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Vol. 14, Issue 3, 858-870, March 2003
Department of Pharmacology, University of Colorado Health
Sciences Center, Denver, Colorado 80111
The molecular mechanisms of clathrin-dependent internalization of
epidermal growth factor receptor (EGFR) are not well understood and, in
particular, the sequence motifs that mediate EGFR interactions with
coated pits have not been mapped. We generated a panel of EGFR mutants
and stably expressed these mutants in porcine aortic endothelial (PAE)
cells. Interestingly, mutations of tyrosine phosphorylation sites 1068 and 1086 that interact with growth-factor-receptor-binding protein Grb2
completely abolished receptor internalization in PAE cells.
Quantitative analysis of colocalization of EGF-rhodamine conjugate
and coated pits labeled with yellow-fluorescent-protein-tagged 
2
subunit of clathrin adaptor complex AP-2 revealed that EGFR mutants
lacking Grb2 binding sites do not efficiently enter coated pits. The
depletion of Grb2 from PAE as well as HeLa cells expressing endogenous
EGFRs by RNA interference substantially reduced the rate of EGFR
internalization through clathrin-dependent pathway, thus providing the
direct evidence for the important role of Grb2 in this process.
Overexpression of Grb2 mutants, in which the SH3 domains were either
deleted or inactivated by point mutations, significantly inhibited EGFR
internalization in both PAE and HeLa cells. These findings indicate
that Grb2, in addition to its key function in signaling through Ras,
has a major regulatory role at the initial steps of EGFR
internalization through clathrin-coated pits. Furthermore, the EGFR
mutant lacking Grb2 binding sites did not efficiently recruit c-Cbl and
was not polyubiquitinated. The data are consistent with the model
whereby Grb2 participates in EGFR internalization through the
recruitment of Cbl to the receptor, thus allowing proper ubiquitylation
of EGFR and/or associated proteins at the plasma membrane.
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