CHAPTER

Analysis of EGF Receptor

Oligomerization by
Homo-FRET
Cecilia de Heus*, Nivard Kagie{, Raimond Heukers*,
16
Paul M.P. van Bergen en Henegouwen*, and Hans C. Gerritsen{
*
Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, Netherlands
{
Molecular Biophysics, Department of Soft Condensed Matter and Biophysics, Science Faculty,
Utrecht University, Utrecht, Netherlands

CHAPTER OUTLINE
Introduction ............................................................................................................ 306
16.1 Theory Homo-FRET Quantification ....................................................................309
16.1.1 Steady-State Fluorescence Anisotropy Imaging ............................. 309
16.1.2 Time-Resolved Fluorescence Anisotropy Imaging........................... 311
16.2 Materials........................................................................................................313
16.2.1 Plasmid Constructs..................................................................... 313
16.2.2 Cell Lines .................................................................................. 315
16.2.3 Microscope ................................................................................ 315
16.2.3.1 Confocal Time-Resolved and Steady-State Fluorescence
Anisotropy Imaging Microscopy ........................................................315
16.3 Methods .........................................................................................................316
16.3.1 Slide Preparation........................................................................ 316
16.3.1.1 Reference Measurements ...................................................316
16.3.1.2 Predimerization Measurements ...........................................316
16.3.1.3 Ligand-induced Oligomerization ..........................................317
16.3.2 Analysis..................................................................................... 317
16.4 Discussion......................................................................................................317
Acknowledgments ................................................................................................... 319
References ............................................................................................................. 320

Abstract
Growth factor receptors are present in the plasma membrane of resting cells as mono-
mers or (pre)dimers. Ligand binding results in higher-order oligomerization of
ligand–receptor complexes. To study the regulation of receptor clustering, several
Methods in Cell Biology, Volume 117
Copyright © 2013 Elsevier Inc. All rights reserved.
ISSN 0091-679X
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305
306 CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET

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 Förster 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 (rinf) 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,
 Introduction 307

the production of phosphatidic acid by phospholipase D2 (PLD2) has been

demonstrated to be required for EGFR clustering (Ariotti et al., 2010). Besides recep-
tor oligomerization and downstream signaling toward cell proliferation, activation of
EGF receptors results in a rapid internalization mainly via clathrin-mediated
endocytosis. The intracellular transport trajectory finally ends up in lysosomes where
the ligand-induced signaling is terminated by degradation of both ligand and recep-
tors (Goh, Huang, Kim, Gygi, & Sorkin, 2010; Sorkin & Goh, 2009).
For studying the clustering of EGF receptors, different experimental techniques
have been developed. Dimerization of EGFR, for example, can be determined using
chemical cross-linking and detection by SDS-PAGE, by co-IP with differentially
tagged EGFR, or by fluorescence complementation (Moriki, Maruyama, &
Maruyama, 2001; Yu, Sharma, Takahashi, Iwamoto, & Mekada, 2002; Zhu, Iaria,
Orchard, Walker, & Burgess, 2003). Studying receptor clustering is more difficult
and demands other experimental strategies. Receptor clustering was initially inves-
tigated using electron microscopy (EM), which can be used at high resolution, en-
abling visualization of gold-labeled EGFR (van Belzen et al., 1988). Analysis of
cluster formation can be done by determining particle distances, either by hand or
automatically using Ripley’s function (Hancock & Prior, 2005). However, limita-
tions exist with respect to determining cluster size, which is dependent on the size
of the gold particle and the antibody (
10 nm) (Hancock & Prior, 2005). Another
approach for determining receptor clustering involves fluorescence light micros-
copy. However, conventional microscopy has a resolution limit of
200 nm, which
makes this technique not very suitable for measuring the formation of small receptor
clusters. To overcome this resolution limit problem, more advanced light microscope
techniques were developed, which do provide information about small cluster sizes.
One of the first examples was described by Gadella and coworkers and included
time-resolved fluorescence microscopy based on Förster resonance energy transfer
(FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM) (Gadella &
Jovin, 1995).
FRET is based on the energy transfer between a donor and an acceptor fluorophore.
For FRET to occur, the donor emission spectrum and acceptor excitation spectrum
need sufficient spectral overlap and the two fluorophores should be within a distance
of
10 nm. Because energy transfer only occurs when fluorophores are within 10 nm
of each other, the detection of FRET can be used to study receptor dimerization or
small cluster formation. There are different methods to detect FRET, for example,
by measuring changes in the donor/acceptor emission intensity ratio, changes in fluo-
rescence lifetime (FLIM), or differences in anisotropy. The donor/acceptor ratio is
determined by measuring the emission of both fluorophores. However, this donor/
acceptor ratio is dependent on the concentrations of both fluorophores. With FLIM,
the time that the donor fluorophore is in its excited state is measured. This lifetime
can be determined by measuring the fluorescence decay of the donor probe after ex-
citation by using a short laser pulse. The fluorescence lifetime changes upon its envi-
ronment and also when energy transfer occurs. This method is less dependent on local
concentrations of both probes and is therefore a more robust way to detect FRET
(Chen, Mills, & Periasamy, 2003; Hofman et al., 2008).
308 CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET

