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Molecular Endocrinology, doi:10.1210/me.2004-0174
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Molecular Endocrinology 18 (11): 2727-2739
Copyright © 2004 by The Endocrine Society

Ectodomain Shedding-Dependent Transactivation of Epidermal Growth Factor Receptors in Response to Insulin-Like Growth Factor Type I

Hesham M. El-Shewy, Francine L. Kelly, Liza Barki-Harrington and Louis M. Luttrell

Departments of Medicine and Biochemistry and Molecular Biology (H.M.E.-S., L.M.L.), Medical University of South Carolina, Charleston, South Carolina 29425; Department of Medicine (F.L.K., L.B.-H.), Duke University Medical Center, Durham, North Carolina 27710; and The Ralph H. Johnson Veterans Affairs Medical Center (H.M.E.-S., L.M.L.), Charleston, South Carolina 29401

Address all correspondence and requests for reprints to: Louis M. Luttrell, Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, 816 Clinical Sciences Building, P.O. Box 250624, Charleston, South Carolina 29425. E-mail: luttrell{at}musc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growth factor (EGF) receptors. Here, we have examined the role of EGF receptors in IGF-I-stimulated ERK1/2 activation in several cultured cell lines. In human embryonic kidney 293 cells, IGF-I triggered proteolysis of heparin binding (HB)-EGF, increased tyrosine autophosphorylation of EGF receptors, stimulated EGF receptor inhibitor (AG1478)-sensitive ERK1/2 phosphorylation, and promoted EGF receptor endocytosis. In a mixed culture system that employed IGF-I receptor null murine embryo fibroblasts (MEFs) (R– cells) to detect paracrine signals produced by MEFs expressing the human IGF-I receptor (R+ cells), stimulation of R+ cells provoked rapid activation of green fluorescent protein-tagged ERK2 in cocultured R– cells. The R– cell response was abolished by either the broad-spectrum matrix metalloprotease inhibitor batimastat or by AG1478, indicating that it resulted from the proteolytic generation of an EGF receptor ligand from adjacent R+ cells. These data suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a general property of IGF-I receptor signaling. In contrast, the contribution of transactivated EGF receptors to IGF-I-stimulated downstream events, such as ERK1/2 activation, varies in a cell type-dependent manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE INSULIN RECEPTOR family consists of three members, the insulin receptor, the insulin-like growth factor type 1 (IGF-I) receptor, and the insulin receptor-related receptor, an orphan whose endogenous ligand is unknown. The three receptors share a common topology, consisting of two entirely extracellular {alpha}-subunits containing the ligand-binding domain, and two ß-subunits that consist of a single transmembrane domain and an intracellular catalytic domain possessing intrinsic ligand-stimulated tyrosine kinase activity. Like classical receptor tyrosine kinases, such as the epidermal growth factor (EGF) and platelet-derived growth factor receptors, stimulation of insulin family receptors leads both to receptor autophosphorylation and to membrane recruitment of signaling proteins containing phosphotyrosine-binding Src-homology 2 (SH2) domains, such as the Ras guanine nucleotide exchange factor complex Grb2/mSos, phospholipase C{gamma}, the p85/p110{alpha} phosphatidylinositol 3-kinase (PI3K) complex, and Src family nonreceptor tyrosine kinases. Unlike the EGF and PDGF receptors, however, autophosphorylated tyrosine residues of insulin family receptors do not appear to directly bind SH2 domain-containing proteins. Rather, the insulin and IGF-I receptors catalyze tyrosine phosphorylation of adapter proteins, such as insulin receptor substrate (IRS) proteins and Shc, which in turn serve as scaffolds for the assembly of multiprotein signal transduction complexes (1, 2).

Substantial data support the hypothesis that the two major tyrosine phosphoprotein scaffolds involved in insulin family receptor signaling, IRS proteins and Shc, play distinct signaling roles. For example, tyrosine phosphorylation of IRS-1 mediates recruitment of the p85/p110{alpha} PI3K complex, leading to both protein kinase B (Akt)-dependent suppression of the BAD/Bcl-X apoptotic pathway (3, 4), and to signals required for insulin-induced translocation of GLUT4 (5, 6). Whereas both IRS-1 and Shc can bind Grb2/mSos, the mitogenic response to insulin or IGF-I often correlates with Shc, but not IRS-1, phosphorylation (7, 8, 9). Furthermore, IRS proteins do not appear to be required for phosphorylation of Shc or for activation of ERK1/2 in many cell types, suggesting that the IRS-1 and Shc pathways function as independent transducers of distinct subsets of insulin responses.

We have previously reported that in COS-7 cells, IGF-I-stimulated tyrosine phosphorylation of Shc and activation of the ERK1/2 cascade occurs predominantly as a result of cross talk between IGF-I and EGF receptors, via a process termed EGF receptor transactivation (10). It has become increasingly apparent that EGF receptors function as a point of convergence for mitogenic signals arising from stimuli as diverse as activation of G protein-coupled (11, 12, 13) and cytokine receptors (14), integrin engagement (15), membrane depolarization (16, 17), and cell stress (18, 19). The best understood mechanism underlying this phenomenon involves stimulus-induced ectodomain shedding, the regulated intramembrane proteolysis of EGF receptor ligand precursors by the disintegrin metalloprotease (ADAM) family matrix metalloproteases (MMPs) (12, 20, 21). The proteolytic release of EGF receptor ligands, such as heparin-binding (HB)-EGF, results in autocrine/paracrine activation of EGF receptors leading to Ras-dependent cell proliferation.

EGF receptor transactivation in response to IGF-I may thus provide a mechanism for transmitting the mitogenic response to IGF-I. Indeed, the mitogenic and antiapoptotic effects of IGF-I in cultured human mammary epithelial cells appear to involve EGF receptor transactivation (22, 23). However, the contribution of EGF receptor transactivation to IGF-I-stimulated ERK1/2 activation seems to vary extensively depending on cell type. In proliferating 3T3-L1 preadipocytes, for example, IGF-I-stimulated ERK1/2 activation involves Shc, but not IRS-1 phosphorylation, yet is insensitive to inhibition of the EGF receptor (24).

