嵌合表皮生长因子疫苗E5T-mSEA的设计及抑瘤活性检测

表皮生长因子受体(Epidermal growth factor receptor,EGFR)及其配体在多种肿瘤细胞中高表达,免疫注射表皮生长因子(Epidermal growth factor,EGF)-载体蛋白所产生的抗体能够干扰EGFR与EGF的相互作用,进而抑制肿瘤生长。本实验室设计了EGF和TGFα的嵌合配体E5T,并将具有免疫增强作用的葡萄球菌肠毒素A(Staphylococcal enterotoxin A,SEA)与之融合。融合蛋白在大肠杆菌内表达后,通过金属螯合亲和层析,得到了纯度较高的E5T-mSEA融合蛋白。将E5T-mSEA免疫小鼠,能够产生高滴度的抗体,该抗体能够同时识别EGF和TGFα。将抗E5T-mSEA免疫血清与肿瘤细胞共培养,在1:10的稀释度下能显著抑制EGFR阳性肿瘤细胞的生长,而对EGFR阴性肿瘤细胞293T的生长没有明显影响。     

Mathematical model for the effects of epidermal growth factor receptor trafficking dynamics on fibroblast proliferation responses.

We apply a mathematical model for receptor-mediated cell uptake and processing of epidermal growth factor (EGF) to analyze and predict proliferation responses to fibroblastic cells transfected with various forms of the EGF receptor (EGFR) to EGF. The underlying conceptual hypothesis is that the mitogenic signal generated by EGF/EGFR binding on the cell surface, via stimulation of receptor tyrosine kinase activity, is attenuated when the receptors are downregulated and growth factor is depleted by endocytic internalization and subsequent intracellular degradation. Hence, the cell proliferation rate ought to depend on receptor/ligand binding and trafficking parameters as well as on intrinsic receptor signal transduction properties. The goal of our modeling efforts is to formulate this hypothesis in quantitative terms. The mathematical model consists of kinetic equations for binding, internalization, degradation, and recycling of EGF and EGFR, along with an expression relating DNA synthesis rate to EGF/EGFR complex levels. Parameter values have been previously determined from independent binding and trafficking kinetic experiments on B82 fibroblasts transfected with wild-type and mutant EGFR. We show that this model can successfully interpret literature data for EGF-dependent growth of NR6 fibroblasts transfected with wild-type EGFR. Moreover, it successfully predicts the literature observation that NR6 cells transfected with a delta 973 truncation mutant EGFR, which is kinase-active but internalization-deficient, require an order of magnitude lower EGF concentration than cells with wild-type EGFR for half-maximal proliferation rate. This result demonstrates that it may be feasible to genetically engineer mammalian cell lines with reduced growth factor requirements by a rational, nonempirical approach. We explore by further model computations the possibility of exploiting other varieties of EGFR mutants to alter growth properties of fibroblastic cells, based on relationships between changes in the primary structure of the EGF receptor and the rates of specific receptor/ligand binding and trafficking processes. Our studies show that the ability to predict cell proliferation as a function of serum growth factors such as EGF could lead to the designed development of cells with optimized growth responses. This approach may also aid in elucidation of mechanisms underlying loss of normal cell proliferation control in malignant transformation, by demonstrating that receptor trafficking dynamics may in some cases play as important a role as intrinsic signal transduction in determining the overall resulting mitogenic response.     

Response to transforming growth factor α (TGFα) and epidermal growth factor (EGF) in hepatocytes: Lower EGF receptor affinity of TGFα is associated with more sustained activation of p42/p44 mitogen-activated protein kinase and greater efficacy in stimulation of DNA synthesis

