We and others have shown that N-cadherin influences the morphology and behavior of epithelial cells (Islam et al. 1996
; Hazan et al. 1997
, Hazan et al. 2000
; Li et al. 1998
). These studies implicate N-cadherin in an epithelial to mesenchymal transition in some cells, but not in others. In squamous epithelial cells, expression of N-cadherin results in downregulation of E-cadherin, which is most likely responsible for the change in cellular morphology. In other cells, such as breast cancer cells, expression of N-cadherin does not alter cell morphology, but does alter cellular behavior by inducing a motile phenotype. In breast cancer cells, expression of E-cadherin remains approximately the same when the cells are forced to express N-cadherin. This suggests that even in cells that express abundant E-cadherin, N-cadherin influences cell behavior. N-cadherin is often expressed by motile cells, such as fibroblasts, and a switch from E-cadherin expression to N-cadherin expression occurs when some cells become motile and/or invasive during normal developmental processes (Edelman et al. 1983
; Hatta and Takeichi 1986
; Zhou et al. 1997
; Huttenlocher et al. 1998
). Thus, it is not unexpected that expression of N-cadherin by tumor cells alters cellular morphology and/or behavior.
The extracellular domain of a cadherin promotes cell–cell adhesion, whereas the cytoplasmic domain serves to link the cadherin to the cytoskeleton via interactions with catenins. These cytosolic interactions are critical to the adhesive function of the cadherin. Linkage to the cytoskeleton is necessary to promote strong cell–cell adhesion and to allow organization of the junction itself. In addition, the catenins have been implicated in signaling events that are thought to regulate the strength of the adhesive activity of the cadherin (for review see Gumbiner 2000
). This led us to propose that the cytoplasmic domain of N-cadherin was responsible for increasing the motility of epithelial cells. When we prepared two chimeric cadherins, one comprised of the extracellular domain of N-cadherin linked to the cytoplasmic domain of E-cadherin (N/E-cadherin) and the other comprised of the extracellular domain of E-cadherin linked to the cytoplasmic domain of N-cadherin (E/N-cadherin), we were surprised to find that it was the extracellular domain of N-cadherin that promoted cell motility. The extracellular domain of cadherins is comprised of five repeat regions with EC1 being the most NH2
-terminal. Most of the known activities of cadherins have been mapped to EC1. The best understood examples are those where cadherin molecules interact with other cadherin molecules. Structure determinations (Shapiro et al. 1995
; Nagar et al. 1996
; Tamura et al. 1998
; Pertz et al. 1999
) and biochemical characterization (Nose et al. 1990
; Ozawa et al. 1990
; Ozawa and Kemler 1990
; Koch et al. 1997
; Shan et al. 2000
) have demonstrated that EC1 is the site of the adhesion interface. Data from several laboratories have suggested that cadherins are displayed on the surface of cells as dimers (Shapiro et al. 1995
; Brieher et al. 1996
; Chitaev and Troyanovsky 1998
; Takeda et al. 1999
; Shan et al. 2000
). Although several differing pictures exist as to how these cis (also called lateral) dimers form and are maintained, the data point to EC1 and EC2 of the cadherins as playing major roles.
In some instances, it has been shown that cadherins can promote cell–cell adhesion via heterophilic interactions, for example N-cadherin can bind to R-cadherin (Inuzuka et al. 1991
), B-cadherin can bind to L-CAM (Murphy-Erdosh et al. 1995
), and cadherin-6B can bind to cadherin-7 (Nakagawa and Takeichi 1995
). Recently, Shimoyama et al. 2000
examined eight different type II cadherins and frequently observed interactions between L cells transfected with different cadherins. Another recent study showed that, in L cells expressing both N- and R-cadherins, the two cadherins formed cis heterodimers that functioned in cell adhesion (Shan et al. 2000
). In this latter case, it was the NH2
terminus of the cadherins that played a role in the formation of the cis heterodimers. It will be interesting to determine if other pairs of cadherins shown to mediate heterophilic cell–cell adhesion are able to form cis heterodimers and what parts of the cadherins are involved. Here, we have shown that the ability of N-cadherin to promote cell motility resides in EC-4. Thus, this activity is distinct from the adhesive function of the cadherin.
In addition to the interaction of cadherins with themselves, various other interacting proteins have been described. The bacterium Listeria monocytogenes
has been shown to use E-cadherin as a receptor. InlA, a surface protein on the bacterium, binds to E-cadherin. Lecuit et al. 1999
showed that changing a single amino acid in EC1 of E-cadherin (proline-16 of EC1) eliminated the binding of InlA and dramatically compromised internalization of Listeria
by cells. In addition to being a target for Listeria
, E-cadherin is the only cadherin that is known to be an integrin ligand. Integrin αE
binds EC1 of E-cadherin, and glutamate-31 of EC1 plays a critical role in the interaction (Karecla et al. 1996
). Since EC1 of cadherins has been shown to play a major role in their biological activities, all of the chimeras used here retained the intact EC1 of N-cadherin.
