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Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
2013 (Engelska)Ingår i: Molecular and Clinical Oncology, ISSN 2049-9469, Vol. 1, nr 2, s. 343-352Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Overexpression and aberrant activity of the epidermal growth factor (EGF) have been observed in various cancer types, rendering it an important target in oncology research. The interaction between EGF and its receptor (EGFR), as well as subsequent internalization, is complex and may be affected by various factors including tyrosine kinase inhibitors (TKIs). By combining real‑time binding curves produced in LigandTracer® with internalization assays conducted at different temperatures and with different TKIs, the processes of ligand binding, internalization and excretion was visualized. SKOV3 cells had a slower excretion rate compared to A431 and U343 cells, and the tested TKIs (gefitinib, lapatinib, AG1478 and erlotinib) reduced the degree of internalization. The kinetic analysis of the binding curves further demonstrated TKI‑dependent balances of EGFR monomer and dimer populations, where lapatinib promoted the monomeric form, while the other TKIs induced dimers. The dimer levels were found to be associated with the apparent affinity of the EGF‑EGFR interaction, with EGF binding stronger to EGFR dimers compared to monomers. This study analyzed how real‑time molecular interaction analysis may be utilized in combination with perturbations in order to understand the kinetics of a ligand‑receptor interaction, as well as some of its associated intracellular processes. Our multiple‑temperature and ‑inhibitor assay setup renders it possible to follow the EGFR monomer, dimer and internalized populations in a detailed manner, allowing for a new perspective of the EGFR biology.

Ort, förlag, år, upplaga, sidor
2013. Vol. 1, nr 2, s. 343-352
Nyckelord [en]
epidermal growth factor, tyrosine kinase inhibitors, internalization, kinetics, dimerization, heterogeneity
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Medicin; Molekylär bioteknik; Medicinsk cellbiologi
Identifikatorer
URN: urn:nbn:se:uu:diva-183868DOI: 10.3892/mco.2012.37OAI: oai:DiVA.org:uu-183868DiVA, id: diva2:564874
Tillgänglig från: 2012-11-05 Skapad: 2012-11-05 Senast uppdaterad: 2018-12-04
Ingår i avhandling
1. Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
Öppna denna publikation i ny flik eller fönster >>Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
2013 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now.

This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates.

In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2013. s. 65
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 854
Nyckelord
Heterogeneity, Kinetics, EGFR, HER2, LigandTracer, Interaction Map, Internalization, Specificity
Nationell ämneskategori
Medicinsk bioteknologi Cell- och molekylärbiologi
Forskningsämne
Medicinsk vetenskap
Identifikatorer
urn:nbn:se:uu:diva-183872 (URN)978-91-554-8570-2 (ISBN)
Disputation
2013-02-15, Rudbeck Hall, Rudbeck Laboratory, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2013-01-24 Skapad: 2012-11-05 Senast uppdaterad: 2018-01-12Bibliografiskt granskad

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Förlagets fulltexthttp://www.spandidos-publications.com/10.3892/mco.2012.37

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Björkelund, HannaGedda, LarsMalmqvist, MagnusAndersson, Karl

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