FRET-FLIM was previously used successfully to determine EGFR cluster forma-

tion by Clayton et al. (2005). EGFR-GFP in combination with anti-phosphotyrosine-
Alexa555 was used to label fixed and permeabilized cells. Using FRET-FLIM in
combination with image correlation spectroscopy (ICS), Clayton et al. found an av-
erage cluster size of 2.2 in the absence of EGF and an average of 3.7 in the presence
of EGF, suggesting the formation of higher-order clusters upon ligand binding.
A limitation of this method is that it assumes the cell to be stationary and therefore
only an average of the different cluster sizes in a cell can be determined instead of
determining the subcellular distribution of the different cluster sizes. To solve this,
Saffarian et al. studied EGFR clustering by a method based on fluorescence intensity
distribution analysis (FIDA) in live cells for quantifying receptor clustering on cell
membranes (Saffarian, Li, Elson, & Pike, 2007). Using this technique, the authors
find an average cluster size of 1.3 in unstimulated cells, which is in contrast to results
from Clayton et al. The difference between the two values might be explained by
different measurement conditions, live cells versus fixed cells, and measuring the
clustering distribution with FIDA instead of determining an average. A limitation
of the FIDA method is that intensity fluctuations might occur during the measure-
ments, which might disturb the clustering measurements. Furthermore, the FIDA
method is relatively slow and may therefore not be suitable for measuring of EGFR
clustering after EGF stimulation because of the fast internalization of EGFR. More
recently, a number and brightness (N&B) analysis technique was used to study
EGFR clustering, which demonstrated the formation of up to pentameric EGFR clus-
ters (Sako, Minoghchi, & Yanagida, 2000). More recently, we introduced a method
based on homo-FRET to analyze EGFR clustering. With this method, EGFR clus-
tering can be accurately measured on intact cells using only a single label
(EGFR-mGFP) (Bader, Hofman, Voortman, en Henegouwen, & Gerritsen, 2009;
Hofman et al., 2010).
For homo-FRET, the same fluorophore for donor and acceptor is used, which has
the advantage that the detection of FRET is not dependent on concentrations of donor
and acceptor. Another advantage of only one fluorophore is that sample preparation
is simplified. However, homo-FRET does require a different quantification method
because homo-FRET does not result in a change in the emission spectrum or in fluo-
rescence lifetime, resulting in FRET to stay unnoticed. The decreased lifetime of the
donor fluorophore in homo-FRET is compensated by the excitation by the acceptor
fluorophore. The here applied analysis of energy transfer is based upon the FRET-
induced loss of anisotropy. When fluorophores are excited with polarized light, only
the subset that is in the right orientation will be excited. In the case of large molecules
that are unable to spin on the time scale of the fluorescence lifetime, the emitted light
by this subset of fluorophores is also polarized, albeit with a slightly different angular
distribution. In the case of two fluorophores in close proximity, there is a high prob-
ability that they are in different orientations. Upon excitation of one fluorophore, the
neighboring one could be excited through FRET, resulting in the emission of light
with this different orientation. Therefore, the energy transfer between two identical
fluorophores with slightly different orientation result in depolarization of the
 16.1 Theory Homo-FRET Quantification 309

