Equilibrium Gel Filtration to Measure Arrestin-3 Binding of Inositol Hexaphosphate

Abstract

Arrestins are multi-functional adapter proteins that orchestrate a vast array of cellular functions including MAPK signaling, receptor trafficking, and transcriptional regulation. Inositol hexaphosphate (IP6) is a naturally occurring phosphorylated carbohydrate that is abundant in almost all plant and mammalian cells. Previously studies showed that IP6 interacts with arrestin proteins, and regulates arrestin-mediated signaling. More interestingly, binding of IP6 affects four arrestin isoforms deferentially. Here, we report an equilibrium gel-filtration method to evaluate the interaction between arrestin and IP6. The interaction of arrestin3-IP6 can be further investigated by the method reported to determine the binding stoichiometry and binding affinities.