title: Analysis of EGF receptor oligomerization by homo-FRET.
abstract: Growth factor receptors are present in the plasma membrane of resting cells as monomers or (pre)dimers. Ligand binding results in higher-order oligomerization of ligand-receptor complexes. To study the regulation of receptor clustering, several experimental techniques have been developed in the last decades. However, many involve invasive approaches that are likely to disturb the integrity of the membrane, thereby affecting receptor interactions. In this chapter, we describe the use of a noninvasive approach to study receptor dimerization and oligomerization. This method is based upon the Förster energy transfer between identical adjacent fluorescent proteins (homo-FRET) and is determined by analyzing the change in fluorescence anisotropy. Homo-FRET takes place within a distance of 10nm, making this an excellent approach for studying receptor-receptor interactions in intact cells. After excitation of monomeric GFP (mGFP) with polarized light, limiting anisotropy values (r(inf)) of the emitted light are determined, where proteins with known cluster sizes are used as references. Dimerization and oligomerization of the epidermal growth factor receptor (EGFR) in response to ligand binding is determined by using receptors that have been fused with mGFP at their C-terminus. In this chapter, we describe the involved technology and discuss the feasibility of homo-FRET experiments for the determination of cluster sizes of growth factor receptors like EGFR.
authors.affiliation: Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, Netherlands.
authors.forename: Cecilia
authors.initials: C
authors.lastname: de Heus
authors.forename: Nivard
authors.initials: N
authors.lastname: Kagie
authors.forename: Raimond
authors.initials: R
authors.lastname: Heukers
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen en Henegouwen
authors.forename: Hans C
authors.initials: HC
authors.lastname: Gerritsen
mesh_terms: D000818:Animals; D016880:Anisotropy; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D041681:NIH 3T3 Cells; D010957:Plasmids; D011485:Protein Binding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C463061:AP20187; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus
keywords: Anisotropy; Clustering; Dimerization; EGFR; FRET; Growth factor; Homo-FRET; Oligomerization; Receptor; mGFP