Analysis of two-dimensional dissociation constant of laterally mobile cell adhesion molecules

We formulate a general analysis to determine the two-dimensional dissociation constant (2D K-d), and use this method to study the interaction of CD2-expressing T cells with glass-supported planar bilayers containing fluorescently labeled CD58, a CD2 counter-receptor. Both CD2 and CD58 are laterally mobile in their respective membranes. Adhesion is indicated by accumulation of CD2 and CD58 in the cell-bilayer contact area; adhesion molecule density and contact area size attain equilibrium within 40 min. The standard (Scatchard) analysis of solution-phase binding is not applicable to the case of laterally mobile adhesion molecules due to the dynamic nature of the interaction. We derive a new binding equation, B/F [(N-t x f)/(K-d x S-cell)] - [(B x p)/K-d], where B and F are bound and free CD58 density in the contact area, respectively; N-t is CD2 molecule number per cell; f is CD2 fractional mobility; S-cell is cell surface area; and p is the ratio of contact area at equilibrium to S-cell. We use this analysis to determine that the 2D K-d for CD2-CD58 is 5.4-7.6 molecules/mu m(2). 2D K-d analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion.