Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes.
After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR.
Department of Anatomy, McGill University, Montreal, Quebec, Canada.
I
I
Wada
W H
WH
Lai
B I
BI
Posner
J J
JJ
Bergeron
D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D015536:Down-Regulation; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D019000:Phosphotyrosine; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D046911:Macromolecular Substances; D008565:Membrane Proteins; D010455:Peptides; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases