Association and colocalization of Eps15 with adaptor protein-2 and clathrin.
Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-alpha results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both alpha-adaptin and clathrin. Upon EGF stimulation, Eps15 and alpha-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.
van Delft
S
S
Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, The Netherlands.
Schumacher
C
C
Hage
W
W
Verkleij
A J
AJ
van Bergen en Henegouwen
P M
PM
D016475:3T3 Cells; D033965:Adaptor Protein Complex alpha Subunits; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002135:Calcium-Binding Proteins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D002966:Clathrin / Q000378:metabolism / Q000502:physiology*; D003034:Coated Pits, Cell-Membrane / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism / Q000502:physiology; D007328:Insulin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions / Q000378:metabolism; D016211:Transforming Growth Factor alpha / Q000494:pharmacology
D033965:Adaptor Protein Complex alpha Subunits; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002135:Calcium-Binding Proteins; D002966:Clathrin; C082729:Eps15 protein, mouse; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors