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.

The ErbB-4 receptor tyrosine kinase has a PDZ domain recognition motif at its carboxyl terminus. The first step in ErbB-4 proteolytic processing is a metalloprotease-dependent cleavage of the receptor ectodomain, which is not influenced by deletion of this motif. Metalloprotease cleavage of ErbB-4 produces a membrane-associated 80-kDa fragment that is a substrate for subsequent ␥-secretase cleavage, which releases the cytoplasmic domain from the membrane and allows nuclear translocation of this fragment. Deletion of the PDZ domain recognition motif does abrogate the ␥-secretase cleavage of ErbB-4. The wild-type 80-kDa ErbB-4 fragment forms an association complex with presenilin, thought to be the catalytic moiety of ␥-secretase activity. However, this association is significantly impaired by loss of the PDZ domain recognition motif from ErbB-4. Deletion of this ErbB-4 motif prevents the nuclear localization of the ErbB-4 cytoplasmic domain. Data also show that the basal cleavage of wild-type ErbB-4 by this proteolytic system can produce a sufficient level of ErbB-4 processing to negatively influence cell growth and that loss of the PDZ domain recognition motif abrogates this response.ErbB-4 is a type I receptor tyrosine kinase that binds heregulin (HRG) 1 (1). HRG also binds to ErbB-3. In addition some ErbB-1 ligands (heparin-binding epidermal growth factor, betacellulin, and epiregulin) are reported to activate ErbB-4.Recently, ErbB-4 was shown to be processed by a novel proteolytic pathway (2), analogous to that which generates cell fate determination responses after activation of the Notch nontyrosine kinase receptor (3). The amyloid precursor protein (APP) is also processed in a similar manner (4). The first step in this pathway is cleavage within the ectodomain close to the plasma membrane. In the case of ErbB-4, this cleavage is stimulatable by TPA (5) and, in some cell lines, by HRG (6). This proteolytic event results in the release of a 120-kDa ErbB-4 fragment into the extracellular milieu and the cellular retention of an 80-kDa membrane-associated fragment. Ectodomain cleavage requires the transmembrane metalloprotease tumor necrosis factor-␣-converting enzyme (7).After ectodomain cleavage of ErbB-4, the cell-associated 80-kDa fragment containing the transmembrane and cytoplasmic domains is cleaved by a ␥-secretase activity releasing the cytoplasmic domain into the cytosol (2, 8). Subsequently, the cytoplasmic domain fragment of ErbB-4 translocates into the nucleus. The function of the 80-kDa fragment in the nucleus is not known, although the carboxyl-terminal region of this fragment does display weak activity in the GAL4 transcription activation assay (2). Importantly, ␥-secretase inhibitors block the induction of cell death by HRG when T47D carcinoma cells are placed in serum-free media (2). The nuclear localization of other receptor tyrosine kinases, including ErbB-1 (9), ErbB-3 (10), and fibroblast growth factor receptor 1 (11), have been described, but these do not appear to involve proteolytic pro...