emission light; the emitted light is more isotropically distributed. Therefore, quan-
tification of the fluorescence anisotropy can be used to determine homo-FRET.
The homo-FRET method can be calibrated using reference measurements on
protein clusters of known size. Subsequently, EGFR cluster sizes can be determined
directly from measured fluorescence anisotropy values. As a tool for the reference mea-
surements, we have used FKBP-mGFP, which dimerizes via its ligand AP20187. Sim-
ilarly, 2xFKBP-mGFP can form oligomers by the addition of AP20187. Clusters made
with the FKBP constructs can therefore be either monomeric in the absence of their
ligand or dimers or oligomers in the presence of ligand. The level of anisotropy of
monomers, dimers, or oligomers can be used as a reference for cluster size measure-
ments. This homo-FRET method can be employed to study the clustering behavior of
EGFR for fundamental research and anticancer drug research. For example, it has been
shown that EGFR can also form inactive dimers in the absence of EGF; these predimers
are formed independently of the C-terminal tail. In contrast, EGF-dependent oligomer-
ization depends on tyrosine kinase activity and more particular on the nine tyrosine
residues in the C-terminal tail of the receptor (Hofman et al., 2010).

16.1 THEORY HOMO-FRET QUANTIFICATION

16.1.1 Steady-state fluorescence anisotropy imaging
In homo-FRET experiments, a linearly polarized excitation laser beam is used to ex-
cite the fluorophores. Next, the intensities of the parallel and perpendicular polarized
fluorescence are detected. Here, parallel and perpendicular are defined with respect
to the polarization direction of the excitation laser. From these intensities of the fluo-
rescence, the anisotropy can be calculated using Eq. (16.1). Upon homo-FRET be-
tween fluorophores, the anisotropy decreases because the energy transfer between
two fluorophores results in emission by a molecule with a different orientation than
the molecule directly excited by the laser (Fig. 16.1) (Bader et al., 2009; Lakowicz,
2006; Runnels & Scarlata, 1995):
Ipar Iper
r ðtÞ ¼ (16.1)
Ipar  2Iper
Besides homo-FRET, other processes can affect the fluorescence anisotropy, such as
rotational diffusion of fluorophores. If the excited fluorophore changes its orientation
in the time period between excitation and emission, the anisotropy will be lowered.
However, in case of a large, slowly rotating molecule like a green fluorescent protein
(GFP), the depolarization due to rotations is negligible (Bader, Hofman, van Bergen
en Henegouwen, & Gerritsen, 2007). The rotation correlation time of fluorescent
proteins is typically in the order of 20 ns, while the fluorescence lifetime is usually
<2.5 ns. The fluorophores in the sample can be separated into two groups, fluoro-
phores that are directly excited by the polarized excitation light and fluorophores that
are indirectly excited by the energy transfer involved in homo-FRET. Runnels and
310 CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET

Emmision of
Excitation with Emmision of directly mGFP after energy
polarized light excited mGFP transfer

par
par

per per

mGFP

Homo-FRET

FIGURE 16.1
Schematic representation of homo-FRET in a dimer of monomeric GFP (mGFP), causing
depolarization of the polarized excitation light.

