Agonist-induced internalization of G-protein coupled receptors (GPCRs) is an important function to maintain homeostatic control in the cell and to regulate cell surface expression of receptors (Ferguson, 2001; Gainetdinov et al., 2004). GPCR internalization primarily occurs through an agonist-driven association with an arrestin protein which functions as a scaffold to target the receptors to clathrin-coated pits and subsequent endosomes (Ferguson, 2001; Gainetdinov et al., 2004). There are four known arrestins, although only two are expressed outside of the visual system (Lefkowitz and Shenoy, 2005). These non-visual arrestins are referred to as arrestin2 and arrestin3, also known as β-arrestin1 and β-arrestin2, respectively. A major area of inquiry has been directed at establishing receptor specificity of the non-visual arrestins and whether or not there is redundancy of function in these molecules. Initial investigations by Oakley et al. (Oakley et al., 2000 and 2001) suggested that there are two classes of GPCRs: class A receptors, which bind arrestin3 with greater affinity than arrestin2, and class B receptors which bind both arrestin2 and arrestin3 with high affinity. Lefkowitz and colleagues have also shown using cells derived from arrestin2 or arrestin3 knockout mice, that internalization of different GPCRs can indeed be selectively mediated by either arrestin2 or arrestin3 (Kohout et al., 2001).
The D2 dopamine receptor (DAR) is an important drug target in neuropsychiatry and understanding its regulation is critical to the development of improved therapies involving the modulation of D2-mediated signaling. While it is known that agonist-induced internalization of the D2 receptor is mediated by arrestins (Ito et al., 1999; Kim et al., 2001), the arrestin specificity of this process is not known with certainty. In heterologous expression systems, the D2 receptor appears to interact equally well with either arresin2 or arrestin3 (Macey et al., 2004, Namkung and Sibley, unpublished observations) suggesting that it might be a class B receptor. In contrast, Macey et al. (2004) have suggested that arrestin2 selectively mediates D2 DAR internalization in neostriatal neurons in culture. Conversely, Caron and colleagues have suggested that the D2 receptor selectively associates with arrestin3 in the brain (Beaulieu et al., 2005). In the current study, we have further investigated the arrestin selectivity of agonist-induced D2 DAR internalization using arrestin3 knockout mice and conclusively find that arrestin3 is required for D2 receptor internalization.