF release and EGFR transactivation are inhibited by CRM197 (4), a non-toxic diphtheria toxin mutant, which selectively binds and inactivates HB-EGF (6). HB-EGF shedding is also sensitive to the non-selective metalloprotease inhibitor, batimistat, indicating that one or more, as yet unknown, matrix metalloproteases function as novel effectors of GPCR signaling (4).

Little is presently known about how GPCRs control metalloprotease activity to induce regulated HB-EGF shedding. In this study, we have developed a mixed culture system to study the mechanism by which alpha 2A-adrenergic receptors (ARs) control HB-EGF shedding in COS-7 cells. In our model system, the release of HB-EGF from donor cells expressing alpha 2AARs is detected by measuring the response in acceptor cells lacking the GPCR. This permits signaling events upstream of HB-EGF shedding to be physically dissociated from those involved in the downstream response to endogenously generated paracrine signals. By selectively introducing dominant negative mutants of putative intermediate proteins into either the donor or acceptor cell pool, we have been able to determine their involvement in either the release of, or response to, HB-EGF. We find that alpha 2AAR-mediated shedding of HB-EGF requires both the release of Gbeta gamma subunits and Src kinase activity, while the response to HB-EGF is dependent upon the EGFR, Src kinases, and clathrin-mediated endocytosis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- LipofectAMINE and all tissue culture reagents were from Life Technologies. CRM197 was purchased from Sigma and pertussis toxin from List Biologicals. PP2 and tyrphostin AG1478 were from Calbiochem. Monoclonal HA.11 anti-hemagglutinin (HA) affinity beads were from Covance. The anti-phospho-ERK1/2 antibody was from Cell Signaling, and the anti-ERK1/2 antibody was from Upstate Biotechnology. Peroxidase-conjugated donkey anti-rabbit secondary antibody was from Amersham Pharmacia Biotech, and SuperSignal chemiluminescence reagent (Pierce) was used to develop blots.

Plasmids-- The plasmid encoding GFP-ERK2 was the gift of K. A. DeFea and N. W. Bunnett (University of California at San Francisco) and that encoding HA-ERK2 was the gift of J. Pouyssegur (University of Nice). The plasmid encoding K97A MEK1 came from E. G. Krebs (University of Washington) and the plasmid encoding c-Src kinase (CSK) came from H. Hanafusa (Rockefeller University). The plasmid encoding GST-beta ARKCT was constructed in our laboratory by excising a BamHI/NotI fragment from pRK5/beta ARKCT and ligating it into pEBG. The effects pEBG/beta ARKCT and pRK5/beta ARKCT were indistinguishable, and these constructs were used interchangeably. All other constructs were prepared in our laboratory.

Cell Culture and Transfections-- COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin in a humified 95% air, 5% CO2 incubator at 37 °C. Transient transfection of COS-7 cells was carried out at 70-80% confluency on the day of transfection. Cells in 10-cm plates were transfected with a total of 5-10 µg of DNA using a 1:6 ratio (w/v) of LipofectAMINE per dish following the manufacturer's instructions. One day following transfections, the cells were trypsinized and transferred to six-well dishes as described. Transfected cells were serum-starved overnight in Dulbecco's modified Eagle's medium supplemented with 10 mM HEPES and 0.1% bovine serum albumin prior to stimulation.