Scarlata (1995) introduced a simple model to calculate cluster sizes from the steady-
state anisotropy (rss) (Eq. 16.2):
1 þ ot ðN  1Þot
rss ¼ rmono þ ret (16.2)
1 þ N ot 1 þ N ot
Here, rmono is the anisotropy of monomers, ret is the anisotropy after energy transfer,
N is the number of molecules in the cluster, and o is defined as

o ¼ ðR0 =RÞ6 =t
where R0 is the Förster distance, R is the distance between two fluorophore, and t the
fluorescence lifetime. The homo-FRET rate o can be determined from time-resolved
anisotropy experiments. In steady-state anisotropy experiments, the rate is either es-
timated or assumed to be much faster than the rate of fluorescence. In the latter case,
ot ! 1 (under these conditions E ¼ 1) and Eq. (16.2) reduces to Eq. (16.3):
1 N1
rss ¼ rmono þ ret (16.3)
N N
In the original work by Runnels and Scarlatta, they presented an example where an-
isotropy after a single energy transfer step is about zero. This further simplifies
Eq. (16.2) to
 16.1 Theory Homo-FRET Quantification 311

FKBP-mGFP 2xFKBP-mGFP

Pentamer
Tetramer
Trimer

Dimer
Dimer
Monomer
Monomer

AP20187 - + - +
FIGURE 16.2
Native PAGE analysis of dimerization constructs. Cells expressing FKBP-mGFP or 2xFKBP-
mGFP, either incubated with AP20187 or mock-treated, were lysed under nonreducing
and nondenaturing conditions. After size separation on a native PAGE gel and blotting to
PVDF membrane, proteins were detected with anti-GFP antibodies.
Adapted from Bader et al. (2009).

1
rss ¼ rmono (16.4)
N
However, our experimental work indicated that the assumptions mentioned earlier
are not correct for our applications. Often, the energy transfer efficiency is not high
enough to ignore and the anisotropy after energy transfer is not equal or close to zero.
Therefore, Eq. (16.3) and (16.4) are not generally applicable. Determination of ret is
not trivial; therefore, we opted for using reference measurements to relate anisotropy
with cluster sizes. This also takes into account effects due to an energy transfer that is
not equal or close to 100%. We employ reference constructs to produce clusters of
known size. To this end, FKBP-mGFP and 2xFKBP-mGFP constructs were used;
they form monomers in the absence of AP20187 but dimers and oligomers, respec-
tively, when AP20187 is added (Fig. 16.2).
Relative steady-state depolarization values due to clustering, r/rmono, of the
reference constructs were determined (Fig. 16.3) (Bader et al., 2009). The value
of r/rmono for dimers was 0.86 and for oligomers 0.78. Using these reference values,
steady-state anisotropy can be converted into an average cluster size on a scale of
N ¼ 1, N ¼ 2, and N  3.

16.1.2 Time-resolved fluorescence anisotropy imaging

In time-resolved fluorescence anisotropy (imaging), the anisotropy decay is
measured. This method provides more detailed information about the homo-FRET
process than steady-state anisotropy measurements. For large molecules or proteins,
the influence of rotation can be neglected and the decay due to homo-FRET can be
described by Eq. (16.5) (Gautier et al., 2001; Tanaka & Mataga, 1979):
312 CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET

FIGURE 16.3
(A) Model for the clustering of FKBP-mGFP constructs when AP2018 is added. (B) Steady-
state fluorescence images and anisotropy images of FKBP-mGFP constructs.
Results adapted from Bader et al. (2009) and Hofman et al. (2010).

rhomoFRET ðtÞ ¼ ðrmono  rinf Þe2ot þ rinf (16.5)

The limiting anisotropy rinf is usually reached within nanoseconds (Fig. 16.4). At rinf,
homo-FRET has occurred multiple times and the probability of emitting a photon is
equal for all fluorophores.
In this case, the limiting anisotropy is a direct measure of the cluster size that is
not affected by the energy transfer rate o, and it can be determined using Eq. (16.6)
(Bader et al., 2007):
rmono N1
rinf ¼ þ ret (16.6)
N N
 16.2 Materials 313

FIGURE 16.4
A schematic presentation of typical anisotropy decay due to homo-FRET. The limiting
anisotropy (rinf) is reached within a few nanoseconds and is dependent on the efficiency of
energy transfer.
Adapted from Bader et al. (2007).

