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• W2038733101 abstract "The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. The EGF receptor tyrosine kinase is a classic receptor tyrosine kinase that mediates cell proliferation in response to a variety of different ligands. Structurally, the receptor possesses an extracellular ligand binding domain and an intracellular tyrosine kinase domain (1.Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells.Nature. 1984; 309: 418-425Crossref PubMed Scopus (1989) Google Scholar). In its inactive state, the receptor appears to exist primarily as a monomer (2.Ferguson K.M. Berger M.B. Mendrola J.M. Cho H.-S. Leahy D.J. Lemmon M.A. EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization.Mol. Cell. 2003; 11: 507-517Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). However, upon binding ligand, the receptor undergoes a substantial conformational change that allows it to dimerize, in a back-to-back orientation, with a second EGF receptor (3.Garrett T.P. McKern N.M. Lou M. Elleman T.C. Adams T.E. Lovrecz G.O. Zhu H.-J. Walker F. Frenkel M.J. Hoyne P.A. Jorissen R.N. Nice E.C. Burgess A.W. Ward C.W. Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor a.Cell. 2002; 110: 763-773Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 4.Ogiso H. Ishitani R. Nureki O. Fukai S. Yamanaka M. Kim J.-H. Saito K. Sakamoto A. Inoue M. Shirouzu M. Yokoyama S. Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains.Cell. 2002; 110: 775-787Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar). Dimerization of the extracellular domains leads to dimerization and activation of the intracellular kinase domain (5.Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar). The active kinase phosphorylates a number of different tyrosine residues in the C-terminal tail of its partner subunit (6.Hsuan J.J. Totty N. Waterfield M.D. Identification of a novel autophosphorylation site (P4) on the epidermal growth factor receptor.Biochem. J. 1989; 262: 659-663Crossref PubMed Scopus (49) Google Scholar, 7.Margolis B.L. Lax I. Kris R. Dombalagian M. Honegger A.M. Howk R. Givol D. Ullrich A. Schlessinger J. All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails.J. Biol. Chem. 1989; 264: 10667-10671Abstract Full Text PDF PubMed Google Scholar, 8.Downward J. Parker P. Waterfield M.D. Autophosphorylation sites on the epidermal growth factor receptor.Nature. 1984; 311: 483-485Crossref PubMed Scopus (468) Google Scholar). This permits the binding of SH2 and PTB domain-containing proteins to the phosphorylated receptor and initiates intracellular signaling events (9.Burgess A.W. EGFR family: structure, physiology, signalling and therapeutic targets.Growth Factors. 2008; 26: 263-274Crossref PubMed Scopus (200) Google Scholar, 10.Hynes N.E. Lane H.A. ErbB receptors and cancer: the complexity of targeted inhibitors.Nat. Rev. Cancer. 2005; 5: 341-354Crossref PubMed Scopus (2679) Google Scholar, 11.Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141: 1117-1134Abstract Full Text Full Text PDF PubMed Scopus (3106) Google Scholar). Although this basic pattern of EGF receptor activation is relatively straightforward, a number of variations serve to make the system significantly more complex. The EGF receptor is a member of the ErbB family of homologous receptors that also includes ErbB2, ErbB3, and ErbB4 (12.Ferguson K.M. Structure-based view of epidermal growth factor receptor regulation.Annu. Rev. Biophys. 2008; 37: 353-373Crossref PubMed Scopus (242) Google Scholar). The ErbB receptors are structurally similar and exhibit comparable mechanisms of activation (5.Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar, 13.Aertgeerts K. Skene R. Yano J. Sang B.-C. Zou H. Snell G. Jennings A. Iwamoto K. Habuka N. Hirokawa A. Ishikawa T. Tanaka T. Miki H. Ohta Y. Sogabe S. 1) Structural analysis of the mechanism of inhibition and allosteric activation of the kinase domain of HER2 protein.J. Biol. Chem. 2011; 286: 18756-18765Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 14.Qiu C. Tarrant M.K. Choi S.H. Sathyamurthy A. Bose R. Banjade S. Pal A. Bornmann W.G. Lemmon M.A. Cole P.A. Leahy D.J. Mechanism of activation and inhibition of the HER4/ErbB4 kinase.Structure. 