Nonvisual arrestin oligomerization and cellular localization are regulated by inositol hexakisphosphate binding

Abstract

Interactions between arrestins and phosphoinositides have been reported to regulate multiple membrane-associated signaling and trafficking events including clathrin-mediated endocytosis and light adaptation in Drosophila. Arrestins have been proposed to have nuclear and cytosolic functions as well, although the ligand dependence of these functions has not been investigated. Here we characterize the structural, molecular, and cellular interactions between arrestin-2 and inositol hexakisphosphate (inositol 1,2,3,4,5,6-hexakisphosphate (IP6)). The crystal structure of the arrestin-2·IP6 complex was solved to 2.9 Å with crystal lattice contacts suggesting two sites on a protein monomer mediating IP6 binding. Mutagenesis coupled to isothermal titration calorimetry and tritiated IP6 binding assays confirmed two-site binding with a low affinity IP6-binding site in the N-domain and a high affinity site in the C-domain. Native gel electrophoresis, gel filtration, and analytical ultracentrifugation demonstrated the ability of IP6 to promote arrestin-2 oligomerization via the two crystallographically defined ligand-binding locations. In addition, analysis in mammalian cells revealed that arrestin-2 not only undergoes homo-oligomerization, but it can also hetero-oligomerize with arrestin-3 in a manner that depends on IP6-binding sites. Mutation of either IP 6-binding site in arrestin-2 disrupted oligomerization while interactions with known binding partners including clathrin, AP-2, and ERK2 were maintained. Subcellular localization studies showed that arrestin-2 oligomers are primarily cytoplasmic, whereas arrestin-2 monomers displayed increased nuclear localization. Thus, by promoting cytosolic oligomerization, IP 6 binding is proposed to be a negative regulator of interactions of arrestin with plasma membrane and nuclear signaling proteins. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.