Exploring Time-Resolved Characterization of the Heterogeneity and Dynamics of Ligand-Receptor Interactions on Living Cells


  • Pavel Barta Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Pharmacology and Toxicology, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic, Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Rudbeck Laboratory, 751 85 Uppsala, Sweden
  • Karl Andersson Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Rudbeck Laboratory, 751 85 Uppsala, Sweden, Ridgeview Instruments AB, Skillsta 4, 74020 Vänge, Sweden
  • Frantisek Trejtnar Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Pharmacology and Toxicology, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic
  • Jos Buijs Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Rudbeck Laboratory, 751 85 Uppsala, Sweden , Ridgeview Instruments AB, Skillsta 4, 74020 Vänge, Sweden




Cetuximab, EGF receptor, Interaction Map, kinetics, panitumumab, tracer.


 The time-resolved interaction analysis was applied on living cells to extract detailed interaction characteristics of two therapeutic antibodies and natural ligand binding to the same receptor expressed on two different human carcinoma cell lines.

The observed differences in the antibody binding characteristics and heterogeneity could be attributed both to differences in antibodies and cell lines. The stability of antibody binding to EGFR on cells is significantly higher than the binding stability to isolated EGFR. This higher stability can be of fundamental importance as it potentially shifts the drug-target residence time into a domain that is limiting in pharmacokinetics and hence is of importance for in vivodrug efficacy.

EGF binding to its receptor was more heterogeneous and it was demonstrated for the first time that time-resolved interaction measurements in combination with Interaction Map analysis could be used to probe the dynamics of a ligand (protein) induced dimerization and/or oligomerization process.


Copeland RA, Pompliano DL, Meek TD. Drug-target residence time and its implications for lead optimization. Nat Rev Drug Discov 2006; 5(9): 730-9. http://dx.doi.org/10.1038/nrd2082

Dahl G, Akerud T. Pharmacokinetics and the drug-target residence time concept. Drug Discov Today 2013; 18(15-16): 697-707. http://dx.doi.org/10.1016/j.drudis.2013.02.010

Abdiche YN, Malashock DS, Pons J. Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors. Protein Sci 2008; 17(8): 1326-35. http://dx.doi.org/10.1110/ps.035402.108

Garrido G, Tikhomirov IA, Rabasa A, Yang E, Gracia E, Iznaga N, et al. Bivalent binding by intermediate affinity of nimotuzumab: a contribution to explain antibody clinical profile. Cancer Biol Ther 2011; 11(4): 373-82. http://dx.doi.org/10.4161/cbt.11.4.14097

Rich RL, Myszka DG. Grading the commercial optical biosensor literature-Class of 2008: 'The Mighty Binders'. J Mol Recognit 2010; 23(1): 1-64. http://dx.doi.org/10.1002/jmr.1004

Rizo J, Rosen MK, Gardner KH. Enlightening molecular mechanisms through study of protein interactions. J Mol Cell Biol 2012; 4(5): 270-83. http://dx.doi.org/10.1093/jmcb/mjs036

Barta P, Malmberg J, Melicharova L, Strandgard J, Orlova A, Tolmachev V, et al. Protein interactions with HER-family receptors can have different characteristics depending on the hosting cell line. Int J Oncol 2012; 40(5): 1677-82.

Bjorkelund H, Gedda L, Andersson K. Avoiding false negative results in specificity analysis of protein-protein interactions. J Mol Recognit 2011; 24(1): 81-9. http://dx.doi.org/10.1002/jmr.1026

Novy Z, Barta P, Mandikova J, Laznicek M, Trejtnar F. A comparison of in vitro methods for determining the membrane receptor expression in cell lines. Nucl Med Biol 2012; 39(7): 893-6. http://dx.doi.org/10.1016/j.nucmedbio.2012.02.007

Rouzet F, Bachelet-Violette L, Alsac JM, Suzuki M, Meulemans A, Louedec L, et al. Radiolabeled fucoidan as a p-selectin targeting agent for in vivo imaging of platelet-rich thrombus and endothelial activation. J Nucl Med 2011; 52(9): 1433-40. http://dx.doi.org/10.2967/jnumed.110.085852

Altschuh D, Bjorkelund H, Strandgard J, Choulier L, Malmqvist M, Andersson K. Deciphering complex protein interaction kinetics using Interaction Map. Biochem Biophys Res Commun 2012; 428(1): 74-9. http://dx.doi.org/10.1016/j.bbrc.2012.10.008

Bjorkelund H, Gedda L, Barta P, Malmqvist M, Andersson K. Gefitinib induces epidermal growth factor receptor dimers which alters the interaction characteristics with (1)(2)(5)I-EGF. PLoS One 2011; 6(9): e24739. http://dx.doi.org/10.1371/journal.pone.0024739

Arteaga CL. Epidermal growth factor receptor dependence in human tumors: more than just expression? Oncologist 2002; 7 Suppl 4: 31-9. http://dx.doi.org/10.1634/theoncologist.7-suppl_4-31

Talavera A, Friemann R, Gomez-Puerta S, Martinez-Fleites C, Garrido G, Rabasa A, et al. Nimotuzumab, an antitumor antibody that targets the epidermal growth factor receptor, blocks ligand binding while permitting the active receptor conformation. Cancer Res 2009; 69(14): 5851-9. http://dx.doi.org/10.1158/0008-5472.CAN-08-4518

Lorenzo GD, Bianco R, Tortora G, Ciardiello F. Involvement of growth factor receptors of the epidermal growth factor receptor family in prostate cancer development and progression to androgen independence. Clin Prostate Cancer 2003; 2(1): 50-7. http://dx.doi.org/10.3816/CGC.2003.n.013

Gullick WJ. The Type 1 growth factor receptors and their ligands considered as a complex system. Endocr Relat Cancer 2001; 8(2): 75-82. http://dx.doi.org/10.1677/erc.0.0080075

Pastore S, Mascia F, Mariani V, Girolomoni G. The epidermal growth factor receptor system in skin repair and inflammation. J Invest Dermatol 2008; 128(6): 1365-74. http://dx.doi.org/10.1038/sj.jid.5701184

Nielsen DL, Kumler I, Palshof JA, Andersson M. Efficacy of HER2-targeted therapy in metastatic breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors. Breast 2013; 22(1): 1-12. http://dx.doi.org/10.1016/j.breast.2012.09.008

Li S, Schmitz KR, Jeffrey PD, Wiltzius JJ, Kussie P, Ferguson KM. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell 2005; 7(4): 301-11. http://dx.doi.org/10.1016/j.ccr.2005.03.003

Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988; 106(3): 761-71. http://dx.doi.org/10.1083/jcb.106.3.761

Citri A, Yarden Y. EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 2006; 7(7): 505-16. http://dx.doi.org/10.1038/nrm1962

Greenwood FC, Hunter WM, Glover JS. The Preparation of I-131-Labelled Human Growth Hormone of High Specific Radioactivity. Biochem J 1963; 89: 114-23.

Bjorkelund H, Gedda L, Malmqvist M, Andersson K. Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach. Mol Clin Oncol 2013; 1(2): 343-52.

Domagala T, Konstantopoulos N, Smyth F, Jorissen RN, Fabri L, Geleick D, et al. Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor. Growth Factors 2000; 18(1): 11-29. http://dx.doi.org/10.3109/08977190009003231

Onell A, Andersson K. Kinetic determinations of molecular interactions using Biacore--minimum data requirements for efficient experimental design. J Mol Recognit 2005; 18(4): 307-17. http://dx.doi.org/10.1002/jmr.745

Rich RL, Myszka DG. Survey of the 2009 commercial optical biosensor literature. J Mol Recognit 2011; 24(6): 892-914. http://dx.doi.org/10.1002/jmr.1138

Nilvebrant J, Kuku G, Bjorkelund H, Nestor M. Selection and in vitro characterization of human CD44v6-binding antibody fragments. Biotechnol Appl Biochem 2012; 59(5): 367-80. http://dx.doi.org/10.1002/bab.1033

Drake AW, Myszka DG, Klakamp SL. Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods. Anal Biochem 2004; 328(1): 35-43. http://dx.doi.org/10.1016/j.ab.2003.12.025

Hunter T, Cooper JA. Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell 1981; 24(3): 741-52. http://dx.doi.org/10.1016/0092-8674(81)90100-8

King AC, Cuatrecasas P. Resolution of high and low affinity epidermal growth factor receptors. Inhibition of high affinity component by low temperature, cycloheximide, and phorbol esters. J Biol Chem 1982; 257(6): 3053-60.