Importantly, the value of rinf only depends on the cluster size of EGFR, while rss also
depends on the homo-FRET efficiency. Therefore, the clustering can be more accu-
rately determined, without the generally unknown contribution to the transfer effi-
ciency, by time-resolved anisotropy imaging. We determined rinf values from
time-resolved measurements on cells expressing EGFR-FKBP-mGFP and EGFR-
2xFKBP-mGFP in the presence and absence of AP20187 (Fig. 16.5). This resulted
in relative changes in limiting anisotropy for dimers r/rinf ¼ 0.82 and for oligomers
r/rinf ¼ 0.72. These relative changes are almost
30% larger than in the steady-state
measurement. The difference is the result of the contribution from the initial part of
the anisotropy decay. This again indicates the advantage of time-resolved measure-
ments over steady-state measurements. Finally, we note that the measurement of the
anisotropy decays allows direct determination of the energy transfer rate o. In the ear-
lier examples, an energy transfer rate o of 70% can be estimated in the case of dimers.
Using the Förster distance R0 for homo energy transfer between two GFPs of about 4.65
(Sharma et al., 2004), a distance of
4 nm is found between two GFPs.

16.2 MATERIALS
16.2.1 Plasmid constructs
For homo-FRET measurements, a monomeric GFP (mGFP) was used as fluorophore
because normal GFP tends to cluster when expressed in high concentrations with a
KD of 110 mM (Zacharias, Violin, Newton, & Tsien, 2002). mGFP has a point mu-
tation at position 206, resulting in a decrease in self-association and therefore more
reliable clustering measurements (Zacharias et al., 2002). This mGFP construct can
314 CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET

EGFR-mGFP EGFR-FKBP-mGFP
EGFR-2xFKBP-mGFP
+AP20187 +AP20187

rinf
B 0.4 0.2

EGFR-FKBP EGFR-FKBP EGFR-2xFKBP

-mGFP -mGFP -mGFP
- + AP20187 + AP20187
FIGURE 16.5
(A) Model for EGFR-FKBP-mGFP clustering in the presence of AP20187. (B) Fluorescence
and time-resolved anisotropy images of EGFR-FKBP-mGFP constructs.
Results adapted from Hofman et al. (2010).
 16.2 Materials 315

be used for control measurements and also for fusion to other proteins to measure
clustering. For the reference measurements, several constructs can be used; for cy-
toplasmic proteins, mGFP is fused to FKBP: 1xFKBP-mGFP or 2xFKBP-mGFP.
FKBP encoding cDNA can be obtained from the vector pC4-Fv1E (pC4-1xFKBP,
Ariad, now the Clontech iDimerize™ Inducible Homodimer System). FKBP can
be dimerized by binding of the ligand AP20187. As a result, 1xFKBP-mGFP will
dimerize, while a 2xFKBP-mGFP construct will form trimers, tetramers, etc.
(Fig. 16.1). For calibration of EGFR clustering, three different constructs were made:
EGFR-mGFP, EFGR-FKBP-mGFP, and EGFR-2xFKBP-mGFP. All these con-
structs were used for transient transfections. For the production of stable cell lines,
the constructs were subcloned into a pcDNA3.1 vector, which contains a Zeocin
resistance gene. All constructs were amplified in E. coli.

16.2.2 Cell lines

All EGFR-expressing cells can be used for measuring EGFR clustering using homo-
FRET. We normally use HeLa and A431 cells and NIH3T3 clone 2.2 cells that do not
express EGFR, which were transfected with human EGFR (Her14 cells). All cells
were cultured at 37  C and 5% CO2 in Dulbecco’s modified eagle’s medium
(PAA, Cambridge) containing 7% fetal bovine serum albumin, 2 mM L-glutamine,
100 U/ml penicillin, and 100 mg/ml streptomycin. For the establishment of stable cell
lines expressing FKBP-mGFP, 2xFKBP-mGFP, EGFR-mGFP, EGFR-FKBP-
mGFP, and EGFR-2xFKBP-mGFP, the cells were transfected with 2 mg plasmid to-
gether with 6 ml FuGENE-6 according to the manufacture’s protocol and then grown
under selective conditions (500 mM Zeocin) for at least 8 weeks.

