Muc4–ErbB2 Complex Formation and Signaling in Polarized CACO-2 Epithelial Cells Indicate That Muc4 Acts as an Unorthodox Ligand for ErbB2

Abstract

Muc4 serves as an intramembrane ligand for the receptor tyrosine kinase ErbB2. The time to complex formation and the stoichiometry of the complex were determined to be <15 min and 1:1 by analyses of Muc4 and ErbB2 coexpressed in insect cells and A375 tumor cells. In polarized CACO-2 cells, Muc4 expression causes relocalization of ErbB2, but not its heterodimerization partner ErbB3, to the apical cell surface, effectively segregating the two receptors. The apically located ErbB2 is phosphorylated on tyrosines 1139 and 1248. The phosphorylated ErbB2 in CACO-2 cells recruits the cytoplasmic adaptor protein Grb2, consistent with previous studies showing phosphotyrosine 1139 to be a Grb2 binding site. To address the issue of downstream signaling from apical ErbB2, we analyzed the three MAPK pathways of mammalian cells, Erk, p38, and JNK. Consistent with the more differentiated phenotype of the CACO-2 cells, p38 phosphorylation was robustly increased by Muc4 expression, with a consequent activation of Akt. In contrast, Erk and JNK phosphorylation was not changed. The ability of Muc4 to segregate ErbB2 and other ErbB receptors and to alter downstream signaling cascades in polarized epithelial cells suggests that it has a role in regulating ErbB2 in differentiated epithelia. INTRODUCTIONErbB2 is a 185-kDa class I receptor tyrosine kinase that is structurally related to the epidermal growth factor receptor EGFR. The ErbB family of receptors includes four members: epidermal growth factor receptor (EGFR, HER1, or c-ErbB1), c-ErbB2 (HER2, p185 neu ), c-ErbB3 (HER3), and c-ErbB4 (HER4) (Riese and Stern, 1998), which share 40 -45% sequence identity (Stein and Staros, 2000). The ErbB receptor extracellular domains are composed of four subdomains, which in order from the N-terminus are known as I (L1), II (CR1), III (L2), and IV (CR2) (Bajaj et al., 1987;Lax et al., 1988;Ward et al., 1995); subdomains I and III and subdomains II and IV are homologous. ErbB ligand binding is mediated primarily by subdomain III, and to a lesser extent by subdomain I (Lax et al., 1989;Khoda et al., 1993). Subdomains II and IV mediate the formation of specific ErbB homo and heterodimers (Garrett et al., 2002;Ogiso et al., 2002). The extracellular domain is followed by a transmembrane domain, a tyrosine kinase domain, and a segment of variable length of ϳ200 amino acids containing several tyrosine phosphorylation sites. On activation, these phosphotyrosines become docking sites for cytoplasmic signaling proteins that, in turn, initiate characteristic downstream signaling events (Klapper et al., 2000). The pathways activated may lead the cell to proliferation, differentiation, or apoptosis (Alroy and Yarden, 1997;Riese and Stern, 1998). All ErbB family members, with the exception of ErbB2, have high-affinity soluble ligands that induce receptor homo-or heterodimer formation and phosphorylation and trigger downstream signaling. The preferred ErbB heterodimer partner is ErbB2, and the ErbB2-ErbB3 pair generates the strongest proliferative signal, even...

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