Although most activities have been mapped to the NH2
-terminal domains, there are several reports suggesting roles for EC3, EC4, and EC5 in cadherin adhesion. Zhong et al. 1999
have characterized a mAb (AA5) recognizing EC5 of C-cadherin that activates adhesion, perhaps by changing the cadherin's organization or altering its interaction with other cellular factors. Sivasankar et al. 1999
have studied the biophysical characteristics of adhesion mediated by layers of oriented recombinant C-cadherin ectodomains. They concluded that complete interdigitation of antiparallel ectodomains (i.e., where EC1 of one molecule interacted with EC5 of the antiparallel partner, EC2 interacted with EC4 of the partner, etc.) gave the strongest interactions. Their data also suggested that ratcheting the molecules one EC domain further apart (such that EC1 interacted with EC4 of its antiparallel partner, etc.) also resulted in an adhesive interaction. In addition, Troyanovsky et al. 1999
have reported that EC3 and EC4 of E-cadherin can mediate cis dimerization under some conditions.
A series of papers from Lilien's laboratory (for review see Lilien et al. 1999
) have suggested that in neural retina cells, the ectodomain of N-cadherin is stably associated with and is a substrate for the cell surface enzyme N
-acetylgalactosaminyphosphotransferase. The interaction of neurocan, a chondroitin sulfate proteoglycan, with N
-acetylgalactosaminyphosphotransferase results in inhibition of N-cadherin–mediated cell adhesion. However, the site(s) on N-cadherin where this interaction takes place is unknown.
Investigators have suggested that N-cadherin can interact with and activate fibroblast growth factor receptors (FGFR) in neurons (Doherty and Walsh 1996
) and ovarian surface epithelial cells (Peluso 2000
). In the ovarian surface epithelial cell system, it has been reported that N-cadherin and FGFR coimmunoprecipitate. To date, this interaction has not been substantiated by other labs. Our laboratory recently showed that N-cadherin–mediated cell motility of breast cancer cells can be decreased by an inhibitor of the FGF-mediated signal transduction pathway, which has been characterized by the Walsh and Doherty labs (Nieman et al. 1999a
). In addition, Hazan et al. 2000
showed that FGF caused a dramatic increase in motility in N-cadherin–expressing cells. The FGFRs contain an HAV sequence (Byers et al. 1992
) that has been proposed to interact with EC4 of N-cadherin. It is interesting to note that the 69–amino acid segment of N-cadherin we have identified here includes the sequences proposed by Doherty and Walsh to interact with the FGFRs. The structure of a portion of FGFR1 bound to FGF2 has been determined (Plotnikov et al. 1999
). The histidine and valine side chains of the HAV sequence in FGFR1 were involved in intradomain contacts and, thus, appear to be unavailable for interacting with partner molecules. Thus, the precise role the FGFR plays in N-cadherin–dependent cell motility is still unknown and it is not clear at this time whether N-cadherin and the FGFR directly interact with one another.
Many studies have shown that N-cadherin promotes cell motility that is dependent on the adhesive function of N-cadherin. The best studied example is that of N-cadherin–dependent neurite extension. In vitro experiments have demonstrated that N-cadherin promotes neurite outgrowth as a purified protein or when it is expressed by transfected cells. Importantly, antibodies that block the adhesive function of N-cadherin block this outgrowth, and it has been suggested that N-cadherin may guide axonal outgrowth in vivo (for review see Grunwald 1996
). In addition, Hazan et al. 1997
suggested that N-cadherin–mediated motility of tumor cells might be due to the interactions of N-cadherin–expressing epithelial cells with N-cadherin–expressing stromal cells. In contrast, the studies presented here, using the 8C11 mAb, provide evidence that N-cadherin may influence the motility of epithelial cells in a manner that is independent of cell–cell adhesion.
Since the 69–amino acid portion of N-cadherin can influence epithelial cell morphology and motility, we compared this portion of human N-cadherin to other cadherins. In this region, mouse and rat N-cadherin are identical to human N-cadherin, whereas 78% of the amino acids in human R-cadherin are identical. The corresponding region of human E-cadherin contains 70 amino acids and is 54% identical to N-cadherin. To further investigate the role this portion of N-cadherin plays in cell motility, we produced a mAb that binds near EC-4 of N-cadherin. When applied to cells in a motility assay, this antibody inhibited cell motility in N-cadherin–expressing cells, but not in N-cadherin–negative cells. In addition, this antibody inhibited motility without inhibiting cell–cell aggregation, providing further evidence that adhesion and motility are two separate properties of the extracellular domain of N-cadherin. It is important to remember that all the chimeras used here were full-length cadherins. Studies are in progress to determine if truncated cadherins can influence cell motility.