The MRC2 gene encodes the constitutively recycling type I transmembrane receptor Endo180 (Wu et al. 1996; Sheikh et al. 2000; Isacke et al. 1990), which is also cited in the literature as urokinase plasminogen activator receptor associated protein (uPARAP) following its identification as part of a trimolecular complex with pro-urokinase plasminogen activator (pro-uPA) and its receptor urokinase plasminogen activator receptor ( uPAR) (Behrendt et al. 2000). Endo180 is a member of the mannose receptor family, which also includes the mannose receptor (MR; CD206), M-type phospholipase A2 receptor, and DEC-205 (CD205) (East and Isacke 2002). The constitutive recycling of Endo180 between clathrin-coated pits at the plasma membrane and intracellular endosomal compartments is dependent on a dihydrophobic endocytosis motif and modulated by a conserved upstream acidic residue (Howard and Isacke 2002). Endo180 internalization is independent of a conserved tyrosine-based motif that is critical role for internalization to the other three family members (Howard and Isacke 2002). Endo180 expression is regulated by transforming growth factor-beta receptor (TGF-beta Receptor) signaling via the Smad transcription factor pathway (Huijbers et al. 2010).
Endo180 Structure and Implications for Signaling
Endo180 Ligands and Implications for Signaling
Endo180 interacts with different subtypes of collagen via its FNII domain (Wienke et al. 2003). For type I collagen this interaction occurs through the interaction of Endo180 with its C-terminal domain (Thomas et al. 2005). The binding of soluble collagen ligands at the cell surface results in their rapid internalization by Endo180 and subsequent transport to lysosomal compartments for degradation (Wienke et al. 2003; Engelholm et al. 2003; East et al. 2003), a function that has been implicated in the tissue remodeling process associated with cancer progression (Huijbers et al. 2010; Kogianni et al. 2009; Wienke et al. 2007) and tumor cell invasion (Huijbers et al. 2010). Another important function for this interaction is the promotion of cell-matrix adhesion (Sturge et al. 2006; Thomas et al. 2005). The intracellular signaling events activated by the engagement of Endo180 with either soluble or fibrillar forms of collagen have not been fully explored. To date, evidence suggests that the enhanced phosphorylation of myosin (regulatory) light chain-2 (MLC2) by the Endo180-Rho-Rho kinase (ROCK) signaling axis is independent of cell adhesion to collagen (Sturge et al. 2006).
Endo180 has been shown to interact with various sugar moeities via calcium-dependent binding to its only functional C-type lectin-like domain, CTLD2 (East et al. 2002, 2003). However, the impact of the lectin activity of Endo180 on its signaling function, or any of its other functions, has not yet been reported.
The trimolecular complex formed between Endo180, uPAR and pro-uPA on the cell surface (Behrendt et al. 2000) is implicated in the enhanced migratory behavior of tumor cells, during which, Endo180 can coordinate the activation of Rho GTPases (Cdc42, Rac, and Rho), cytoskeletal remodeling events (filopodial, lamellipodial, and stress fiber dynamics), cell-matrix adhesion turnover and cell–cell adhesion disassembly (Sturge et al. 2003, 2006; Takahashi et al. 2010) (see section “Endo180-Dependent Signaling in Cell Migration” for further details). MR-dependent chemotactic responses in myoblasts (Jansen and Pavlath 2006) suggests that a common biological role exists for this related receptor; however, the molecular basis underlying the function of MR as a chemotaxis receptor and the chemotactic signaling events that are activated downstream of MR have not been determined.
Endo180–Dependent Signaling in Cell Migration
Ectopic overexpression of Endo180 in receptor null tumor cells results in their enhanced cell migration and chemotaxis (Sturge et al. 2003). In accordance, the genetic silencing of Endo180 by small interference RNA (siRNA) in invasive human tumor cell lines that express high endogenous levels of Endo180 (Sturge et al. 2003, 2006), or the targeted deletion of Endo180 in murine embryonic fibroblasts (Engelholm et al. 2003; East et al. 2003), results in reduced cell migration and adhesion. As stated previously there are three major signaling pathways that have been identified as downstream targets that can be modulated by Endo180 and can promote cell migration.
