It has been shown that the epidermal growth factor receptor (EGFR) can directly interact with F-actin via an actin-binding domain (ABD) located at amino acid residues 984–996. In this study, we investigate the interaction of the EGFR with actin using an actin cosedimentation assay in a reconstituted cell free system. We find that soluble components of the cytosol from A431 cells dramatically enhance the EGFR/actin interaction. This result is found for EGFR purified by EGFR1 immunoprecipitation, over an EGFR1 affinity column or over a wheat germ agglutinin column. This EGFR/actin interaction is shown to be both cytosol protein concentration and actin concentration dependent. Several lines of evidence suggest that this interaction is not mediated by the previously defined ABD of the EGFR but rather requires both the kinase domain and a portion of the C-terminal regulatory domain of the EGFR. Our results suggest the binding of EGFR to F-actin is under the regulation of one or multiple soluble cytosolic proteins through a mechanism that involves the complete tertiary structure of the cytoplasmic domain of the EGFR, but not the previously defined ABD. Altering the extent of the phosphorylation of the EGFR does not change the ability of the receptor to precipitate with F-actin, suggesting that the interaction between the EGFR and actin is independent of the phosphorylation of the EGFR. Intriguingly, the actin co-precipitated EGFR shows a decreased level of phosphorylation compared with the receptor in the supernatant in an actin cosedimentation assay. In addition, depletion of some F-actin-binding proteins from the cytosol enhances the EGFR involved protein tyrosine phosphorylation in the same assay. Cell imaging experiments show that disruption of the actin cytoskeleton in the intact cell using latrunculin B inhibits EGF stimulated internalization of the EGFR. These results suggest that the actin cytoskeleton system plays an important role in the regulation of the EGFR function. A hypothesis for the regulation of the signaling of the EGFR by the actin cytoskeleton is thus proposed for the future study.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-3530 |
| Date | 01 January 2001 |
| Creators | Tang, Jun |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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