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 Kd), 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=[(Ntxf)/(KdxScell)]-[(Bxp)/Kd], where B and F are bound and free CD58 density in the contact area, respectively; Nt is CD2 molecule number per cell; f is CD2 fractional mobility; Scell is cell surface area; and p is the ratio of contact area at equilibrium to Scell. We use this analysis to determine that the 2D Kd for CD2-CD58 is 5.4-7.6 molecules/microm2. 2D Kd analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion.
Zhu
De-Min
DM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Hematology Division, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Dustin
Michael L
ML
Cairo
Christopher W
CW
Golan
David E
DE
D018801:CD2 Antigens / Q000378:metabolism*; D018968:CD58 Antigens / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*
D018801:CD2 Antigens; D018968:CD58 Antigens; D015815:Cell Adhesion Molecules