Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.

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Abstract. We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAbl08), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a eDNA encoding the full-length human EGF receptor gene, and fully responsive to EGE The binding to saturation of mAbl08 to the surface of these cells at 4°C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGE The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37°C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 106 M-~-s -~) than did low-affinity receptor (2.5 × 105 M-Ls-~), whereas the off-rate constants, measured at 4°C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors.At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAbl08 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca 2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAbl08 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.

Serum response factor (SRF) is a mammalian transcription factor that binds to the serum response element in the enhancer of the c-los proto-oncogene and thus may mediate serum-induction of c-los transcription. We report here that the DNA binding activity of recombinant SRF made in Eschericlffa coil can be greatly enhanced by incubation of the protein with HeLa cell nuclear extract. The enhancing activity is ATP or GTP dependent and cohaetionates with a protein kinase that phosphorylates SRF on a specific tryptic peptide. Coincubation with phosphatase blocks the enhancing activity, further suggesting that the enhanced binding activity is due to phosphorylation. The specific tryptic phosphopeptide phosphorylated in vitro is also phosphorylated in vivo, demonstrating that this phosphorylation is physiologically important. We have localized the phosphorylation site by a small deletion mutant. Finally, we show that the kinase activity is provided by casein kinase II (CKII) or a close variant. The potential role of CKII as either a regulatory or constitutive modifier of SRF in vivo will be discussed.

SUMMARYThe expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EG F receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas.