Single amino acid substitutions in conserved extracellular domains of E-cadherin differ in their functional consequences

The calcium-dependent homophilic cell adhesion molecule E-cadherin typicall y connects epithelial cells. The extracellular portion of the mature transm embrane protein consists of five homologous domains. The four sequences lin king these domains contain the structural amino acid motif DXXD that is tho ught to be involved in direct calcium binding. In gastric cancer patients m utations affecting this motif between the second and third domain are frequ ently seen. In order to determine the functional significance of similar se quence alterations with regard to their location, we analyzed single amino acid substitutions changing the DXXD motif to DXXA in each linker region ac cording to a mutation found in gastric cancer (D370A). The cDNA sequences c oding for DQND, DVLD and DVND were changed (D257A, D479A, D590A, respective ly) and stably expressed in E-cadherin negative MDA-MB-435S mammary carcino ma cells. We found that the D257A and D370A mutations result in abnormal pr otein localization, changes in the actin cytoskeleton, markedly reduced hom ophilic cell adhesion, and altered cell morphology. Unexpectedly, the tumor -associated D370A mutation but not the D257A mutation induced increased cel l motility. The D479A mutation only had slight functional consequences wher eas cells expressing the D590A mutant did not differ from cells expressing the wild-type molecule. Although the putative calcium binding motif DXXD is located at repetitive positions in the extracellular portion of E-cadherin , our results indicate that it has different functions depending on the loc ation. Remarkably, tumor cells select for mutations in the most critical do mains resulting both in loss of function (decreased cell adhesion) and in g ain of function (increased cell motility). Since multiple DXXD motifs are t ypically seen in other cadherins, our structure-function study is relevant for this gene family in general. (C) 2001 Academic Press.