The epidermal growth factor (EGF) receptor mediates the effects of both EGF and transforming growth factor α (TGFα). Recent data suggested that EGF acts as a partial agonist/antagonist in hepatocytes, TGFα exerting a larger maximal stimulation of DNA synthesis than EGF. To further study the mechanisms involved in mediating the different effects of EGF and TGFα, we have examined receptor binding of the two growth factors and their action on the p42/p44 mitogen-activated protein (MAP) kinase activity in hepatocytes. Single-ligand concentration curves and competition experiments showed that the binding affinity to a common population of surface binding sites was about 20-fold lower for TGFα than for EGF. MAP kinase activity responded to EGF and TGFα with different kinetics. While the two agents produced almost identical acute (5 min) stimulation (peak about fivefold), TGFα produced a more sustained MAP kinase activity than EGF. The difference between EGF and TGFα was still detectable 24 h after growth factor addition. The results show that in hepatocytes a lower receptor affinity of TGFα, as compared to EGF, is associated with a more sustained activation of the MAP kinase and a greater efficacy in the stimulation of DNA synthesis. This suggests that differential interaction of these two agents with the EGF receptor results in differences in the downstream events elicited at a given level of receptor occupancy. The data also are compatible with a role of a prolonged MAP kinase activity in the mitogenic effects of EGF and TGFα. J. Cell. Physiol. 175:10–18, 1998. © 1998 Wiley-Liss, Inc.     

Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

The classic mode of G protein‐coupled receptor (GPCR)‐mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)‐mediated cleavage of plasma membrane‐anchored EGFR ligands. Herein, we show that the Gαs‐activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E₂ (PGE₂) transactivate EGFR through increased cell‐surface delivery of the EGFR ligand transforming growth factor‐α (TGFα) in polarizing madin‐darby canine kidney (MDCK) and Caco‐2 cells. This is achieved by PKA‐mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα‐containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE₂ rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser‐223, a process that is facilitated by the molecular scaffold A‐kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell‐surface delivery of TGFα and increased EGFR activation. Thus, GPCR‐triggered, PKA/AKAP12/NKD2‐regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.      

Growth factor/growth factor receptor loops in autocrine growth regulation of human prostate cancer DU145 cells

Autocrine growth factors produced by epithelial cells mediate the development and proliferation of neoplastic human prostate tissue. Various approaches have been used to down-regulate neoplastic growth of prostate cancer using natural flavonoids, soluble receptors, pseudo-ligands, monoclonal antibodies and tyrosine kinase inhibitors (tyrphostins). Selected growth factor/growth factor receptor loops (mainly TGFα/EGFR and IGFs/IGFIR) have been proposed as regulators of prostate cancer cell growth. We have previously determined that blockade of IGFIR or VEGF2R signaling pathways by tyrphostin AG1024 and SU1498 inhibits autocrine growth and viability of DU145 cells in vitro. Recently, we compared the activity of AG1024 and SU1498 with the inhibiting effect of tyrphostin A23 (a selective inhibitor of EGFR). The results described in this paper confirm that DU145 cells do not produce IGFI or EGF. In contrast, DU145 cells produce a great amount of VEGF, much more than TGFα (about 60-fold), and VEGF may be the real autocrine growth factor of the investigated cells. The results indicate that the growth of DU145 may be regulated by at least three autocrine loops: TGFα/EGFR, IGFII/IGFIR and VEGF/VEGFR2. Neither AG1024 nor SU1498 affected the production of TGFα substantially, which excludes the possibility that IGFRs or VEGFR2 inhibitors arrest the growth of these cells by inhibition of synthesis and/or secretion of TGFα. The obtained data indicate that all tree investigated tyrphostins (AG1024, SU1498 and A23) inhibit signal transmission by Akt (PKB), ERK(1/2), Src and STAT in a similar manner. A comparison of the effects of the investigated tyrphostins indicates that TGFα, IGFII and VEGF stimulate cell growth by affecting the same signaling pathway. The hypothesis was confirmed by the effect of the investigated tyrphostins on activation of EGFR. All these inhibitors decreased phosphorylation of EGFR to the same extent, and after the same time of incubation with cell culture. These results strongly suggest that stimulation of EGFR kinase is the main step in the initiation of mitogen signaling in DU145 cells, regardless of the type of ligand (TGFα, IGFs or VEGF) and their specific receptors.     

Structural basis for epidermal growth factor receptor function

The receptor for epidermal growth factor (EGF) has been the subject of intense study primarily as a consequence of the pioneering studies of Cohen on growth factors and also because of its homology to the transforming protein encoded by the avian oncogene v-erbB, which is a truncated receptor, and its consequent role in cancer. Although similar structural mutation of the EGF receptor has not yet been found in human tumours, aberrant overexpression of both EGF receptors and c-erbB2, a closely related putative receptor [1], have been found to occur in squamous cell carcinomas and glial tumours, and mammary carcinomas respectively [2–4]. In addition to EGF, the related polypeptides transforming growth factor α (TGFα) and vaccinia virus growth factor [5] are also ligands for the EGF receptor. Expression of TGFα occurs during embryonal development and in specific adult tissues; it may also play a role in cellular transformation (reviewed in Ref. 6). These important properties, as well as the potential roles of both TGFα and EGF in wound repair, have emphasized the need to understand EGF receptor structure, function and regulation. This review discusses the structural properties of the EGF receptor and how these can be related to receptor function and regulation.     

Effect of epidermal growth factor receptor internalization on regulation of the phospholipase C-gamma1 signaling pathway

The epidermal growth factor receptor (EGFR) ligands, epidermal growth factor (EGF), and transforming growth factor-alpha (TGFalpha) elicit differential postendocytic processing of ligand and receptor molecules, which impacts long-term cell signaling outcomes. These differences arise from the higher affinity of the EGF-EGFR interaction versus that of TGFalpha-EGFR in the acidic conditions of sorting endosomes. To determine whether EGFR occupancy in endosomes might also affect short-term signaling events, we examined activation of the phospholipase C-gamma1 (PLC-gamma1) pathway, an event shown to be essential for growth factor-induced cell motility. We found that EGF continues to stimulate maximal tyrosine phosphorylation of EGFR following internalization, while, as expected, TGFalpha stimulates markedly less. The resulting higher level of receptor activation by EGF, however, did not yield higher levels of phosphatidylinositol (4,5)-bisphosphate (PIP2) hydrolysis over those stimulated by TGFalpha. By altering the ratio of activated receptors between the cell surface and the internalized compartment, we found that only cell surface receptors effectively participate in PLC function. In contrast to PIP2 hydrolysis, PLC-gamma1 tyrosine phosphorylation correlated linearly with the total level of Tyr(P)-EGFR stimulated by either ligand, indicating that the functional deficiency of internal EGFR cannot be attributed to an inability to interact with and phosphorylate signaling proteins. We conclude that EGFR signaling through the PLC pathway is spatially restricted at a point between PLC-gamma1 phosphorylation and PIP2 hydrolysis, perhaps because of limited access of EGFR-bound PLC-gamma1 to its substrate in endocytic trafficking organelles.     

Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-α and epidermal growth factor in vivo

Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) are members of the EGF family of growth factors. They have a common receptor, the EGF receptor. This belongs to the tyrosine kinase group of receptors called the ErbB receptor family. Other members are ErbB-2, ErbB-3, and ErbB-4. Binding of either ligand to the receptor elicits an increase in tyrosine kinase activity, resulting in the autophosphorylation of the receptor followed by a phosphorylation cascade of other tyrosine kinase substrates including mitogen-activated protein kinase (MAPK). TGF-α and EGF have been shown to stimulate cell division in the olfactory epithelium in vitro and may regulate cell division in vivo. To investigate whether exogenous TGF-α or EGF has a functional effect on the olfactory mucosa in vivo, 12.5–50 μg of each growth factor was administered to rats via the carotid artery. After 2 min, olfactory mucosa and liver samples were collected, homogenized, and immunoprecipitated with antibodies to the ErbB receptors. The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblotting. Using phosphotyrosine antibody, the receptors were probed for phosphorylation. Activation of MAPK was also investigated using MAPK antibody. Exogenous TGF-α activated EGFR, ErbB-2 and MAPK, whereas EGF activated only the EGFR. TGF-α was a more potent activator of EGFR than EGF. Neither ligand had an effect on ErbB-3 and ErbB-4 receptors. These effects were absent in the control animals which received the same solution without the growth factor. These results are consistent with the notion that binding of TGF-α to EGFR may play a role in olfactory cell division in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 199–210, 1998     

Epidermal growth factor (EGF) receptor density controls mitogenic activation of normal rat kidney (NRK) cells by EGF

