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Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase
1Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Abstract:
EGF-receptor (EGF-R) tyrosine kinase is required for the down-regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, 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 that lack autophosphorylation sites also requires an active tyrosine kinase. This suggests that a tyrosine kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Addition of a soluble EGF-R tyrosine kinase fully and specifically restores the recruitment of kinase-deficient EGF-R into coated pits providing a powerful functional assay for identification of these substrate(s).
Insights
Ligand-induced activation of the epidermal growth factor receptor (EGF-R) tyrosine kinase is crucial for its efficient internalization into coated pits. This process requires kinase activity, suggesting a role for non-receptor tyrosine kinase substrates.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- The epidermal growth factor receptor (EGF-R) tyrosine kinase plays a role in receptor downregulation.
- The precise role of ligand-induced EGF-R tyrosine kinase activation in internalization and lysosomal targeting remains debated.
Purpose of the Study:
- To investigate whether ligand-induced activation of EGF-R tyrosine kinase is necessary for its recruitment into coated pits.
- To elucidate the role of EGF-R tyrosine kinase activity in receptor endocytosis.
Main Methods:
- Development of a cell-free assay to specifically measure EGF-R recruitment into coated pits.
- Utilized wild-type, kinase-deficient, and mutant EGF-R constructs lacking autophosphorylation sites.
- Assessed the effect of soluble EGF-R tyrosine kinase on receptor recruitment.
Main Results:
- EGF binding to wild-type receptors efficiently promotes sequestration into coated pits.
- Kinase-deficient EGF-R and receptors lacking cytoplasmic domains show inefficient sequestration.
- Sequestration of EGF-R mutants lacking autophosphorylation sites also requires an active tyrosine kinase.
- Addition of soluble EGF-R tyrosine kinase fully restores recruitment of kinase-deficient receptors.
Conclusions:
- Efficient, ligand-induced recruitment of EGF-R into coated pits necessitates an active tyrosine kinase.
- This process likely involves tyrosine kinase substrates distinct from the EGF-R itself.
- The study provides a functional assay for identifying these critical EGF-R tyrosine kinase substrates.