2008; 16: 460-467Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). As a result of these similarities, the ErbB receptors can interact with each other to form heterodimers. Although most combinations of ErbB receptors can form, in general, it appears that ErbB2 is the preferred heterodimerization partner (15.Graus-Porta D. Beerli R.R. Daly J.M. Hynes N.E. ErbB2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.EMBO J. 1997; 16: 1647-1655Crossref PubMed Scopus (1298) Google Scholar, 16.Karunagaran D. Tzahar E. Beerli R.R. Chen X. Graus-Porta D. Ratzkin B.J. Seger R. Hynes N.E. Yarden Y. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.EMBO J. 1996; 15: 254-264Crossref PubMed Scopus (586) Google Scholar, 17.Tzahar E. Waterman H. Chen X. Levkowitz G. Karunagaran D. Lavi S. Ratzkin B.J. Yarden Y. A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.Mol. Cell Biol. 1996; 16: 5276-5287Crossref PubMed Scopus (864) Google Scholar). This is noteworthy because ErbB2 is the only ErbB receptor that does not appear to bind a ligand (18.Qian X. LeVea C.M. Freeman J.K. Dougall W.C. Greene M.I. Heterodimerization of epidermal growth factor receptor and wild-type or kinase-deficient Neu: a mechanism of interreceptor kinase activation and transphosphorylation.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 1500-1504Crossref PubMed Scopus (141) Google Scholar, 19.Wada T. Qian X.L. Greene M.I. Intermolecular association of the p185neu protein and EGF receptor modulates EGF receptor function.Cell. 1990; 61: 1339-1347Abstract Full Text PDF PubMed Scopus (334) Google Scholar). In addition to the variation in dimerization partners, the EGF receptor also possesses seven different cognate ligands (for a review, see Ref. 20.Wilson K.J. Gilmore J.L. Foley J. Lemmon M.A. Riese 2nd, D.J. Functional selectivity of EGF family peptide growth factors: implications for cancer.Pharmacol. Ther. 2009; 122: 1-8Crossref PubMed Scopus (166) Google Scholar), including EGF, TGFα, betacellulin (BTC), 2The abbreviations used are: BTCbetacellulinAREGamphiregulin. heparin-binding EGF, amphiregulin (AREG), epiregulin, and epigen. Of these EGFs, TGFα, AREG, and epigen bind only to the EGF receptor, whereas BTC, heparin-binding EGF, and epiregulin also bind to ErbB4 (20.Wilson K.J. Gilmore J.L. Foley J. Lemmon M.A. Riese 2nd, D.J. Functional selectivity of EGF family peptide growth factors: implications for cancer.Pharmacol. Ther. 2009; 122: 1-8Crossref PubMed Scopus (166) Google Scholar). These seven ligands can also be subdivided on the basis of their affinity for the EGF receptor. EGF, TGFα, BTC, and heparin-binding EGF exhibit affinities of 0.1–1 nm for the EGF receptor, whereas AREG, epiregulin, and epigen exhibit affinities 10- to 100-fold lower than this (20.Wilson K.J. Gilmore J.L. Foley J. Lemmon M.A. Riese 2nd, D.J. Functional selectivity of EGF family peptide growth factors: implications for cancer.Pharmacol. Ther. 2009; 122: 1-8Crossref PubMed Scopus (166) Google Scholar, 21.Jones J.T. Akita R.W. Sliwkowski M.X. Binding specificities and affinities of EGF domains for ErbB receptors.FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (319) Google Scholar). Therefore, there is variation in both receptor specificity and receptor affinity. betacellulin amphiregulin. Despite the fact that the seven EGF receptor ligands all bind to and activate the same EGF receptor, they are capable of inducing different biological effects, even within the same cell (for a review, see Ref. 20.Wilson K.J. Gilmore J.L. Foley J. Lemmon M.A. Riese 2nd, D.J. Functional selectivity of EGF family peptide growth factors: implications for cancer.Pharmacol. Ther. 2009; 122: 1-8Crossref PubMed Scopus (166) Google Scholar). For example, in 32D cells expressing the EGF receptor, TGFα and AREG stimulate higher levels of cell proliferation than EGF and heparin-binding EGF (22.Wilson K.J. Mill C. Lambert S. Buchman J. Wilson T.R. Hernandez-Gordillo V. Gallo R.M. Ades L.M. Settleman J. Riese 2nd, D.J. EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling.Growth Factors. 2012; 30: 107-116Crossref PubMed Scopus (80) Google Scholar). In human fibroblasts, EGF stimulates cell migration through a mechanism involving p70S6K, whereas TGFα stimulates migration via phospholipase C (23.Ellis I.R. Schor A.M. Schor S.L. EGF and TGF-a motogenic activities are mediated by the EGF receptor via distinct matrix-dependent mechanisms.Exp. Cell Res. 2007; 313: 732-741Crossref PubMed Scopus (33) Google Scholar). In β-HC9 cells, EGF stimulates MAP kinase activation via ras, whereas BTC-stimulated MAP kinase activation occurs independently of ras (24.Saito T. Okada S. Ohshima K. Yamada E. Sato M. Uehara Y. Shimizu H. Pessin J.E. Mori M. Differential activation of epidermal growth factor (EGF) receptor downstream signaling pathways by βcellulin and EGF.Endocrinology. 2004; 145: 4232-4243Crossref PubMed Scopus (79) Google Scholar), and AREG, but not EGF, activates NF-κB in SUM149 cells (25.Streicher K.L. Willmarth N.E. Garcia J. Boerner J.L. Dewey T.G. Ethier S.P. Activation of a nuclear factor-κB /interleukin-1 positive feedback loop by amphiregulin in human breast cancer cells.Mol. Cancer Res. 2007; 5: 847-861Crossref PubMed Scopus (55) Google Scholar). In some cases, these differences in biological effects have been correlated with differences in the phosphorylation of specific sites on the C-terminal tail of the EGF receptor (22.Wilson K.J. Mill C. Lambert S. Buchman J. Wilson T.R. Hernandez-Gordillo V. Gallo R.M. Ades L.M. Settleman J. Riese 2nd, D.J. EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling.Growth Factors. 2012; 30: 107-116Crossref PubMed Scopus (80) Google Scholar, 24.Saito T. Okada S. Ohshima K. Yamada E. Sato M. Uehara Y. Shimizu H. Pessin J.E. Mori M. Differential activation of epidermal growth factor (EGF) receptor downstream signaling pathways by βcellulin and EGF.Endocrinology. 2004; 145: 4232-4243Crossref PubMed Scopus (79) Google Scholar). However, the molecular basis for these differences remains largely unexplored. In this work, we use luciferase fragment complementation imaging and radioligand binding to probe the interaction of the EGF receptor and ErbB2 subunits after stimulation with EGF, TGFα, BTC, and AREG. The results suggest that differences in ligand-induced receptor-receptor interactions, along with variations in dimerization kinetics, likely contribute to the different biological effects induced by the binding of different growth factors to the EGF receptor. EGF was purchased from Biomedical Technologies. TGFα was from Leinco. Human BTC was from Prospec, and AREG was from Leinco. Antibodies to the EGF receptor, pTyr-845, pTyr-992, pTyr-1045, pTyr-1068, and pTyr-1221 were from Cell Signaling Technology. The antibody against pTyr-1173 was from Thermo Scientific. Antibodies against ErbB2 were from Millipore. FetalPlex was from Gemini Bio-Products. Na125I was from PerkinElmer Life Sciences. 125I-EGF was synthesized using the method of Doran and Spar (26.Doran D.M. Spar I.L. Oxidative iodine monochloride iodination technique.J. Immunol. Methods. 1980; 39: 155-163Crossref PubMed Scopus (35) Google Scholar). The c'698-EGF receptor fused to the N-terminal half of firefly luciferase (NLuc) or the C-terminal half of luciferase (CLuc) was generated by introducing a BsiWI site into the EGF receptor after Ala-698 in pcDNA3.1 Zeo. The NheI to BsiWI fragment was then isolated and ligated into the pBI Tet vector or pcDNA3.1 Zeo vector cut with BsiWi and NheI and encoding the NLuc or CLuc fragments, respectively. The c'709-ErbB2-NLuc construct was generated in a similar fashion by introducing a BsiWI site into ErbB2 after Ala-709 and ligating an NheI to BsiWI fragment into the pBI Tet vector containing the NLuc fragment. The generation of CHO cells constitutively expressing C-terminally truncated forms of the EGF receptor (after residue 645) or ErbB2 (truncated after residue 678) and fused to the NLuc or CLuc fragments on a Tet-inducible plasmid has been described previously (27.Li Y. Macdonald-Obermann J. Westfall C. Piwnica-Worms D. Pike L.J. Quantitation of the effect of ErbB2 on EGF receptor binding and dimerization.J. Biol. Chem. 2012; 287: 31116-31125Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The construction of the kinase-dead K721A-EGFR-NLuc and K721A-EGFR-CLuc cell lines has also been described previously (28.Macdonald-Obermann J.L. Piwnica-Worms D. Pike L.J. The mechanics of EGF receptor/ErbB2 kinase activation revealed by luciferase fragment complementation imaging.