Dopamine D1 receptor interaction with arrestin3 in neostriatal neurons

Abstract

Dopamine D1 receptor interactions with arrestins have been characterized using heterologously expressed D1 receptor and arrestins. The purpose of this study was to investigate the interaction of the endogenous D1 receptor with endogenous arrestin2 and 3 in neostriatal neurons. Endogenous arrestin2 and 3 in striatal homogenates bound to the C-terminus of the D1 receptor in a glutathione-S-transferase (GST) pulldown assay, with arrestin3 binding more strongly. The D1 C-terminus and, to a lesser extent, the third cytoplasmic loop also bound purified arrestin2 and 3. In neostriatal neurons, 2, 5, and 20 min agonist treatment increased the colocalization of the D1 receptor and arrestin3 immunoreactivity without altering the colocalization of the D1 receptor and arrestin2. Further, agonist treatment for 5 and 20 min caused translocation of arrestin3, but not arrestin2, to the membrane. The binding of arrestin3, but not arrestin2, to the D1 receptor was increased as assessed by coimmunoprecipitation after agonist treatment for 5 and 20 min. Agonist treatment of neurons induced D1 receptor internalization (35-45%) that was maximal within 2-5 min, a time-course similar to that of the increase in colocalization of the D1 receptor with arrestin3. These data indicate that the D1 receptor preferentially interacts with arrestin3 in neostriatal neurons. Keywords: arrestin, D1 dopamine receptor, G proteincoupled receptor, receptor internalization. Arrestin plays a critical role in the desensitization of G protein-coupled receptors (GPCRs) by binding to GPCR kinase-phosphorylated receptors, resulting in dissociation of the G protein from the receptor. Arrestin then targets the receptor to clathrin-coated pits for internalization and either degradation or recycling back to the membrane for resensitization (Pippig et al. 1995;Tsao et al. 2001). Arrestin can also promote the stable interaction of signaling proteins with the GPCR (Luttrell et al. 2001).Based on their rates of resensitization and relative affinities for arrestin2 and 3, GPCRs have been classified into two groups, class A and B. Residues within the C-terminal tail of the receptor appear to contribute to the stability of the receptorarrestin complex, which determines the rate of resensitization of the GPCR (Oakley et al. 2000). Class A receptors dissociate from arrestin relatively fast and are rapidly dephosphorylated and recycled back to the membrane. Class A receptors also have a higher affinity for arrestin3 than 2 (Shenoy and Lefkowitz 2003). Class B receptors remain associated with arrestin during internalization, which makes the recycling and dephosphorylation of the receptor occur more slowly, and have a similar affinity for arrestin2 and 3 (Shenoy and Lefkowitz 2003). The dopamine D1 receptor has been designated a class A GPCR, along with the b 2 -adrenoceptor, whereas the neurokinin NK-1 and vasopression V2 receptors are examples of class B GPCRs (Oakley et al. 2000).Desensitization and internalization of the dopamine D1 receptor have been described i...

Distinct Cellular and Subcellular Distributions of G Protein-Coupled Receptor Kinase and Arrestin Isoforms in the Striatum

“…For example, dopamine D1 and D3 and A1 adenosine receptors preferentially localized to striatonigral, or direct, MSN, whereas D2 dopamine and 2A adenosine are segregated to striatopallidal, or indirect, MSN [24]–[29], see also [30] and references therein]. In the rat striatum, the D1 dopamine receptor preferentially interacts with arrestin-3 [31], whereas the D2 receptor - with arrestin-2 [32], although there is no difference in the arrestin levels between the D1- and D2-expressing MSN. Interestingly, in vitro D2 receptor binds both purified arrestin isoforms equally well [32].…”

Modulating μ-Opioid Receptor Phosphorylation Switches Agonist-dependent Signaling as Reflected in PKCϵ Activation and Dendritic Spine Stability

“…On the one hand, the most critical regions on GPCRs for G protein coupling are the second intracellular loop (IL), the N terminus, and the C terminus of IL3 (30). On the other hand, in addition to the C terminus facilitating the binding of ␤-arrestin by mediating phosphorylation (31), the IL2, the N terminus, and the C terminus of the IL3 are also essential for the binding of ␤-arrestin (32)(33)(34)(35)(36). Due to this overlap of binding sites, receptor phosphorylation can be suggested as regulating the competition between G proteins and ␤-arrestin for the binding site on GPCRs (7).…”

Manipulation of Very Few Receptor Discriminator Residues Greatly Enhances Receptor Specificity of Non-visual Arrestins

“…tor-specific Mutants-Both non-visual arrestins are fairly promiscuous, capable of interacting with many of the GPCRs tested so far (14,17,20,22,25,38,39). However, arrestin-3 appears to have a wider receptor repertoire and binds several GPCRs with higher affinity than arrestin-2 (17,40,41).…”