12th Carl Zeiss sponsored workshop on ... - Institut Fresnel

LIGAND-STIMULATED EGFR CAUSES LIPID DOMAIN-DEPENDENT HIGHER
ORDER AGGREGATION AND INTERNALIZATION OF ERBB2
László
Ujlaky-Nagy,
Árpád Szöır,
János Szöllısi,
György Vereb
University of Debrecen, Dept. of Biophysics and Cell Biology, H-4032 Debrecen (Hungary)
vereb@dote.hu
Members of the ErbB receptor tyrosine kinase family, ErbB1 (EGFR), ErbB2, ErbB3 and
ErbB4, form homo- and heterodimers or higher oligomers with each other which promote cell
survival and proliferation [1]. Generally, overexpression of the ligandless ErbB2 is thought to
drive homodimer formation in the membrane of tumor cells. However, ErbB2 can also act as a
heterodimerizing partner for other family members, and serve as a signal amplifier for their
ligand-bound forms, which may have clinical relevance in tumors expressing multiple ErbBs
[2]. We have used fluorescence correlation spectroscopy to investigate molecular events of
ErbB1-ErbB2 transactivation in single living SKBR3 breast tumor cells. ErbB2 was labeled
with A488 Fabs. Fluorescence fluctuation traces were taken from single submicron sized spots
in the upper membrane of cells and used to construct fluorescence autocorrelation spectra.
These were fitted using a model accounting for triplet state, diffusion of dissociated Fabs, as
well as cytoplasmic and membrane (lateral, 2D) diffusion of ErbB2 molecules or their
aggregates. Correlation spectra were obtained every 2.5 min for 1 hour. After 20 min, half of
the samples were stimulated with 50 nM EGF to detect time-dependent changes of ErbB2
diffusion. Controls showed a time dependent fluctuation of membrane diffusion times in the
120-200 ms range (182±9 ms, mean±SEM). This was paralleled with internalization and
recycling to the surface of receptors as measured by flow cytometry. Membrane diffusion of
ErbB2 was divided into at least two components: a faster component characterizing
monomers or smaller aggregates, and a slower component related to higher oligomers or
larger clusters. The corresponding diffusion times were 94±3 ms and 360±18 ms. Codiffusion
of ErbB2 with ErbB2 upon labeling with a mixture of A488 and A647 fluorophors
was detected by fluorescence cross-correlation spectroscopy (FCCS) mainly for the slower
component indicating that the faster component might be biased towards monomers.
Responses to EGF stimulation were vastly heterogeneous in timing, but manifested
unanimously in an increase of characteristic average membrane diffusion time to maximally
550±119 ms. The average onset of response was 5.5 min post stimulus and slowed-down
diffusion was maintained for ~30 min. EGF-induced transactivation did not change the faster
component (100±19 ms), but lead to the increased diffusion times of the slower one (from 360
ms to 764±66 ms), at the same time decreasing its fractional proportion from 0.55 to 0.2,
while the fast component increased from 0.37 to 0.65. This may be an indirect indication that
larger aggregates are needed for internalization and the receptors are recycled as monomers or
smaller clusters afterwards. Consequences of the increased ErbB1-ErbB2 interaction could be
detected by microscopic fluorescence resonance energy transfer as well as from increased
ErbB1 and ErbB2 phosphorylation in Western blots and were decreased by extraction of
membrane cholesterol with methyl-ß-cyclodextrine. This treatment also accelerated the
diffusion time of ErbB2 to 83±16 ms, indicating that the slower component (higher
aggregates) corresponds to microscopic lipid rafts. Given the low expression of ErbB1 in
SKBR3 cells, the mechanism of ErbB1-ErbB2 transactivation can be interpreted as ErbB1
serving as signal antennae that upon ligand binding recruit ErbB2 to form increasing size
activated aggregates on lipid raft platforms that eventually get internalized.
[1] Y. Yarden, M. X. Sliwkowski, Nat Rev Mol Cell Biol. 2 (2001) 127-137.
[2] A. Citri., Y. Yarden, Nat Rev Mol Cell Biol. 7 (2006) 505-516.
12 th <strong>Zeiss</strong> <strong>sponsored</strong> FCS <strong>workshop</strong> – Cargèse, Corsica, France – <strong>12th</strong> -16th October 2009