Epidermal growth factor: relationship between receptor down regulation in cultured NRK cells and epidermal growth factor enhancement of phosphorylation of a 170000 molecular weight membrane protein in vitro.
Incubation of confluent nondividing NRK cells in serum-free media with unlabeled epidermal growth factor (EGF) leads to a reduction in the specific binding capacity for 125I-labeled EGF. This modulation of the binding capacity for 125I-labeled EGF by unlabeled EGF, termed receptor down regulation, was dependent on EGF concentration and time. Membranes from untreated NRK cells have a phosphorylating system which catalyzed in vitro the phosphorylation of numerous membrane components; this phosphorylating system was stimulated by EGF. Although EGF enhanced the phosphorylation of many membrane proteins, one major component with Mr 170K and a minor band of Mr 150K were primarily affected. A comparison of the membrane phosphoproteins of untreated and down-regulated cells by in vitro phosphorylation and NaDodSO4 gel electrophoresis revealed that down regulation of EGF receptors results in a specific decrease in 32P phosphorylation of the 170K- and 150K-dalton components to subsequent stimulation with EGF in vitro. We further characterized the modulation of phosphorylation of the 170K protein by down regulation with EGF and found it to be dependent on EGF concentration and time. These studies demonstrated a correlation between the loss of 125I-labeled EGF binding activity by the cells and the loss of the vitro EGF-dependent 32P phosphorylation of the 170K-dalton membrane protein. In addition, the results suggest that the major 170K Mr phosphoprotein band is a component of the receptor for EGF which is a substrate of the phosphorylation reaction.
D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors; D007668:Kidney / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010766:Phosphorylation; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*
D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D066246:ErbB Receptors