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• W2078112275 abstract "Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR) family. Many cell types express multiple EGFR family members (including EGFR, HER2, HER3, and/or HER4) that interact to form an array of homo- and heterodimers. Differential trafficking of these receptors should strongly affect signaling through this system by changing substrate access and heterodimerization efficiency. Because of the complexity of these dynamic processes, we used a quantitative and computational model to understand their integrated operation. Parameters characterizing EGFR and HER2 interactions were determined using experimental data obtained from mammary epithelial cells constructed to express different levels of HER2, enabling us to estimate receptor-specific internalization rate constants and dimer uncoupling rate constants. Significant novel results obtained from this work are as follows: first, that EGFR homodimerization and EGFR/HER2 heterodimerization occur with comparable affinities; second, that EGFR/HER2 heterodimers traffic as single entities. Furthermore, model predictions of the relationship of HER2 expression levels to consequent distribution of EGFR homodimers and EGFR/HER2 heterodimers suggest that the levels of HER2 found on normal cells are barely at the threshold necessary to drive efficient heterodimerization. Thus, altering HER2 concentrations, either overall or local, could provide an effective mechanism for regulating EGFR/HER2 heterodimerization and may explain why HER2 overexpression found in some cancers has such a profound effect on cell physiology. Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR) family. Many cell types express multiple EGFR family members (including EGFR, HER2, HER3, and/or HER4) that interact to form an array of homo- and heterodimers. Differential trafficking of these receptors should strongly affect signaling through this system by changing substrate access and heterodimerization efficiency. Because of the complexity of these dynamic processes, we used a quantitative and computational model to understand their integrated operation. Parameters characterizing EGFR and HER2 interactions were determined using experimental data obtained from mammary epithelial cells constructed to express different levels of HER2, enabling us to estimate receptor-specific internalization rate constants and dimer uncoupling rate constants. Significant novel results obtained from this work are as follows: first, that EGFR homodimerization and EGFR/HER2 heterodimerization occur with comparable affinities; second, that EGFR/HER2 heterodimers traffic as single entities. Furthermore, model predictions of the relationship of HER2 expression levels to consequent distribution of EGFR homodimers and EGFR/HER2 heterodimers suggest that the levels of HER2 found on normal cells are barely at the threshold necessary to drive efficient heterodimerization. Thus, altering HER2 concentrations, either overall or local, could provide an effective mechanism for regulating EGFR/HER2 heterodimerization and may explain why HER2 overexpression found in some cancers has such a profound effect on cell physiology. In the EGFR 1The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; HER2, human epidermal growth factor receptor 2; mAb, monoclonal antibody. family, endocytic trafficking processes can strongly influence cell responses to EGF family ligands. Many cell types express multiple EGFR family members that can interact to form an array of homo- and heterodimers (1Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Google Scholar). Regulation of the distribution of these receptors among cell compartments can significantly modulate the overall signaling through this system by changing access to heterodimerization partners. Because of the potential complexity of EGFR family interactions associated with concomitant receptor trafficking and signaling, application of quantitative experimental and computational modeling techniques to its analysis should be very useful. The EGFR family (EGFR/HER1/ErbB-1, HER2/ErbB-2/neu, HER3/ErbB-3, and HER4/ErbB-4) of receptor tyrosine kinases consists of four highly related receptors each with a unique set of functional properties. Following