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Summary Abnormalities of the EGF receptor and/or the related ERBB2 receptor occur in a significant proportion of cases of human breast cancer and are important influences in the behaviour of this tumour type.In this report we demonstrate by nucleic acid analysis and immunohistochemistry that the recently recognised third member of this gene Costa et al., 1988). Overexpression of the ERBB2 protein is also a marker of poor prognosis for node-positive disease (Slamon et al., 1987) and probably also for node-negative disease (Perren, 1991). More exciting, however, is the accumulating evidence that the ERBB2 receptor protein has potential as a target for antibody-directed therapy (Shepard et al., 1991) and other approaches such as inhibition of receptor dimerisation and tyrosine kinase function (Gullick 1990b Thirty six biopsy samples of non-neoplastic breast tissue

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Antisera were prepared against three synthetic peptides with amino acid sequences identical to those surrounding the three major autophosphorylation sites of the epidermal growth factor (EGF) receptor. The affinity‐purified antibodies reacted strongly in an enzyme‐linked immunosorbent assay against the immunizing peptide but showed little cross‐reaction with the other two phosphorylation site peptides. EGF receptors labelled by autophosphorylation could be specifically precipitated by each of the phosphorylation site antibodies. The antibodies recognised EGF receptors labelled at each of the autophosphorylation sites, indicating that they could bind to the immunizing sequences irrespective of their states of phosphorylation. The antibodies were able to inhibit EGF receptor autophosphorylation without affecting EGF‐stimulated tyrosine kinase activity towards exogenous peptide substrates, suggesting that the kinase and autophosphorylation sites were in distinct domains. Immunofluorescent staining of A431 cells showed that the autophosphorylation site sequences resided inside the cell. The autophosphorylation sites were shown to be within a domain of 20 000 mol. wt. which could be cleaved from the receptor through limited proteolysis by the calcium‐dependent protease, calpain. The position of cleavage of the EGF receptor by the protease was mapped to lie between residues 996 and 1059. These results are discussed in the context of a model for the structure and function of the human EGF receptor.

Down-regulation of protein kinase C induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined in Swiss 3T3, V79, MDBK and C6 cells by Western blotting. Variations in the rate of down-regulation caused by treatment with 100 nM-TPA were observed; TPA treatment for 5 h caused maximal down-regulation in V79 cells, whereas TPA treatment for 10 h or 30 h was needed for maximal down-regulation of protein kinase C in MDBK or Swiss 3T3 cells respectively. The decrease in amount of immunologically detectable protein kinase C was 30% in MDBK cells and 100% in V79 and Swiss 3T3 cells. MDBK and C6 cells could be completely depleted of protein kinase C by treatment with 250 nM-TPA. In C6 cells, after treatment with 500 nM-TPA, an 80% loss of protein kinase C was seen over 10 h. Measurement of the numbers of phorbol-ester-binding sites remaining in each cell line when protein kinase C was maximally down-regulated indicated that in MDBK and Swiss 3T3 cells loss of phorbol-ester-binding sites paralleled loss of protein kinase C, whereas in V79 and C6 cells no such correlation was observed.