A direct radioimmunoassay for human epidermal growth factor receptor using 32P-autophosphorylated receptor

Abstract

A simple and reproducible radioimmunoassay of the epidermal growth factor (EGF) receptor which uses 32P-labeled EGF receptor and anti-receptor monoclonal antibodies is reported. In vitro phosphorylation of A431 cell membranes with [γ- 32P]ATP in the presence of 20% dimethyl sulfoxide (which stimulates autophosphorylation of the EGF receptor) and 10 μ m Na 3VO 4 (a potent inhibitor of phosphotyrosyl protein phosphatase) provides radiolabeled EGF receptor for radioimmunoassay without further purification. The most selective phosphorylation of the EGF receptor is achieved at ATP concentrations of 0.1 – 0.2 μ m, which corresponds to the reported K m value for the autophosphorylation reaction of the EGF receptor (W. Weber, P. J. Bertics, and G. N. Gill, 1984, J. Biol. Chem., 259, 14631–14939). The incorporation of 32P into EGF receptors increases in proportion to the increase of ATP concentration up to 6 mol of labeled phosphate at 2.0 μ m ATP. The label is entirely on tyrosine residues. The cell membranes can be stored at −70°C for 3 months without loss of immunoreactivity and autophosphorylating activity. Standard curves for the radioimmunoassay were constructed employing either A431 cell membranes or whole cell homogenates containing a known amount of EGF receptor. The assay can detect 7 × 10 10 EGF receptor molecules or 20 ng of the receptor protein, and can quantitatively distinguish the difference in EGF receptor numbers between A431 cells and 29E2 and KB cells with 10-fold and 15-fold fewer receptors than A431 cells, respectively. 29E2 cells and KB cells express twofold more immunoreactive EGF receptors than EGF-binding sites. In contrast, A431 cells possess the same number of immunoreactive sites and receptor sites for EGF binding. To assess total EGF receptor expression, it is necessary to use a method which detects EGF receptors regardless their intrinsic kinase activity, or capacity to bind EGF. This radioimmunoassay detects immunoreactive receptor molecules, even those which do not bind EGF.