Normal rat kidney (NRK) fibroblasts are immortalized cells that are strictly dependent on externally added growth factors for proliferation. When cultured in the presence of epidermal growth factor (EGF) as the only growth stimulating hormone, these cells have a normal phenotype and undergo density-dependent growth inhibition. It has been postulated that this density-arrest results from a decrease of EGF receptor levels below a threshold level which makes these cells unresponsive to stimulation by EGF. In the present study, we show that NRK cells, made quiescent by serum-deprivation at submaximum density, are mitogenically still responsive to EGF, but show enhanced mitogenic stimulation after 8 hr pre-treatment with either transforming growth factor β (TGFβ) or retinoic acid (RA), while prostaglandin F_2α (PGF_2α) and bradykinin (BK) enhance the mitogenic stimulation by EGF only slightly under these conditions. Addition of TGFβ or RA results in an increase of both ¹²⁵I-EGF-binding capacity and EGF receptor mRNA levels. Using flow cytometric analysis, we show that pre-treatment with TGFβ or RA increases the percentage of cells entering the cell cycle as a function of time. Furthermore, pre-treatment of the cells with TGFβ or RA increases the rate of mitogen-activated protein kinase (MAPK) phosphorylation by EGF. PGF_2α and BK also increase EGF receptor levels, but only with delayed kinetics. These results show that already in serum-deprived quiescent NRK cells, EGF receptor levels limit EGF-induced mitogenic stimulation. This observation provides further evidence for the regulating role of the EGF receptor in density-dependent growth control of NRK cells. J. Cell. Physiol. 174:9–17, 1998. © 1998 Wiley-Liss, Inc.     

Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor‐alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB‐EGF (heparin‐binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C‐terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala⁴¹, Glu⁴⁴, and His⁴⁵. These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.     

Differential Effects of EGFR Ligands on Endocytic Sorting of the Receptor

Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-α causes receptor recycling. TGF-α therefore leads to continuous signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-α, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking.
We have compared the effect of six different ligands on endocytic trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-α and epiregulin lead to complete receptor recycling. EGF leads to lysosomal degradation of the majority but not all EGFRs. Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling. The Cbl ubiquitin ligases, especially c-Cbl, are responsible for EGFR ubiquitination after stimulation with all ligands, and persistent EGFR phosphorylation and ubiquitination largely correlate with receptor degradation.     

A differential requirement for the COOH-terminal region of the epidermal growth factor (EGF) receptor in amphiregulin and EGF mitogenic signaling

The epidermal growth factor receptor (EGFR) mediates the actions of a family of bioactive peptides that include epidermal growth factor (EGF) and amphiregulin (AR). Here we have studied AR and EGF mitogenic signaling in EGFR-devoid NR6 fibroblasts that ectopically express either wild type EGFR (WT) or a truncated EGFR that lacks the three major sites of autophosphorylation (c'1000). COOH-terminal truncation of the EGFR significantly impairs the ability of AR to (i) stimulate DNA synthesis, (ii) elicit Elk-1 transactivation, and (iii) generate sustained enzymatic activation of mitogen-activated protein kinase. EGFR truncation had no significant effect on AR binding to receptor but did result in defective GRB2 adaptor function. In contrast, EGFR truncation did not impair EGF mitogenic signaling, and in c'1000 cells EGF was able to stimulate the association of ErbB2 with GRB2 and SHC. Elk-1 transactivation was monitored when either ErbB2 or a truncated dominant-negative ErbB2 mutant (ErbB2-(1-813)) was overexpressed in cells. Overexpression of full-length ErbB2 resulted in a strong constitutive transactivation of Elk-1 in c'1000 but only slightly stimulated Elk-1 in WT or parental NR6 cells. Conversely, overexpression of ErbB2-(1-813) inhibited EGF-stimulated Elk-1 transactivation in c'1000 but not in WT cells. Thus, the cytoplasmic tail of the EGFR plays a critical role in AR mitogenic signaling but is dispensable for EGF, since EGF-activated truncated EGFRs can signal through ErbB2.     

Transforming growth factor alpha.

Transforming growth factor alpha (TGFα) is a close relative of epidermal growth factor (EGF), the first polypeptide mitogen discovered in 1962 (Cohen, 1962). TGFα, like EGF, exerts its effect on cells through binding to the EGF-Receptor (EGF-R). Here we review the molecular and cell biology of TGFα before proceeding to describe our own work on signaling molecules induced in response to activation of the EGF-R.     

