Perspective

Erbin: Sorting Out ErbB2 Receptors or Giving Ras a Break?

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Science's STKE  09 Sep 2003:
Vol. 2003, Issue 199, pp. pe37
DOI: 10.1126/stke.2003.199.pe37

Abstract

Erbin is a member of the leucine-rich repeat and PDZ domain (LAP) family. Originally cloned as an epidermal growth factor receptor (EGFR)-associated protein involved in receptor sorting and cell polarization, erbin has now been shown to inhibit EGF signaling by preventing the activation of the Raf-1 kinase by Ras. This discovery provides new insights into the rapidly expanding roles of adaptor and scaffolding proteins in the regulation of receptor signaling. It also highlights the complexity of cellular signaling networks in which the tasks of individual components are determined by the specific functional context.

EGF Receptor Signaling and ERK Activation

The need of cells to communicate with each other and to interact with their environment has led to the evolution of intricate signal transduction systems that use biochemical reactions to sense, process, and integrate external cues into distinct biological responses. A major challenge for modern biology is to unravel how the adequacy and fidelity of these responses is achieved. This endeavor is daunting because the "one gene, one protein, one function" relationship has broken down. The human genome has far too few genes to account for our complexity. Hence, one gene often functions in more than one pathway, and multiple pathways interact in combinatorial fashions to achieve distinct responses.

One of the best-understood signaling systems is the epidermal growth factor receptor (EGFR). The EGFR system comprises a family of four related transmembrane proteins, termed ErbB1 to ErbB4, with intracellular tyrosine kinase domains and extracellular ligand binding domains. The existence of multiple ligands, together with the ability of the different EGFRs to homo- and heterodimerize, sets the stage for the combinatorial assembly of diverse signaling platforms. These platforms consist of adaptor and signaling proteins that are recruited to specific docking sites on autophosphorylated receptor dimers.

The Ras-Raf-MEK [mitogen-activated protein kinase kinase or extracellular signal-regulated kinase (ERK) kinase]-ERK pathway is a major downstream signaling pathway that emanates from all EGFR complexes. This pathway consists of a small guanosine triphosphatase (GTPase), Ras, and a three-tiered kinase cascade, Raf-MEK-ERK. Ras is a membrane protein that cycles between inactive, guanosine diphosphate (GDP)-bound, and active, GTP-bound, conformations. EGFR activation promotes the exchange of GDP for GTP by recruiting guanine nucleotide exchange factors (GEFs) into the vicinity of Ras. Activated Ras-GTP binds Raf-1 with high affinity and translocates it to the cell membrane, where Raf-1 is activated. Activated Raf-1 phosphorylates and activates MEK, which in turn phosphorylates and activates ERK. ERK musters more than 70 substrates, including nuclear transcription factors. The specificity of the biological response depends on the intensity, duration, and localization of ERK activity. For instance, in PC12 cells, sustained activation of ERK drives differentiation, whereas transient activation leads to proliferation. Although the basic biochemistry of the EGFR pathway is well understood, much less is known about the fine-tuning of this pathway and how cross talk to other pathways is regulated. A recent paper by Huang et al. (1) sheds new light on the former issue and raises some provocative possibilities regarding the latter. The authors show that the ErbB2 binding protein erbin suppresses ERK activation by interfering with the activation of Raf-1 by Ras-GTP.

Erbin, a Member of the LAP Family

Erbin was originally identified as a binding partner of ErbB2. Its identification founded the leucine-rich repeat and PDZ domain (LAP) family, which is characterized by 16 leucine-rich repeats (LRRs) at the N terminus and one to four PDZ domains at the C terminus (2). The other LAP family members (densin-180, LET-413, and Scribble) are all involved in determining cell polarity (3). Cell polarity is essential for the function of cells that are located at physical or functional interfaces. For instance, epithelial cells in the digestive tract have an apical membrane that points toward the lumen of the gut and absorbs nutrients through numerous microvilli. In contrast, the basolateral membrane mediates the contacts to neighboring epithelial cells at the sides of the cell and to the basement membrane at the base. The apical and basolateral membranes are separated from each other by specialized membrane domains, termed "tight junctions" and "adherens junctions," that seal the space between the cells and fasten neighboring cells together. Mutations of the Caenorhabditis elegans LET-413 or Drosophila melanogaster Scribble genes disrupt this polarization, causing epithelial disorganization and complex failures in organ morphogenesis during development (3).

