Ectodomain Cleavage of ErbB-4

Abstract

Ectodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646 -657 was incubated with recombinant tumor necrosis factor-␣-converting enzyme, fragments representing residues 646 -651 and 652-657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulindependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule.ErbB-4 is a member of the ErbB receptor tyrosine kinase family, which also includes the epidermal growth factor receptor (ErbB-1), ErbB-2, and ErbB-3 (1). ErbB-4 and ErbB-3 bind the neuregulin (heregulin) family of growth factors, and ErbB-4 also recognizes certain growth factors in the epidermal growth factor family of ErbB-1 agonists, such as betacellulin, epiregulin, and heparin-binding epidermal growth factor. ErbB-4 heterodimerizes with ErbB-2 as do ErbB-1 and ErbB-3; however, ErbB-4 can also signal through the formation of ErbB-4 homodimers.Within the receptor tyrosine kinase family, ErbB-4 is uniquely processed (2) by a proteolytic pathway that is known to occur with certain other transmembrane proteins, such as Notch (3), the low density lipoprotein receptor-related protein (4), the amyloid precursor protein (APP) 1 (5), and the adhesion molecules CD44 (6, 7) and E-cadherin (9). The first step in this pathway involves the release of the ErbB-4 ectodomain by a cleavage that produces two fragments as follows: a 120-kDa ectodomain fragment and an 80-kDa membrane-associated fragment, designated m80 (9). The latter fragment contains the ErbB-4 transmembrane domain and the entire cytoplasmic region, including the tyrosine kinase domain. Ectodomain cleavage of ErbB-4 in cells occurs at a low constitutive or basal level (10) that can be increased by TPA (9) or by heregulin or other growth factors that bind ErbB-4 (11). Also, cleavage is potentiated by the addition to cells of pervanadate, a tyrosine phosphatase inhibitor that provokes ErbB-4 tyrosine phosphorylation (12). The ectodomain cleavage of ErbB-4 is sensitive to metalloprotease inhibitors (10) and does not occur in cells genetically deficient in tumor necrosis factor-␣-converting enzyme (TACE), a ...

Secretase-dependent Tyrosine Phosphorylation of Mdm2 by the ErbB-4 Intracellular Domain Fragment

“…Treatment of cells with heregulin (8) or 12-O-tetradecanoylphorbol-13-acetate (9) initiates a metalloprotease-dependent ectodomain cleavage of ErbB-4 between His-651 and Ser-652 (10), placing this initial cleavage site eight residues prior to the transmembrane domain, which is typical of an ␣-secretase activity. Ectodomain cleavage or ErbB-4 is abrogated in TACE (ADAM17) null cells (11), and recombinant TACE is able to cleave a peptide representing ErbB-4 residues 646 -657 between His-651 and Ser-652 (10). Therefore, it seems likely that tumor necrosis factor alpha converting enzyme (TACE) executes this cleavage in vivo.…”

The ErbB-4 s80 intracellular domain is a constitutively active tyrosine kinase

“…The initial cleavage occurs within the ErbB-4 ectodomain between His651 and Ser652, placing this cleavage site eight residues outside the transmembrane domain (Cheng et al, 2003). It seems likely that this cleavage is executed by the transmembrane metalloprotease TACE (ADAM17), as TACE null cells fail to initiate TPA-provoked cleavage (Rio et al, 2000) and recombinant TACE cleaves a peptide representing ErbB-4 residues 646-657 between His651 and Ser652 (Cheng et al, 2003).…”

“…Reports that ErbB-1 cytoplasmic domain interactions exist (Chantry, 1995;Murali et al, 1996;Stamos et al, 2002;Landau et al, 2004;Aifa et al, 2005) and our observation that the m80 fragment of ErbB-4 associates with intact ErbB-2 (Cheng et al, 2003), led to a test of the capacity of the s80 ICD to self-associate. Cos7 cells were cotransfected with cDNAs that encode the ErbB-4 s80 fragment tagged with FLAG or GFP at the NH 2 -terminus.…”

“…The products of ErbB-4 ectodomain cleavage are an ectodomain fragment of 120 kDa and membrane-associated 80 kDa fragment (m80) that begins with Ser652 and includes the transmembrane and cytoplasmic domains of ErbB-4 (Vecchi et al, 1996;Cheng et al, 2003). The m80 fragment serves as substrate for a second cleavage event that is mediated by the g-secretase component presenilin, a polytopic transmembrane protein, which cleaves substrates within the transmembrane domain.…”

Antitumor and Antiangiogenic Activities of BMS-690514, an Inhibitor of Human EGF and VEGF Receptor Kinase Families

“…As HER4 has recently been implicated in cancer cell signaling as well, the potency of HER4 inhibition by BMS-690514 was also characterized in a cell assay. As reported previously, HER4 is subject to proteolytic cleavage (39,40). Both the full-length and cleaved receptors are extensively phosphorylated on tyrosines when overexpressed (Fig.…”