The ERBB receptors have a crucial role in morphogenesis and oncogenesis. We have identified a new PDZ protein we named ERBIN (ERBB2 interacting protein) that acts as an adaptor for the receptor ERBB2/HER2 in epithelia. ERBIN contains 16 leucine-rich repeats (LRRs) in its amino terminus and a PDZ (PSD-95/DLG/ZO-1) domain at its carboxy terminus, and belongs to a new PDZ protein family. The PDZ domain directly and specifically interacts with ERBB2/HER2. ERBIN and ERBB2/HER2 colocalize to the lateral membrane of human intestinal epithelial cells. The ERBIN-binding site in ERBB2/HER2 has a critical role in restricting this receptor to the basolateral membrane of epithelial cells, as mutation of the ERBIN-binding site leads to the mislocalization of the receptor in these cells. We suggest that ERBIN acts in the localization and signalling of ERBB2/HER2 in epithelia.

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure-function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.

Integrity of epithelial tissues relies on the proper apical-basolateral polarity of epithelial cells. Members of the LAP (LRR and PDZ) protein family such as LET-413 and Scribble are involved in maintaining epithelial cell polarity in Caenorhabditis elegans and Drosophila melanogaster, respectively. We previously described Erbin as a mammalian LET-413 homologue interacting with ERBB2/HER2, an epidermal growth factor receptor family member. Erbin and ERBB2/HER2 are located in the basolateral membranes of epithelial cells. We show here that Erbin interacts with p0071 (also called plakophilin-4), an armadillo repeat protein linked to the cytoskeleton. Erbin binds to p0071 in vitro and in vivo in a PDZ domain-dependent manner, and both proteins colocalized in desmosomes of epithelial cells. Using a dominant negative approach, we found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. We propose that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis.

Identification of protein complexes associated with the ERBB2/HER2 receptor may help unravel the mechanisms of its activation and regulation in normal and pathological situations. Interactions between ERBB2/ HER2 and Src homology 2 or phosphotyrosine binding domain signaling proteins have been extensively studied. We have identified ERBIN and PICK1 as new binding partners for ERBB2/HER2 that associate with its carboxyl-terminal sequence through a PDZ (PSD-95/ DLG/ZO-1) domain. This peptide sequence acts as a dominant retention or targeting basolateral signal for receptors in epithelial cells. ERBIN belongs to the newly described LAP (LRR and PDZ) protein family, whose function is crucial in non vertebrates for epithelial homeostasis. Whereas ERBIN appears to locate ERBB2/ HER2 to the basolateral epithelium, PICK1 is thought to be involved in the clustering of receptors. We show here that ERBIN and PICK1 bind to ERBB2/HER2 with different mechanisms, and we propose that these interactions are regulated in cells. Since ERBIN and PICK1 tend to oligomerize, further complexity of protein networks may participate in ERBB2/HER2 functions and specificity.The ERBB2/HER-2 gene is overexpressed in about 30% of human breast cancers and is also frequently altered in carcinoma of lungs and kidneys. The ERBB2 protein belongs to the large family of receptors with tyrosine kinase activity (RTK) 1 and more precisely to the ERBB subfamily of four receptors which includes the EGF receptor. Upon dimerization and activation, it phosphorylates many substrates including itself. This step induces a network of protein interactions. For example, phosphotyrosine binding and SH2 domains found in cytosolic proteins such as GRB2 and SHC adaptors bind to phosphorylated ERBB2 and lead to the activation of the RAS pathway (1, 2). RTKs also interact with cytoplasmic proteins in a nonphosphorylated dependent manner by means of PDZ protein modules found in adaptor proteins (3-8).PDZ domains are 90 -100-amino acid domains that interact with short peptides usually found at the carboxyl terminus of proteins. Proteins bearing a S/TXV motif, such as the glutamate receptors (NMDAR) and K ϩ channels, interact with class I PDZ domains found in LIN-7 and PSD-95 (9, 10). The second class of PDZ domains including the LIN-2/CASK PDZ domain prefers a carboxyl-terminal ⌿X⌿ sequence, where ⌿ is a hydrophobic residue. A third class of PDZ domain including the NOS PDZ domain binds to DXV peptides and PDZ domains (11,12). Within the RTK superfamily, several members have a canonical PDZ domain binding site in carboxyl-terminal position, i.e. two ERBB receptors (ERBB2/HER2 and ERBB4/ HER4), MUSK (a muscle-specific receptor), and several EPH receptor family members involved in developmental processes such as axon guidance. Kim and colleagues have demonstrated the importance of PDZ domain proteins for RTKs function in nonvertebrates. LET-23, the homolog of ERBB receptors in C. elegans, is localized to the basolateral epithelial membrane upon interaction with LIN-7, a PDZ...