Immunoprecipitation and Immunoblotting-- Serum-starved transfected cells in six-well dishes were pretreated with the appropriate concentrations of inhibitors as indicated in the figure legends. Additionally, all cells were pretreated with the beta -adrenergic receptor antagonist, propranolol (1 µM), to block the activation of endogenous beta  receptors in COS-7 cells. Cells were exposed to agonist at 37 °C for the times indicated in the figure legends, washed once with ice-cold phosphate-buffered saline, lysed in 300 µl of glycerol lysis buffer (5 mM HEPES, 250 mM NaCl, 10% (v/v) glycerol, 0.5% Nonidet P-40, 2 mM EDTA, 100 µM NaV04, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin), and clarified by centrifugation. Before immunoprecipitation, 50 µl of the cell lysate was transferred to a separate tube and an equal volume of 2× Laemmli sample buffer was added to provide samples for the determination of GFP-ERK2 phosphorylation and plasmid expression. HA-ERK2 was immunoprecipitated using 20 µl of a 50% slurry of anti-HA affinity beads that had been mixed with an equal volume of CL4B-Sepharose and rotated overnight at 4 °C. Immune complexes were washed twice with cold glycerol lysis buffer and denatured in 2× Laemmli sample buffer. Immunoprecipitated proteins were resolved by protein electrophoresis on 4-20% SDS-polyacrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (PerkinElmer Biosystems). Phospho-ERK1/2 was detected using a 1:3000 dilution of a rabbit polyclonal phospho-ERK1/2 specific antibody (New England Biolabs), and total ERK1/2 was detected using a 1:2000 dilution of an ERK1/2 antibody (Upstate Biotechnology). Blots were probed with a 1:5000 dilution of a donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech). Proteins were visualized using Supersignal chemiluminescence reagent (Pierce), and the autoradiographs were quantitated using a Fluor-S MultiImager (Bio-Rad).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha 2AARs Employ At Least Two Distinct Mechanisms to Activate the ERK Cascade-- Fig. 1A schematically depicts the mixed culture system employed to dissociate transactivation-dependent ERK activation from EGFR-independent ERK activation after alpha 2AAR stimulation. Two populations of COS-7 cells were prepared by transient transfection. Donor cells expressed the alpha 2AAR, a receptor that is not endogenously expressed in COS-7 cells, along with HA epitope-tagged ERK2 (HA-ERK2), to allow ERK phosphorylation in the receptor-expressing population to be independently assayed. Acceptor cells in which the alpha 2AAR was not introduced were transfected with green fluorescent protein-tagged ERK2 (GFP-ERK2) to uniquely identify the ERK pool in those cells. Following transfection, the transfected cell populations were mixed and plated at high density using a ratio of donor/acceptor cells of 1:1.5. Following agonist stimulation, cell lysates were prepared, and anti-HA immunoprecipitation and subsequent immunoblotting were used to isolate the HA-ERK2 expressed in the donor cells. GFP-ERK2 phosphorylation in the acceptor cell pool was determined by immunoblotting of the whole cell lysates, since GFP-ERK2 could be resolved from the endogenous ERK pool based upon its slower electrophoretic mobility. This system permitted the simultaneous monitoring of ERK activation in the two separate populations of cells from the same dish.


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Fig. 1.   Co-culture system for measuring ERK phosphorylation simultaneously in two populations of cells, donor cells that express the alpha 2AAR and co-cultured acceptor cells that do not. A, schematic representation of the co-culture system. Donor COS-7 cells in 10-cm dishes were transfected with 50 ng of alpha 2AAR/pRK5 along with 1 µg of HA-ERK2/pCDNA3, whereas acceptor COS-7 cells were transfected with 1 µg of ERK2/pEGFP, which encodes a GFP-tagged form of ERK2 as described under "Experimental Procedures." One day after transfection, cells were trypsinized, and the donor and acceptor cells were cultured together in 6-well dishes at high density. Cells were serum-starved overnight before stimulation with 1 µM UK14304. Cells were then harvested and analyzed as described under "Experimental Procedures." Lysates and immunoprecipitations were analyzed with both phospho-ERK1/2 and total ERK1/2 antibodies to ensure that ERK expression was not affected by the presence of coexpressed plasmids. B, time course of alpha 2AAR-stimulated ERK phosphorylation in donor and acceptor cells. Co-cultured donor and acceptor cells were stimulated for the indicated times with 1 µM UK14304 at 37 °C and then harvested as indicated under "Experimental Procedures." The experiment shown is representative of four independent experiments.

As shown in Fig. 1B, stimulation of co-cultures with the selective alpha 2AR agonist UK14304 leads to a time-dependent increase in phospho-ERK2 immunoreactivity both in donor cells and in acceptor cells. The time course of ERK2 phosphorylation was similar in both populations, with maximal activation occurring within 5 min of agonist application. The acceptor cell response was more transient, however, with ERK2 phosphorylation returning to near basal levels within 30 min, whereas ERK2 phosphorylation was maintained in the donor cells for at least 2 h. Control experiments (not shown) demonstrated that UK14304 failed to induce ERK2 phosphorylation in COS-7 cells in the absence of transfected alpha 2AAR and that responses in the acceptor cells were only seen when the cells were cultured at high density. These data are consistent with a paracrine response in the acceptor cells that is mediated in response to the release of a soluble factor from the donor cells.

To determine the role of endogenous EGFRs in alpha 2AAR-mediated ERK2 activation, we examined the extent to which ERK activation in both donor and acceptor cells was sensitive to inhibitors of HB-EGF shedding and of EGFR signaling. Fig. 2 shows the results of experiments in which co-cultures were pretreated with either tyrphostin AG1478, a specific inhibitor of the EGFR tyrosine kinase (7), or with CRM197, a non-toxic diphtheria toxin analog that selectively binds to the EGF-like domain of HB-EGF (6). In the alpha 2AAR-expressing donor cells, maximally efficacious concentrations of CRM197 and tyrphostin AG1478 each inhibited only about 30% of UK14304-stimulated ERK activation (black bars). In contrast, the acceptor cell response in the same co-culture was almost completely blocked by either inhibitor (white bars). Whereas the alpha 2AAR-stimulated ERK response in the donor cells was only partially sensitive to tyrphostin AG1478, comparable EGF-stimulated ERK activated was essentially completely inhibited by tyrphostin AG1478 (data not shown). These data suggest that alpha 2AARs can employ at least two mechanisms to stimulate ERK2 phosphorylation in COS-7 cells, a direct pathway that is independent of HB-EGF shedding and the EGFR, and a transactivation-dependent pathway that requires both HB-EGF shedding and EGFR activation. In cells expressing the alpha 2AAR, the EGFR-independent pathway predominates. Pretreating cells with CRM197 and tyrphostin AG1478 in combination did not result in any further reduction in the donor cell response, indicating that HB-EGF shedding can account for all of the transactivation-dependent ERK activation in both donor and acceptor cells (data not shown).


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Fig. 2.   alpha 2AAR-stimulated ERK activation in donor and acceptor cells is differentially sensitive to inhibitors of HB-EGF shedding and EGFR tyrosine kinase activity. A, cells that were transiently transfected as described were pretreated with either 250 nM tyrphostin AG1478 (AG) or 10 µg/ml CRM197 for 20 min prior to a 5 min stimulation with 1 µM UK14304 (UK) at 37 °C. A representative experiment demonstrating the effects of AG1478 and CRM197 on ERK2 activation in cells that express alpha 2AAR (HA IP (Donor Cells)) or in co-cultured cells that do not express the alpha 2AAR (LYSATE (Acceptor Cells)) (A). B, results shown are the mean ± S.E. of seven independent experiments that were normalized to the percent maximal response found in cells that were not pretreated with any inhibitor.

In HB-EGF expressing CHO-K1 cells, shedding of HB-EGF in response to phorbol esters is sensitive to the MEK inhibitor, PD98059 (8). Similarly, in neutrophils, N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated L-selectin shedding is blocked by inhibitors of ERK or p38 MAP kinase signaling (9). These data suggest that MAP kinase activation may be required to initiate metalloprotease-dependent shedding. In contrast, EGFR activation in response to Gi- and Gq/11-coupled receptor activation in fibroblasts is insensitive to PKC inhibitors (4). To test whether in our system, ERK activity is required for alpha 2AAR-stimulated HB-EGF shedding, we transiently transfected the dominant inhibitory MEK1 inhibitor, K97A MEK1, into the donor cells and measured ERK activation in both the donor and the acceptor cells. As shown in Fig. 3, expression of K97A MEK1 in donor cells markedly attenuated ERK2 activation in the donor cell pool but had no effect on ERK2 activation in the acceptor cells. These data support the hypothesis that the EGFR-independent and HB-EGF-dependent pathways of ERK2 activation employed by alpha 2AAR are mechanistically independent. Tyrphostin AG1478 and CRM197 treatment blocks HB-EGF-mediated ERK activation in acceptor cells while not affecting the transactivation-independent component of the donor cell response (Fig. 2). Conversely, expression of K97A MEK1 in donor cells blocks the donor, but not the acceptor, cell response, indicating that ERK2 activation in the donor cells is not required for HB-EGF shedding (Fig. 3).


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Fig. 3.   Effect of dominant negative K97AMEK1 on alpha 2AAR-stimulated ERK activation in donor and acceptor cells. Cells were transfected as described in the legend to Fig. 1 with the addition of 5 µg of the MEK1 dominant inhibitory construct K97A MEK1 transfected into the donor cells. Following co-culture, donor and acceptor cells were stimulated with 1 µM UK14304 (UK) for 5 min at 37 °C prior to harvesting. Results shown are the mean ± S.E. of five independent experiments and were normalized to the percent maximal response of cells that were not transfected with K97A MEK1.

Gbeta gamma Subunits, Src Kinases, and Clathrin-mediated Endocytosis Are Involved in alpha 2AAR-mediated EGFR Transactivation-- In the co-culture system, alpha 2AAR-mediated ERK activation in acceptor cells reflects the release of HB-EGF from the donor cell pool. For ERK activation to occur in acceptor cells, signaling events leading to the release of HB-EGF must occur in donor cells, whereas events involved in the response to HB-EGF must occur in the acceptor cells. Thus, by introducing dominant inhibitory mutants of putative intermediate proteins selectively into either donor or acceptor cells, intermediates upstream and/or downstream of HB-EGF shedding and EGFR activation can be unambiguously identified.

To determine the role of heterotrimeric G protein subunits in alpha 2AAR-mediated EGFR transactivation, we assayed the effects of pertussis toxin and the Gbeta gamma subunit sequestrant polypeptide beta ARKCT on the ability of acceptor cells to respond to alpha 2AAR stimulation. As shown in Fig. 4, both pertussis toxin pretreatment and expression of the beta ARKCT peptide in donor cells inhibited ERK activation in both the donor cell and acceptor cell populations. Expression of beta ARKCT in acceptor cells had no effect upon alpha 2AAR-mediated ERK2 phosphorylation in either the donor or acceptor cells (not shown). These data indicate that consistent with prior reports (3), Gbeta gamma subunits derived from pertussis toxin-sensitive Gi/o proteins are necessary for alpha 2AAR-mediated transactivation of the EGFR.


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Fig. 4.   Effect of pertussis toxin and beta ARKCT expression on alpha 2AAR-stimulated ERK activation in donor and acceptor cells. For the pertussis toxin experiments, donor cells were transfected with plasmids encoding the alpha 2AAR and HA-ERK2 as described. The co-cultured donor and acceptor cells were treated for 16 h with 100 ng/ml pertussis toxin prior to agonist stimulation. To test the inhibitory effects of beta ARKCT on alpha 2AAR-stimulated ERK activation in the donor and the acceptor cells, donor cells were transfected with plasmids encoding the alpha 2AAR, HA-ERK2, and 5-10 µg of a plasmid encoding beta ARKCT. For both the pertussis toxin and beta ARKCT experiments, cells were exposed to 1 µM UK14304 (UK) for 5 min at 37 °C prior to harvesting the cells. Results shown are the mean ± S.E. of seven experiments with pertussis toxin and nine experiments with beta ARKCT. The result in each experiment was normalized to the response in cells that were not exposed to any inhibitor.

Src family nonreceptor tyrosine kinases are also known to play a role in GPCR-mediated ERK activation via transactivated EGFR (2, 3, 10). Uncertainty remains, however, as to whether these kinases are involved in regulating the release of HB-EGF or in the downstream response of the transactivated EGFR. As shown in Fig. 5A, treatment of co-cultures with the Src kinase inhibitor PP2 inhibits alpha 2AAR-mediated ERK2 phosphorylation in both donor and acceptor cells. To distinguish between the potential roles of Src kinases upstream or downstream of HB-EGF shedding, two different expressible Src inhibitors were selectively transfected into either the donor or the acceptor cells. As shown in Fig. 5B, the dominant inhibitory mutant K298M c-Src, when expressed in donor cells, significantly inhibited alpha 2AAR-stimulated ERK activation in both the donor and in the acceptor cells. When K298M c-Src is expressed in acceptor cells, alpha 2AAR-stimulated ERK activation in the acceptor cells is attenuated, whereas ERK activation in the donor cells is unaffected. As shown in Fig. 5C, comparable results were obtained when donor and acceptor cells were transfected with the physiological c-Src inhibitor, CSK, which inhibits c-Src activity by phosphorylating the regulatory carboxyl-terminal tyrosine of Src family kinases (11). These data are consistent with a role for Src kinase activity in both the direct and EGFR transactivation-dependent pathways. Moreover, in the transactivation pathway, c-Src is required both for alpha 2AAR-mediated HB-EGF shedding and for HB-EGF-dependent ERK activation via the transactivated EGFR, because inhibiting Src kinase activity in either the donor cell or the acceptor cell blocks the acceptor cell response.


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Fig. 5.   Effect of c-Src inhibitors on alpha 2AAR-stimulated ERK activation in donor and acceptor cells. A, cells transfected as described were pretreated with 5 µM PP2 for 20 min prior to stimulation with 1 µM UK14304 (UK) for 5 min at 37 °C. Results shown are the mean ± S.E. of five independent experiments that were normalized to the percent maximal response found in cells that were not pretreated with PP2. B, cells were transfected with 3-5 µg of the dominant inhibitory mutant K298M c-Src transfected in either donor cells or in acceptor cells. Cells were stimulated with 1 µM UK14304 for 5 min at 37 °C prior to harvesting. Results shown are the mean ± S.E. of four independent experiments and were normalized to the percent maximal response of cells that were not transfected with K298M c-Src. C, cells were transfected with 5 µg of plasmid DNA encoding the c-Src inhibitory kinase (CSK) in either the donor cells or in the acceptor cells. Cells were stimulated with 1 µM UK14304 for 5 min at 37 °C prior to harvesting. Results shown are the mean ± S.E. of seven independent experiments and were normalized to the percent maximal response of cells that were not transfected with CSK.

In HeLa cells, the ability of EGFR to mediate activation of the ERK cascade has previously been shown to involve clathrin-mediated endocytosis (12). Unlike many GPCRs, the alpha 2AAR does not undergo agonist-induced internalization (13). However, in alpha 2A-expressing COS-7 cells, UK14304 stimulation causes both EGFR internalization and ERK2 activation that is sensitive to inhibitors of clathrin-mediated endocytosis (14). Thus, we have previously proposed that inhibitors of clathrin-mediated endocytosis inhibit ERK activation not at the level of the GPCR itself but rather downstream of the EGFR (14). To test this hypothesis definitively, a dominant inhibitory form of dynamin I (K44A dynamin I) was expressed in either donor or acceptor cells, and the effect on ERK2 phosphorylation in the acceptor cells was measured. As shown in Fig. 6, K44A dynamin I expression in donor cells did not affect ERK activation in acceptor cells, whereas transfecting K44A dynamin I into the acceptor cells attenuated the alpha 2AAR-stimulated ERK activation. These data not only dissociate the early steps of alpha 2AAR activation from the requirement for clathrin-mediated endocytosis but also demonstrate that in the transactivation pathway dynamin-dependent endocytosis is required downstream of the EGFR.


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Fig. 6.   Effect of K44A dynamin I expression in donor or acceptor cells on alpha 2AAR-stimulated ERK activation in acceptor cells. Cells were transfected with 5 µg of plasmid DNA encoding either wild-type or K44A dynamin I (K44A DynI), an inhibitor of clathrin-mediated endocytosis, in donor or in acceptor cells. Cells were stimulated with 1 µM UK14304 for 5 min at 37 °C prior to harvesting. Results shown are the mean ± S.E. of seven independent experiments and were normalized to the percent maximal response in the acceptor cells when either the donor or the acceptor cells were transfected with wild-type dynamin I.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

With the system developed in these experiments, the transactivation pathway to ERK activation can be isolated from other pathways of ERK activation and intermediate steps localized as either upstream or downstream of HB-EGF shedding and the EGFR. Fig. 7 depicts a model of alpha 2AAR-stimulated ERK activation that is consistent with our data. In response to alpha 2AAR stimulation, ERK activation proceeds simultaneously via both a direct pathway and a transactivation-dependent pathway. The initial steps of both pathways are indistinguishable and involve both Gbeta gamma subunits released from pertussis toxin-sensitive G proteins and Src kinase activity. Following c-Src activation, the direct and the transactivation pathways diverge. The direct pathway leads to MEK activation and ERK phosphorylation that is independent of EGFR kinase activity. In the transactivation pathway, Src kinase activity is required for the induction of HB-EGF shedding, which is mediated by one or more as yet unidentified matrix metalloproteases. Consistent with previous reports (2, 3, 14), both c-Src activity and dynamin-dependent endocytosis are required downstream of the transactivated EGFR. Thus, Src kinases are apparently involved both upstream of HB-EGF shedding and downstream of the EGFR in the pathway of transactivation-dependent ERK activation.


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Fig. 7.   Model of alpha 2AAR-mediated ERK activation that proceeds via both a direct pathway and via a transactivation-dependent pathway. In the donor cells, both the direct pathway and the transactivation-dependent pathway contribute to alpha 2AAR-mediated ERK activation, whereas in the acceptor cells, alpha 2AAR-stimulated ERK activation proceeds only via HB-EGF-dependent EGFR transactivation. Both pathways require Gbeta gamma subunits released from pertussis toxin-sensitive G proteins and Src kinase activity. The pathways, however, diverge downstream of c-Src. The response of acceptor cells to HB-EGF is dependent upon both Src kinase activity and dynamin function.

To date, the mechanisms underlying Gbeta gamma activation of Src kinases in response to alpha 2AAR stimulation remain unknown. In the case of the beta 2-adrenergic receptor (15), the CXCR1 receptor (16), and the NK-1 receptor (17), the binding of Src family kinases to beta -arrestin results in recruitment of an activated tyrosine kinase to the GPCR following agonist stimulation. Other mechanisms of GPCR-mediated Src activation must exist, however. For example the beta 3-adrenergic receptor, which does not interact with beta -arrestins, recruits Src kinase via a direct interaction between the kinase and intracellular domains of the receptor (18). Like the beta 3-adrenergic receptor, alpha 2AARs do not undergo clathrin-mediated endocytosis (14, 18), and the mechanism for alpha 2AAR-stimulated c-Src activation remains enigmatic.

Previously, HB-EGF shedding has been shown to be downstream of phorbol 12-myristate 13-acetate (PMA)-stimulated ERK activation (8, 9). However, in our system, expression of K97A MEK1 in donor cells almost completely abolished ERK activation in the donor cells without affecting ERK activation in the acceptor cells. Although a role for ERK activation in alpha 2AAR-stimulated HB-EGF shedding cannot be entirely ruled out, our data strongly suggest that the direct and transactivation pathways diverge downstream of c-Src but upstream of MEK activation and that ERK activation probably does not contribute to alpha 2AAR-stimulated HB-EGF shedding in COS-7 cells.

An attractive hypothesis is that c-Src in the donor cells is a proximal regulator of the unknown metalloprotease responsible for HB-EGF shedding. Several metalloproteases of the ADAM family, which have been shown to regulate shedding of other EGFR ligands, including transforming growth factor alpha , have proline-rich Src-homology (SH3) binding domains in their cytoplasmic tails. Src family members are known to bind many of their substrates via SH3 domain interactions. Recently, activation of a c-Src family kinase was shown to regulate the ADAM-dependent shedding of the L1 adhesion molecule (19). In this case, L1 adhesion molecule shedding is regulated by two distinct pathways, a protein kinase C-dependent pathway that is ERK-dependent and a pervanadate pathway that is dependent on c-Src activation but independent of ERK activation. L1 shedding, then, may be analogous to HB-EGF shedding in which there are at least two pathways, an ERK-dependent pathway downstream of PKC activation and an ERK-independent pathway that involves c-Src activation. In the case of the alpha 2AAR, it is not ERK activation but rather activation of c-Src that is the major pathway contributing to HB-EGF shedding. Potentially, then, the metalloprotease responsible for GPCR-stimulated HB-EGF shedding may contain proline-rich SH3 binding domains.

