Characterization of epidermal growth factor receptor in human endometrial cells in culture.
The aim of the study was to determine the binding characteristics of the epidermal growth factor (EGF) receptor in isolated human endometrial glands and stromal cells in culture. Stromal cells and glands were obtained from endometrial tissue by collagenase dispersion followed by sieve filtration. They were plated into 24-well multiwell plates in Ham's F10 medium supplemented with 5% fetal calf serum and used at 70-80% confluence. Scatchard analysis revealed a single class of high-affinity binding sites in both cell types with apparent dissociation constants of 1.17 +/- 0.6 (n = 15) and 1.20 +/- 0.3 (n = 8) nmol 1-1 for stromal cells and glands, respectively. The concentration of receptors was higher in stromal cells than in glands, 719 +/- 377 (n = 16) and 310 +/- 177 (n = 8) fmol mg-1 protein, respectively. Epidermal growth factor labelled with 125I was displaced from the receptor by EGF and transforming growth factor alpha, but not insulin, insulin-like growth factor, fibroblast growth factor, or platelet-derived growth factor. Binding was shown to be dependent on time and temperature. Downregulation of the receptor was demonstrated by preincubating cells with 5 nmol EGF I-1, which reduced receptor concentrations by 75%. 12-O-Tetradecanoylphorbol-13-acetate decreased the affinity of the receptor for EGF changing the dissociation constant from 1.8 to 3.9 nmol l-1. A suitable system for investigating the regulation of this receptor in human endometrium was established.
Watson
H
H
Unit of Metabolic Medicine, St Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London, UK.
Franks
S
S
Bonney
R C
RC
D002478:Cells, Cultured; D015536:Down-Regulation; D004717:Endometrium / Q000737:chemistry* / Q000166:cytology; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000032:analysis* / Q000378:metabolism; D005260:Female; D006801:Humans; D011485:Protein Binding; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000378:metabolism
D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D013755:Tetradecanoylphorbol Acetate