ligand binding, EGFR family receptors interact to form an array of homo- and heterodimers each with a characteristic repertoire of downstream signaling molecules (1Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Google Scholar). EGFR and HER2 are by far the most studied and have gained particular attention in the process of tumorigenesis. HER2 is commonly postulated to be the “preferred dimerization partner” of all EGFR family receptors (2Tzahar E. Waterman H. Chen X. Levkowitz G. Karunagaran D. Lavi S. Ratzkin B.J. Yarden Y. Mol. Cell. Biol. 1996; 16: 5276-5287Google Scholar, 3Qian X. LeVea C.M. Freeman J.K. Dougall W.C. Greene M.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1500-1504Google Scholar, 4Graus-Porta D. Beerli R.R. Daly J.M. Hynes N.E. EMBO J. 1997; 16: 1647-1655Google Scholar). HER2 behaves much like a receptor subunit, as it binds none of the eight reported EGF family ligands (EGF, transforming growth factor-α, betacellulin, amphiregulin, heparin-binding EGF, epiregulin, and the neuregulins (NRG and NRG-2)) with high affinity (5Riese D.J. Stern D.F. Bioessays. 1998; 20: 41-48Google Scholar). Overexpression of both the EGFR and HER2 has been associated with cell transformation and tumorigenesis. HER2 is overexpressed in 25–30% of all breast and ovarian cancers and is emerging as an important player in many other cancers as well (6Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell Biol. 2001; 2: 127-137Google Scholar). HER2 overexpression correlates with poor prognoses in breast cancer (7Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Science. 1987; 235: 177-182Google Scholar), and its effects in the process of tumorigenesis have been fairly well documented. Increased HER2 expression contributes to cell transformation, anchorage-independent cell growth, increased proliferation and mitogenic sensitivity, as well as increased tumor cell migration and invasiveness (8DiFiore P.P. Pierce J.H. Kraus M.H. Segatto O. King C.R. Aaronson S.A. Science. 1987; 237: 178-182Google Scholar, 9Yu D. Hung M.-C. Bioessays. 2000; 22: 673-680Google Scholar, 10Spencer K.S.R. Graus-Porta D. Leng J. Hynes N. Klemke R.L. J. Cell Biol. 2000; 148: 385-397Google Scholar, 11Brandt B.H. Roetger A. Dittmar T. Nikolia G. Seeling M. Merschjann A. Nofer J.-R. Dehmer-Moller G. Junker R. Assmann G. Zaenker K. FASEB J. 1999; 13: 1939-1950Google Scholar). Receptor overexpression could potentially influence cell behavior in multiple ways. The presence of excess receptors could recruit additional signaling molecules, resulting in an increase in signal amplitude. Alternatively, excess receptors could saturate and interfere with processes involved in receptor down-regulation and signal attenuation (12French A.R. Sudlow G.P. Wiley H.S. Lauffenburger D.A. J. Biol. Chem. 1994; 269: 15749-15755Google Scholar, 13Wiley H.S. J. Cell Biol. 1988; 107: 801-810Google Scholar). This second phenomenon is a receptor trafficking effect that may affect the duration of the signaling by interfering with receptor degradation and ligand dissociation. Studies have shown that trafficking defects in receptors can facilitate cell transformation (14Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Google Scholar), suggesting that signal duration may be more of a determining factor in mitogenic sensitivity than signal amplitude, particularly at physiological ligand concentrations. It appears likely that receptor expression levels are directly connected to their trafficking behavior which, in turn, affects signaling. The trafficking behavior of the EGFR, in the absence of other family members, has been well characterized (15Carpenter G. Bioessays. 2000; 22: 697-707Google Scholar, 16Lauffenburger D.A. Linderman J.J. Receptors. Oxford University Press, Inc., New York1993: 73-132Google Scholar). Binding of EGFR ligands to the EGFR promotes receptor homodimerization and phosphorylation of cytoplasmic tyrosine residues that initiate signaling cascades (5Riese D.J. Stern D.F. Bioessays. 