The enzyme-mediated activation of radical source reaction: a new approach to identify partners of a given molecule in membrane microdomains

Important biological events associated with plasma membranes, such as signal transduction, cell adhesion, and protein trafficking, are mediated through the membrane microdomains. However, it is difficult to assess the issue of how they assemble under physiological conditions. We developed a new approach to identify partners of a given molecule on the cell surface in living cells. The important feature of this system, termed as enzyme-mediated activation of radical source, is that activation of cross-linking reagent arylazide-biotin tag can be accomplished not by ultraviolet light, but by an enzyme, horseradish peroxidase. By using this method, we found that many kinds of receptor tyrosine kinases are associated with beta 1 integrin whereas a few receptor tyrosine kinases are associated with ganglioside GM1 in HeLa S3 cells. This system is a comprehensive approach to identify interactions between cell surface molecules under living conditions. The advantages of this approach are as follows: (i) easy, high throughput, and without the need for special equipment, (ii) applicable to systematic approaches such as proteomic analysis, (iii) applicable to studies on the interactions among not only proteins but also glycans and lipids. The biochemical approach although the enzyme-mediated activation of radical source reaction will provide a new insight into a wide range of research concerning cis-interaction between biomolecules on the cell surface in living cells.