We propose to investigate the relationship among structural variation, molecular property, and cellular function of CD16 and to use CD16 as a model to investigate receptor-mediated cell-cell adhesion. CD16 is a Fc receptor for immunoglobulin 6 (Fc-gammaRIII) that is vital to the immune system. It is one of only four known eukaryotic cell surface receptors that naturally use two types of membrane anchor: the hydrophobic transmembrane polypeptide chain and the glycosyl phosphatidylinositol linkage. For this reason, CD16 represents an excellent experimental system to study this structural difference notwithstanding that results of this study will be directly relevant to the understanding of the immune system function. The present-project will focus on the CD16-mediated cell adhesion because it is a primary step in most of the CD16 functions. Receptor properties which may be affected by membrane anchor and which are important to cell adhesion include the strength of an individual receptor- ligand crossbridge, the mechanism of molecular detachment, the affinity of a receptor for its ligands, and the lateral mobility of the receptor on the cell membrane. The complex dependence of cell adhesion on properties of the receptor, the ligand and the cell can be broken down into three levels: a single pair of receptor and ligand, the interplay of multiple coupled crossbridges, and the interaction of crossbridges with the cell surface. The availability of ligands, monoclonal antibodies, cDNAs, and cell lines expressing isoforms of CD16 allows us to use this real system of physiological importance as an ideal model to study cell adhesion because all key variables can be measured in one single system. Using mechanism-based mathematical models, experiments can now be designed such that these variables are carefully controlled and the effects of their variations on the CD16-mediated cell adhesion are systematically dissected. The following specific aims are designed to test the hypothesis that the structural variations of CD16 affect its adhesion functionality through the effects on the receptor properties. I. To determine the effects of structural variations of the CD 16 molecule on the crossbridge strength, detachment mechanism, and binding affinity of its interactions with ligands by 1) measuring the crossbridge strengths at a per molecular bond level, 2) identifying the detachment mechanisms, and 3) correlating the crossbridge strengths and detachment mechanisms with the structural variations of CD16 and its affinities for the ligands. II. To measure the dependence of the number of CD16-ligand crossbridges supporting cell adhesion on various properties of CD16, its ligand, and the cells expressing them by 1) quantifying the integrated effects of CD16 expression, ligand density, and binding affinity on the crossbridge number, and 2) determining the relative contributions of crossbridge lateral migration and interfacial roughness smoothening to the fracture energy amplification during contact separation. The proposed research will contribute to our understanding as to how the membrane anchor of Fc receptors affects their properties, their interactions with ligands, and the ability of such interactions to induce effector functions.
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