The demonstration that LAP proteins are required for epithelial cell polarization triggered the search for the molecular mechanisms involved. Several possibilities are plausible: They could form a barrier to separate apical from basolateral membrane components. They could direct sorting of the transport vesicles that constantly cycle between different cellular compartments and deliver proteins to their appropriate destination. Or they could anchor proteins at their intended location in the appropriate membrane compartment. Let-23, the worm homolog of the EGFR, which is tethered to the basolateral membrane by a complex of three PDZ domain proteins, provides a paradigm for the last of these mechanisms (4). Borg et al. have suggested that erbin plays a similar role for ErbB2 (2). ErbB2 is preferentially localized to the basolateral membrane of epithelial cells (2, 5), and this localization is important for its proper function (6). Borg et al. (2) showed that the erbin PDZ domain selectively binds to ErbB2 and that deletion of the erbin binding site in an ErbB1-ErbB2 chimeric molecule compromises the proper localization of the chimera to the basolateral membrane. This conclusion was challenged by Dillon et al. (5), who showed that targeting of ErbB2 to basolateral membranes is erbin independent. This discrepancy may reflect not only the use of different ErbB2 expression proteins, but also the intricacies of cell polarization, which could use a combination of mechanisms to ensure the vital task of correct protein sorting.

Erbin, an Inhibitor of the Raf-MEK-ERK Pathway

A recent publication by Huang et al. (1) adds a new twist to our understanding of erbin function. The authors show that erbin can inhibit the activation of ERK by interfering with the binding of Raf-1 to activated Ras. Interestingly, this function was not confined to EGFR signaling, because erbin could also inhibit neuronal differentiation in response to nerve growth factor. Use of activated and dominant negative Ras and Raf mutants showed that the erbin block lies between Ras and Raf-1. Ras can be locked in a constitutively activated GTP-bound state by point mutations that impair the hydrolysis of the associated GTP. Such mutations result in an oncogenic Ras protein that is a hallmark of many human cancers. Erbin bound selectively to mutationally activated Ras and blocked its interaction with Raf-1, thereby abolishing Ras activation of Raf-1 and the ERK pathway (1). Inhibition of the ERK pathway required the LRRs, but not the PDZ domain. LLRs mediate protein interactions and have previously been identified as Ras binding motifs, for instance, in Sur-8 and RSP1 [Ras suppressor protein 1, also known as RSU1 (Ras suppressor 1)]. Sur-8 consists entirely of LRRs. It serves as an adaptor that enhances the interaction between Ki-Ras and Raf-1, thereby facilitating Raf-1 activation (7). Rsp1 was originally identified as a suppressor of Ras transformation. However, subsequent studies showed that Rsp1 interfered with only some aspects of Ras function, such as c-Jun N-terminal kinase activation, but enhanced others, such as ERK activation (8). Thus, LRRs can exert a finely diversified regulation of Ras signaling by selectively inhibiting or enabling Ras binding to various partners. Given the ever-increasing number of identified Ras binding proteins, such gatekeepers are likely to coordinate the activation of downstream signaling pathways. Erbin may have a similar role, as it did not inhibit EGF-induced Akt phosphorylation (1). Interestingly, the association between erbin and Ras-GTP could not be detected in a yeast two-hybrid system; this suggests that the interaction is indirect or needs a ternary partner for stabilization (1).

More Functions for Erbin?

