Visualization of CD2 interaction with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area.
Many adhesion receptors have high three-dimensional dissociation constants (Kd) for counter-receptors compared to the KdS of receptors for soluble extracellular ligands such as cytokines and hormones. Interaction of the T lymphocyte adhesion receptor CD2 with its counter-receptor, LFA-3, has a high solution-phase Kd (16 microM at 37 degrees C), yet the CD2/LFA-3 interaction serves as an effective adhesion mechanism. We have studied the interaction of CD2 with LFA-3 in the contact area between Jurkat T lymphoblasts and planar phospholipid bilayers containing purified, fluorescently labeled LFA-3. Redistribution and lateral mobility of LFA-3 were measured in contact areas as functions of the initial LFA-3 surface density and of time after contact of the cells with the bilayers. LFA-3 accumulated at sites of contact with a half-time of approximately 15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Kd for the CD2/LFA-3 interaction was 21 molecules/microns 2, which is lower than the surface densities of CD2 on T cells and LFA-3 on most target or stimulator cells. Thus, formation of CD2/LFA-3 complexes should be highly favored in physiological interactions. Comparison of the two-dimensional (membrane-bound) and three-dimensional (solution-phase) KdS suggest that cell-cell contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Kd and the intermembrane distance at the site of contact. LFA-3 molecules in the contact site were capable of lateral diffusion in the plane of the phospholipid bilayer and did not appear to be irreversibly trapped in the contact area, consistent with a rapid off-rate. These data provide insights into the function of low affinity interactions in adhesion.
Dustin
M L
ML
Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Ferguson
L M
LM
Chan
P Y
PY
Springer
T A
TA
Golan
D E
DE
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D018801:CD2 Antigens / Q000502:physiology*; D018968:CD58 Antigens / Q000502:physiology*; D002448:Cell Adhesion; D002460:Cell Line; D006801:Humans; D007365:Intercellular Junctions / Q000502:physiology* / Q000648:ultrastructure; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D016399:Lymphoma, T-Cell; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018801:CD2 Antigens; D018968:CD58 Antigens; D008051:Lipid Bilayers; D008081:Liposomes