Such findings suggest that IGF-I-stimulated EGF receptor transactivation occurs only in certain cell types. However, an alternative hypothesis is that the agonist-induced proteolytic release of EGF receptor ligands is a general property of IGF-I receptor signaling, but that transactivated EGF receptors make a variable contribution to individual downstream signals, such as ERK1/2 activation, depending on the cell-specific availability of alternative signaling routes. To discriminate between these alternatives, we have determined the contribution of EGF receptors to IGF-I-stimulated ERK1/2 activity in several cell lines. We find that although the sensitivity of IGF-I-stimulated ERK1/2 activation to EGF receptor inhibition varies widely, even cells that exhibit little or no transactivation-dependent ERK1/2 activation are capable of catalyzing HB-EGF proteolysis and stimulating EGF receptor activation in response to IGF-I. Using a mixed cell culture system that employs IGF-I receptor null cells as sensors for the paracrine release of EGF receptor ligands in response to IGF-I, we demonstrate that stimulation of a cell line in which IGF-I-stimulated ERK1/2 activation is EGF receptor independent nonetheless leads to EGF receptor-dependent ERK1/2 activation in adjacent cells. These data support the hypothesis that IGF-I receptor activation leads to the production of EGF receptor ligands that can affect ERK1/2 activity in both an autocrine and paracrine manner.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Different Cell Lines Vary in the Contribution of EGF Receptors to IGF-I-Stimulated ERK1/2 Phosphorylation
The existence of conflicting reports in the literature (10, 22, 23, 24) suggests that EGF receptor transactivation makes a variable contribution to IGF-I receptor-mediated ERK1/2 activation in different cell types. To assess the extent of this variability, we determined whether IGF-I-stimulated phosphorylation of endogenous ERK1/2 was sensitive to the EGF receptor kinase inhibitor tyrphostin AG1478 in four fibroblast lines: Rat-1, human embryonic kidney (HEK)-293, COS-7, and C57BL/6 MEFs. As shown in Fig. 1Go, pretreatment with AG1478 significantly inhibited IGF-I-stimulated ERK1/2 phosphorylation in three of the four lines, with the response in C57BL/6 MEFs exhibiting no AG1478 sensitivity. Furthermore, the extent of inhibition in the AG1478-sensitive cells varied from greater than 80% in Rat-1 cells to approximately 30% in HEK-293 cells. These data, which are consistent with previous reports, support the hypothesis that IGF-I receptors can employ multiple mechanisms to activate the ERK1/2 cascade.



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  		Fig. 1. Cell Type Variation in the Sensitivity of IGF-I-Stimulated ERK1/2 Activation to Tyrphostin AG1478

Serum-starved monolayers of rat-1 fibroblasts (A), COS-7 cells (B), HEK-293 cells (C), and C57BL/6 MEFs (D) were preincubated for 15 min in the presence or absence of 100 nM AG1478, before stimulation for 5 min with 10 nM human IGF-I or 1 ng/ml EGF. Basal, IGF-I-, and EGF-stimulated ERK1/2 phosphorylation were determined as described. In each panel, a representative anti-phospho ERK1/2 immunoblot is shown above a graph depicting mean ± SE for three independent experiments. NS, Not stimulated; WC, whole cell; IB, immunoblot.

 
IGF-I Stimulates HB-EGF Proteolysis, EGF Receptor Phosphorylation, and EGF Receptor Internalization in HEK-293 Cells
The ERK1/2 MAPKs are a downstream convergence point for external stimuli transiting multiple signal transduction cascades (25). Thus, insensitivity of IGF-I-stimulated ERK1/2 activation to inhibition of the EGF receptor does not exclude the possibility that EGF receptor transactivation is occurring even in cells where alternative pathways of ERK1/2 activation predominant. To initially address this hypothesis, we examined whether HEK-293 cells, in which IGF-I-stimulated ERK1/2 phosphorylation is only partially sensitive to AG1478 treatment, possess the ability to transactivate EGF receptors in response to IGF-I.

To determine whether stimulation of endogenous IGF-I receptors causes the proteolysis of EGF receptor ligand precursors, we assayed for stimulus-induced cleavage of influenza virus hemagluttinin (HA)/myc-HB-EGF in transiently transfected HEK-293 cells. As shown in Fig. 2Go, stimulation with IGF-I resulted in the proteolysis of approximately 40% of the cellular pool of HA/myc-HB-EGF within 5–10 min of stimulation. Treatment with phorbol myristate acetate (PMA), which causes protein kinase C (PKC)-dependent HB-EGF cleavage through activation of the MMP ADAM9 (26, 27) caused loss of 60% of the tagged HB-EGF pool with a similar time course.



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  		Fig. 2. IGF-I- and PMA-Stimulated Proteolysis of HA/myc-Tagged HB-EGF in Transfected HEK-293 Cells

Serum-starved monolayers of HEK-293 cells transiently expressing HA/myc-HB-EGF were stimulated for the indicated times with 10 nM IGF-I or 100 nM PMA. Intact HA/myc-HB-EGF was immunopreciptiated from detergent solubilized cell lysates, and HB-EGF cleavage was detected by immunoblotting for residual intact HA/myc-HB-EGF. Shown are representative anti-HA epitope immunoblots from IGF-I-stimulated (upper immunoblot) and PMA-stimulated (lower immunoblot) cells. Graphs depict mean ± SE for three independent experiments. *, Less than nonstimulated; P < 0.05. IP, Immunoprecipitate; IB, immunoblot.

 
We next determined whether IGF-I stimulation led to EGF receptor activation. As shown in Fig. 3AGo, 5-min exposure to IGF-I, EGF, or HB-EGF increased phosphorylation of Tyr-1068 of the endogenous HEK-293 cell EGF receptors, a major EGF receptor autophosphorylation site. The IGF-I-induced increase was approximately 10–20% of that provoked by saturating concentrations of EGF or HB-EGF. Another hallmark of activated EGF receptors is receptor internalization. Indeed, transactivation of EGF receptors in response to stimulation of serotonin 5-hydroxy tryptamine receptors causes the rapid internalization of endogeous EGF receptors in cultured renal mesangial cells (28). To determine whether IGF-I exposure stimulated EGF receptor internalization, we measured the loss of cell surface EGF receptors in response to IGF-I or EGF. As shown in Fig. 3BGo, 30 min treatment with EGF resulted in the loss of greater than 90% of cell surface EGF receptors. Consistent with the less robust increase in endogenous EGF receptor autophosphorylation in response to IGF-I, IGF-I stimulation caused internalization of about 20% of the EGF receptor pool.



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  		Fig. 3. IGF-I- and EGF-Stimulated Autophosphorylation and Internalization of Endogenous EGF Receptors in HEK-293 Cells

A, Untransfected serum-starved HEK-293 cells were stimulated for 5 min with 10 nM IGF-I, 1 ng/ml EGF, or 1 ng/ml HB-EGF. The upper immunoblot depicts the expression of endogeous EGF receptor in whole cell lysates, whereas the lower immunoblot depicts representative increases in EGF receptor autophosphorylation detected using phospho-Tyr1068-specific anti-EGF receptor IgG. B, Serum-starved HEK-293 cells were stimulated for 30 min with 10 nM IGF-I or 1 ng/ml EGF, after which internalization of endogenous EGF receptors was determined by measuring the percent loss of cell surface binding of [125I]-EGF. Data shown represent the mean ± SE for four independent experiments. *, Less than control, P < 0.05. NS, Not stimulated; WC, whole cell; IB, immunoblot.

 
To further examine EGF receptor signaling and trafficking in response to IGF-I, we employed cells stably expressing a green fluorescent protein (GFP)-tagged EGF receptor (29). As shown in Fig. 4AGo, expression of GFP-EGF receptor to levels approximately 2-fold over endogenous EGF receptor expression had no effect on expression of the endogenous IGF-I receptor ß-subunit. GFP-EGF receptor expression did modestly increase basal phosphorylation of Tyr-1068 of both the endogenous and GFP-tagged EGF receptors. As with the endogenous EGF receptor, IGF-I stimulation for 5 min resulted in an increase in EGF receptor autophosphorylation to a level about 10–15% of that observed in response to a maximally efficacious concentration of EGF.



