ErbB-4 is a transmembrane receptor tyrosine kinase that regulates cell proliferation and differentiation. After binding of its ligand heregulin (HRG) or activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA), the ErbB-4 ectodomain is cleaved by a metalloprotease. We now report a subsequent cleavage by gamma-secretase that releases the ErbB-4 intracellular domain from the membrane and facilitates its translocation to the nucleus. gamma-Secretase cleavage was prevented by chemical inhibitors or a dominant negative presenilin. Inhibition of gamma-secretase also prevented growth inhibition by HRG. gamma-Secretase cleavage of ErbB-4 may represent another mechanism for receptor tyrosine kinase-mediated signaling.

~z~I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37~ the cell-bound ~25I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound ~25I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0~ cell-bound nSI-hEGF is not degraded but slowly dissociates from the cell.The data are consistent with a mechanism in which ~zSI-hEGF initially is bound to the cell surface and subsequently is internalized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-

Some gliomas, melanomas and squamous carcinomas have large numbers of EGF receptors which could, in these cases, be used for targeting with toxic agents. We investigated whether EGF could be conjugated to dextran, which is a suitable carrier for toxic agents, without losing its ability to bind to the receptor. Dextran of 20 kDa molecular weight was activated with I-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) yielding highly active pyridinium-isourea derivatives. EGF was coupled to the activated dextran through the amino terminus and glycine was added to block residual activity. The EGF-dextran conjugate was, after purification on Sephadex G25 and Sephacryl200 columns, tested for its receptor binding properties on human malignant glioma, U343MGaC I2:6, cells. The conjugate inhibited binding of "%EGF in a competitive assay, showing that the binding was receptor-specific. Dextran conjugated with glycine, without EGF, had no inhibitory effect. The conjugate was radiolabelled either on the EGF part with lZ5l or on the dextran part with Wglycine, and the internalization patterns were compared to the internalization of '251-EGF. The radioactivity of the conjugates remained cell-associated for more than 20 hr, regardless of whether the radioactivity was on the EGF or on the dextran part, while the radioactivity of unconjugated EGF rapidly disappeared from the cells. Most of the cellassociated radioactivity was, at all analysed time intervals, located intracellularly. Thus, it seems promising to use dextran, conjugated with EGF, as a carrier of, for example, toxic radioactive nuclides. MATERIAL AND METHODS DextranDextran (Sigma, St. Louis, MO, industrial grade, catalogue number D-4626) with a molecular weight of about 20 kDa (average 19,5) was used. Epidermal growth factorEpidermal growth factor, EGF, tissue-culture grade, of murine origin [from Janssen, Beerse, Belgium or from Sigma (E 4127)] was used in all experiments.4T0 whom correspondence and reprint requests should be sent. guided irradiation of brain glioma by arterial infusion of radioactive monoclonal antibody against epidermal growth factor receptor and blood group A antigen. Brit. med.

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SUMMARY In several growth factor receptors, the intracellular juxtamembrane (JM) region participates in autoinhibitory interactions that must be disrupted for tyrosine kinase activation. Using alanine scanning mutagenesis and crystallographic approaches, we define a domain within the JM region of the epidermal growth factor receptor (EGFR) that instead plays an activating – rather than autoinhibitory – role. Mutations in the C-terminal 19 residues of the EGFR JM region abolish EGFR activation. In a crystal structure of an asymmetric dimer of the tyrosine kinase domain, the JM region of an acceptor monomer makes extensive contacts with the C-lobe of a donor monomer, thus stabilizing the dimer. We describe how an uncharacterized lung cancer mutation in this JM activation domain (V665M) constitutively activates EGFR by augmenting its capacity to act as an acceptor in the asymmetric dimer. This JM mutant promotes cellular transformation by EGFR in vitro and is tumorigenic in a xenograft assay.