β-Arrestins (β-arrs) were originally appreciated for the roles they play in the desensitization and internalization of G protein-coupled receptors (GPCRs). They are also now known to act as molecular scaffolds, providing control in multiple signalling pathways. Through their scaffolding properties, β-arrs dynamically regulate the activity and/or subcellular distribution of protein partners giving rise to an appropriate cellular response. There are two β-arr isoforms, namely, β-arr1 and β-arr2, which share high sequence homology and structural conservation. While the β-arrs often display conserved overlapping roles, decisive differences between the isoforms also exist. A striking example of this is the subcellular distribution of the β-arr isoforms. While β-arr1 is distributed both in cytoplasmic and nuclear compartments, β-arr2 displays an apparent cytoplasmic distribution. Both β-arrs are actively imported into the nucleus, but β-arr2 is constitutively exported by a leptomycin B-sensitive pathway due to a nuclear export signal in its C-terminus that is absent in β-arr1. β-arr2 therefore undergoes constitutive nucleocytoplasmic shuttling enabling the displacement of nuclear binding cargoes, such as Mdm2. Here, we describe methods to explore the differential nucleocytoplasmic shuttling capacities of the β-arrs.
Keywords: Mdm2; Nuclear export signal; Nucleocytoplasmic trafficking; p53; β-Arrestin.