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  		Fig. 4. IGF-I- and EGF-Stimulated Tyrosine Phosphorylation and Internalization of GFP-Tagged EGF Receptor in HEK-293 Cells

A, Serum-starved HEK-293 cells stably expressing GFP-EGF receptor were stimulated for 5 min with 10 nM IGF-I, 1 ng/ml EGF or 1 ng/ml HB-EGF. The upper immunoblots depict the expression of endogenous and GFP-tagged EGF receptors (left panel) and endogenous IGF-I receptor ß subunit (right panel), in untransfected (WT) and GFP-EGF receptor-expressing HEK-293 cells. The lower immunoblot depicts representative increases in endogenous and GFP-tagged EGF receptor autophosphorylation detected using phospho-Tyr1068-specific anti-EGF receptor IgG. B, GFP-EGF receptor-expressing HEK-293 cells were preincubated for 15 min in the presence or absence of 100 nM AG1478, before stimulation for 5 min with 10 nM IGF-I or 1 ng/ml EGF. The upper immunoblot depicts representative changes in the phosphotyrosine content of proteins detected in whole cell lysates. Also shown are the positions of GFP-EGF receptor and IGF-I receptor ß-subunit determined by reprobing stripped antiphosphotyrosine immunoblots using anti-GFP and anti-IGF-I receptor ß-subunit IgG, respectively. The lower immunoblot depicts ERK1/2 phosphorylation determined from the same samples. C, GFP-EGF receptor-expressing HEK-293 cells were preincubated for 15 min in the presence or absence of 100 nM AG-1478 or 5 µM batimastat (BB-94), before stimulation for 5 min with 10 nM IGF-I or 1 ng/ml EGF. The phosphotyrosine content of GFP-EGF receptors was determined from antiphosphotyrosine immunoblots of GFP-EGF receptor immunoprecipitates. A representative immunoblot is shown above a graph depicting mean ± SE for three independent experiments. *, Less than control, P < 0.05. D, GFP-EGF receptor-expressing HEK-293 cells were stimulated for 30 min with 10 nM IGF-I or 1 ng/ml EGF, before fixation and determination of the cellular distribution of GFP-EGF receptor by confocal fluorescence microscopy. Shown are representative confocal fields from one of three independent experiments that gave similar results. NS, Not stimulated; WC, whole cell; IB, immunoblot.

 
Because the GFP-EGF receptor migrates with an apparent molecular mass of about 210 kDa, we were able to visualize IGF-I- and EGF-stimulated phosphorylation of GFP-EGF receptor in antiphosphotyrosine immunoblots of whole cell lysates without interference from endogenous 180-kDa tyrosine phosphoproteins. Figure 4BGo depicts representative antiphosphotyrosine and anti-phospho-ERK1/2 immunoblots of whole cell lysates after stimulation with IGF-I or EGF. As shown, 5-min exposure to IGF-I increased tyrosine phosphorylation of the 210-kDa band corresponding to the GFP-EGF receptor fusion protein, as well as endogenous proteins of approximately 180 and 95 kDa. The 95-kDa phosphoprotein band corresponded to the IGF-I receptor ß-subunit, and thus reflected agonist-induced autophosphorylation of the IGF-I receptor catalytic domain. Stimulation with EGF induced robust tyrosine phosphorylation of GFP-EGF receptor, without increasing phosphorylation of the 180- and 95-kDa bands. Treatment with AG1478 blocked GFP-EGF receptor phosphorylation and inhibited ERK1/2 activation in response to both IGF-I and EGF. Significantly, tyrosine autophosphorylation IGF-I receptor ß-subunit was unaffected in AG1478-treated cells, indicating that the effect of AG1478 on ERK1/2 phosphorylation was not the result of nonspecific inhibition of IGF-I receptor autophosphorylation.

Figure 4CGo compares the effects of AG1478 and the broad-spectrum MMP inhibitor, batimastat, on IGF-I- and EGF-stimulated tyrosine phosphorylation of the GFP-EGF receptor. IGF-I stimulation induced a 5-fold increase in basal GFP-EGF receptor phosphorylation that was significantly reduced by both inhibitors. These data are consistent with the model that IGF-I stimulation transactivates the EGF receptor via the MMP-dependent generation of endogenous EGF receptor ligands that activate the intrinsic kinase activity of the EGF receptor. In contrast, EGF-stimulated GFP-EGF receptor phosphorylation was sensitive to AG1478, but unaffected by batimastat. These results are again predictable because EGF receptor phosphorylation in response to exogenously supplied EGF would require intact receptor kinase activity but should be independent of MMP activity.

Use of GFP-EGF receptor also permitted us to visualize the cellular redistribution of EGF receptors after stimulation with IGF-I and EGF using confocal fluorescence microscopy. As shown in Fig. 4DGo, GFP-EGF receptor was uniformly distributed along the plasma membrane of the transfected HEK-293 cells in the absence of stimulation. After 30 min exposure to either IGF-I or EGF, GFP-EGF receptors redistributed into large endosomal vesicles representing early endosomes (30). Thus, stimulation of HEK-293 cells with IGF-I produced autophosphorylation and internalization of GFP-EGF receptors that was similar to, but less robust than, the effect of an exogenously applied saturating dose of EGF.

IGF-I Stimulation Causes Paracrine EGF Receptor-Dependent ERK1/2 Activation by Stimulating the Release of Endogenous EGF Receptor Ligands
To definitively test the hypothesis that IGF-I stimulation causes the release of endogenous EGF receptor ligands, we devised a mixed cell culture system in which IGF-I receptor-null R– MEFs were employed as biological sensors to detect the paracrine release of EGF receptor ligands from IGF-I receptor-expressing R+ MEFs (31). As shown in Fig. 5AGo, the R+ MEFs overexpressed human IGF-I receptor approximately 50-fold compared with the level of endogenous murine IGF-I receptor expression in C57BL/6 MEFs. Interestingly, the R– MEFs lacking IGF-I receptors exhibited up-regulated expression of endogenous EGF receptors compared with control C57BL/6 MEFs. When the IGF-I receptor was overexpressed in the R– line, EGF receptor expression was down-regulated to below the level of detection by immunoblot. As shown in Fig. 5BGo, stimulation with 1 nM IGF-I did not cause activation of endogenous ERK1/2 in R– cells when cultured alone. R– MEFs did activate ERK1/2 in response to 1 ng/ml exogenously applied EGF, indicating that they possess an intact EGF receptor signaling network. Figure 5CGo depicts the responses of R+ MEFs under identical conditions. In these cells, IGF-I induced robust ERK1/2 phosphorylation. Like wild-type C57BL/6 MEFs, IGF-I-stimulated ERK1/2 phosphorylation in R+ cells was completely insensitive to tyrphostin AG1478. Consistent with the down-regulation of EGF receptors in the setting of IGF-I receptor overexpression, the R+ cells were themselves minimally responsive to EGF.