Endo180–Dependent Activation of Cdc42
The requirement for Endo180 in MDA-MB-231 metastatic breast cancer cells to sense a chemotactic gradient of pro-uPA was demonstrated by genetic silencing of Endo180 by siRNA in Dunn chemotaxis chamber assays (Sturge et al. 2003). The role of Endo180 in this chemotactic response was supported by a gain in directional migration toward a gradient of pro-uPA following its overexpression in non-invasive MCF7 breast cancer cells. These changes in chemotactic behavior are directly linked to altered dynamics of pro-uPA-dependent Cdc42 activation, which is suppressed by Endo180 silencing in MDA-MB-231 cells, enhanced in MCF7 cells that overexpress Endo180 and display increased filopodial protrusions in response to pro-uPA (Sturge et al. 2003). The activation of Cdc42 and detection of a pro-uPA gradient in cells that express an Endo180 mutant receptor that cannot be internalized indicates that the chemosensory function of Endo180 is independent of receptor endocytosis (Sturge et al. 2003).
Endo180–Dependent Activation of Rac
The rapid activation of Rac by uPA in MCF7 cells that overexpress Endo180 and the requirement for the internalization of Endo180 to activate Rac and promote an increase in migratory speed suggest the mechanism of Rac activation by Endo180 is distinct from that required for its activation of Cdc42 (Sturge et al. 2003). The localization of Endo180 to lamellipodia (Sturge et al. 2003) and the requirement of Endo180 for lamellipodial protrusion formation (Takahashi et al. 2010) indicates that it plays a critical role in Rac-dependent signaling, which drives cytoskeletal remodeling and cell migration.
Endo180–Dependent Activation of Rho-ROCK-MLC2
Endo180 is required for the diphosphorylation of serine 19/threonine 18 of MLC2 in MG63, MDA-MB-231, BE and HT-1080 cells and these signals are enhanced by the overexpression of Endo180 in MCF7 cells. Endo180 is also required for the phosphorylation of myosin light chain phosphatase-1 (MYPT1) and LIMK, which are common downstream targets of ROCK. Functional studies demonstrated that the Rho-ROCK-MLC2 diphosphorylation signaling axis is spatially localized by Endo180–containing endosomes for the promotion of focal adhesion disassembly at the rear of migrating cells (Sturge et al. 2006).
Phosphorylation of Endo180
Endo180 is phosphorylated by purified protein kinase C (PKC) in vitro and can be phosphorylated in vivo by treating cells with phorbol esters (Sheikh et al. 2000; Isacke et al. 1990). 32Pi-labeling revealed that Endo180 contains at least three residues, which are constitutively phosphorylated in Flow 2000 embryonic lung diploid fibroblasts under normal growth conditions (Sheikh et al. 2000; Isacke et al. 1990). Treatment of Flow 2000 cells with phorbol 12-myristate 13-acetate (PMA) increased the phosphorylation of one of these sites and revealed two additional phosphorylation sites. All three PMA responsive phosphorylation sites have been identified as serine residues. PKC purified from rat brain showed a strong preference to only one of these three phosphorylation sites in vitro. Endo180 has also been tested in vitro for a kinase activity against itself and a number of common substrates with negative results (Sheikh et al. 2000; Isacke et al. 1990). Endo180 has several putative phosphorylation sites in its cytoplasmic tail (see Fig. 2) but their functional role has not been confirmed.
Endo180 is an endocytic receptor that can signal to regulate the overall and tempero-spatial activation of the small Rho GTPases, Cdc42, Rac, and Rho (Sturge et al. 2003, 2006). Future work is required to: (a) elucidate the factors responsible for Rho GTPase activation by Endo180; (b) identify the intracellular partners of Endo180; (c) determine how conformational changes in the receptor ectodomain impact on receptor function and signaling, with a particular focus on the impact of collagen and uPA/uPAR-binding; and (d) investigate the influence of Endo180 phosphorylation on downstream signal transduction pathways.
- Takahashi S, Yamada-Okabe H, Hamada K, Ohta S, Kawase T, Yoshida K, et al. Downregulation of uPARAP mediates cytoskeletal rearrangements and decreases invasion and migration properties in glioma cells. J Neurooncol. 2010. doi: 10.1007/s11060-010-0398-z.