INTERCADHERIN INTERACTIONS IN EPITHELIAL CELLS

E-钙粘附素属于经典钙粘附素的多基因家族，在特殊的细胞间连接中充当结构跨膜元件，称为黏附连接。基于E-钙粘附素的黏附对于上皮组织的正常发育和维持至关重要。然而，尽管它们很重要，但人们对这些连接组装所涉及的分子机制知之甚少。目前的建议是基于我们的发现，E-钙粘附素可以自结合形成三个暴露在细胞表面的不同的复合体。通过结晶学分析预测了其中的两个，即E-钙粘素分子通过残基Trp156形成二聚体的侧向复合体(Trp156依赖的侧向复合体)和反平行(粘附性)复合体。鉴定的第三个复合体是横向Trp156-独立复合体。它是由培养上清液中的细胞外钙离子耗尽引起的。重要的是，只有黏附复合体的形成依赖于细胞质中的E-钙粘蛋白结构域。此外，表皮生长因子(EGF)显著增加了这些复合体的数量。这三个不同的复合体很可能代表粘连连接组装中的连续步骤。因此，本提案的总体目标是评估这一假设。为此，我们将研究这些复合体中的每一个对粘连连接组装的贡献。将评估依赖于Trp156的侧向复合体和连接素在粘附性复合体形成中的作用。我们将确定E-钙粘附素的哪个决定因素参与Trp156-独立的相互作用。此外，我们计划检查点突变或特定多肽使该决定簇失活是否会影响黏附复合体聚集成黏附连接。该提案的最后部分侧重于了解EGF对粘连复合体形成的影响。这项建议中描述的实验将进一步加深我们对上皮形态发生的分子机制的理解。

E-cadherin belongs to a multigene family of classic cadherins that serve as structural transmembrane elements in specialized intercellular junctions termed adherens junctions. E-cadherin-based adhesion is critical for normal development and maintenance of epithelial tissue. Despite their importance, however, little is known about the molecular mechanisms involved in the assembly of these junctions. The present proposal is based on our finding that E-cadherin can self- associate to form three distinct complexes exposed on the cell surface. Two of these complexes, the lateral complex in which E-cadherin molecules form dimers via residue Trp156 (Trp156-dependent lateral complex), and the antiparallel (adhesive) complex have been predicted by crystallographic analysis. The third complex identified is the lateral Trp156-independent complex. It is triggered by a depletion of extracellular Ca2+ ions from culture medium. Importantly, only the formation of adhesive complexes depends on cytoplasmic E-cadherin domain. Furthermore, epidermal growth factor (EGF) strongly increases the amount of these complexes. It is likely that the three distinct complexes represent consecutive steps in adherens junction assembly. The broad goal then, of the present proposal is to evaluate this hypothesis. To this end, we will study the contribution of each of these complexes to adherens junction assembly. The role of the lateral Trp156-dependent complexes and catenins in the formation of the adhesive complexes will be evaluated. We will identify which determinant of E-cadherin is involved in Trp156- independent interactions. Also, we plan to examine whether inactivation of this determinant by point mutations, or by specific peptides, affects clustering of the adhesive complexes into adherens junctions. The final part of the proposal is focused on understanding the effect of EGF on the adhesive complex formation. The experiments described in this proposal will further our understanding of the molecular mechanisms responsible for epithelial morphogenesis.