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  		Fig. 5. Effect of IGF-I and EGF on ERK1/2 Phosphorylation in IGF-I Receptor Null (R–) and Human IGF-I Receptor-Expressing (R+) MEFs

A, Expression of endogenous EGF receptor (upper panel) and IGF-I receptor ß subunit (lower panel) was determined from whole cell lysates of control C57BL/6 (WT), R–, and R+ MEFs. B, Serum-starved monolayers of R– MEFs were preincubated for 15 min in the presence or absence of 100 nM AG1478, before stimulation for 5 min with 1 nM IGF-I or 1 ng/ml EGF. Basal and IGF-I-, and EGF-stimulated ERK1/2 phosphorylation were determined as described. C, Serum-starved monolayers of R+ MEFs were preincubated for 15 min in the presence or absence of 100 nM AG-1478, before stimulation for 5 min with 1 nM IGF-I or 1 ng/ml EGF. Basal, IGF-I, and EGF-stimulated ERK1/2 phosphorylation were determined as described. In panels B and C, a representative anti-phospho ERK1/2 immunoblot is shown above a graph depicting mean ± SE for three independent experiments. NS, Not stimulated; WC, whole cell; IB, immunoblot.

 
To test whether stimulation of the IGF-I receptors in R+ MEFs, which are minimally responsive to EGF, nonetheless caused them to release EGF receptor ligands that could act on adjacent cells, R+ MEFs were cultured in a 50:50 mixture with EGF responsive, but IGF-I unresponsive, R– MEFs. To discriminate R– MEF responses in the mixed culture, the R– MEFs were transiently transfected with GFP-tagged ERK2 before mixing with untransfected R+ cells. Expression of GFP-ERK2, which migrates with an apparent molecular mass of approximately 70 kDa, permitted simultaneous visualization of both endogenous ERK1/2 and GFP-ERK2 in samples derived from mixed monolayers. Responses in the R– cell population were then measured as increases in GFP-ERK2 phosphorylation in whole cell lysate immunoblots. The GFP-ERK2 chimera has previously been shown to undergo growth factor-stimulated phosphorylation and nuclear translocation in a manner analogous to endogenous ERK1/2 (32). This experimental model is depicted schematically in Fig. 6AGo.



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  		Fig. 6. Stimulation of IGF-I Receptors Expressed in R+ MEFs Leads to Paracrine Activation of GFP-ERK2 Expressed in IGF-I Receptor Null R– MEFs in a Mixed Culture System

A, Schematic depiction of IGF-I receptor mediated paracrine signaling in a mixed cell culture containing IGF-I receptor-expressing in R+ MEFs (donor) and IGF-I receptor null R– MEFs (acceptor). B, Representative immunoblots depicting the time course of IGF-I-stimulated phosphorylation of endogenous ERK1/2 and transiently expressed GFP-ERK2 in R– and R+ MEFs cultured alone or in a 50:50 mixed culture. Serum-starved monolayers were stimulated for the indicated times with 3 nM IGF-I. Phosphorylation of endogenous ERK1/2 (lower bands) and GFP-ERK2 (upper band in R– and R–/R+ cultures) were determined in immunoblots of whole cell lysates as described. C, Graph comparing the time course of endogenous ERK1/2 phosphorylation in R+ cells cultured alone (open circles), with that of GFP-ERK2 in R– cells cultured either alone (open squares) or in mixed culture with R+ MEFs (closed circles). Shown are mean ± SE for three independent experiments. *, Greater than R– alone; P < 0.05. WC, Whole cell; IB, immunoblot.

 
Figure 6Go, B and C, depicts the time courses of ERK1/2 and GFP-ERK2 phosphorylation in R–, R+, and mixed R–/R+ cultures in response to 3 nM IGF-I. In isolated GFP-ERK2 expressing R– MEFs, IGF-I stimulation had no significant effect on endogenous ERK1/2 phosphorylation over the 30 min duration of the experiment. Because of the low basal phosphorylation state of GFP-ERK2, which increases the sensitivity of the assay, modest IGF-I-stimulated increases in GFP-ERK2 phosphorylation were detectable in R– MEFs, probably reflecting the presence of endogenous insulin or IGF-II receptors in the IGF-I receptor null MEFs. In contrast, IGF-I stimulation of isolated R+ MEFs caused a robust increase in endogenous ERK1/2 phosphorylation that peaked within 5 min of stimulation and declined gradually thereafter. When mixed R–/R+ cell cultures were stimulated with IGF-I, a substantial enhancement in GFP-ERK2 phosphorylation was observed. The time course of IGF-I-stimulated GFP-ERK2 phosphorylation paralleled the change in ERK1/2 phosphorylation in the endogenous pool. The increase in IGF-I-stimulated GFP-ERK2 phosphorylation in IGF-I receptor-null R– MEFs growing in the presence of IGF-I receptor-expressing R+ cells is consistent with a paracrine response in R– MEFs arising from the action of IGF-I on adjacent R+ MEFs.

Figure 7Go compares the dose dependence of IGF-I-stimulated endogenous ERK1/2 phosphorylation in R+ cells cultured alone with that of GFP-ERK2 expressed in R– MEFs in mixed R–/R+ cultures. The EC50 for IGF-I-stimulated ERK1/2 phosphorylation in R+ MEFs was 2.0 nM, vs. 6.3 nM for IGF-I-stimulated GFP-ERK2 phosphorylation in R– MEFs in the mixed cultures. Given that the affinity of insulin and IGF-II receptors for IGF-I is approximately two orders of magnitude lower than for their native ligand, these data are consistent with the hypothesis that IGF-I binding to its receptor expressed in R+ MEFs mediated the IGF-I-stimulated increase in GFP-ERK2 phosphorylation in the R– MEFs.



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  		Fig. 7. Dose Dependence of IGF-I-Stimulated Activation of Endogenous ERK1/2 in R+ MEFs and IGF-I-Stimulated Paracrine Activation of GFP-ERK2 in R– Cells in Mixed R–/R+ Cultures

Serum-starved R+ MEFs cultured alone (upper immunoblot), and GFP-ERK2-expressing R– MEFs in mixed R–/R+ cultures (lower immunoblot) were stimulated for 5 min with the indicated concentrations of IGF-I. Phosphorylation of endogenous ERK1/2 and GFP-ERK2 were determined in immunoblots of whole cell lysates as described. The graph depicts mean ± SE for three independent experiments. WC, Whole cell; IB, immunoblot.

 
To determine the mechanism underlying the paracrine activation of GFP-ERK2 in R–/R+ cultures, we tested whether the response was sensitive to inhibitors of EGF receptor transactivation. As shown in Fig. 8Go, IGF-I-stimulated GFP-ERK2 phosphorylation in R–/R+ cultures was blocked by pretreatment with either the MMP inhibitor, batimastat, or the EGF receptor inhibitor, AG1478. IGF-I stimulation of endogenous ERK1/2 in the mixed culture was not significantly affected because this response largely reflects ERK1/2 activation in the R+ MEFs, which is EGF receptor independent (Fig. 4BGo). EGF stimulation produced robust phosphorylation of GFP-ERK2 in R–/R+ cultures that was sensitive to AG1478, but insensitive to batimastat, consistent with the direct action of exogenously applied EGF on the R– MEF population. EGF stimulation of endogenous ERK1/2 in the mixed culture resembled the GFP-ERK2 response. Because R+ cells are largely unresponsive to direct application of EGF, the effect of EGF on ERK1/2 phosphorylation in R–/R+ cultures, like the effect on GFP-ERK2, reflects its action on the EGF-responsive R– MEFs. These data strongly support the hypothesis that R+ cells can induce paracrine activation of the ERK1/2 cascade in nearby cells through the MMP inhibitor-sensitive generation of EGF receptor ligands that transactivate EGF receptors on R– MEFs.



