Abstract
N-cadherin comprises five homologous extracellular domains, a transmembrane, and a cytoplasmic domain. The extracellular domains of N-cadherin play important roles in homophilic cell adhesion, but the contribution of each domain to this phenomenon has not been fully evaluated. In particular, the following questions remain unanswered: what is the minimal domain combination that can generate cell adhesion, how is domain organization related to adhesive strength, and does the cytoplasmic domain serve to facilitate extracellular domain interaction? To address these issues, we made serial constructs of the extracellular domains of N-cadherin and produced various cell lines to examine adhesion properties. We show that the first domain of N-cadherin alone on the cell surface fails to generate adhesive activity and that the first two domains of N-cadherin form the "minimal essential unit" to mediate cell adhesion. Cell lines expressing longer extracellular domains or N-cadherin wild type cells formed larger cellular aggregates than those expressing shorter aggregates. However, adhesion strength, as measured by a shearing test, did not reveal any differences among these aggregative cell lines, suggesting that the first two domains of N-cadherin cells generate the same strength of adhesive activity as longer extracellular domain cells. Furthermore, truncations of the first two domains of N-cadherin are also sufficient to form cisdimerization at an adhesive junction. Our findings suggest that the extracellular domains of N-cadherin have distinct roles in cell adhesion, i.e. the first two domains are responsible for homophilic adhesion activity, and the other domains promote adhesion efficiency most likely by positioning essential domains relatively far out from the cell surface.
Highlights
N-cadherin comprises five homologous extracellular domains, a transmembrane, and a cytoplasmic domain
Our findings suggest that the extracellular domains of N-cadherin have distinct roles in cell adhesion, i.e. the first two domains are responsible for homophilic adhesion activity, and the other domains promote adhesion efficiency most likely by positioning essential domains relatively far out from the cell surface
Truncated N-cadherins Are Appropriately Expressed at the Cell Surface—We generated serial constructs of N-cadherin, which included different EC domains linked to the natural transmembrane and cytoplasmic domains (NEC)
Summary
Antibodies—A polyclonal antibody against the cytoplasmic domain of N-cadherin was generated in rabbits. To obtain single or double transfectants, the cells were transfected by using Superfect (Qiagen; 1 g of each cadherin in pCXN2 vector in single or double transfection), and cultured in selective medium containing 800 g/ml of G418 (Invitrogen). Adhesion Strength Assays—To study adhesion strength in chimeric N-cadherin-transfected cell lines, plastic Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) were first coated with 10 g/ml protein A in a coating buffer (0.2 carbonate-bicarbonate (pH 9.4)), incubated at 4 °C overnight, washed with HCMF several times, and blocked with 1% BSA/HCMF for 1 h room temperature. Immunoblotting and Immunoprecipitation—For membrane fractionation, cell lines transfected with N-cadherin truncated constructs were harvested in ice-cold PBS and centrifuged at 1000 ϫ g, and the sediment was washed in PBS.
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