Differentiation of mammary epithelium in vivo requires signaling through prolactin-andHER4 (expressed from a cDNA construct) localized to the nucleus, activated STAT5A, and induced three-dimensional lumen formation. Nuclear localization of exogenous s80 HER4 required intact kinase activity of s80 HER4 , as did activation of STAT5A. In contrast, nuclear localization of s80 HER4 and STAT5A activation did not require the 16-amino acid region of the ErbB4 intracellular domain specific to the Cyt-1 isoform of ErbB4, and absent in the Cyt-2 isoform. These results suggest that s80 HER4 formation contributes to ErbB4-dependent differentiation of mammary epithelial cells.

The ErbB receptor tyrosine kinase family has often been associated with increased growth of breast epithelial cells, as well as malignant transformation and progression. In contrast, ErbB4/HER4 exhibits unique attributes from a two step proteolytic cleavage which releases an 80 kilodalton, nuclear localizing, tyrosine kinase to a signal transduction mechanism that slows growth and stimulates differentiation of breast cells. This review provides an overview of ErbB4/HER4 in growth and differentiation of the mammary epithelium, including its physiologic role in development, the contrasting growth inhibition/tumor suppression and growth acceleration of distinct ErbB4/HER4 isoforms and a description of the unique cell cycle regulated pattern of nuclear HER4 ubiquitination and destruction.

In general, epidermal growth factor receptor family members stimulate cell proliferation. In contrast, at least one HER4 isoform, JM-a/Cyt1, inhibits cell growth after undergoing a two-step proteolytic cleavage that first produces a membrane-anchored 80-kDa fragment (m80 HER4 ) and subsequently liberates a soluble 80-kDa fragment, s80 HER4 . Here we report that s80 HER4 Cyt1 action increased the expression of WWP1 (for WW domain-containing protein 1), an E3 ubiquitin ligase, but not other members of the Nedd4 E3 ligase family. The HER4 Cyt1 isoform contains three proline-rich tyrosine (PY) WW binding motifs, while Cyt2 has only two. WWP1 binds to all three Cyt1 PY motifs; the interaction with PY2 found exclusively in Cyt1 was strongest. WWP1 ubiquitinated and caused the degradation of HER4 but not of EGFR, HER2, or HER3. The HER4-WWP1 interaction also accelerated WWP1 degradation. Membrane HER4 (full length and m80 HER4 , the product of the first proteolytic cleavage) were the preferred targets of WWP1, correlating with the membrane localization of WWP1. Conversely s80 HER4 , a poorer WWP1 substrate, was found in the cell nucleus, while WWP1 was not. Deletion of the C2 membrane association domain of WWP1 allowed more efficient s80 HER4 degradation, suggesting that WWP1 is normally part of a membrane complex that regulates HER4 membrane species levels, with a predilection for the growth-inhibitory Cyt1 isoform. Finally, WWP1 expression diminished HER4 biologic activity in MCF-7 cells. We previously showed that nuclear s80 HER4 is ubiquitinated and degraded by the anaphase-promoting complex, suggesting that HER4 ubiquitination within specific cellular compartments helps regulate the unique HER4 signaling capabilities.The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTKs) consists of EGFR/HER1/ ErbB1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4. All family members have a ligand-binding ectodomain, a single transmembrane segment, an intracellular tyrosine kinase domain, and a tyrosine-rich carboxy terminus. Ten ligands from two related ligand families, the epidermal growth factor (EGF) and heregulin/neuregulin families, bind to EGFR family RTKs. Ligand-dependent homo-and heterodimerization results in kinase activation and cross-phosphorylation. Although HER1 and HER4 are capable of both homo-and heterodimerization under most conditions, HER2 and HER3 are obligate heterodimeric partners, since HER2 does not bind any known ligand, and HER3 is devoid of intrinsic kinase activity. The activated receptor complexes exhibit multiple signaling capabilities including stimulation of canonical mitogenactivated protein kinase, phosphatidylinositol 3-kinase, and/or STAT signaling pathways (10,41,47).All four EGFR family members play a role in regulating cardiovascular, neuronal, and epithelial development. Null mutations of any of the EGFR family members result in embryonic lethality. Despite these similarities, HER4 exhibits unique biological activities that set it apart from the rest of the EGFR fam...

Unlike the proliferative action of other epidermal growth factor (EGF) receptor family members, HER4/ErbB4 is often associated with growth-inhibitory and differentiation signaling. These actions may involve HER4 two-step proteolytic processing by intramembraneous gamma-secretase, releasing the soluble, intracellular 80-kDa HER4 cytoplasmic domain, s80HER4. We demonstrate that pharmacological inhibition of either gamma-secretase activity or HER4 tyrosine kinase activity blocked heregulin-dependent growth inhibition of SUM44 breast cancer cells. We next generated breast cell lines stably expressing GFP-s80HER4 [green fluorescent protein (GFP) fused to the N terminus of the HER4 cytoplasmic domain, residues 676-1308], GFP-CT(HER4) (GFP fused to N terminus of the HER4 C-terminus distal to the tyrosine kinase domain, residues 989-1308), or GFP alone. Both GFP-s80HER4 and GFP-CTHER4 were found in the nucleus, but GFP-s80HER4 accumulated to a greater extent and sustained its nuclear localization. s80HER4 was constitutively tyrosine phosphorylated, and treatment of cells with a specific HER family tyrosine kinase inhibitor 1) blocked tyrosine phosphorylation; 2) markedly diminished GFP-s80HER4 nuclear localization; and 3) reduced signal transducer and activator of transcription (STAT)5A tyrosine phosphorylation and nuclear localization as well as GFP-s80HER4:STAT5A interaction. Multiple normal mammary and breast cancer cell lines, stably expressing GFP-s80HER4 (SUM44, MDA-MB-453, MCF10A, SUM102, and HC11) were growth inhibited compared with the same cell line expressing GFP-CTHER4 or GFP alone. The s80HER4-induced cell number reduction was due to slower growth because rates of apoptosis were equivalent in GFP-, GFP-CTHER4-, and GFP-s80HER4-expressing cells. Lastly, GFP-s80HER4 enhanced differentiation signaling as indicated by increased basal and prolactin-dependent beta-casein expression. These results indicate that surface HER4 tyrosine phosphorylation and ligand-dependent release of s80HER4 are necessary, and s80HER4 signaling is sufficient for HER4-dependent growth inhibition.

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