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  		Fig. 8. Tyrphostin AG1478 and MMP Inhibitor Sensitivity of IGF-I-Stimulated Paracrine Activation of GFP-ERK2 in R– Cells in Mixed R–/R+ Cultures

Serum-starved GFP-ERK2-expressing R– MEFs in mixed culture with R+ MEFs were preincubated for 15 min in the presence or absence of 100 nM AG1478 or 5 mM batimastat (BB-94), before stimulation for 5 min with 3 nM human IGF-I or 1 ng/ml EGF. Phosphorylation of endogenous ERK1/2 and GFP-ERK2 were determined in immunoblots of whole cell lysates as described. A representative anti-phospho ERK1/2 immunoblot is shown above a graph depicting mean ± SE for three independent experiments. *, Less than NS; P < 0.05. NS, Not stimulated; WC, whole cell; IB immunoblot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our results suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a property shared by many IGF-I-responsive cells. Using two different model systems, we are able to demonstrate that IGF-I receptor activation can trigger proteolysis of EGF receptor ligand precursors, cause MMP inhibitor-sensitive activation of the intrinsic tyrosine kinase activity of the EGF receptor, induce EGF receptor internalization, and initiate a paracrine signaling cascade that activates the ERK pathway in adjacent cells through EGF receptor transactivation. In the mixed culture system, IGF-I-stimulated release of soluble EGF receptor ligands can be detected even when the ligand donor (R+ MEFs) is itself minimally responsive to direct application of EGF.

In contrast, the contribution of EGF receptor transactivation to IGF-I-stimulated activation of the ERK1/2 cascade varies widely between cell types. Of the cell lines employed in this study, three (Rat 1a, COS-7, and HEK-293) exhibited significant sensitivity to the EGF receptor inhibitor tyrphostin AG1478, whereas a fourth (C57BL/6 MEFs) did not. IGF-I receptor-null MEFs overexpressing the human IGF-I receptor (R+ cells) also exhibited AG1478-insensitive ERK1/2 activation in response to IGF-I. Interestingly, IGF-I-stimulated ERK1/2 activation tended to be less robust in the more AG1478-sensitive cells lines, and more robust in cells that exhibited less AG1478 sensitivity. This is consistent with the hypothesis that multiple mechanisms exist for regulation of ERK1/2 activity, and that in cells where alternative mechanisms predominate, EGF receptor transactivation represents only a minor component of the total ERK1/2 activation signal.

The link between receptor tyrosine kinases and the small G protein, Ras, is the GRB2-mSos Ras guanine nucleotide exchange factor complex. GRB2-mSos can bind a number of phosphorylated scaffolds in addition to EGF receptors and Shc, among them IRS-1 (33), Gab1 (34), and focal adhesion kinase (35). Thus, IGF-I receptor-mediated phosphorylation of IRS-1 should be able to directly support Ras-dependent signaling, independent of IGF-I receptor-mediated EGFR transactivation. Previous work has shown that the relative contributions of Shc and IRS-1 to insulin-stimulated ERK1/2 activation varies between cell types and is probably determined by the relative levels of expression of the two adapter proteins (7, 8, 36). In cells that express insulin family receptors and IRS-1 at relatively low levels, activation of Ras and ERK1/2 is mediated primarily via Shc phosphorylation (7, 8), and in COS-7 cells the principal mechanism underlying IGF-I-stimulated Shc phosphorylation appears to be EGF receptor transactivation (10).

Each of the known endogenous EGF receptor ligands, which include EGF, TGF{alpha}, HB-EGF, amphiregulin, betacellulin, and epiregulin (37), is synthesized as a transmembrane precursor that undergoes regulated proteolysis to produce a soluble growth factor. Because ligands such as HB-EGF remain associated with the membrane glycocalyx and are likely to achieve significant concentrations only over short distances from their source, the hormonally stimulated production of endogenous EGF receptor ligands has been difficult to detect directly (38, 39, 40). Nonetheless, using techniques such as measuring the degradation or secretion of epitope-tagged ligand precursors (26), treating native cells with MMP inhibitors or catalytically inactive variants of Diptheria toxin (10, 41), or employing mixed culture systems to detect paracrine signals (41, 42, 43), it has been possible to demonstrate that the autocrine/paracrine release of soluble EGF-like ligands accounts for EGF receptor transactivation by such diverse stimuli as phorbol esters (27, 44, 45), ionizing radiation (46), and activation of G protein-coupled (41, 42, 47), and IGF-I (10) receptors.

Regulated intramembrane proteolysis of EGF receptor ligand precursors is mediated by members of the ADAM family of MMPs (48). Nonetheless, the mechanisms of receptor dependent metalloprotease regulation are poorly understood. PKC-dependent HB-EGF cleavage in response to phorbol esters reportedly involves the metalloprotease ADAM 9 (27, 44), cell adhesion (26) and MAPK activity (26, 47). In contrast, HB-EGF cleavage in response to activation of Gi- and Gq/11-coupled G protein-coupled receptors involves neither PKC nor ADAM 9 (41). In the heart, a related MMP, ADAM 12, catalyzes G protein-coupled receptor-mediated HB-EGF shedding (49). Several of the ADAMs, notably ADAM-9, -10, -12, -15, -17, and -19, possess consensus SH3 domain binding motifs within their short intracellular domains that might mediate interaction with Src kinases, and Src kinase activity has been implicated in the regulation of MMP-dependent ectodomain shedding (42). Further work will be required to specifically identify the intermediates involved in IGF-I receptor-mediated proteolysis of EGF receptor ligands.

Several previous studies have demonstrated codependence of IGF-I and EGF receptor-mediated signals in the control of cell proliferation and anchorage-independent growth (50, 51, 52). Simultaneous overexpression of IGF-I and EGF receptors in BALB/c3T3 cells confers the ability to grow for prolonged periods in serum-free medium, whereas overexpression of IGF-I receptor alone does not (50). Similarly, MEFs expressing wild-type levels of IGF-I receptor along with overexpressed EGF receptors can grow in EGF alone and form colonies in soft agar, but IGF-I receptor null MEFs overexpressing EGF receptors cannot (51). In MEFs, IGF-I receptor expression is required for prolonged activation of the ERK1/2 cascade in response to EGF (52). The explanation for these phenomena appears to be that EGF stimulation increases the expression of IGF-I mRNA and stimulates secretion of IGF-I into the medium (50). Collectively, these data suggest that EGF growth responses in vitro may, in part, derive from transactivation of IGF-I receptors through the transcriptionally regulated secretion of IGF-I.

Our finding of IGF-I-stimulated release of EGF receptor ligands adds another dimension to the cross talk between IGF-I and EGF receptors. If IGF-I stimulation promotes the release of EGF-like ligands, whereas EGF stimulation increases IGF-I transcription and secretion, the potential exists for an autostimulatory loop controlling cell proliferation. Although the physiologic relevance of such a mechanism is unknown, the in vitro data would be consistent with such a model. If so, the finding that IGF-I receptors can mediate ERK1/2 activation via autocrine/paracrine release of HB-EGF suggests the intriguing prospect that it may be possible to selectively block the proliferative or hypertrophic effects of IGF-I by targeting the extracellular components of the feedback loop without adversely affecting metabolic effects of IGF-I or insulin that are mediated through IRS protein phosphorylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Recombinant human IGF-I and PMA were purchased from Sigma Chemical Co. (St. Louis, MO). EGF, HB-EGF, and tyrphostin AG1478 were from Calbiochem (San Diego, CA). Batimastat, BB-94, was generously provided by A. Ullrich (Max Planck Institute for Biochemistry, Martinsried, Germany). [125I]-EGF was from DuPont NEN (Boston, MA). Tissue culture media and supplements were from Invitrogen Life Technologies (Carlsbad, CA). LipofectAMINE and Plus reagents were purchased from Invitrogen Life Technologies. FuGene 6 was from Roche Diagnostics (Indianapolis, IN). Rabbit polyclonal anti-phospho-ERK1/2 IgG was from Cell Signaling (Beverly, MA). Monoclonal anti-ERK1/2 IgG, polyclonal phospho-Tyr1068-specific anti-EGF receptor IgG, and monoclonal antihuman EGF receptor (R1) IgG were from Upstate Biotechnology (Lake Placid, NY). Monoclonal anti-IGF-I receptor ß subunit IgG, rabbit polyclonal anti-HA epitope IgG, and rabbit polyclonal anti-GFP IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-HA affinity agarose was from CoVance Research Products (Denver, PA). Horseradish peroxidase-conjugated monoclonal antiphosphotyrosine (PY20) IgG was purchased from Transduction Laboratories (Lexington, KY). Horseradish peroxidase conjugated donkey antirabbit IgG and donkey antimouse IgG were from Jackson ImmunoResearch (West Grove, PA).

cDNA Constructs
The pCR3.1 expression plasmid encoding HB-EGFTM-HA/Myc (26) was from M. Klagsbrun (Harvard Medical School, Boston, MA). The pEGFP-N1 expression plasmids encoding GFP-EGF receptor (29) and GFP-ERK2 (32) were provided by A. Sorkin (University of Colorado Health Sciences Center, Denver, CO) and K. A. DeFea and N. Bunnett (University of California at San Francisco, San Francisco, CA), respectively.

Cell Culture and Transfection
Rat-1a fibroblasts, COS-7 cells, and HEK-293 cells were obtained from the American Type Culture Collection (Manassas, VA). Spontaneously immortalized murine embryo fibroblasts (MEFs) derived from C57 BL/6 embryos were a gift from R. Lefkowitz (Duke University, Durham, NC), and were prepared as described (53). Clonal HEK-293 cells stably expressing the GFP-EGF receptor were isolated after transfection using LipofectAMINE and selection with neomycin. MEFs derived from IGF-I receptor null embryos (R– cells), as well as R– cells stably expressing the human IGF-I receptor (R+ cells), were generous gifts from R. Baserga (Thomas Jefferson University, Philadelphia, PA), and were prepared as described (31).

HEK-293 cells were grown in Eagle’s MEM with Earle’s salt supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin. Rat 1a fibroblasts, COS-7 cells, and MEF lines were grown in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin. Transient transfection of HEK-293 cells was performed using FuGene 6. Subconfluent monolayers in 100-mm dishes were transfected in growth medium using 6 µg of pCR3.1 HB-EGFTM-HA/Myc and 36 µl Fugene 6 according to the manufacturer’s instructions. Transient transfection of R– cells was performed using LipofectAMINE and Plus reagents. Confluent cells (50–60%) in 100-mm dishes were refed with fresh growth medium, then transfected by the dropwise addition of 10 µg of pEGFP-N1-GFP-ERK2 premixed with LipofectAMINE and Plus reagents in 1 ml of plain DMEM. Transient transfection efficiency using either technique was 70–80% as assessed by visualizing GFP expression under fluorescence microscopy. After 24–48 h, transfected cells were split into six- or 12-well plates as appropriate, then incubated overnight in serum-free growth medium supplemented with 10 mM HEPES (pH 7.4), 0.1% BSA, and 100 U/ml penicillin and streptomycin before stimulation.

Proteolysis of HA/myc Tagged-HB-EGF
Proteolytic cleavage of HB-EGF was measured as the stimulus-induced loss of HA epitope from detergent lysates of HEK-293 cells transiently expressing HA/myc-HB-EGF. Serum-starved cells were stimulated with IGF-I or PMA as described in the figure legend, washed with ice cold PBS, solubilized in 1.0 ml of glycerol lysis buffer [50 mM HEPES, 50 mM NaCl, 10% (vol/vol) glycerol, 0.5% (vol/vol) Nonidet P-40, 2 mM EDTA, 100 µM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, 2.5 µg/ml aprotinin], and clarified by centrifugation. Clarified lysates were agitated overnight at 4 C with 20 µl of 50% slurry of monoclonal anti-HA affinity agarose to immunoprecipitate intact HA-epitope-tagged HB-EGF. Immune complexes containing intact HA/myc-HB-EGF were resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane for immunoblotting. Immunoblots were performed using a 1:1000 dilution of rabbit polyclonal anti-HA IgG, with horseradish peroxidase-conjugated polyclonal donkey antirabbit IgG as secondary antibody. Immunoprecipitated proteins on PVDF filters were visualized by enzyme-linked chemiluminescence using SuperSignal (Pierce Biotechnology, Inc. Rockford, IL) chemiluminescence reagent and immunoblots were quantified using a Fluor-S MultiImager (BioRad, Hercules, CA).

EGF Receptor Phosphorylation
EGF receptor autophosphorylation and whole cell tyrosine phosphorylation were assayed in immunoblots performed on whole cell lysates. Serum-starved cultures in six-well plates were stimulated as described in the figure legends. Monolayers were washed with 4 C PBS before lysis in 200 µl of Laemmli sample buffer, and approximately 20 µg of cell protein from each sample was resolved by SDS-PAGE. Autophosphorylated EGF receptors were detected using polyclonal phospho-Tyr1068-specific anti-EGF receptor IgG, with horseradish peroxidase-conjugated polyclonal donkey antirabbit IgG as secondary antibody. Whole cell and EGF receptor tyrosine phosphorylation was detected using horseradish peroxidase-conjugated monoclonal antiphosphotyrosine (PY20) IgG. PVDF filters were stripped of Ig and reprobed using monoclonal anti-IGF-I receptor ß-subunit IgG, monoclonal antihuman EGF receptor IgG, or polyclonal anti-GFP IgG to confirm the identity of major phosphoprotein bands.

