Abstract
When human epidermoid carcinoma A431 cells labeled with 32Pi to steady state specific activity were treated either with epidermal growth factor (EGF) or with active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate, labeling of 160 kDa EGF receptors isolated by immunoprecipitation with monoclonal anti-EGF receptor IgG was increased 2- to 3-fold. These treatments produced no significant increase in 32Pi labeling of acid-precipitable material present in detergent extracts of the cells. Phosphoamino acid analysis of radiolabeled EGF receptors isolated from these cells revealed several differences: the relative abundance of phosphotyrosine in EGF receptors was increased in cells treated with EGF, but decreased in cells treated with TPA; the overall relative abundance of phosphothreonine in EGF receptors was decreased in cells treated with EGF, but remained constant within the limits of experimental detection in cells treated with TPA. Two-dimensional mapping of the radiolabeled phosphopeptides produced from EGF receptors isolated by immunoprecipitation and treated with trypsin revealed 9 independent labeled regions, 2 of which contained phosphothreonine and were present only in EGF- or TPA-treated cells. These two phosphopeptide regions were more highly labeled in cells treated with TPA than with EGF.
Highlights
When human epidermoid carcinoma A431 cells la- tiallypurifiedreceptor preparations(4).In cells, EGFenbeled with ”Pi to steady state specific activity were hances phosphorylationof EGF receptors at tyrosine residues, treated either with epidermal growth factor (EGF) or but far more predominantly a t serine and threonineresidues, with activephorbol ester tumor promoters such as 12- and phosphopeptide mapping reveals a complex array of re
EGF receptor IgG was increased 2- to %fold. These diated processes, including those mediating communication treatments produced no significant increase in 32Pi labeling of acid-precipitable material present in detergent extracts of the cells.Phosphoamino acid analysis of radiolabeled EGF receptors isolated from these cells revealed several differences: the relatiavbeundanceof phosphotyrosine in EGF receptors was increased in cells treated with EGF, but decreased in cells treated with tetradecanoylphorbol 13-acetate (TPA); theoverallrelative abundance of phosbetween EGF receptors and othelirgand-receptor systems
Platelet-derived growth factor decreases EGF receptor downregulation occurring in response to epidermal growth factor (5).Fibroblast growth factor (6),vasopressin ( 7 ),and phorbol esters active in tumor promotion sucahs 12-O-tetradecanoylphothreonine in EGF receptors was decreased in cells phorbol 13-acetate (8-13) reduce EGF receptoraffinity in treated with EGF, but remained constant within the mitogenically responsive cells by binding to sites other than limits of experimental detection in cells treated with EGF receptors
Summary
Isolation of 32P~-labeleEdGF Receptors from A431 Celk-A431 cells were labeled to steady state specific activity with 32Pi(1)on 10-cm dishes, chilled, washed twice with ice-cold Hepes-buffered saline; and EGF receptors were solubilized in 0.5-1.0 ml of ice-cold RIPA buffer of thetotal radioactivity intheEGF receptor band was precipitated by the amount of clone 528 anti-EGF receptor IgG used routinely (Fig.[1]).At these precipitationyields, both antibody preparations precipitatedmore radioactivelylabeled EGF receptor from TPA-treated (Fig. 1, lanes b and d) than (23) modified to contain 100 p~ sodium vanadate (24) and 5 mM fromuntreated (lanesa and c) cells. Receptors by Trypsin A~tion-~~Pi-labeled EGrFeceptors migrating at 160 kDa were eluted from acrylamide gel lanes in 0.35 ml of 50 mM NH4HC03 containing0.1% SDS and 5%2-mercaptoethanol, and were digested with 50 pgof TPCK-treated trypsin as described by ulated by TPA was approximately the sameregardless of the antibody preparation used for receptor isolation. Phosphoamino Acid Analysis-EGF receptors eluted from electrophoretic gels a s described above or individual phosphopeptides scraped from cellulose thin layer plates andeluted with 100 pl of 100 mM formic acid were digested in 6 N HC1 a t 100 "C for 3 h in. TPA-treated cells were precipitated by clone 528 anti-EGF receptor IgG
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