Subcellular localization of the EGF receptor maturation process.
The glycosylation and the processing of the epidermal growth factor (EGF) receptor are suggested to play a crucial role(s) in the activation of ligand binding activity. To examine whether the receptor acquires EGF binding activity in the endoplasmic reticulum (ER) or in the Golgi complex, we carried out parallel kinetic analysis of the EGF binding activity and the intracellular transport of the newly synthesized receptor by immunoprecipitation with the anti-EGF receptor antibody B4G7 using the EGF receptor hyperproducing cell line NA. The kinetic analysis revealed that a receptor capable of binding EGF appeared after 30 to 60 min labeling with [35S]methionine. Pulse-chase experiments also indicated that the receptor capable of binding EGF appeared after a 30-min pulse with a 30-min chase. Subcellular fractionation analysis indicated that the newly synthesized receptor was present in the Golgi complex after labeling with [35S]methionine for 30 min. After a 30-min chase, the Mr 170K receptor appeared in the Golgi complex and plasma membrane. Thus, these results together indicated that after a 30-min pulse incubation a fraction of the EGF receptors have been transported from the ER to the Golgi complex; however, the receptor is unable to bind EGF. Although the EGF receptor appeared on the cell surface after a 30-min pulse with a 30-min chase, only half of the receptors are capable of binding EGF. Therefore, the EGF receptor acquires ligand binding activity at a late stage of the maturation process, most likely in the Golgi complex.
Gamou
S
S
Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
Shimagaki
M
M
Minoshima
S
S
Kobayashi
S
S
Shimizu
N
N
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D011233:Precipitin Tests
D004815:Epidermal Growth Factor; D066246:ErbB Receptors