Previous reports have implicated Src kinase activity in the cellular response to transactivated EGFRs. Activation of c-Src downstream of the EGFR may be involved in regulating catalytic activity of the EGFR and/or regulating dynamin-dependent endocytosis of the EGFR or a downstream intermediate. c-Src has been shown to phosphorylate two residues on the EGFR that increase the catalytic activity of the EGFR and regulate EGF-stimulated mitogenesis (20). GPCR-induced tyrosine phosphorylation of the adapter proteins Shc and Gab1 (2, 3) requires the activity of both the EGFR and Src kinases. c-Src may also regulate internalization of the EGFR by phosphorylating two components of the clathrin-dependent endocytosis system, clathrin and dynamin. Mutations of two tyrosines in dynamin that are phosphorylated by c-Src diminish EGFR internalization and ERK activation (21). Similarly, blocking EGF-stimulated c-Src-dependent phosphorylation of clathrin also inhibits EGFR internalization (22).

This model of transactivation-dependent ERK activation clearly dissociates endocytosis of the GPCR from the requirement for dynamin-dependent endocytosis in transactivation-dependent ERK activation. Our data demonstrate that all of the early steps in transactivation-mediated ERK activation up to and including activation of the metalloprotease are independent of clathrin-mediated endocytosis. In the acceptor cells, dynamin-dependent endocytosis is required either at or downstream of EGFR phosphorylation. Previous studies have demonstrated that, in the case of the beta 2-adrenergic receptor, all of the steps leading up to and including Raf activation were intact in cells expressing a dominant inhibitory form of dynamin (23, 24), suggesting that the Raf-MEK interface is the step regulated by dynamin-dependent endocytosis.

While helping to clarify the mechanisms whereby GPCRs mediate activation of the ERK cascade, our data do not provide insight into the functional significance of the direct and transactivation-dependent pathways. Why should such seeming redundancy exist? One possibility is that the consequences of ERK activation are determined to a significant degree by the mechanism by which they are activated. Activation of the ERK cascade via different pathways may provide a mechanism for regulating either the time course or spatial distribution of ERK activity, resulting in distinctly different consequences for the cell. Some evidence for this is provided by the finding that wild-type PAR-2 receptors, which cause beta -arrestin-dependent activation of a cytosolic pool of ERK1/2, do not mediate mitogenic responses in KNRK cells. In contrast, mutant PAR-2 receptors that cannot bind beta -arrestin, but still activate ERK1/2 through a calcium-dependent pathway, stimulate nuclear translocation of the ERK and provoke a proliferative response (25). Further experimentation with the system presented here should provide insight into the relevance of GPCR-mediated ERK activation in a host of physiological and pathophysiological conditions.

    ACKNOWLEDGEMENTS

We thank Donna Addison, Mary Holben, and Julie Turnbough for excellent secretarial assistance and Francine Roudabush for valuable technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL16037 (to R. J. L.) and DK55524 (to L. M. 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.

Dagger These authors contributed equally to this work.

Investigator of the Howard Hughes Medical Inst. To whom correspondence should be addressed. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: Lefko001@receptor-biol.duke.edu.

Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M101303200

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; EGFR, epidermal growth factor receptor; HA, hemagglutinin; MAP, mitogen-activated protein; CSK, c-Src kinase; GFP, green fluorescent protein; HB-EGF, heparin-binding EGF; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RTK, receptor tyrosine kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Daub, H., Weiss, F. U., Wallasch, C., and Ullrich, A. (1996) Nature 379, 557-560[CrossRef][Medline] [Order article via Infotrieve]
2. Daub, H., Wallasch, C., Lankenau, A., Herrlich, A., and Ullrich, A. (1997) EMBO J. 16, 7032-7044[Abstract/Free Full Text]
3. Luttrell, L. M., Della Rocca, G. J., van Biesen, T., Luttrell, D. K., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 4637-4644[Abstract/Free Full Text]
4. Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C., and Ullrich, A. (1999) Nature 402, 884-888[CrossRef][Medline] [Order article via Infotrieve]
5. Raab, G., and Klagsbrun, M. (1997) Biochim. Biophys. Acta 1333, F179-99[CrossRef][Medline] [Order article via Infotrieve]
6. Mitamura, T., Higashiyama, S., Taniguchi, N., Klagsbrun, M., and Mekada, E. (1995) J. Biol. Chem. 270, 1015-1019[Abstract/Free Full Text]
7. Levitzki, A., and Gazit, A. (1995) Science 267, 1782-1788[Medline] [Order article via Infotrieve]
8. Gechtman, Z., Alonso, J. L., Raab, G., Ingber, D. E., and Klagsbrun, M. (1999) J. Biol. Chem. 274, 28828-28835[Abstract/Free Full Text]
9. Fan, H., and Derynck, R. (1999) EMBO J. 18, 6962-6972[Abstract/Free Full Text]
10. Eguchi, S., Numaguchi, K., Iwasaki, H., Matsumoto, T., Yamakawa, T., Utsunomiya, H., Motley, E. D., Kawakatsu, H., Owada, K. M., Hirata, Y., Marumo, F., and Inagami, T. (1998) J. Biol. Chem. 273, 8890-8896[Abstract/Free Full Text]
11. Matsuda, M., Mayer, B. J., Fukui, Y., and Hanafusa, H. (1990) Science 248, 1537-1539[Medline] [Order article via Infotrieve]
12. Vieira, A. V., Lamaze, C., and Schmid, S. L. (1996) Science 274, 2086-2089[Abstract/Free Full Text]
13. Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720[Abstract/Free Full Text]
14. Pierce, K. L., Maudsley, S., Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1489-1494[Abstract/Free Full Text]
15. Luttrell, L. M., Ferguson, S. S. G., Daaka, Y., Miller, W. E., Maudsley, S., Della Rocca, G. J., Lin, F. T., Kawakatsu, H., Owada, K., Luttrell, D. K., Caron, M. G., and Lefkowitz, R. J. (1999) Science 283, 655-661[Abstract/Free Full Text]
16. Barlic, J., Andrews, J., Kelvin, A., Bosinger, S., DeVries, M., Xu, L., Dobransky, T., Feldman, R., Ferguson, S., and Kelvin, D. (2000) Nat. Immunol. 1, 227-233[CrossRef][Medline] [Order article via Infotrieve]
17. DeFea, K. A., Vaughn, Z. D., O'Bryan, E. M., Nishijima, D., Dery, O., and Bunnett, N. W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11086-11091[Abstract/Free Full Text]
18. Cao, W., Luttrell, L. M., Medvedev, A. V., Pierce, K. L., Daniel, K. W., Dixon, T. M., Lefkowitz, R. J., and Collins, S. (2000) J. Biol. Chem. 275, 38131-38134[Abstract/Free Full Text]
19. Gutwein, P., Oleszewski, M., Mechtersheimer, S., Agmon-Levin, N., Krauss, K., and Altevogt, P. (2000) J. Biol. Chem. 275, 15490-15497[Abstract/Free Full Text]
20. Biscardi, J. S., Maa, M. C., Tice, D. A., Cox, M. E., Leu, T. H., and Parsons, S. J. (1999) J. Biol. Chem. 274, 8335-8343[Abstract/Free Full Text]
21. Ahn, S., Maudsley, S., Luttrell, L. M., Lefkowitz, R. J., and Daaka, Y. (1999) J. Biol. Chem. 274, 1185-1188[Abstract/Free Full Text]
22. Wilde, A., Beattie, E. C., Lem, L., Riethof, D. A., Liu, S. H., Mobley, W. C., Soriano, P., and Brodsky, F. M. (1999) Cell 96, 677-687[Medline] [Order article via Infotrieve]
23. Kranenburg, O., Verlaan, I., and Moolenaar, W. H. (1999) J. Biol. Chem. 274, 35301-35304[Abstract/Free Full Text]
24. Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688[Abstract/Free Full Text]
25. DeFea, K. A., Zalevsky, J., Thoma, M. S., Dery, O., Mullins, R. D., and Bunnett, N. W. (2000) J. Cell Biol. 148, 1267-1281[Abstract/Free Full Text]


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