Dopamine D3 Receptors Are Down-regulated following Heterologous Endocytosis by a Specific Interaction with G Protein-coupled Receptor-associated Sorting Protein-1

Abstract

GPCR signaling is tightly regulated by several mechanisms, including receptor trafficking. Once activated, many GPCRs are phosphorylated by GPCR kinases, recruit arrestins, and are internalized into clathrin-coated pits via a dynamin-dependent mechanism. Indeed, such a mechanism has been reported for both the D 1 (1, 2) and the D 2 (3, 4) dopamine receptors. Furthermore, following endocytosis, D 1 and D 2 differ in their post-endocytic fate. Specifically, D 1 receptors are recycled back to the plasma membrane where they may bind ligand once again, whereas D 2 receptors are targeted to the lysosomes for degradation (5). Targeted degradation of the D 2 receptor is facilitated through an interaction of the D 2 receptor with the GPCR-associated sorting protein GASP-1 (5). GASP-1 binds to distinct GPCRs from several other families as well, including the ␦-opioid receptor (6), the CB1 receptor (7), the bradykinin B 2 receptor (8), and the virally encoded chemokine receptor US28 (9), all of which are targeted for degradation after endocytosis. Members of these families that do not bind GASP-1, as well as many other GPCRs that do not bind GASP (10), are recycled rather than degraded after endocytosis. Post-endocytic degradation of GPCRs by GASP-1 has been shown to have behavioral relevance in vivo. For example, GASP-1 knock-out mice show reduced tolerance to the cannabinoid WIN5,212-2 as a consequence of reduced CB1 down-regulation (11). In addition, GASP-1 knockout mice also show reduced sensitization to the locomotor activating effects of repeated cocaine as a consequence of reduced D 2 receptor degradation (12).Unlike the D 1 and D 2 receptors, D 3 receptors do not endocytose in response to activation by dopamine (13,14). Instead, endocytosis of the D 3 receptor appears to occur through a heterologous mechanism mediated by PKC (15), requiring both the ␤ and ␦ PKC isoforms, actin-binding motif 280 and filamin A (16). This endocytosis is dependent on dynamin, indicating a clathrin component, it but appears to be GPCR kinase-and arrestin-independent (16).Importantly, no prior study has examined the post-endocytic fate of the D 3 receptor. Here, we report that the D 3 receptor is targeted for degradation following endocytosis in response to phorbol ester activation of PKC and that it does

β-Arrestin-Dependent Dopaminergic Regulation of Calcium Channel Activity in the Axon Initial Segment

“…Interestingly, β-arrestin-dependent endocytosis of D3R occurs only if D3R is phosphorylated by PKC (Cho et al, 2007; Thompson and Whistler, 2011). Together, these observations suggest that signaling to Ca V 3 could be arrestin mediated.…”

Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

“…For example, dopamine receptor recycling is largely a CME dependent process [79][80][81][82] , and dopamine D2 receptor surface expression is regulated by CME 83 . Thus, CME directly regulates dopamine receptor numbers at the postsynaptic site, known to be an important determinant of dopamine signal strength.…”

Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

“…In our personal experience, the radioligand-binding assay is more convenient and accurate when compared with other methodologies. Receptor internalization can also be measured by fluorescence (Thompson and Whistler, 2011) or cell-surface biotinylation (Vickery and von Zastrow, 1999); however, these two methods can result in either difficultly in selectively quantifying receptors on the cell membrane and in the cytosol or the necessity to supplement with immunoprecipitation and immunoblot assays, which could introduce large variations in the measurements. When receptor sequestration accompanies conformational changes and short-distance trafficking within the plasma membrane, resulting in failure to bind hydrophilic ligands (pharmacological sequestration; Fig.…”