PURIFICATION AND OPTIMIZATION OF FUNCTIONAL RECONSTITUTION ON THE SURFACE OF LEUKEMIC-CELL LINES OF GPI-ANCHORED FC-GAMMA RECEPTOR-III

Purified glycosyl phosphatidyl inositol (GPI)-anchored cell surface pr oteins can be reincorporated spontaneously into the cell membrane by i ncubating the cells with these proteins. This unique property provides a novel way of introducing cell surface receptors on live cell membra nes without the use of gene transfection. Since any classical transmem brane cell surface protein can be converted to a GPI anchored protein by recombinant techniques, this method provides a means of studying ec todomain associated receptor functions on various cell types. Moreover , in some circumstances, it can be used to correct deficient cellular functions resulting from lack of cell surface protein expression. Usin g GPI-anchored Fc gamma receptor III (CD16B), a low affinity Fc gamma receptor, we have systematically studied the optimal conditions for re constitution of a functional receptor on nucleated cells. CD16B is pur ified to homogeneity from neutrophil lysates by single step immunoaffi nity chromatography. The purified CD16B is functionally active as evid enced by its ability to bind IgG opsonized erythrocytes. CD16B incorpo ration on nucleated cells is temperature dependent with an optimum of 37 degrees C. The level of expression of incorporated CD16B is also de pend on the concentration of CD16B available and the duration of incub ation. The incorporated CD16B retains its ability to bind ligand and a lso mediates endocytosis of the bound ligand. In summary, our results demonstrate that purified, functionally active GPI-anchored receptors can be expressed on desired cells in a controlled manner and retain so me functional properties.