Preferential Interaction between the Dopamine D2 Receptor and Arrestin2 in Neostriatal Neurons

Abstract

Dopamine D2 receptor interactions with arrestins and arrestindependent internalization have been characterized using heterologously expressed D2 receptor and arrestins. The purpose of this study was to investigate D2 receptor interaction with endogenous arrestins. Arrestin2 and arrestin3 in striatal homogenates bound to the third cytoplasmic loop of the D2 receptor, and purified arrestin2 and arrestin3 bound to the second and third loops and C terminus of the D2 receptor, in a glutathione S-transferase pull-down assay. In NS20Y neuroblastoma cells expressing an enhanced green-fluorescent protein-tagged D2 receptor (D2-EGFP), 2-h D2 agonist stimulation enhanced the colocalization of D2-EGFP with endogenous arrestin2 and arrestin3. These results suggest that the D2 receptor has the intrinsic ability to bind both nonvisual arrestins.Agonist treatment of D2-EGFP NS20Y cells induced D2 receptor internalization (36 -46%) that was maximal within 20 min, but that was prevented by small interfering RNA-induced depletion of arrestin2 and arrestin3. In neostriatal neurons, 2-h agonist treatment selectively increased the colocalization of the endogenous D2 receptor with arrestin2, whereas receptor colocalization with arrestin3 was reduced. Agonist stimulation caused translocation of arrestin2, but not arrestin3, to the membrane in neurons and selectively enhanced the coimmunoprecipitation of the D2 receptor and arrestin2. All three measures of receptor/arrestin interaction (colocalization, translocation, and coprecipitation) demonstrated selective agonist-induced interaction between the D2 receptor and arrestin2 in neurons.Receptor desensitization is a phenomenon in which receptor responsiveness decreases after continued or repeated stimulation with an agonist. After termination of agonist stimulation, desensitization is followed by resensitization, the reinstatement of the ability to respond to ligands (Krupnick and Benovic, 1998). For G protein-coupled receptors (GPCRs), trafficking of the receptor through various subcellular compartments is an important part of desensitization and resensitization. A model of GPCR desensitization, best characterized for the ␤ 2 -adrenergic receptor, has been developed in which the agonist-activated GPCR is phosphorylated by GPCR kinases (GRKs) or second-messenger-dependent kinases such as protein kinase A (Krupnick and Benovic, 1998). Phosphorylation by GRKs enhances the interaction of the GPCR with additional proteins, termed arrestins. Binding of arrestin causes rapid desensitization of the receptor by inhibiting receptor binding to G proteins and also targets the receptor to clathrin-coated pits for internalization and either degradation or resensitization (Pippig et al., 1995;Tsao et al., 2001). Arrestin can also act as a scaffolding protein, promoting the stable association of signaling proteins with the receptor (Luttrell et al., 2001).The rate of GPCR resensitization depends on the stability of the receptor/arrestin complex. Receptors that dissociate from arrestin near the...

The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

“…Thus, all reported affinity measurements are in good agreement with the rest of the data, clearly indicating the multi-site nature of the arrestin-receptor interaction. Qualitative studies of direct arrestin binding to various elements of different receptors have implicated i3 of the m2 and m3 muscarinic, the α2A-adrenergic receptors (Wu et al, 1997), and the 5-HT2A receptor (Gelber et al, 1999), i3 and the C-terminus of delta-opioid and the C-terminus of kappa-opioid receptors (Cen et al, 2001b), as well as i2, i3, and the C-terminus of dopamine D2 (Macey et al, 2004) and D1 receptors (Macey et al, 2005). Where the binding of a combination of elements was tested (e.g., i3+C-terminus of the delta-opioid receptor (Cen et al, 2001b), it was found to be additive, suggesting that these elements bind to different sites on arrestin.…”

Identification of Two Functionally Distinct Endosomal Recycling Pathways for Dopamine D2Receptor

“…For this reason, most antipsychotics currently in use are DRD2 antagonists. The function of DRD2 is determined by its levels on the plasma membrane (PM) (Lin et al, 2001Binda et al, 2002;Free et al, 2007;Kim et al, 2008a,b;Tirotta et al, 2008;Genedani et al, 2010), and DRD2 endocytosis plays an important role in its desensitization following DA stimulation (Kim et al, 2001;Jeanneteau et al, 2004;Kabbani et al, 2004;Macey et al, 2004;Namkung and Sibley, 2004;Sugiura et al, 2004;Bartlett et al, 2005;Genedani et al, 2005;Torvinen et al, 2005;Paspalas et al, 2006;Iizuka et al, 2007;Kim, 2008;Xiao et al, 2009;Celver et al, 2010;Shimokawa et al, 2010). However, the fate of DRD2 following DA-induced endocytosis remains controversial.…”

Endosomal location of dopamine receptors in neuronal cell cytoplasm

“…It is interesting to note that the dopamine transporter protein, which regulates dopamine receptor activation, is known to be internalized into endosomes by a clathrindependent mechanism (Sorkina et al Gulledge and Jaffe 1998). Unlike Ntera-2 cells in culture, post-mortem rat brain is exposed to very high levels of released dopamine within a few minutes of death (Vulto et al 1988) and agonistinduced internalization of dopamine receptors can also be a very rapid process (Vickery and von Zastrow 1999; Macey et al 2004). It is possible that receptors have been internalized as a result of this process and this may partly explain why their endosomal location has not been described previously.…”