16.2.3 Microscope
16.2.3.1 Confocal time-resolved and steady-state fluorescence
anisotropy imaging microscopy
A confocal microscope (Nikon C1, Japan) was equipped with a 473 nm solid-state
diode laser (Becker & Hickl, BDL-473-SMC) with a pulse width of 60 ps and a rep-
etition rate of 80 MHz. The laser light was polarized by a linear polarizer (Meadow-
lark, Frederick, CO, USA) and focused by a 60 water immersion objective (N.A.
1.2, Nikon). The high NA objectives, like the one used here, result in reduced values
for the anisotropy. This does not, however, affect the cluster sizes when reference
constructs are used to calibrate cluster sizes. Fluorescence emission was selected
by a 515/30 nm band-pass filter. The emitted light was then split in a parallel and
a perpendicular channel with respect to the excitation light by a broadband polarizing
beam splitter cube (PBS, OptoSigma, Santa Ana, CA, USA). Signals were detected
with two high quantum efficiency photomultiplier tubes (Hamamatsu H7422P-40)
connected to two time-gated fluorescence lifetime imaging modules (LIMO (De
Grauw & Gerritsen, 2001), Nikon Instruments BV, Badhoevedorp, the Netherlands).
For each pixel in the image, the LIMOs collect photons in four 2 ns-wide consecutive
316 CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET

time gates. Two synchronized LIMOs are used to capture the time-resolved anisot-
ropy decays of each polarization direction. An acquisition time of 3 ms per pixel was
chosen and a threshold of 300 counts was applied. At this dwell time, the maximum
number of counts per pixel amounted to
4500. All images covered an area of
50  50 mm at 160  160 pixels.
Determination of the time-resolved anisotropy using the two separate detection
channels requires careful synchronization in time and correction for their difference
in sensitivity. The former was achieved by using reference dye with fast decay time
(aqueous solution of Rose Bengal, t ¼ 70 ps). The correction factor for the difference
in transmission (Ctr) between both channels was determined by recording an anisot-
ropy image of an aqueous solution of fluorescein. In this case, fast rotation of the
fluorophore will result in emission that is completely depolarized before the second
gate opens. Consequently, the anisotropy in the last three gates will be zero, and
Ipar ¼ Ctr Iper. Finally, a correction was applied to correct for small difference in gate
width between the parallel and perpendicular channel. These differences were deter-
mined using a sample containing 10 mM GFP monomers in 50/50 glycerol/buffer. In
this solution, rotation of the fluorophore is much slower than the fluorescence life-
time. Consequently, the anisotropy will remain constant at the r0 level. For every
gate, a correction factor was determined that ensures that the anisotropy in this gate
is identical to the steady-state anisotropy. The absence of concentration-dependent
homo-FRET in the GFP solution was checked by lowering the concentration; no in-
crease in anisotropy was observed. Steady-state anisotropy images were obtained by
summation of the intensities in all four gates.

16.3 METHODS
16.3.1 Slide preparation
16.3.1.1 Reference measurements
NIH3T3 2.2 cells stably transfected with cDNA encoding the indicated reference
protein were seeded on glass coverslips and the day after treated with 1 mM
AP20187 for 1–2 h at 37  C to allow controlled dimerization and oligomerization. Af-
ter treatment, cells were fixed with 4% paraformaldehyde (PFA) and autofluorescence
was quenched by 100 mM glycine/PBS. Finally, the coverslips were embedded in 10%
polyvinyl-alcohol (Mowiol), and the anisotropy values were determined and used as
reference for the determination of cluster size. Samples can be stored at 4  C until
further use.

