E-cadherin is a key cell–cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin–based AJs.

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of transinteracting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited nontrans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin-and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120 ctn , and strengthened the binding of p120 ctn to E-cadherin, eventually reducing non-transinteracting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits nontrans-interacting E-cadherin endocytosis through afadin, Rap1, and p120 ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs. Adherens junctions (AJs)1 are the principal mediators of cell-cell adhesion in epithelial cells and highly dynamic structures that turn over rapidly. E-cadherin is the major component of AJs in epithelial cells (1-4). E-cadherin first forms cis-dimers on the cell surface of the same cells, followed by formation of trans-dimers (trans-interactions) on the cell surface of two neighboring cells, through the extracellular region in a Ca 2ϩ -dependent manner, eventually causing cell-cell adhesion (5-7). The cytoplasmic region is linked to the actin cytoskeleton through ␣-and ␤-catenins (8), which strengthen the cell-cell adhesion activity of E-cadherin (4, 8, 9). p120 ctn directly binds to the juxtamembrane region of E-cadherin (10, 11). The definitive role of p120 ctn remains unknown, but several lines of evidence for its role have been reported: Mutations of the p120 ctn binding region of E-cadherin inhibit transport of de novo synthesized E-cadherin to the plasma membrane in L fibroblasts (12); knock down of p120 ctn reduces cell surface E-cadherin, presumably by enhancing its endocytosis (13); and p120 ctn binds to kinesin and promotes cell surface trafficking of cadherins (14,15).Nectin is a recently emerged Ca 2ϩ -independent immunoglobulin (Ig)-like cell-cell adhesion molecule that forms AJs cooperatively with cadherin (16,17). Nectin comprises a family of four members, nectin-1, -2, -3, and -4. Each member first forms homo-cis-dimers and then homo-or hetero-trans-dimers (trans-interactions) through the extracellular region in a Ca 2ϩ -independent manner, indu...

Neurotransmitter is released from nerve terminals by Ca2+-dependent exocytosis through many steps. SNARE proteins are key components at the priming and fusion steps, and the priming step is modulated by cAMP-dependent protein kinase (PKA), which causes synaptic plasticity. We show that the SNARE regulatory protein tomosyn is directly phosphorylated by PKA, which reduces its interaction with syntaxin-1 (a component of SNAREs) and enhances the formation of the SNARE complex. Electrophysiological studies using cultured superior cervical ganglion (SCG) neurons revealed that this enhanced formation of the SNARE complex by the PKA-catalyzed phosphorylation of tomosyn increased the fusion-competent readily releasable pool of synaptic vesicles and, thereby, enhanced neurotransmitter release. This mechanism was indeed involved in the facilitation of neurotransmitter release that was induced by a potent biological mediator, the pituitary adenylate cyclase-activating polypeptide, in SCG neurons. We describe the roles and modes of action of PKA and tomosyn in Ca2+-dependent neurotransmitter release.

In epidemiological studies, moderate alcohol consumption has been consistently associated with a reduced risk of myocardial infarction (MI). About half of Japanese show an extremely high sensitivity to alcohol (ethanol), which is due to a missense mutation from glutamic acid (Glu) to lysine (Lys) at codon 487 in an isoenzyme of aldehyde dehydrogenase (ALDH2) with a low Km. We obtained a preliminary result that subjects homozygous for the Lys 487 allele had higher risk for myocardial infarction. The purpose of the present study was to assess this hypothesis by employing a larger cohort of subjects with MI. The experimental group consisted of 342 male subjects with demonstrated MI who were selected randomly from our outpatient clinic. As controls, we employed 1,820 male subjects with no cardiovascular complications who were selected from the Suita Study. All subjects provided their written informed consent to participate in the genetic analyses. Subjects with MI were older and had higher body mass index, higher prevalence of diabetes mellitus, higher prevalence of smoking habit, higher prevalence of the Lys/Lys genotype (homozygous for Lys 487 allele), and lower high density lipoprotein (HDL) cholesterol level (HDL-C). The ALDH2 genotype affected the level of alcohol consumption, and HDL-C. Multiple logistic analyses indicated that the odds ratio of the Lys/Lys genotype to the Lys/Glu+Glu/Glu genotype was 1.56 (p=0.0359). Inclusion of HDL-C as one of the independent variables downplayed the importance of the ALDH2 genotype. This may indicate that the ALDH2 genotype affects MI via its effects on HDL-C. In conclusion, the ALDH2 Lys/Lys genotype is a risk factor for myocardial infarction in Japanese men due to its influence on HDL cholesterol level.

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