Abstract
Epidermal growth factor (EGF) receptors extracted with Triton X-100 from human skin fibroblasts and A431 epidermoid carcinoma cells rapidly lose EGF-binding activity precipitable with polyethylene glycol. The presence of concanavalin A which can cross-link and, thereby, aggregate the receptors, allowed quantitative recovery of the lost EGF-binding activity. Scatchard analysis of EGF binding of Triton X-100-solubilized receptors showed that A431 cells and skin fibroblasts possess approximately 1.5 X 10(6) and 7 X 10(4) EGF-binding sites/cell, respectively, which exhibit similar affinities for the ligand. The heavy isotope density-shift method was employed to determine whether differences in rates of receptor synthesis or decay account for the large difference in number of receptors/cell between the two cell types. After shifting cells to medium containing heavy (15N, 13C, and 2H) amino acids, light and heavy receptors, solubilized from total cellular membranes, were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that A431 cells synthesize EGF receptors at a rate 12 times faster than skin fibroblasts and that the half-life for receptor decay of A431 cells is somewhat longer (t1/2 = 16 h) than that (t1/2 = 9 h) of fibroblasts. Down-regulation of cell surface and total cellular EGF-binding capacity in A431 cells occurs with a t1/2 of 2-3 h and results in a 70-83% decrease in receptor level in 12 h. Scatchard analysis revealed that these changes in EGF binding were due to an alteration of receptor number and not EGF-binding affinity. Rates of EGF receptor synthesis and inactivation/decay were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of EGF receptor down-regulation. Down-regulation, however, caused a decrease in receptor half-life from 16 to 4.5 h. These results indicate that EGF-dependent regulation of EGF receptor level in A431 cells involves an alteration of the rate of receptor inactivation.
Highlights
A431 epidermoid carcinoma cells rapidly lose EGF- of the receptor by the ligand for 12-16 h [5]; these effects binding activity precipitable with polyethylene glycol. include induction of DNA, RNA, and protein synthesis and
Scat- receptors occupied, the magnitude of the effect is likely to chard analysis of EGF binding of Triton X-100-solubi- depend both upon the concentration of t h e ligand and the lized receptors showed that A431 cells and skin fibroblasts possess approximately 1.5 X 10‘ and 7 X lo4EGF
In vivo studies have shown that the decreased lar membranes, were resolved by isopycnic banding on cellularsensitivity to certain hormones, notably insulin,in density gradients and quantitated.It was demon- variousdisease states is associated with a reduced level of strated that A431 cells synthesize EGF receptors at a functional cell surface hormone receptors[11]
Summary
Cell-SurfaCe '*'I-EGF bindlogCapacity was detemlnedwth cell mnolayer ~ t h a t had. ~ rrvlo ~ b~eelny SubJected t o &he rapld EGF debindln gp mtocol( below ). Cell-SurfaCe '*'I-EGF bindlogCapacity was detemlnedwth cell mnolayer ~ t h a t had. ~ rrvlo ~ b~eelny SubJected t o &he rapld EGF debindln gp mtocol( below ). Cell mnolayers were incubated overnight a t4 1 $ 5 w i t h 3 ml Krebs-Ringepr hosphatebuffer containing the dpumuriate COnCentratlOn of I-EGF O T '"I-EGF plusI ~ g / m lu nlabeled EGF ( fornonrpecifl cb inding ) .A f t e rr emvin gt h e med,um, the cells yere washed 5 tiwS. I n 0.5 m l of 20 Rw HEPE8,'pH 7.5, bhffer. The. I-EGF-receptor compIexcs were precipitated by theaddltlonof 1 ml O f 20 rN HEPES buffer , pH 7 . 5 ,contalnlng 0.1% bovine gamglobulin and 1 31 O f 21.3% ( v / y ~ 5 polyethyleneglycol 8000 (Jlrher) ~n buffer. Nonrpeciflcbinding,deflned dlthat observed Inthe Presence O f 1 ug p e r ml O f unlabeled EGF formrt experiments Orinthe presence O f a 100-fold excels unlabeled EGF f o rS catchar dp l o t s , was w b t r & d fmm total blndlngtoobtainrpec?flcblnding
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