1998; 20: 41-48Google Scholar, 17van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar). Phosphorylated receptors are rapidly internalized by clathrin-coated pit endocytosis resulting in both short and long term loss of receptor activity. Once internalized, receptors and ligands are sorted in endosomes and either targeted toward lysosomal degradation or follow the recycling pathway back to the surface (16Lauffenburger D.A. Linderman J.J. Receptors. Oxford University Press, Inc., New York1993: 73-132Google Scholar, 17van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar, 18Sorkin A. Waters C.M. Bioessays. 1993; 15: 375-382Google Scholar). Ligand-induced degradation of receptors can be impaired both at the level of endocytosis and/or endosomal sorting. Disruption of endocytosis has been shown to increase EGF-dependent proliferation (14Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Google Scholar). Overexpression of EGFR has also been shown to impair their degradation. Endocytic and endosomal sorting machinery have both been shown to exhibit saturation at high receptor levels, apparently due to limiting levels of the regulatory molecules involved in these processes (12French A.R. Sudlow G.P. Wiley H.S. Lauffenburger D.A. J. Biol. Chem. 1994; 269: 15749-15755Google Scholar, 13Wiley H.S. J. Cell Biol. 1988; 107: 801-810Google Scholar, 18Sorkin A. Waters C.M. Bioessays. 1993; 15: 375-382Google Scholar, 19Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Google Scholar). The association of HER2 with a number of different pathologies could be due to a host of molecular level effects. Following dimerization with another (ligand-bound) EGFR family member, HER2 becomes phosphorylated and is then able to recruit a distinct repertoire of signaling molecules including effectors both overlapping and distinct from EGFR-associated ones (1Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Google Scholar). In addition to the signal amplification role played by HER2, a second effect is elicited at the level of trafficking. Overexpression of HER2 affects the normal trafficking behavior of the EGFR, disrupting the processes that control receptor degradation (20Worthylake R. Opresko L.K. Wiley H.S. J. Biol. Chem. 1999; 274: 8865-8874Google Scholar). The precise trafficking behavior of HER2 remains unclear. The internalization rates of HER2, HER3, and HER4 have been studied through the construction of chimeras consisting of the EGFR extracellular domain and different HER cytoplasmic domains. All EGFR/ErbB chimeras are internalized severalfold more slowly than the EGFR (21Sorkin A. DiFiore P.P. Carpenter G. Oncogene. 1993; 8: 3021-3028Google Scholar, 22Baulida J. Kraus M.H. Alimandi M. DiFiore P.P. Carpenter G. J. Biol. Chem. 1996; 271: 5251-5257Google Scholar, 23Muthuswamy S. Gilman M. Brugge J.S. Mol. Cell. Biol. 1999; 19: 6845-6957Google Scholar). EGF-induced HER2 down-regulation has also been reported (20Worthylake R. Opresko L.K. Wiley H.S. J. Biol. Chem. 1999; 274: 8865-8874Google Scholar). However, other investigators have failed to observe any EGF-induced HER2 internalization or EGFR/HER2 internalization (24Wang Z. Zhang L. Yeung T.K. Chen X. Mol. Biol. Cell. 1999; 10: 1621-1636Google Scholar). In recent work, we have demonstrated the internalization of HER2 in the absence of EGF and its accelerated internalization following EGF stimulation (25Hendriks B.S. Opresko L.K. Wiley H.S. Lauffenburger D.A. Cancer Res. 2003; 63: 1130-1137Google Scholar). Additionally, we found that overexpression of HER2 decreases the internalization rate of EGF. A quantitative evaluation of EGFR and HER2 internalization in the context of previous literature data, however, has not been done. In this work, we examine the endocytic portion of the trafficking pathway in detail with the aim of quantitatively understanding how EGFR and HER2 interact in the process of internalization. We found the following: (i) EGFR/HER2 heterodimers are internalized as single entities, with other models of internalization being inconsistent with literature data (25Hendriks B.S. Opresko L.K. Wiley H.S. Lauffenburger D.A. Cancer Res. 2003; 63: 1130-1137Google Scholar); (ii) EGFR/HER2 heterodimers have a comparable dimerization affinity as EGFR/EGFR homodimers, thus the notion of HER2 as a preferred dimerization partner should be re-assessed; and (iii) there appears to be a threshold level of HER2 above which heterodimerization is maximal but increased HER2 expression is still able to alter signaling through longer term effects. Reagents and Cell Culture—Antibody 13A9 against the EGFR (26Winkler M.E. O'Connor L. Winget M. Fendly B. Biochemistry. 1989; 28: 6373-6378Google Scholar), mAbs 7C2 and 2C4 against HER2 (27Fendly B.M. Winget M. Hudziak R.M. Lipari M.T. Napier M.A. Ullrich A. Cancer Res. 1990; 50: 1550-1558Google Scholar, 28Sliwkowski M.X. Schaefer G. Akita R.W. Lofgren J.A. Fitzpatrick V.D. Nuijens A. Fendly B.M. Cerione R.A. Vandlen R.L. Carraway III, K.L. J. Biol. Chem. 1994; 269: 14661-14665Google Scholar), and the Fab fragment of monoclonal 7C2 were gifts from Genentech. The human mammary epithelial cell lines MTSV1–7 and ce2 have been described previously (29D'Souza B. Berdichevsky F. Kyprianou N. Taylor-Papadimitriou J. Oncogene. 1993; 8: 1797-1806Google Scholar) and were provided as a generous gift from Dr. Joyce Taylor-Papadimitriou. These cells were grown in Dulbecco's modified Eagle's medium (Flow Laboratories) containing 10% calf serum (Hyclone) supplemented with 1 μm insulin and 5 μm dexamethasone. ErbB-2 expression in ce2 cells was maintained by the addition of 500 μg/ml G418 (Sigma). Antibodies and EGF were iodinated with IODO-BEADS (Pierce) according to the manufacturer's directions to specific activities of 2.7 × 106 cpm/pmol (mAb), 8 × 105 cpm/pmol for the Fab fragment of 7C2, and 1.6 × 106 cpm/pmol for EGF. Binding Analysis—Numbers of EGFR and HER2 molecules on the cell surface were determined by steady state analysis (30Wiley H.S. Cunningham D.D. Cell. 1981; 25: 433-440Google Scholar). Cells were incubated with concentrations from 6.7 × 10–11 to 2 × 10–8m for 3.5 h at 37 °C. The relative amount of antibody associated with the cell surface was determined by acid stripping, and the data were analyzed as described previously (20Worthylake R. Opresko L.K. Wiley H.S. J. Biol. Chem. 1999; 274: 8865-8874Google Scholar). Specific internalization rates (ke) for the EGFR were determined as described (31Lund K.A. Opresko L.K. Starbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Google Scholar) using 17 nm ligand and a 5-min incubation period. Specific internalization rates for the labeled mAbs were determined using a concentration of 1.3 nm antibody and 2-min intervals for a total of 10 min. Values were calculated as regression slope of the integral surface-associated ligand against the amount internalized (31Lund K.A. Opresko L.K. Starbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Google Scholar). Constitutive Case—Our model was designed to output results that can be compared with the internalization experiments used to generate the data (25Hendriks B.S. Opresko L.K. Wiley H.S. Lauffenburger D.A. Cancer Res. 2003; 63: 1130-1137Google Scholar, 31Lund K.A. Opresko L.K. Starbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Google Scholar, 32Wiley H.S. Cunningham D.D. J. Biol. Chem. 1982; 257: 4222-4229Google Scholar). The constitutive internalization of EGFR and HER2 is modeled with a set of coupled mass action kinetic equations. Consistent with published reports, we assume that a constitutive level of EGFR and HER2 homo- and heterodimerization takes place (33Penuel E. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 2002; 277: 28468-28473Google Scholar, 34Mendrola J.M. Berger M.B. King M.C. Lemmon M.A. J. Biol. Chem. 2002; 277: 4704-4712Google Scholar, 35Yu X. Sharma K.D. Takahashi T. Iwamoto R. Mekada E. Mol. Biol. Cell. 2002; 13: 2547-2557Google Scholar). As diagrammed in Fig. 1 (in the absence of any stimulation), EGFRs are allowed three states: free EGFR (R1), homodimerized with another EGFR (R1R1), or heterodimerized with HER2 (R1R2). HER2 is allowed similar freedom: free HER2 (R2), homodimerized with another HER2 (R2R2), or heterodimerized with EGFR (R1R2). The behavior of each species with regard to internalization is characterized by an internalization rate constant. Each dimerization and uncoupling act is characterized by its own, but not necessarily unique, kinetic rate constant. Ligand-stimulated Case—By using the constitutive internalization model as a basis, ligand-induced interactions were added (diagrammed in Fig. 1). We have included the complete set of binary interactions between the EGFR and HER2 with and without EGF. Higher order oligomerization of receptors is neglected, as this is a first