Cell Surface Localization of α3β4 Nicotinic Acetylcholine Receptors Is Regulated by N-Cadherin Homotypic Binding and Actomyosin Contractility

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central and peripheral nervous system and are localized at synaptic and extrasynaptic sites of the cell membrane. However, the mechanisms regulating the localization of nicotinic receptors in distinct domains of the cell membrane are not well understood. N-cadherin is a cell adhesion molecule that mediates homotypic binding between apposed cell membranes and regulates the actin cytoskeleton through protein interactions with the cytoplasmic domain. At synaptic contacts, N-cadherin is commonly localized adjacent to the active zone and the postsynaptic density, suggesting that N-cadherin contributes to the assembly of the synaptic complex. To examine whether N-cadherin homotypic binding regulates the cell surface localization of nicotinic receptors, this study used heterologous expression of N-cadherin and alpha 3 beta 4 nAChR subunits C-terminally fused to a myc-tag epitope in Chinese hamster ovary cells. Expression levels of alpha 3 beta 4 nAChRs at cell-cell contacts and at contact-free cell membrane were analyzed by confocal microscopy. alpha 3 beta 4 nAChRs were found distributed over the entire surface of contacting cells lacking N-cadherin. In contrast, N-cadherin-mediated cell-cell contacts were devoid of alpha 3 beta 4 nAChRs. Cell-cell contacts mediated by N-cadherin-deleted proteins lacking the beta-catenin binding region or the entire cytoplasmic domain showed control levels of alpha 3 beta 4 nAChRs expression. Inhibition of actin polymerization with latrunculin A and cytochalasin D did not affect alpha 3 beta 4 nAChRs localization within N-cadherin-mediated cell-cell contacts. However, treatment with the Rho associated kinase inhibitor Y27632 resulted in a significant increase in alpha 3 beta 4 nAChR levels within N-cadherin-mediated cell-cell contacts. Analysis of alpha 3 beta 4 nAChRs localization in polarized Caco-2 cells showed specific expression on the apical cell membrane and colocalization with apical F-actin and the actin nucleator Arp3. These results indicate that actomyosin contractility downstream of N-cadherin homotypic binding regulates the cell surface localization of alpha 3 beta 4 nAChRs presumably through interactions with a particular pool of F-actin.