To investigate the possible biological function of the lateral "strand dimer" observed in crystal structures of a D1 domain extracellular fragment from N-cadherin, we have undertaken site-directed mutagenesis studies of this molecule. Mutation of most residues important in the strand dimer interface abolish the ability of N-cadherin to mediate cell adhesion. Mutation of an analogous central residue (Trp-2) in E-cadherin also abrogates the adhesive capacity of that molecule. We also determined the crystal structure of a Ca2+-complexed two-domain fragment from N-cadherin. This structure, like its E-cadherin counterpart, does not adopt the strand dimer conformation. This suggests the possibility that classical cadherins might stably exist in both dimeric and monomeric forms. Data from several laboratories imply that lateral dimerization or clustering of cadherins may increase their adhesivity. We suggest the possibility that the strand dimer may play a role in this activation.
The relationship between adhesive interactions across the synaptic cleft and synaptic function has remained elusive. At certain CNS synapses, pre- to postsynaptic adhesion is mediated at least in part by neural (N-) cadherin. Here, we demonstrate that upon depolarization of hippocampal neurons in culture by K+ treatment, or application of NMDA or alpha-latrotoxin, synaptic N-cadherin dimerizes and becomes markedly protease resistant. These properties are indices of strong, stable, enhanced cadherin-mediated intercellular adhesion. N-cadherin retained protease resistance for at least 2 hr after recovery, while other surface molecules, including other cadherins, were completely degraded. The acquisition of protease resistance and dimerization of N-cadherin is not dependent on new protein synthesis, nor is it accompanied by internalization of N-cadherin. By immunocytochemistry, we found that high K+ selectively induces surface dispersion of N-cadherin, which, after recovery, returns to synaptic puncta. N-cadherin dispersion under K+ treatment parallels the rapid expansion of the presynaptic membrane consequent to the massive vesicle fusion that occurs with this type of depolarization. In contrast, with NMDA application, N-cadherin does not disperse but does acquire enhanced protease resistance and dimerizes. Our data strongly suggest that synaptic adhesion is dynamically and locally controlled, and modulated by synaptic activity.
Classical cadherins form parallel cis-dimers that emanate from a single cell surface. It is thought that the cis-dimeric form is active in cell–cell adhesion, whereas cadherin monomers are likely to be inactive. Currently, cis-dimers have been shown to exist only between cadherins of the same type. Here, we show the specific formation of cis-heterodimers between N- and R-cadherins. E-cadherin cannot participate in these complexes. Cells coexpressing N- and R-cadherins show homophilic adhesion in which these proteins coassociate at cell–cell interfaces. We performed site- directed mutagenesis studies, the results of which support the strand dimer model for cis-dimerization. Furthermore, we show that when N- and R-cadherins are coexpressed in neurons in vitro, the two cadherins colocalize at certain neural synapses, implying biological relevance for these complexes. The present study provides a novel paradigm for cadherin interaction whereby selective cis-heterodimer formation may generate new functional units to mediate cell–cell adhesion.
N-cadherin is up-regulated in aggressive breast carcinomas, but its mechanism of action in vivo remains unknown. Transgenic mice coexpressing N-cadherin and polyomavirus middle T antigen (PyVmT) in the mammary epithelium displayed increased pulmonary metastasis, with no differences in tumor onset or growth relative to control PyVmT mice. PyVmT-N-cadherin tumors contained higher levels of phosphorylated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) than PyVmT controls, and phosphorylated ERK staining was further increased in pulmonary metastases. Tumor cell isolates from PyVmT-N-cadherin mice exhibited enhanced ERK activation, motility, invasion, and matrix metalloproteinase-9 (MMP-9) expression relative to PyVmT controls. MAPK/ERK kinase 1 inhibition in PyVmT-N-cadherin cells reduced MMP-9 production and invasion but not motility. Furthermore, inactivation of fibroblast growth factor receptor in PyVmT-N-cadherin cells reduced motility, invasion, and ERK activation but had no effect on PyVmT cells. Thus, de novo expression of N-cadherin in mammary ducts enhances metastasis of breast tumors via enhanced ERK signaling. [Cancer Res 2007;67(7):3106-16]
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