Receptor-mediated cell adhesion is fundamental to a wide variety of biological processes. Interactions between Fc gamma receptors (Fc- gamma-Rs) and immunoglobulin G (IgG) are key mechanisms through which antibody effector functions are mediated. These molecules are clearly defined and have been well characterized biochemically. As such, they represent an excellent biological system for biophysical studies of receptor-ligand interactions notwithstanding that the results of these studies will be directly relevant to the understanding of immune functions. The proposed research thus has a dual objective: to elucidate the structure-function relationship of two low-affinity Fc-gamma-Rs (CD16 and CD32) and to use these as a model system to address fundamental issues in the biophysics of cell adhesion. The former include the effects of the membrane anchor on the ligand binding kinetics of CD16b and CD32a, the dissection of the critical amino acid that gives rise to the affinity difference between CD16a and CD16b, and the cross- regulation between CD16b and CD32a. The latter include development of measurement methods, construction of mathematical models, and characterization of interfacial adhesive dynamics. The focus is kinetic and equilibrium constants of receptor-ligand interactions when both molecules are linked to apposing surfaces. These constants are essential determinants of cell adhesion, as they govern how likely, how rapidly and how strongly cells bind, as well as how long they remain bound in the presence and absence of external forces. Our central hypothesis is that the effective kinetic rates and affinities depend not only on the intrinsic binding reaction, but also on how the receptors and ligands are presented by the surfaces, how the two surfaces are brought into contact, and how the membranes are aligned to form an ordered contact area. This hypothesis will be tested by systematic studies organized in three specific aims: 1. Quantify the effects of structural variations in CD16 and CD32 on their ligand-binding kinetics; 2. Determine the kinetics of Fc-gamma- R-IgG interactions using the flow chamber; and 3. Measure the kinetics of Fc-gamma-R-IgG interactions by a new contact area FRAP (fluorescence recovery after photobleaching) method. The results of this project promise to improve our understanding of the biophysical basis of the Fc-gamma-R-IgG interaction-mediated cell adhesion at the molecular level. It may also provide guidance to the development of Fc-gamma-R- based drugs for treatment of inflammatory disorders and auto-immune diseases.
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