approximation of possible EGF/EGFR/HER2 interactions. Additionally, HER3 and HER4 were left out of our analysis because we were examining the effects of EGF stimulation and they are typically expressed at lower levels. In addition to the constitutive receptor species (R1, R1R1, R1R2, R2, and R2R2) and their interactions, EGFRs now bind ligand (L) to form complexes (R1L). Complexes may homodimerize with another complex to form doubly bound EGFR homodimers (LR1R1L) or heterodimerize with HER2 to form EGF·EGFR/HER2 heterodimers (R1R2L). Additionally, the existence of singly bound EGFR homodimers (R1R1L) is permitted, and each species may be formed in any order. For example, singly bound EGFR homodimers can be formed by ligand dissociation from doubly bound EGFR homodimers or by ligand binding to unbound EGFR homodimers or by dimerization of an empty EGFR with an EGF·EGFR complex. As before, each receptor species may have a unique internalization rate constant, and every interaction is reversible with its own set of kinetic rate constants. Superimposed on the surface level receptor interactions are two additional processes: (i) the binding of radiolabeled antibodies to HER2, corresponding to the actual experiment; and (ii) the internalization of each receptor species. Under resting conditions, each receptor species is assumed to be at steady state, wherein rates of receptor internalization are perfectly balanced by rates of receptor synthesis and degradation. Thus, internalization and recycling of unlabeled receptor species can be left out of the model. Receptor species that have been bound by EGF, an antibody or antibody Fab fragment, however, are internalized at a rate specific to each species to an inside compartment. Recycling of each species is assumed to be negligible over the 7.5-min time scale of the model/experiment (30Wiley H.S. Cunningham D.D. Cell. 1981; 25: 433-440Google Scholar). Inside and surface data are generated with the model and manipulated in the same manner as the experimental data to calculate the observed internalization rate constants for specific EGFR and HER2 expression levels. The model parameters are list in Table I. The overall observed internalization rate for EGF or Fab-bound species is essentially a weighted sum of the individual internalization rate of each EGF- or Fab-bound receptor species. Model equations corresponding to the interactions diagrammed in Fig. 1 with Fab binding superimposed are listed under the Appendix of the Supplemental Material.Table IInternalization model parametersModel parameterDescriptionValueBinding parameterskonEGF association9.7 × 107 (M min)-1koffEGF dissociation0.24 min-1konFabFab association1.4 × 107 (M min)-1koffFabFab dissociation0.30 min-1Internalization rate constants (estimated from experimental data)keR1Unoccupied EGFR internalization rate constant0.08 min-1keR2-HER2 internalization rate constant, No EGF0.01 min-1keR2+HER2 internalization rate constant, + EGF0.03 min-1keR1R2EGFR/HER2 unbound heterodimer internalization rate constant0.04 min-1keR1LEGF·EGFR complex internalization rate constant0.28 min-1keR1R2LEGF·EGFR/HER2 bound heterodimer internalization rate constant0.10 min-1Dimerization/uncoupling parameters (fit to experimental data)kcaSet to diffusion-limited value.Receptor dimerization rate constant1 × 10-3 (#/cell min)-1kuR1R1EGFR/EGFR unbound homodimer uncoupling rate constant10 min-1kuR1R2EGFR/HER2 unbound heterodimer uncoupling rate constant10 min-1kuR2R2HER2/HER2 homodimer uncoupling rate constant1 min-1kuR1R2LEGF·EGFR/HER2 bound heterodimer uncoupling rate constant0.1 min-1kuLR1R1LEGF·EGFR/EGFR·EGF homodimer uncoupling rate constant0.1 min-1a Set to diffusion-limited value. Open table in a new tab Parameter Estimation and Determination—The model parameters can be divided into three categories: (i) binding parameters, which govern EGF and antibody binding and dissociation; (ii) internalization rate constants, which describe the internalization rate of each receptor species; and (iii) dimerization rate constants, which characterize the dimerization and uncoupling of EGFR and HER2 in various states. In the case of our experiments, we used Fab fragments of anti-HER2 antibodies. Fab binding (konFab) and dissociation (koffFab) were experimentally measured