16.3.1.2 Predimerization measurements

NIH3T3 2.2 cells were transfected with cDNA encoding either mGFP or EGFR-mGFP.
The cells were seeded on 18 mm glass coverslips and allowed to adhere overnight. Cells
were fixed with 4% PFA followed by the quenching of autofluorescence with 100 mM
glycine/PBS for 10 min. The coverslips were embedded in Mowiol and stored at 4 or
20  C until further use. However, determination of anisotropy immediately after
 16.4 Discussion 317

embedment is preferred. These treatments were previously shown not to influence the
clustering behavior (Bancroft & Stevens, 1982). To exclude any effect of the fused
mGFP, a nice control is to express EGFR-mGFP in cells that contain endogenous EGFR
like A431 and Her14. In these cells, hetero-predimers between EGFR-mGFP and en-
dogenous EGFR will cause the anisotropy to be similar to the mGFP control.

16.3.1.3 Ligand-induced oligomerization

Cells were seeded on an 18 mm glass coverslip. The day after, cells were
serum-starved overnight in medium containing 0.5% serum. After serum starvation,
cells were treated with 8 nM EGF for 10 min at 37  C. Then, cells were fixed
with 4% PFA for 20 min at room temperature and autofluorescence was quenched with
100 mM glycine/PBS for 10 min. Finally, the coverslips were embedded in Mowiol
and used for measurement immediately or stored at 4 or 20  C until further use.

16.3.2 Analysis
In the steady-state anisotropy experiments, cluster sizes can be determined by direct
comparison of the measured anisotropy values with the reference anisotropies found
from the reference measurements on the FKBP constructs. Here, the steady-state an-
isotropies are calculated by integrating the intensities of all four time gates for both
polarization directions. In addition, the difference in transmission between the two
polarization channels can be corrected for by multiplying Iper by Ctr. In the time-
resolved anisotropy experiments, rinf values are extracted from the time-gated fluo-
rescence anisotropy images. Cluster sizes can again be obtained by comparison with
the reference measurements on FKBP constructs. The anisotropy values in the last
gates are, in good approximation, equal to rinf, provided that E is high enough. This
condition is met for the three last gates provided that E > 0.5. Four-gate anisotropy
decays are created by binning the intensities Ipar and Iper per gate in regions of inter-
est. In the anisotropy imaging experiments, a threshold of Ipar,inf þ 2Iper,inf > 300
counts was applied to all images. In theory, this number of counts corresponds to
a standard deviation in the anisotropy of 0.05 (Bader et al., 2009; Hofman et al.,
2010). An example of cluster size images derived from homo-FRET images is shown
in Fig. 16.6A, where a resting cell before EGF stimulation is shown. Here, cluster
sizes are small, but a clear population of predimers is visible;
40% of the EGFR
is present as dimer. After stimulating the cell with EGF for 10 min average, cluster
sizes have increased up to >2; and a significant fraction of the clusters are of size >3
(Fig. 16.6B).

16.4 DISCUSSION
Studying EGFR dimerization and oligomerization is important to completely under-
stand the signaling and trafficking mechanism of EGFR. EGFR dimerization has
been studied by several biochemical studies like chemical cross-linking, co-IP with
318 CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET

A B

EGFR-mGFP EGFR-mGFP
+EGF

NAV=1 NAV=2 NAV ³ 3

FIGURE 16.6
(A) Homo-FRET-based cluster size image before stimulation with EGF and (B) after
stimulation with 8 nM EGF.
Results adapted from Hofman et al. (2010).

differentially labeled EGFR, and also crystallography to determine the monomer and
dimer structure. Although there is already a lot known about the dimerization of
EGFR, the mechanism of oligomerization remains obscure. Advanced microscopy
techniques were developed to study EGFR oligomerization; initially EM with
gold-labeled EGFR was used. The cluster size that can be determined by EM depends
on the labeling of EGFR, in other words the size of the gold particles and antibody
used (10 nm). Advanced fluorescence microscopy techniques were developed like
FRET-FLIM, FIDA, and N&B analysis. These techniques are able to measure small
cluster sizes and are complementary techniques. FRET-FLIM is rapid and accurate,
while FIDA is able to measure cluster size in life cells. FIDA measures the clustering
on the plasma membrane of cells and is a slow method; therefore, it is not suitable for
measuring EGF-induced EGFR clustering in time. EGFR will internalize before the
FIDA method is able to determine the cluster sizes on the plasma membrane. N&B
uses the same principle as fluorescence correlation spectroscopy (FCS) measure-
ments and measures the distribution and association states of molecules in live cells
(Nagy, Claus, Jovin, & Arndt-Jovin, 2010). This technique demonstrated the
concentration-dependent predimer formation. In contrast, FIDA is able to give infor-
mation about the distribution of cluster sizes within a single pixel. FIDA and FCS,
however, need high illumination intensities for accurate measurements, which can
cause photodamage to the proteins in the cell. To study in more detail the EGF-
induced EGFR clustering, FRET was used and more specifically the homo-FRET
method. With this method, small EGFR clusters can be accurately determined and
the cluster size distribution in one cell can be determined within single pixels.
The advantage of homo-FRET instead of hetero-FRET is that the detection of FRET
is not dependent on the concentrations of donor and acceptor, which simplifies the
sample preparation. Homo-FRET is detected by a change in anisotropy, which is a
different method than used for measuring hetero-FRET. Anisotropy is a measure for
 Acknowledgments 319

the depolarization of the linear polarized excitation beam. To be able to convert the
obtained anisotropy values into cluster sizes, we use reference proteins like EGFR-
FKBP-mGFP constructs, which form clusters of known sizes when AP20187 is
added. With this method, cluster sizes of EGFR in the order of N ¼ 1, N ¼ 2, or
N  3 can be determined. A conventional fluorescence microscope can be converted
in a homo-FRET-detecting system when a set of polarizers is integrated and suffi-
cient signal is generated. The limitation of this method is that it is up till now only
able to distinguish monomers, dimers, and oligomers. This method is not able to
measure the precise amount of molecules in higher-order clusters. Homo-FRET
was used to study the predimer formation of EGFR and showed to be receptor
concentration-independent by homo-FRET measurements. This is in contrast to what
was found by the N&B analyses (Hofman et al., 2010; Nagy et al., 2010). This dif-
ference might be explained by the different sample preparations as homo-FRET was
measured in fixed cells and N&B analysis in live cells. We note that the N&B method
does not include the immobile fraction of the receptors, which is known to exist. An-
other explanation for the difference could be that the N&B analysis cannot distinguish
the cluster size distribution within a single pixel and homo-FRET can. It might be that
the amount of predimers changes upon receptor concentration but that there are still
predimers formed when there are low EGFR concentrations present. This is just one
example of contradicting results of EGFR clustering measurements. Combining these
different methods is required to overcome these contradictions. Most probably,
the most ideal way to measure clustering is by combining FRET studies with high-
resolution microscopy methods like FIDA, N&B, EM, or others because in that situ-
ation, you measure both the larger cluster organizations and the smaller subclusters
using homo-FRET. The super resolution microscopy technique STED would be a
good possibility to combine with FRET because the resolution of STED can be up
to 20 nm, which is slightly higher than homo-FRET, which makes it possible when
the two methods are combined to measure small clusters simultaneously with larger
clusters. STED can also be applied in life cells and gives clustering information about a
whole cell, which is not the case with, for example, single-molecule tracking where
only a set of clusters can be analyzed (Pellett et al., 2011).
In this chapter, we have described the homo-FRET method for measuring EGFR
clustering and compared them with other experimental techniques. This method can
equally be applied for other proteins, provided their functioning is not affected by the
fusion to mGFP. Essential are the reference proteins, which are mGFP fused to FKBP
for cytoplasmic proteins and FKBP fused to a receptor can be used as reference for
the analysis of receptor clustering. Homo-FRET has the advantage of simple sample
preparation and accurate cluster size determination. However, homo-FRET is not
able to detect larger cluster organization.

Acknowledgments
The authors wish to thank Dr. Erik G. Hofman and Dr. Arjan N. Bader for their fruitful discussions.
320 CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET

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