EGF Receptor Internalization
Stimulus-induced internalization of endogenous EGF receptors was measured as the loss of cell surface [125I]-EGF binding as previously described (28). Briefly, serum-starved HEK293 cells grown in six-well plates were incubated with IGF-I, EGF or vehicle for 30 min at 37 C. All subsequent washes and incubations were performed at 4 C. Monolayers were washed twice with PBS, once with acid wash buffer [50 mM glycine, 100 mM NaCl (pH 3.0)] to remove any cell surface bound EGF, and three times with PBS. Cells were then incubated with 50 pM [125I]-EGF for 90 min in HEPES binding medium [DMEM, 40 mM HEPES, 0.1% BSA (pH 7.4)] to bind EGF receptors remaining on the plasma membrane. Cells were washed three times with PBS, dissolved in 1 ml of 1 M NaOH, and bound counts measured by scintillation counting. Nonspecific binding, which represented less than 10% of the total binding in vehicle-treated cells, was determined in wells containing 100 ng/ml unlabeled EGF and was subtracted from total binding to yield specific [125I]-EGF binding.

Internalization of the GFP-EGF receptor was assayed by confocal fluorescence microscopy. Serum-starved cells in collagen-coated 35-mm glass bottom dishes were stimulated as described in figure legend, washed with PBS, fixed with 10% paraformaldehyde for 30 min at room temperature, and again washed with PBS before examination. Confocal microscopy was performed using a Zeiss LSM510 laser scanning microscope using a Zeiss 63 x 1.4 numerical aperture water immersion lens with 488-nm excitation and 515- to 540-nm emission filters. (Carl Zeiss MicroImaging, Inc., Thornwood, NY).

ERK1/2 Phosphorylation
Phosphorylation of endogenous ERK1/2 and transiently expressed GFP-ERK2 were determined by immunoblotting using phosphorylation site-specific antisera. Serum-starved cultures in 12-well plates were incubated in the presence or absence of inhibitors for 15 min, before stimulation as described in the figure legends. After stimulation, monolayers were washed with 4 C PBS, and lysed in 200 µl of Laemmli sample buffer. For the determination of total cellular ERK1/2 and phospho-ERK1/2, aliquots containing approximately 20 µg of cell protein were resolved by SDS-PAGE. ERK1/2 and phospho-ERK2 were detected by protein immunoblotting using polyclonal anti-ERK1/2 and anti-phospho-ERK1/2 antisera, respectively, with horseradish peroxidase-conjugated polyclonal donkey antirabbit IgG used as secondary antibody. Immune complexes were visualized by enzyme-linked chemiluminescence, and quantified using a Fluor-S MultiImager. In each experiment, equal loading of ERK1/2 protein was confirmed by probing parallel immunoblots using anti-ERK1/2 IgG.


   ACKNOWLEDGMENTS
 
The authors thank Vincent Capizzi (Duke University, Durham, NC) for excellent technical assistance.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants DK58283 (to L.M.L.).

Abbreviations: ADAM, A disintegrin and metalloprotease; EGF, epidermal growth factor; ERK1/2, extracellular signal-regulated kinases 1 and 2; G protein, guanine nucleotide-binding protein; GFP, green fluorescent protein; HA, influenza virus hemagluttinin; HB, heparin-binding; HEK, human embryonic kidney; IRS, insulin receptor substrate; MEF, murine embryo fibroblast; MMP, matrix metalloprotease; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol myristate acetate; PVDF, polyvinylidene difluoride; SH2, src homology domain 2; SH3, src homology domain 3.