to be 1.4 × 107 (m min)–1 and 0.30 min–1 (data not shown). As suggested in the literature, Fab binding was assumed to have no effect on receptor dimerization/uncoupling, EGF binding/dissociation, or receptor internalization. EGF binding (kon) and dissociation (koff) for 184A1 human mammary epithelial cells were reported to be 9.7 × 107 (m min)–1 and 0.24 (min)–1, respectively (25Hendriks B.S. Opresko L.K. Wiley H.S. Lauffenburger D.A. Cancer Res. 2003; 63: 1130-1137Google Scholar). Many of the species-specific internalization rate constants can be estimated from the experimental data. If one assumes that free HER2 and HER2 homodimers internalize at the same rate, their internalization rate constant should be reflected by the case where HER2 expression is much greater than EGFR expression. In the absence of EGF, the internalization rate constant of free HER2 (keR2–) is reflected by the asymptote approached at high HER2 expression levels (0.01 min–1), approximately the rate of membrane turnover (36Burke P.M. Wiley H.S. J. Cell. Physiol. 1999; 180: 448-460Google Scholar). In the presence of EGF, there is an increase in membrane turnover, and the internalization rate of free HER2 (keR2+) is roughly 0.03 min–1. The internalization rate of free EGFR (keR1) has been reported to be 0.08 min–1 (25Hendriks B.S. Opresko L.K. Wiley H.S. Lauffenburger D.A. Cancer Res. 2003; 63: 1130-1137Google Scholar). As with HER2, we assume that unbound EGFR homodimers internalize with the same rate constant as free EGFR. Unbound EGFR/HER2 heterodimers are likely to internalize at a rate between that of HER2 homodimers and EGFR homodimers. We estimate the internalization rate constant of unligated EGFR/HER2 heterodimers (keR1R2) to be 0.04 min–1. The internalization rates of EGF·EGFR complexes, complex homodimers, and singly bound EGFR homodimers are assumed to internalize with the same first order rate constant (keR1L). This parameter represents the case where no HER2 is present and can be extrapolated to the y intercept of the graph of EGFR internalization versus HER2 expression level, yielding a value of ∼0.28 min–1. The internalization rate constant for bound EGFR/HER2 heterodimers (keR1R2L) is represented by the asymptote of EGFR internalization with increasing HER2 expression. From the EGFR internalization data, at the highest level of HER2 expression, the internalization rate constant of HER2 heterodimers is estimated to be 0.10 min–1. All receptor dimer species are estimated to form at the diffusion-limited values, with a coupling rate constant (kc) of 10–3 (n/cell min)–1 (37Shea L.D. Omann G.M. Linderman J.J. Biophys. J. 1997; 73: 2949-2959Google Scholar, 38Mahama P.A. Linderman J.J. Biophys. J. 1994; 67: 1345-1357Google Scholar). The uncoupling rate constants of each dimer species (kuR1R1, kuR1R2, and kuR2R2) were fit to the HER2 internalization data in the absence of EGF. Holding these parameters fixed, the uncoupling rate constants for EGFR complex homodimers and bound heterodimers (kuLR1R1L and kuR1R2L) were fit to the either the EGFR internalization data or the HER2 + EGF internalization data. These values are intended as order of magnitude estimates of the various uncoupling rate constants. Computations—Mathematical equations corresponding to the model described above were coded into MATLAB version 6.5 (Mathworks, Natick, MA) and solved using ODE solver ode23s. Parameters were fit to experimental data using the lsqcurvefit routine from the Optimization Toolbox. Recent work has highlighted the importance of receptor internalization in determining the distribution and induced degradation of EGFR in response to EGF stimulation (25Hendriks B.S. Opresko L.K. Wiley H.S. Lauffenburger D.A. Cancer Res. 2003; 63: 1130-1137Google Scholar). HER2 has emerged as a modulator of EGFR internalization presumably through heterodimerization. Here we explored the importance of heterodimerization in the internalization of both EGFR and HER2 as a function of HER2 expression level using previously published experimental data and a computational model of EGFR and HER2 internalization. Parameter Determination from Experimental Data—Experimental data demonstrating the effect of increasing HER2 expression on the internalization rate constants of HER2 and EGF is derived from Ref. 25Hendriks B.S. Opresko L.K. Wiley H.S. Lauffenburger D.A. Cancer Res. 2003; 63: 1130-1137Google Scholar and shown in Fig. 2A. In the absence of EGF, HER2 was internalized slowly at a rate comparable with membrane turnover. The addition of EGF accelerated the internalization of HER2. The internalization rate of EGF showed a marked decrease with increasing HER2 expression, whereas HER2 internalization under similar conditions showed a mild decline in internalization with increasing HER2 expression. In Fig. 2B is experimental data (25Hendriks B.S. Opresko L.K. Wiley H.S. Lauffenburger D.A. Cancer Res. 2003; 63: 1130-1137Google Scholar) showing the effect of preincubation with heterodimerization-blocking antibodies (2C4) on the internalization rate constant of EGF. Blocking heterodimerization abrogated any HER2-dependent effect on EGF internalization. The significance of these results has been explored previously from a global, whole cell perspective. Here we seek to explore the mechanistic basis for these findings in order to test different models of internalization, determine which receptor species are dominant under different conditions, and elucidate which interactions dictate internalization behavior. The distribution of receptors between different signaling states (homodimers versus heterodimers, for example) should provide insight into the circumstances that contribute to aberrant cell behavior. Models of Receptor Internalization—The general framework for our internalization models is shown in Fig. 1. Every binary interaction between EGFR and HER2 with and without EGF addition is included. Individual models differ with regard to certain parameter values or assumptions regarding species-specific behavior. The first internalization model that we consider is one in which each dimer species is sufficiently stable to be internalized as a single entity, hereafter referred to as the “coupled internalization” model. This model is fit to the data by first fitting uncoupling rate constants (kuR1R1, kuR1R2, and kuR2R2) to the data in the absence of EGF. Holding those parameters fixed, the remaining uncoupling rate constants (kuLR1R1L and kuR1R2L) are fit to the EGFR data and used to predict the HER2 + EGF internalization data (shown in Fig. 2A). Nearly identical results are obtained if one fits to the HER2 + EGF data and predicts the EGFR internalization data (data not shown). The parameter values determined are shown in Table I. We simulated the effect of disrupting heterodimerization on the EGF internalization rate constant by increasing the bound heterodimer uncoupling rate constants (kuR1R2L), as shown in Fig. 2B. Model predictions are shown together with experimental data for EGF internalization in the presence of an antibody (2C4) that blocks EGFR/HER2 heterodimerization (28Sliwkowski M.X. Schaefer G. Akita R.W. Lofgren J.A. Fitzpatrick V.D. Nuijens A. Fendly B.M. Cerione R.A. Vandlen R.L. Carraway III, K.L. J. Biol. Chem. 1994; 269: 14661-14665Google Scholar). An alternative model of internalization might be one in which dimer species are transiently stable so that only individual receptors and complexes are internalized. We simulated this by setting the uncoupling rate constants to sufficiently large values such that no significant degree of dimerization occurs. This model required no fitting of parameters and clearly does not capture the trends seen in the experimental data (Fig. 3A). Another possibility is a model proposed by Wang et al. (24Wang Z. Zhang L. Yeung T.K. Chen X. Mol. Biol. Cell. 1999; 10: 1621-1636Google Scholar), in which they assert that heterodimers do not internalize. To simulate this model, we have set the internalization rate constant of heterodimers (keR1R2L) to zero and fit the same parameters as with the original coupled internalization model. When this" @default.
• W2078112275 created "2016-06-24" @default.
• W2078112275 creator A5035474346 @default.
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• W2078112275 date "2003-06-01" @default.
• W2078112275 modified "2023-10-15" @default.
• W2078112275 title "Quantitative Analysis of HER2-mediated Effects on HER2 and Epidermal Growth Factor Receptor Endocytosis" @default.
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