The solution structures of epidermal growth factor and transforming growth factor alpha

The structures of human epidermal growth factor (EGF) and human transforming growth factor alpha (TGFα) have been determined in solution using nuclear magnetic resonance techniques. The features of each structure are described and similarities and differences between them are discussed. The structures are combined with information from sequence homologies to produce a model of the receptor-recognition sites of EGF and TGFα, which can be tested in a site-directed mutagenesis programme. The model assists in explaining previous observations of sequence-activity relationships. The TGFα and EGF structures also serve as models for homologous modules in other extracellular proteins.     

Epidermal growth factor and transforming growth factor alpha bind differently to the epidermal growth factor receptor

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) compete with each other for binding to the EGF receptor. These two growth factors have similar actions, but there are distinguishable differences in their biological activities. It has never been clear how this one receptor can mediate different responses. A monoclonal antibody to the EGF receptor (13A9) has been identified which has only small effects on the binding of EGF to the EGF receptor, but which has very large effects on the binding of TGF alpha to the EGF receptor; 5 micrograms/mL antibody has been shown to totally block 0.87 microM TGF alpha from binding to purified EGF receptor and to lower both the high- and low-affinity binding constants of TGF alpha binding to EGF receptor on A431 cells by about 10-fold. The 13A9 antibody causes a 2.5-fold stimulation of the tyrosine kinase activity of partially purified EGF receptor, compared to a 4.0-fold stimulation of the tyrosine kinase activity by EGF under the same conditions. The data suggest either that the antibody stabilizes a conformation of the EGF receptor which is not favorable for TGF alpha binding or that it blocks a part of the surface of the receptor which is necessary for TGF alpha binding but not EGF binding.     

Amphiregulin exosomes increase cancer cell invasion

Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGFα or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24?AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1?colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche.     

The membrane-anchoring domain of epidermal growth factor receptor ligands dictates their ability to operate in juxtacrine mode

All ligands of the epidermal growth factor (EGF) receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparin-binding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF, still required proteolytic release for activity, whereas ligands with the membrane-anchoring domain of HB-EGF could elicit full biological activity while still membrane anchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus. However, cell-mixing experiments and fluorescence resonance energy transfer studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.     

Epidermal growth factor: the receptor and its function

Epidermal growth factor (EGF) is a small polypeptide hormone with mitogenic properties in vivo and in vitro. EGF elicits biologic responses by binding to a cell surface receptor which is a transmembrane glycoprotein containing a cytoplasmic protein tyrosine kinase. EGF responses are mediated by ligand binding and activation of this intrinsic protein kinase. The receptor can be phosphorylated by other protein kinases, and this may regulate receptor function. Stimulation of the receptor tyrosine kinase activity by ligand binding must regulate the activity of an as yet undefined molecule(s) responsible for transmitting a mitogenic signal to the nucleus.     

Regulation of epidermal growth factor receptor internalization by G protein-coupled receptors

The epidermal growth factor (EGF) receptor (EGFR) plays a central role in regulating cell proliferation, differentiation, and migration. Cellular responses to EGF are dependent upon the amount of EGFR present on the cell surface. Stimulation with EGF induces sequestration of the receptor from the plasma membrane and its subsequent downregulation. Recently, internalization of the EGFR was also shown to be required for mitogenic signaling via the activation of MAP kinases. Therefore, mechanisms regulating internalization of the EGFR represent an important facet for the control of cellular response. Here, we demonstrate that EGFR is removed from the cell surface not only following stimulation with EGF, but also in response to stimulation of G protein-coupled lysophosphatidic acid (LPA) and beta2 adrenergic (beta2AR) receptors. Using a FLAG epitope-tagged EGFR to quantitate receptor internalization, we show that incubation with EGF, LPA, or isoproterenol (ISO) causes the time-dependent loss of cell surface EGFR. Internalization of EGFR by these ligands involves the tyrosine kinase activity of the receptor itself and c-Src, as well as the GTPase activity of dynamin. Unexpectedly, we find that internalization of the EGFR by EGF is dependent upon Gbetagamma and beta-arrestin proteins; expression of minigenes encoding the carboxyl terminii of the G protein-coupled receptor kinase 2, or beta-arrestin1, attenuates LPA-, ISO-, and EGF-mediated internalization of EGFR. Thus, G protein-coupled receptors can control the function of the EGFR by regulating its endocytosis.