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 137-142Crossref PubMed Scopus (54) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FetalPlex and maintained in an incubator at 37 °C in 5% CO2. CHO cells were plated into 96-well black-walled dishes 2 days prior to use. Immediately before the assay, cells were transferred to Dulbecco's modified Eagle's medium without phenol red but with 1 mg/ml bovine serum albumin and 50 mm Hepes (pH 7.4). Cells were then incubated with 0.6 mg/ml D-luciferin for 20 min at 37 °C prior to the addition of growth factor and the start of imaging. Cell radiance (photons/second/centimeter squared/Steradian) was measured every 30 s for 25 min using a cooled charge-coupled device camera and the IVIS50 imaging system. Assays were performed in quintuplicate. Data were fit to single or double exponential model curves using GraphPad Prism 6. Cells constitutively expressing ∼300,000 EGF receptors/cell with ErbB2 on a Tet-inducible promoter were plated onto 6-well dishes 48 h prior to use (27.Li Y. Macdonald-Obermann J. Westfall C. Piwnica-Worms D. Pike L.J. Quantitation of the effect of ErbB2 on EGF receptor binding and dimerization.J. Biol. Chem. 2012; 287: 31116-31125Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium in the absence or presence of 1 µg/ml doxycycline to induce the expression of ErbB2. This concentration of doxycycline induced the expression of approximately ∼1.5 × 106 ErbB2 molecules/cell. Before use, cells were washed in chilled phosphate-buffered saline and cooled to 4 °C prior to the addition of 30 pm 125I-EGF in Ham's F12 medium containing 50 mm Hepes (pH 7.2) and 5 mg/ml bovine serum albumin. Increasing concentrations of unlabeled EGF, TGFα, BTC, or AREG were added to the wells in triplicate. After incubation overnight at 4 °C, plates were washed three times in cold phosphate-buffered saline. Monolayers were dissolved in 1 ml 1 N NaOH and counted in a Beckman γ counter. Assays were done in triplicate. Data were fit to the equation for log(inhibitor) versus response (variable slope) using GraphPad Prism 6. The significance of the differences between the EC50 values in the absence and presence of ErbB2 was based on the p value assigned to those differences by Prism 6. CHO cells constitutively expressing the EGF receptor and stably transfected with ErbB2 on a Tet-inducible plasmid were plated in 6-well dishes and grown for 2 days before use. When desired, 50 ng/ml doxycycline was added to the growth medium. Immediately before the assay, cells were transferred into warmed Ham's F12 medium containing 25 mm Hepes (pH 7.2) and 1 mg/ml bovine serum albumin and stimulated with growth factor for the indicated time. Plates were incubated at 37 °C, and the assay was stopped by washing in ice-cold phosphate-buffered saline followed by the addition of radioimmune precipitation assay buffer. Monolayers were scraped into the radioimmunoprecipitation assay buffer, and cells were solubilized by passage through a fine-gauge needle. After pelleting unsolubilized material, equal amounts of protein were analyzed on SDS-polyacrylamide gels, and proteins were identified by Western blotting. Results were quantitated using ImageJ software. We first compared the biological effects of EGF, TGFα, BTC, and AREG in CHO cells that constitutively expressed ∼300,000 EGF receptors/cell and contained ErbB2 on a tetracycline-inducible promoter. This allowed us to determine the effect of the four different growth factors in cells containing only EGF receptors or in cells containing both the EGF receptor and ErbB2. To compare the biological effects of these four growth factors, CHO cells grown in the absence or presence of doxycycline were stimulated with a saturating concentration of EGF, TGFα, BTC, or AREG and assayed by Western blotting for phosphorylation of the EGF receptor and ErbB2. The Western blot analyses are shown in Fig. 1, A and B, left panels, and are quantitated in the right panels. As can be seen in Fig. 1A, in cells expressing only the EGF receptor, all four ligands stimulated maximal tyrosine phosphorylation of the EGF receptor within 2 min. However, EGF, TGFα, and BTC each stimulated about twice as much phosphorylation as AREG. This phosphorylation was transient because it had declined by 5 min after growth factor treatment. The effects of AREG appeared to decline somewhat more slowly than those of the other three growth factors. A similar phenomenon was seen in cells expressing both the EGF receptor and ErbB2 (Fig. 1B). Again, phosphorylation was maximal by 2 min, and EGF, TGFα, and BTC stimulated about twice as much phosphorylation of both the EGF receptor and ErbB2 as AREG. Therefore, AREG is a partial agonist for the phosphorylation of both EGFR homodimers and EGFR/ErbB2 heterodimers. Replicate lysates at the 2-min time point in Fig. 1, A and B, were subjected to Western blotting with phosphosite-specific antibodies to determine whether there was differential phosphorylation of sites by the different growth factors. As shown in Fig. 1C, AREG stimulated less phosphorylation than EGF, TGFα, or BTC at essentially all sites on the EGF receptor, except for pTyr-1173. At this site, AREG-stimulated phosphorylation was comparable or higher in EGF receptor homodimers but less in heterodimers compared with the other three growth factors. AREG also stimulated less phosphorylation of ErbB2 at pTyr-1221. These data demonstrate that AREG ultimately induces a quantitatively different biological response than EGF, TGFα, and BTC in these cells. It is possible that this difference in phosphorylation was due to differences in down-regulation of the EGF receptor by the four growth factors. Therefore, we examined the ability of the four different ligands to induce down-regulation of the EGF receptor. Cells were incubated for the indicated times with a saturating dose of the growth factor and then washed with low-pH buffer to remove surface-bound ligand. Residual cell surface EGF receptors were then detected via 125I-EGF binding. As shown in Fig. 2A, in cells expressing only the EGF receptor, all four ligands induced a similar, ∼65% decrease in cell surface EGF receptors over the time course of our assay. Expression of ErbB2 is known to reduce the ligand-stimulated internalization of the EGF receptor (29.Haslekås C. Breen K. Pedersen K.W. Johannessen L.E. Stang E. Madshus I.H. The inhibitory effect of ErbB2 on epidermal growth factor-induced formation of clathrin-coated pits correlates with retention of epidermal growth factor receptor-ErbB2 oligomeric complexes at the plasma membrane.Mol. Biol. Cell. 2005; 16: 5832-5842Crossref PubMed Scopus (79) Google Scholar, 30.Worthylake R. Opresko L.K. Wiley H.S. ErbB2 amplification inhibits down-regulation and induces constitutive activation of both ErbB2 and epidermal growth factor receptors.J. Biol. Chem. 1999; 274: 8865-8874Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). This was apparent in cells expressing both the EGF receptor and ErbB2 (Fig. 2B), where each ligand induced only an ∼30% decrease in cell surface EGF receptors. Because all four ligands appear to induce the same extent of down-regulation in both cell lines, the differences observed in our phosphorylation assays do not appear to be associated with differences in ligand-induced internalization of the EGF receptor. One possible explanation for the partial agonism of AREG in the phosphorylation assay is that it is less effective than the other growth factors at inducing the formation of EGF receptor dimers. We have previously used luciferase fragment complementation assays to measure the formation of EGF receptor homodimers and EGFR/ErbB2 heterodimers (31.Macdonald-Obermann J.L. Adak S. Landgraf R. Piwnica-Worms D. Pike L.J. Dynamic analysis of the epidermal growth factor (EGF) receptor-ErbB2-ErbB3 protein network by luciferase fragment complementation imaging.J. Biol. Chem. 2013; 288: 30773-30784Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 32.Yang K.S. Ilagan M.X. Piwnica-Worms D. Pike L.J. Luciferase fragment complementation imaging of conformational changes in the EGF receptor.J. Biol. Chem. 2009; 284: 7474-7482Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Therefore, we used this method to compare dimer formation induced by the four different ligands. Initially, we performed luciferase fragment complementation imaging using EGF receptors and ErbB2 that had been C-terminally truncated at the end of