Characterization of Ligand-Induced Endocytosis of EGF Receptors.
Abstract
Down-regulation of activated EGF-receptors (EGF-R) requires their tyrosine kinase (Y-kinase) activity. However, controversy existed as to whether ligand-induced activation of the EGF-R Y-kinase was required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay which selectively measures the recruitment of EGF-R into coated pits. While EGF bound to wild-type receptors is efficiently sequestered in coated pits, sequestration of kinase- deficient receptors occurs inefficiently and at the same basal rate of endocytosis of unoccupied receptors or receptors lacking any cytoplasmic domain. Sequestration of deletion mutants of the EGF-R which lack autophosphorylation sites also requires an active Y-kinase. Our results directly establish that the EGF-R Y-kinase is required for internalization. They further suggest that a kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Finally we have developed a powerful functional assay for identification of these I substrate(s) based on our finding that the addition of a soluble EGF-R Y-kinase fully and specifically restores the recruitment of kinase-deficient EOF-R into coated pits.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 17, 1995
- Accession Number
- ADA300531
Entities
People
- Christophe Lamaze
- Sandra L. Schmid
Organizations
- Scripps Research