The above findings reveal two seemingly contradictory aspects of erbin: (i) as an ERK pathway suppressor protein, and (ii) as a protein that ensures the proper localization and function of the ErbB2 receptor (which activates ERK). Superficially, this paradox is reminiscent of the Sprouty protein family, which can inhibit or enhance EGFR signaling according to the cellular context. The EGFR phosphorylates Sprouty on a tyrosine residue, creating a docking site for two key regulators of EGFR signaling, Grb-2 and Cbl. Grb-2 is a positive effector that is involved in Ras activation, whereas Cbl is a negative regulator that triggers the degradation of the EGFR. By competing with the EGFR for Grb-2 and Cbl, phosphorylated Sprouty can either impair or enhance EGFR signaling, respectively. Moreover, Sprouty can bind to Raf-1 and prevent its activation (9). Erbin preferentially binds to the inactive ErbB2 receptor and is phosphorylated in response to ErbB2 activation (2). However, the role of this phosphorylation is not known. The erbin sequence contains several regions with homology to SH2 domains, and phosphorylation could generate a docking site for transferring erbin from ErbB2 to another binding partner and perhaps another pathway. It certainly would be instructive to follow the kinetics of erbin binding to ErbB2 and Ras and to correlate these kinetics with changes in erbin phosphorylation, Ras-GTP formation, and ERK activation. It would also be interesting to test whether erbin affects the association of Ras with other effectors. Huang et al.'s (1) data suggest that erbin selectively suppresses ERK activation. This raises the intriguing possibility that erbin could shift Ras signaling from ERK to other effectors and could function as a switch between different Ras effectors rather than as a mere inhibitor.

The fact that Ras inhibition maps to the LRRs at the N terminus of erbin and ErbB2 binding maps to the PDZ domain at the C terminus suggests that erbin has dual functionality (Fig. 1), raising the question of whether these two functions are independent of each other. The fact that these motifs are united in one protein may argue in favor of a link, although many proteins can serve independent functions in different pathways. However, an emerging theme is that signaling pathways are physically, and as a consequence functionally, connected by scaffolding and adaptor proteins that often use modular interaction motifs to tie pathways together (10). The AKAPs (adenosine kinase-associated proteins), which serve as signaling hubs by assembling components from different pathways (11), are a classical example of this. Erbin has several other binding partners, mainly proteins that are components of adherens junctions [such as the p120 catenin family proteins p0071 (12-14), δ-catenin, and ARVCF (15)] and proteins involved in cell attachment to substrates [such as β4-integrin and bullous pemphigoid antigen 1 (16), a constituent of hemidesmosomes]. These associations are mediated by the erbin C terminus that contains the PDZ domain, which would leave the LRRs free to interact with Ras and link Ras signaling pathways to cell adhesions and cell junctions.

Fig. 1.

Erbin is a multifunctional protein. Solid arrows indicate direct binding; broken arrows indicate indirect binding. Interacting proteins are in gray boxes; the putative functions of the interactions are in tan shapes. The details are described in the text, except those pertaining to erbin interactions with Smads (18) and PSD-95 (19). ARVCS, Armadillo protein deleted in velo-cardiofacial syndrome.

Ras signaling subverts epithelial cell polarity by disrupting cell-cell contacts and adherens junctions, mainly through activation of the ERK pathway (17). Erbin localizes to adherens junctions (17), and one function could be to stabilize these junctions by preventing localized ERK signaling. Activated Rho family proteins direct the localization of erbin, along with p0071 and β-catenin, to adherens junctions (17). These Ras-related proteins promote the assembly of adherens junctions and colocalize with erbin. Thus, erbin guided by Rho proteins could chaperone adhesion proteins to their proper destinations. So far, no direct interaction between erbin and Rho family proteins has been observed (17). However, the Ras family is large, and erbin may have yet undetected binding partners in this family. Another challenge will be to define the composition of erbin complexes and determine how their assembly is regulated. It is unlikely that erbin interacts with its many partners simultaneously, especially as most of them use the PDZ domain for binding. Thus, distinct erbin complexes with different functions are likely to exist. In its short life in the scientific literature, erbin has already produced a bewildering array of functions; hence, it is safe to predict that erbin's cornucopia will hold more surprises.

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