Yarden Y, Schlessinger J. Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry 1987; 26(5): 1443-51. http://dx.doi.org/10.1021/bi00379a035

Zhou M, Felder S, Rubinstein M, Hurwitz DR, Ullrich A, Lax I, et al. Real-time measurements of kinetics of EGF binding to soluble EGF receptor monomers and dimers support the dimerization model for receptor activation. Biochemistry 1993; 32(32): 8193-8. http://dx.doi.org/10.1021/bi00083a020

Odaka M, Kohda D, Lax I, Schlessinger J, Inagaki F. Ligand-binding enhances the affinity of dimerization of the extracellular domain of the epidermal growth factor receptor. J Biochem 1997; 122(1): 116-21. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a021718

Clayton AH, Tavarnesi ML, Johns TG. Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding. Biochemistry 2007; 46(15): 4589-97. http://dx.doi.org/10.1021/bi700002b

Gadella TW, Jr., Jovin TM. Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. J Cell Biol 1995; 129(6): 1543-58. http://dx.doi.org/10.1083/jcb.129.6.1543

Moriki T, Maruyama H, Maruyama IN. Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain. J Mol Biol 2001; 311(5): 1011-26. http://dx.doi.org/10.1006/jmbi.2001.4923

Needham SR, Hirsch M, Rolfe DJ, Clarke DT, Zanetti-Domingues LC, Wareham R, et al. Measuring EGFR separations on cells with ~10 nm resolution via fluorophore localization imaging with photobleaching. PLoS One 2013; 8(5): e62331. http://dx.doi.org/10.1371/journal.pone.0062331

Pang X, Zhou HX. Activation of signaling receptors: do ligands bind to receptor monomer, dimer, or both? BMC Biophys 2013; 6: 7. http://dx.doi.org/10.1186/2046-1682-6-7

Red Brewer M, Yun CH, Lai D, Lemmon MA, Eck MJ, Pao W. Mechanism for activation of mutated epidermal growth factor receptors in lung cancer. Proc Natl Acad Sci U S A 2013; 110(38): E3595-604. http://dx.doi.org/10.1073/pnas.1220050110

Macdonald JL, Pike LJ. Heterogeneity in EGF-binding affinities arises from negative cooperativity in an aggregating system. Proc Natl Acad Sci U S A 2008; 105(1): 112-7. http://dx.doi.org/10.1073/pnas.0707080105

Rees AR, Gregoriou M, Johnson P, Garland PB. High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion. EMBO J 1984; 3(8): 1843-7.

Russo AJ. Decreased Epidermal Growth Factor (EGF) Associated with HMGB1 and Increased Hyperactivity in Children with Autism. Biomark Insights 2013; 8: 35-41. http://dx.doi.org/10.4137/BMI.S11270

Hurwitz DR, Emanuel SL, Nathan MH, Sarver N, Ullrich A, Felder S, et al. EGF induces increased ligand binding affinity and dimerization of soluble epidermal growth factor (EGF) receptor extracellular domain. J Biol Chem 1991; 266(32): 22035-43.

Kankanala S. Studies of epidermal growth factor receptor targeted compounds using surface plasmon resonance. [Master Thesis]: Virginia Commonwealth University; 2009.

van Erp NP, Gelderblom H, Guchelaar HJ. Clinical pharmacokinetics of tyrosine kinase inhibitors. Cancer Treat Rev 2009; 35(8): 692-706. http://dx.doi.org/10.1016/j.ctrv.2009.08.004

Bjorkelund H, Gedda L, Andersson K. Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context. PLoS One 2011; 6(1): e16536.

Moasser MM, Basso A, Averbuch SD, Rosen N. The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells. Cancer Res 2001; 61(19): 7184-8.

Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: an emerging feature of cancer. Nat Rev Cancer 2008; 8(11): 835-50. http://dx.doi.org/10.1038/nrc2521

Seshacharyulu P, Ponnusamy MP, Haridas D, Jain M, Ganti AK, Batra SK. Targeting the EGFR signaling pathway in cancer therapy. Expert Opin Ther Targets 2012; 16(1): 15-31. http://dx.doi.org/10.1517/14728222.2011.648617




How to Cite

Pavel Barta, Karl Andersson, Frantisek Trejtnar, & Jos Buijs. (2014). Exploring Time-Resolved Characterization of the Heterogeneity and Dynamics of Ligand-Receptor Interactions on Living Cells. Journal of Analytical Oncology, 3(2),  94–104. https://doi.org/10.6000/1927-7229.2014.03.02.4