Abstract
The interaction of the T cell glycoprotein CD2 and its ligand CD58 is important for T cell interaction with antigen-presenting and target cells. The binding interaction is of low affinity and has a fast off-rate (>5 s-1) in solution. However, solution measurements may not accurately predict the behavior of molecules in an adhesive contact area. Interaction between T cells that express CD2 and glass-supported planar bilayers containing purified and fluorescently labeled CD58 leads to accumulation of CD58 (fluorescence) in the cell/bilayer contact area. CD58 molecules accumulated within the contact area in excess of the CD58 density in the bilayer outside the contact area can be considered as bound by cell surface CD2. Here, this phenomena and fluorescence photobleaching recovery were utilized to determine whether CD2-CD58 bonds are transient in contact areas. Fluorescent CD58 molecules accumulated in the T cell-bilayer interface were completely bleached. The bleached CD58 molecules accumulated in the contact area were rapidly replaced by fluorescent CD58 that diffused into the contact area from adjacent bilayer regions outside the contact area. Rapid recovery of the accumulated fluorescence directly demonstrates that the CD2-CD58 bonds are dissociating and that the dissociation leads to partner exchange, rather than rebinding of the same CD2-CD58 pairs. This suggests that the solution off-rate provides an accurate description of CD2-CD58 interaction in contact areas. Accumulated fluorescent IgG in contacts between K562 cells expressing low affinity Fc receptors and planar bilayers with fluorescent IgG bound to hapten-derivitized phospholipids displayed slower recovery than CD58 by a factor of 10. This suggests that the Fc receptor-IgG interaction has a longer lifetime than the CD2-CD58 interaction. These findings have implications for the mechanism of signaling by CD2 and the mechanism of cell detachment from large numbers of transient interactions.
Highlights
Specific binding of membrane receptors across a small gap between neighboring cells is an exquisitely specific mode of communication
We conclude that the dissociation of CD2 from CD58 was followed by diffusion of the free CD58 away from the CD2 receptor so that it can bind another CD2 molecule or diffuse out of the contact area. This property was not regulated by cell activation or parallel ligation of CD2 with the CD2.1 antibody. To determine if this kind of dynamic behavior was associated with another low affinity receptor system, we studied the low affinity Fc receptor II expressed on K562 cells
The novel finding of this report is that the CD2-CD58 bond displays striking dynamics in contact areas
Summary
Specific binding of membrane receptors across a small gap between neighboring cells is an exquisitely specific mode of communication. The accumulation of CD58 in contacts between T cells and bilayers containing fluorescently labeled CD58 has been used to determine the density of CD2-CD58 complexes as a function of free CD58 density. In the CD2-CD58 system, the interaction can drive striking accumulation of CD58 in the contact area between a cell and an artificial bilayer containing fluorescently labeled CD58, which is observed by fluorescence microscopy.
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