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Epidermal growth factor receptor interaction with clathrin adaptors is mediated by the Tyr974-containing
A Sorkin1, M Mazzotti, T Sorkina
1Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Abstract:
The carboxyl-terminal regulatory domain of the epidermal growth factor (EGF) receptor is essential for its endocytosis and interaction with the clathrin-associated protein complex AP-2. To identify AP-2 binding motif in the receptor, several single and multiple-point mutations within the region between residues 966 and 977 of the human EGF receptor were made, and the mutant receptors were expressed in NIH3T3 cells. Mutation of tyrosine 974 alone or together with surrounding residues and the deletion of residues 973-975 essentially eliminated AP-2 co-immunoprecipitation with the EGF receptor. Furthermore, a synthetic peptide corresponding to receptor residues 964-978 blocked AP-2 association with the wild-type EGF receptor. These data suggest that AP-2 has only one high-affinity binding site in the EGF receptor composed of Tyr974-containing motif. Receptor mutants that did not bind AP-2 displayed a lower rate of internalization, down-regulation, and turnover compared to wild-type receptors when expressed at high levels. However, similar receptor mutants expressed at low levels were internalized and down-regulated as efficiently as wild-type receptors. Internalization of the mutant receptors lacking the high-affinity binding site for AP-2 was inhibited by K+-depletion of the cells, indicating that their endocytosis required intact coated pits. We suggest that whereas one mechanism of EGF receptor recruitment into coated pits involves high-affinity binding of AP-2 to Tyr974-containing motif, another pathway may be mediated by weak receptor/AP-2 interactions or by proteins other than AP-2.
Insights
The epidermal growth factor (EGF) receptor
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The carboxyl-terminal regulatory domain of the epidermal growth factor (EGF) receptor is crucial for its endocytosis and interaction with the clathrin-associated protein complex AP-2.
- Identifying the specific binding motif for AP-2 within the EGF receptor is key to understanding receptor trafficking.
Purpose of the Study:
- To pinpoint the AP-2 binding motif within the human EGF receptor.
- To investigate the functional consequences of disrupted AP-2 binding on EGF receptor endocytosis and regulation.
Main Methods:
- Site-directed mutagenesis was employed to create single and multiple-point mutations in the human EGF receptor (residues 966-977).
- Mutant receptors were expressed in NIH3T3 cells, and AP-2 co-immunoprecipitation was performed.
- Synthetic peptides corresponding to EGF receptor residues were used to block AP-2 association.
- Internalization, down-regulation, and turnover rates of wild-type and mutant receptors were assessed under varying expression levels and cellular conditions (K+-depletion).
Main Results:
- Mutation of tyrosine 974, or deletion of residues 973-975, significantly impaired AP-2 co-immunoprecipitation, indicating a critical role for this motif.
- A synthetic peptide (residues 964-978) effectively blocked AP-2 association with the wild-type EGF receptor.
- Receptor mutants lacking high-affinity AP-2 binding showed reduced internalization and down-regulation at high expression levels but were efficient at low levels.
- Internalization of AP-2 binding-deficient mutants was dependent on intact coated pits, as shown by inhibition upon K+-depletion.
Conclusions:
- A high-affinity AP-2 binding site in the EGF receptor is primarily mediated by a motif containing Tyr974.
- Disruption of this high-affinity binding site affects EGF receptor endocytosis and down-regulation, particularly at higher receptor expression levels.
- EGF receptor recruitment into coated pits can occur via high-affinity AP-2 binding or alternative pathways involving weaker interactions or other proteins.