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Analysis of the influences of the E5 transforming protein on kinetic parameters of epidermal growth factor binding and metabolism
Author(s) -
Waters Christopher M.,
Overholser Knowles A.,
Sorkin Alexander,
Carpenter Graham
Publication year - 1992
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041520206
Subject(s) - internalization , epidermal growth factor , receptor , transfection , cell culture , dissociation constant , chemistry , reaction rate constant , ligand (biochemistry) , 3t3 cells , epidermal growth factor receptor , microbiology and biotechnology , biology , kinetics , biophysics , biochemistry , physics , quantum mechanics , genetics
The E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21–32, 1989). To determine whether E5 affects EGF:receptor interactions, we analyzed the kinetics of 125 I‐EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (k a ), dissociation (k d ), internalization (k e ), recycling (k r ), and degradation (k h ). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125 I‐EGF association k a was significantly decreased in E5/ER cells, but was apparently occupancy‐independent in both cell lines. The 125 I‐EGF dissociation rate constant k d was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization k e were also found to be occupancy‐dependent, although at a given level of occupancy k e was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant k r . The 125 I‐EGF degradation rate constant k h was 30% lower in E5 transformed cells, and was occupancy‐independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor. © 1992 Wiley‐Liss, Inc.