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PMID:14702346

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Citation

Stoorvogel, W, Kerstens, S, Fritzsche, I, den Hartigh, JC, Oud, R, van der Heyden, MA, Voortman, J and van Bergen en Henegouwen, PM (2004) Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain. J. Biol. Chem. 279:11562-9

Abstract

Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the second transport step requires a domain of the EGFR that encompasses residues 985-996 and was previously found to interact with actin. Deletion of domain 989-994 (Delta989-994 EGFR) did not interfere with EGFR uptake but completely abrogated its degradation. In contrast, both uptake and degradation were affected for K721A EGFR, a kinase-deficient EGFR mutant. To measure intracellular EGFR sorting, we developed a novel cell fractionation assay toward which cells were co-transfected for chicken hepatic lectin, a receptor for agialoglycoproteins. These cells were incubated with agialofetuin-coupled colloidal gold, which was targeted to lysosomes after receptor-mediated endocytosis. Compartments within the lysosomal pathway gained buoyant density because of the presence of colloidal gold and could be isolated from cell homogenates by ultracentrifugation through a high-density sucrose cushion. In contrast to endocytosed wild type EGFR, both Delta989-994 EGFR and K721A EGFR were largely not retrieved in gold-containing endocytic compartments. These results are supported with morphological data. We conclude that sorting of endocytosed EGFR into the degradation pathway requires both its kinase activity and actin-binding domain.

Links

PubMed Online version:10.1074/jbc.M308449200

Keywords

Actins/metabolism; Animals; Binding Sites; Cell Compartmentation; Humans; Ligands; Lysosomes/metabolism; Mice; Microscopy, Fluorescence; NIH 3T3 Cells; Protein Transport; Receptor, Epidermal Growth Factor/chemistry; Receptor, Epidermal Growth Factor/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:EGFR_original

GO:0005768: endosome

IDA: Inferred from Direct Assay: :

C


HUMAN:EGFR_original

GO:0051015: actin filament binding

IDA: Inferred from Direct Assay: :

F


HUMAN:EGFR_original

GO:0030122: AP-2 adaptor complex

TAS: Traceable Author Statement: :

C


HUMAN:EGFR_original

GO:0004713: protein tyrosine kinase activity

IMP: Inferred from Mutant Phenotype:

F


HUMAN:EGFR_original

GO:0005768: endosome

IDA: Inferred from Direct Assay:

C


HUMAN:EGFR_original

GO:0051015: actin filament binding

IDA: Inferred from Direct Assay:

F


HUMAN:EGFR_original

colocalizes_with

GO:0030122: AP-2 adaptor complex

TAS: Traceable Author Statement:

C


HUMAN:EGFR

enables

GO:0051015: actin filament binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:EGFR

part_of

GO:0005768: endosome

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:EGFR

enables

GO:0004713: protein tyrosine kinase activity

ECO:0000315: mutant phenotype evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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