Quantifying the impact of membrane microtopology on effective two-dimensional affinity

Just as interactions of soluble proteins are affected by the solvent, membr ane protein binding is influenced by the surface environment. This is parti cularly true for adhesion receptors because their function requires tightly apposed membranes. We sought to demonstrate, and further, to quantify the possible scale of this phenomenon by comparing the effective affinity and k inetic rates of an adhesion receptor (CD16b) placed in three distinct envir onments: red blood cells (RBCs), detached Chinese hamster ovary (CHO) cells , and K562 cells. Effective affinity reflects both the intrinsic receptor-l igand kinetics and the effectiveness of their presentation by the host memb ranes. Expression of CD16b, a low affinity Fc gamma receptor, was establish ed by either transfection or spontaneous insertion via its glycosylphosphat idylinositol anchor. Binding to IgG-coated RBCs, measured using a micropipe tte method, indicated a 50-fold increase in effective affinity for receptor s on RBCs over CHO and K562 cells, whereas the off rates were similar for a ll three. Electron microscopy confirmed that specific tight contacts were b road in RBC RBC conjugates but sparse in CHO-RBC conjugates, We suggest tha t through modulation of surface roughness the cytoskeleton can greatly impa ct the effectiveness of adhesion molecules, oven those with no cytoplasmic structures. Implications for locomotion and static adhesion are discussed.