Received for publication April 27, 2004. Accepted for publication July 16, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Medema RH, Bos JL 1993 The role of p21ras in receptor tyrosine kinase signaling. Crit Rev Oncog 4:615–661[Medline]
  2. White MF 1997 The insulin signalling system and the IRS proteins. Diabetologia 40(Suppl 2):S2–S17
  3. Franke TF, Kaplan DR, Cantley LC 1997 PI3K: downstream AKTion blocks apoptosis. Cell 88:435–437[CrossRef][Medline]
  4. Del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G 1997 Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278:687–689[Abstract/Free Full Text]
  5. Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M 1994 Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem 269:3568–3573[Abstract/Free Full Text]
  6. Hara K, Yonezawa K, Sakaue H, Ando A, Kotani K, Kitamura T, Kitamura Y, Ueda H, Stephens L, Jackson TR 1994 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci USA 91:7415–7419[Abstract/Free Full Text]
  7. Sasaoka T, Draznin B, Leitner JW, Langlois WJ, Olfesky JM 1994 Shc is the predominant signaling molecule coupling insulin receptors to activation of guanine nucleotide releasing factor and p21ras-GTP formation. J Biol Chem 269:10734–10738[Abstract/Free Full Text]
  8. Sasaoka T, Rose DW, Jhun BH, Saltiel AR, Draznin, Olfesky JM 1994 Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J Biol Chem 269:13689–13694[Abstract/Free Full Text]
  9. Boney M, Gruppuso PA, Faris RA, Frackelton Jr AR 2000 The critical role of Shc in insulin-like growth factor-I-mediated mitogenesis and differentiation in 3T3–L1 preadipocytes. Mol Endocrinol 14:805–813[Abstract/Free Full Text]
  10. Roudabush FL, Pierce L, Maudsley S, Khan KD, Lin F-T, Luttrell LM 2000 Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells. J Biol Chem 275:22583–22589[Abstract/Free Full Text]
  11. Luttrell LM, Daaka Y, Lefkowitz RJ 1999 Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol 11:177–183[CrossRef][Medline]
  12. Hackel PO, Zwick E, Prenzel N, Ullrich A 1999 Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Curr Opin Cell Biol 11:184–189[CrossRef][Medline]
  13. Carpenter G 1999 Employment of the epidermal growth factor receptor in growth factor-independent signaling pathways. J Cell Biol 146:697–702[Abstract/Free Full Text]
  14. Yamaguchi T, Ueki K, Tobe K, Tanemoto H, Sekine N, Wada M, Honjo M, Takahashi M, Takahashi T, Harai H, Tushima T, Akanuma Y, Fujita T, Komuro I, Yazaki Y, Kadowaki T 1997 Tyrosine phosphorylation of the EGF receptor by the kinase Jak2 is induced by growth hormone. Nature 390:91–96[CrossRef][Medline]
  15. Moro L, Venturino M, Bozzo C, Silengo L, Altruda F, Beguinot L, Tarone G, Defillippi P 1998 Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival. EMBO J 17:6622–6632[CrossRef][Medline]
  16. Rosen LB, Greenberg ME 1996 Stimulation of growth factor receptor signal transduction by activation of voltage-sensitive calcium channels. Proc Natl Acad Sci USA 93:1113–1118[Abstract/Free Full Text]
  17. Zwick E, Daub H, Aoki N, Yamaguchi-Aoki Y, Tinhofer I, Maly K, Ullrich A 1997 Critical role of calcium-dependent epidermal growth factor receptor transactivation in PC12 cell membrane depolarization and bradykinin signaling. J Biol Chem 272:24767–24770[Abstract/Free Full Text]
  18. Rosette C, Karin M 1996 Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science 274:1194–1197[Abstract/Free Full Text]
  19. Goldkorn T, Balaban AN, Shannon M, Matsukuma K 1997 EGF receptor phosphorylation is affected by ionizing radiation. Biochim Biophys Acta 1358:289–299[Medline]
  20. Zwick E, Hackel PO, Prenzel N, Ullrich A 1999 The EGF receptor as central transducer of heterologous signalling systems. Trends Pharmacol Sci 20:408–412[CrossRef][Medline]
  21. Gschwind A, Zwick E, Prenzel N, Leserer M, Ullrich A 2001 Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene 20:1594–1600[CrossRef][Medline]
  22. Gilmore AP, Valentijn AJ, Wang P, Ranger AM, Bundred N, O’Hare MJ, Wakeling A, Korsmeyer SJ, Streuli CH 2002 Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor. J Biol Chem 277:27643–27650[Abstract/Free Full Text]
  23. Ahmad T, Farnie G, Bundred NJ, Anderson NG 2004 The mitogenic action of insulin-like growth factor I in normal human mammary epithelial cells requires the epidermal growth factor receptor tyrosine kinase. J Biol Chem 279:1713–1719[Abstract/Free Full Text]
  24. Boney CM, Sekimoto H, Gruppuso PA, Frackelton Jr AR 2001 Src family tyrosine kinases participate in insulin-like growth factor I mitogenic signaling in 3T3–L1 cells. Cell Growth Differ 12:379–386[Abstract/Free Full Text]
  25. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH 2001 Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22:153–183[Abstract/Free Full Text]
  26. Gechtman Z, Alonso JL, Raab G, Ingber DE, Klagsbrun M 1999 The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading. J Biol Chem 274:28828–28835[Abstract/Free Full Text]
  27. Izumi Y, Hirata M, Hasuwa H, Iwamoto R, Umata T, Miyado K, Tamai Y, Kurisaki T, Sehara-Fujisawa A, Ohno S, Mekada E 1998 A metalloprotease-disintegrin, MDC9/meltrin-{gamma}/ADAM9 and PKC{delta} are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J 17:7260–7272[CrossRef][Medline]
  28. Grewal JS, Luttrell LM, Raymond JR 2001 G protein-coupled receptors desensitize and down-regulate epidermal growth factor receptors in renal mesangial cells. J Biol Chem 276:27335–27344[Abstract/Free Full Text]
  29. Carter RE, Sorkin A 1998 Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera. J Biol Chem 273:35000–35007[Abstract/Free Full Text]
  30. Hunyady L, Baukal AJ, Gaborik Z, Olivares-Reyes JA, Bor M, Szaszak M, Lodge R, Catt KJ, Balla T 2002 Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor. J Cell Biol 157:1211–1222[Abstract/Free Full Text]
  31. Sell C, Rubini M, Rubin R, Liu JP, Efstratiadis A, Baserga R 1993 Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type 1 insulin-like growth factor receptor. Proc Natl Acad Sci USA 90:11217–11221[Abstract/Free Full Text]
  32. DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett NW 2000 ß-Arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol 148:1267–1281[Abstract/Free Full Text]
  33. Myers Jr MG, Wang LM, Sun X, Zhang Y, Yenush L, Schlessinger J, Pierce JH, White MF 1994 Role of IRS-1-GRB-2 complexes in insulin signaling. Mol Cell Biol 14:3577–3587[Abstract/Free Full Text]
  34. Nishida K, Hirano T 2003 The role of Gab family scaffolding adapter proteins in the signal transduction of cytokine and growth factor receptors. Cancer Sci 94:1029–1033[CrossRef][Medline]
  35. Parsons JT 2003 Focal adhesion kinase: the first ten years. J Cell Sci 116:1409–1416[Abstract/Free Full Text]
  36. Rose DW, Saltiel AR, Majumdar M, Decker SJ, Olefsky JM 1994 Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction. Proc Natl Acad Sci USA 91:797–801[Abstract/Free Full Text]
  37. Riese DJ II, Stern DF 1998 Specificity within the EGF family/ErbB receptor family signaling network. Bioessays 20:41–48[CrossRef][Medline]
  38. Daub H, Weiss FU, Wallasch C, Ullrich A 1996 Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 379:557–560[CrossRef][Medline]
  39. Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T 1998 Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 273:88960–88968
  40. Tsai W, Morielli D, Peralta EG 1997 The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity. EMBO J 16:4597–4605[CrossRef][Medline]
  41. Prenzel N, Zwick E, Daub E, Leserer M, Abraham R, Wallasch C, Ullrich A 1999 EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 402:884–888[Medline]
  42. Pierce KL, Tohgo A, Ahn S, Field ME, Luttrell LM, Lefkowitz RJ 2001 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. J Biol Chem 276:23155–23160[Abstract/Free Full Text]
  43. Ahmed I, Gesty-Palmer D, Drezner MK, Luttrell LM 2003 Transactivation of the epidermal growth factor receptor mediates parathyroid hormone and prostaglandin F2 {alpha}-stimulated mitogen-activated protein kinase activation in cultured transgenic murine osteoblasts. Mol Endocrinol 17:1607–1621[Abstract/Free Full Text]
  44. Goishi K, Higashiyama S, Klagsbrun M, Nakano N, Umata T, Ishikawa M, Mekada E, Taniguchi N 1995 Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity. Mol Biol Cell 6:967–980[Abstract]
  45. Dong J, Wiley HS 2000 Trafficking and proteolytic release of epidermal growth factor receptor ligands are modulated by their membrane-anchoring domains. J Biol Chem 275:557–564[Abstract/Free Full Text]
  46. Dent P, Reardon DB, Park JS, Bowers G, Logsdon C, Valerie K, Schmidt-Ullrich R 1999 Radiation-induced release of transforming growth factor {alpha} activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death. Mol Biol Cell 10:2493–2506[Abstract/Free Full Text]
  47. Fan H, Derynck R 1999 Ectodomain shedding of TGF-{alpha} and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades. EMBO J 18:6962–6972[CrossRef][Medline]
  48. Schlondorff J, Blobel CP 1999 Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding. J Cell Sci 112:3603–3617[Abstract]
  49. Asakura M, Kitakaze M, Takashima S, Liao Y, Ishikura F, Yoshinaka T, Ohmoto H, Node K, Yoshino K, Ishiguro H, Asanuma H, Sanada S, Matsumura Y, Takeda H, Beppu S, Tada M, Hori M, Higashiyama S 2002 Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat Med 8:35–40[CrossRef][Medline]
  50. Pietrzkowski Z, Sell C, Lammers R, Ullrich A, Baserga R 1992 Roles of insulin-like growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells. Mol Cell Biol 12:3883–3889[Abstract/Free Full Text]
  51. Burgaud JL, Baserga R 1996 Intracellular transactivation of the insulin-like growth factor I receptor by an epidermal growth factor receptor. Exp Cell Res 223:412–419[CrossRef][Medline]
  52. Swantek JL, Baserga R 1999 Prolonged activation of ERK2 by epidermal growth factor and other growth factors requires a functional insulin-like growth factor 1 receptor. Endocrinology 140:3163–3169[Abstract/Free Full Text]
  53. Kohout TA, Lin FS, Perry SJ, Conner DA, Lefkowitz RJ 2001 ß-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking. Proc Natl Acad Sci USA 98:1601–1606[Abstract/Free Full Text]



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Mol. Pharmacol., January 1, 2007; 71(1): 259 - 275.
[Abstract] [Full Text] [PDF]