Analysis of EGF Receptor Oligomerization by Homo-FRET - Methods in Cell Biology - page 298

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 non-
invasive approach to study receptor dimerization and oligomerization. This method
is based upon the F¨rster energy transfer between identical adjacent fluorescent pro-
teins (homo-FRET) and is determined by analyzing the change in fluorescence an-
isotropy. Homo-FRET takes place within a distance of 10 nm, 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 exper-
iments for the determination of cluster sizes of growth factor receptors like EGFR.
INTRODUCTION
The epidermal growth factor receptor (EGFR), also called ErbB1 or Her1, is a mem-
ber of the ErbB single-pass transmembrane tyrosine kinase receptor family ( Ullrich
& Schlessinger, 1990; Yarden & Sliwkowski, 2001 ). Activation of EGFR and its
family members is involved in cell growth, cell proliferation, and migration. Many
cancer types show overexpression or deregulation of EGFR, and it is therefore a
well-studied receptor and an attractive anticancer drug target ( Oliveira, van
Bergen en Henegouwen, Storm, & Schiffelers, 2006; Sorkin & Goh, 2009 ). EGFR
is composed of an extracellular domain, a transmembrane domain, and an intracel-
lular C-terminal tail containing the tyrosine kinase and several sites involved in post-
translational modifications and signaling ( Jorissen et al., 2003 ). The extracellular
part of EGFR contains four domains of which domain I and III are involved in ligand
binding and domain II in receptor dimerization. More than 20 different ligands are
known for receptors from the ErbB family of which EGF is the most studied one.
Ligand binding induces conformational changes of the ectodomain, which results
in not only receptor dimerization but also even receptor oligomerization or clustering
( Clayton et al., 2005 ). As a consequence of these changes, cross-phosphorylation of
its C-terminal tail occurs ( Jura et al., 2009 ). Interestingly, EGFR can form homo- or
heterodimers with other ErbB family members and this already happens in resting
cells. These inactive dimers on the plasma membrane are the so-called receptor pre-
dimers. Although the structure of receptor dimers is clear from crystal structures, the
mechanism of receptor oligomerization remains obscure ( Ferguson, 2008 ). Crystal
structures have shown head to head interactions between receptors, suggesting the
involvement of these sequences in oligomerization. Recently, also kinase activity
was shown to be required for receptor clustering, which suggests the involvement
of signaling in this process ( Clayton et al., 2005; Hofman et al., 2010 ). For example,