the transmembrane domain and thus lacked the entire intracellular domain (31.Macdonald-Obermann J.L. Adak S. Landgraf R. Piwnica-Worms D. Pike L.J. Dynamic analysis of the epidermal growth factor (EGF) receptor-ErbB2-ErbB3 protein network by luciferase fragment complementation imaging.J. Biol. Chem. 2013; 288: 30773-30784Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 32.Yang K.S. Ilagan M.X. Piwnica-Worms D. Pike L.J. Luciferase fragment complementation imaging of conformational changes in the EGF receptor.J. Biol. Chem. 2009; 284: 7474-7482Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Therefore, in these truncated constructs, dimerization was mediated only by interactions between the extracellular and/or transmembrane domains of the receptor subunits. For these experiments, the N-terminal half (NLuc) or C-terminal half (CLuc) of firefly luciferase was fused to the C terminus of the truncated EGFR (ΔC-EGFR) or the truncated ErbB2 (ΔC-ErbB2). A flexible linker was added between the truncated receptor and the NLuc or CLuc fragments to facilitate complementation. The appropriate pairs of receptors were then stably expressed in CHO cells. We first determined the dose response to all four ligands for complementation between ΔC-EGFR-NLuc and ΔC-EGFR-CLuc (Fig. 3). In this system, each ligand stimulated an initial rapid increase in light production, which then slowed toward a plateau after 10–15 min. A similar pattern was observed for complementation between ΔC-ErbB2-NLuc and ΔC-EGFR-CLuc (Fig. 4).FIGURE 4Luciferase complementation in CHO cells stably expressing ΔC-EGFR-CLuc and inducible ΔC-ErbB2-NLuc. CHO cells were plated into 96-well dishes and treated with doxycycline to induce the expression of the ΔC-ErbB2-NLuc. Cells were treated with the indicated concentrations of EGF (A), TGFα (B), BTC (C), or AREG (D) and assayed for luciferase fragment complementation as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) On the basis of these dose-response curves, the concentration of each growth factor that gave the highest level of complementation was selected and compared directly with the others in a luciferase complementation assay in cells coexpressing ΔC-EGFR-NLuc and ΔC-EGFR-CLuc (Fig. 5A) or ΔC-ErbB2-NLuc and ΔC-EGFR-CLuc (Fig. 5B). In both the homodimer (Fig. 5A) and the heterodimer (Fig. 5B), stimulation with EGF, TGFα, and BTC induced approximately twice as much complementation as the optimal dose of AREG. This is consistent with the results of the receptor autophosphorylation assays and suggests that the smaller biological effect of AREG may be due to a decreased ability to generate dimerized receptors compared with the other three growth factors. The lower level of dimerization induced by AREG is not simply due to the truncated nature of the receptor in which these assays were done. As shown in Fig. 6, A and B, AREG also stimulated lower levels of complementation in homodimers and heterodimers composed of receptors that were truncated after the intracellular juxtamembrane domain of the receptors (c'698-EGFR and c'709-ErbB2), known to form an antiparallel helical dimer (33.Jura N. Endres N.F. Engel K. Deindl S. Das R. Lamers M.H. Wemmer D.E. Zhang X. Kuriyan J. Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment.Cell. 2009; 137: 1293-1307Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Furthermore, the same pattern was observed when the full-length, kinase-dead version of the EGF receptor was used (Fig. 6, C and D). Therefore, AREG appears to function as a partial agonist with respect to the induction of dimer formation, regardless of receptor structure. As is apparent from the dose-response curves of AREG in FIGURE 3, FIGURE 4, at very high doses, the luciferase activity exhibits an unusual rapid rise and fall, followed by a slow rise. This suggests that receptor dimerization involves a multistep proc" @default.
• W2038733101 created "2016-06-24" @default.
• W2038733101 creator A5059328000 @default.
• W2038733101 creator A5070180381 @default.
• W2038733101 date "2014-09-01" @default.
• W2038733101 modified "2023-10-12" @default.
• W2038733101 title "Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation" @default.
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