Role of the regulatory domain of the EGF-receptor cytoplasmic tail in selective binding of the clathrin-associated complex AP-2.
BACKGROUND
After stimulation of a cell by the mitogenic epidermal growth factor (EGF), the EGF receptor (EGF-R) is cleared from the cell surface in order to turn off receptor signaling. This internalization is mediated via clathrin-coated pits and coated vesicles, and ultimately the receptors are delivered to the lysosome and destroyed. It is believed that clathrin-associated protein complexes or adaptors (APs) link the entrapment of EGF-R and other nutrient and growth-factor receptors to the formation of the clathrin-coated pit. Two classes of APs are known--AP-2, found at the plasma membrane, and AP-1, found in the trans-Golgi network. Activated EGF-R associates with AP-2s at the plasma membrane, but the mechanism responsible for this association is not known. Here, we investigate, in vivo and in vitro, three aspects of the interaction between APs and EGF-R: firstly, we ask whether EGF-R at the plasma membrane distinguishes between AP-1 and AP-2; secondly, we ask which part of the receptor's cytoplasmic tail is responsible for binding; finally, we ask whether autophosphorylation by EGF-R is essential for the interaction.


RESULTS
We demonstrate that EGF-R displays a selective association for AP-2 over AP-1 in vivo, and that this preferential interaction can also be detected using surface plasmon resonance in vitro. Using a truncated mutant and a kinase-dead mutant of EGF-R, we show that the regulatory domain of the cytoplasmic tail is essential for the recruitment of AP-2 in vivo and that this domain is required for association between purified AP-2 and EGF-R in vitro. Finally, we demonstrate, in vivo and in vitro, that tyrosine auto-phosphorylation by the receptor is not an essential pre-condition for the recruitment of AP-2.


CONCLUSIONS
EGF-R binds selectively to AP-2s, and the regulatory domain of its cytoplasmic tail is required for this interaction. The lack of correlation between receptor autophosphorylation and AP-2 recruitment suggests that activation of the EGF-R kinase stimulates endocytosis by the phosphorylation of a factor distinct from EGF-R itself, as also proposed by others based on experiments measuring receptor traffic and entrapment.
Boll
W
W
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Gallusser
A
A
Kirchhausen
T
T
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding
D033942:Adaptor Proteins, Vesicular Transport; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D066246:ErbB Receptors