One biological issue for 2D binding of receptor-ligand interactions is how molecular structures determine the functionality of cell adhesions. Different species of ligands bind to their receptors with varied kinetic rates and affinities. For example, E-selectin dissociates 2-fold faster from HL-60 cells expressing PSGL-1 than Colo-205 cells expressing both
PSGL-1 and sLex carbohydrates (27). CD16A (FcγRIII) binds to rabbit IgG with 4-fold higher affinity than human IgG (24). Even with same species, different isoforms or constructs of interacting receptors reports distinct binding mechanisms to their ligands. For example, glycosylpho- sphatidylinositol-anchored CD16A binds faster and with higher affinities to human and rabbit IgGs but slower and with lower affinity to murine IgG2a as compared to trans- membrane-anchored CD16A (26). L- or P-selectin trans- fected preB cells roll more irregularly on and dissociate faster from the substrate of immobilized single tyrosine replacement of PSGL-1 than wild-type PSGL-1 molecules (63). Under static conditions, PSGL-1-expressing neutrophils adhere equivalently to cells expressing wild-type P-selectin that contains 9 CRs units and to P-selectin constructs with as few as two CRs. However, P-selectin requires at least 5 CRs to mediate optimal rolling of flowing neutrophils under shear conditions (64).
Surface presentation of interacting receptors affects the 2D kinetics of receptor-ligand interactions. One surface environmental factor is the orientation of the binding pockets of adhesion receptors. For example, randomizing the orientation of selectin or rabbit IgG lowers 2D affinities of selectin vs PSGL-1 or rabbit IgG vs CD16A interactions by reducing the forward rates but not the reverse rates (Figures 3a and 3b). In contrast, the soluble antibody binds with similar three-dimensional affinities to cell-bound P-selectin constructs regardless of their orientation (28). HL-60 cells adhere to short E-selectin constructs with two or less CRs when they are captured by a nonblocking monoclonal antibody adsorbed on the plastic surface (65). Cell adhesion to fibronectin is regulated by surface chemistries that alter fibronectin adsorption (66). Cells expressing an L-selectin construct that replaces its EGF domain with the EGF domain of P-selectin roll better on L-selectin ligands than cells expressing wild-type L-selectin, probably because an altered orientation of Lec domain enhances the association rate with surface ligands (67). Another surface environmental factor is the length of the binding pockets above the cell membrane. For example, lowering selectin ligand- and antibody-binding domain above the cell membrane lowers 2D affinities of selectin vs PSGL-1 or rabbit IgG vs CD16A interactions by reducing the forward rates but not the reverse rates (Figures 3c and 3d). In contrast, the soluble antibody binds with similar three-dimensional affinities to cell-bound P-selectin constructs regardless of their length (28). Extending the binding site of CD58 above the cell membrane in the stalk (~15 nm) enhances its binding to CD2 on T lymphocytes (68). K562 cells bearing a glycosulfopeptide 2-GSP-6 modeled after the binding domain of PSGL-1, which is attached to the membrane-distal region of a nonbinding molecule ~50 nm above the cell surface, roll more stably on P-selectin than cells bearing 2-GSP-6 randomly attached to cell surface proteins (69). HL-60 cells bind to immobilized full-length E-selectin that contains all six CRs but not to shorter E-selectin constructs with two or less CRs (65).
Membrane microtopology and cell deformation onto which the interacting receptors are anchored also influence the 2D receptor-ligand kinetics. For example, Fcγ receptor CD16B binds to IgG with a 50-fold higher in effective 2D affinity AcKa for receptors anchored on smooth RBCs than those on rough CHO and K562 cells, whereas the reverse rates are similar for all three (29). Rough cells may initially mediate the point contact, whereas smooth cells initiate the area contact, which results in the differences in contact area Ac. As of another example, PSGL-1 localized on the tips of elastic microvilli slows neutrophils rolling on P-selectin substrate and lowers reverse rate, as compared to PSGL-1 coupled onto rigid polystyrene microbeads. However, fixed neutrophils with less deformation support relatively faster rolling and dissociation than resting neutrophils (70).
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