Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

Adaptor
protein Grb2 binds phosphotyrosines in the epidermal growth
factor (EGF) receptor (EGFR) and thereby links receptor activation
to intracellular signaling cascades. Here, we investigated how recruitment
of Grb2 to EGFR is affected by the spatial organization and quaternary
state of activated EGFR. We used the techniques of image correlation
spectroscopy (ICS) and lifetime-detected Förster resonance
energy transfer (also known as FLIM-based FRET or FLIM–FRET)
to measure ligand-induced receptor clustering and Grb2 binding to
activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected
with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR
(EGFR–eGFP). Following stimulation of the cells with EGF, we
detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP
clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP
per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP
had an average cluster size of 1 ± 0.3 EGFR molecules per punctum.
In the absence of EGF, there was no association between EGFR–eGFP
and Grb2–mRFP. To interpret these data, we extended our recently
developed model for EGFR activation, which considers EGFR oligomerization
up to tetramers, to include recruitment of Grb2 to phosphorylated
EGFR. The extended model, with adjustment of one new parameter (the
ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster
size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1%
of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2.
Together, our experimental and modeling results further implicate
tetrameric EGFR as the key signaling unit and call into question the
widely held view that dimeric EGFR is the predominant signaling unit.