Protein kinase Cβ is a modulator of the dopamine D2 autoreceptor‐activated trafficking of the dopamine transporter

Abstract

The strength and duration of extracellular dopamine concentrations are regulated by the presynaptic dopamine transporter (DAT) and dopamine D2 autoreceptors (D2autoRs). There is a functional interaction between these two proteins. Activation of D2autoRs increases DAT trafficking to the surface whereas disruption of this interaction compromises activities of both proteins and alters dopaminergic transmission. Previously we reported that DAT expression and activity are subject to modulation by protein kinase Cβ (PKCβ). Here, we further demonstrate that PKCβ is integral for the interaction between DAT and D2autoR. Inhibition or absence of PKCβ abolished the communication between DAT and D2autoR. In mouse striatal synaptosomes and transfected N2A cells, the D2autoR-stimulated membrane insertion of DAT was abolished by PKCβ inhibition. Moreover, D2autoR-stimulated DAT trafficking is mediated by a PKCβ-ERK (extracellular signal-regulated kinase) signaling cascade where PKCβ is upstream of ERK. The increased surface DAT expression upon D2autoR activation resulted from enhanced DAT recycling as opposed to reduced internalization. Further, PKCβ promoted accelerated DAT recycling. Our study demonstrates that PKCβ critically regulates D2autoR-activated DAT trafficking and dopaminergic signaling. PKCβ is a potential drug target for correcting abnormal extracellular dopamine levels in diseases such as drug addiction and schizophrenia.

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…However, further work exploring PKC isoform specificity for DAT regulation points to a greater complexity of enzymes and actions. Thus, studies conducted with the PKCb specific inhibitor LY379196 [8((dimethylamino)methyl)-6,7,8,9,10,11-hexahydro-5,21:12,17-dimetheneo-18H-dibenzo(i,o) pyrrolo (3,4-1)(1,8)diazacyclohexandecine-18,10(19H) dione] as well as kinase knockout mice support a role for PKCb in DAT regulation, although distinct from that observed in studies with phorbol ester stimulation, Chen et al (2013) observed that treatment of mouse striatal synaptosomes with LY279196 blocked the ability of the D2 agonist quinpirole to increase DAT surface levels and DA uptake, suggesting a role for PKCb in supporting elevated surface expression. LY279196 also blocked quinpirole-induced increases in p-extracellular signal-related kinase 1/2 (ERK1/2).…”

“…As noted earlier, progress in the study of PKC mechanisms that regulate DAT will likely depend on the identification of receptor-mediated signaling pathways that rely on PKC signaling. As noted above, PKCb isoforms have been reported to play a role in D2 receptor-mediated trafficking of DAT (Chen et al, 2013), possibly connected to DAT via ERK1/2 (see below). Additionally, Page et al (2001) reported that an mGluR agonist decreases DA uptake in rat striatal synaptosomes, an effect that can be blocked by the PKC inhibitor …”

“…Considering work by Chen et al (2013) discussed above seems to indicate that PKCb may act to elevate DAT surface levels, it is possible that phosphorylation of DAT by PKC activation actually supports DAT surface expression, despite the net decrease in surface levels upon nonspecific PKC activation by b-PMA. In support of this, b-PMA treatment increases phosphorylation of Thr53, a residue whose phosphorylation seems to promote DAT surface expression (Foster et al, 2012).…”

“…In support of this, b-PMA treatment increases phosphorylation of Thr53, a residue whose phosphorylation seems to promote DAT surface expression (Foster et al, 2012). Based on work from Chen et al (2013) that showed that PKCb appears to function upstream of ERK1/2, which are strong candidates for targeting Thr53 (Gorentla et al, 2009), it is possible that PKCb may act through ERK1/2 to increase Thr53 phosphorylation and therefore positively regulate DAT, whereas other PKC isoforms act to downregulate DAT activity, potentially through direct phosphorylation of the transporter or other interacting proteins. Caution is needed with respect to interpretations from heterologous expression systems; however, because these contexts may not accurately reflect the endogenous environment in which DAT interacts with PKC signaling pathways, including DAT-associated proteins.…”

Temporal Pattern of Cocaine Intake Determines Tolerance vs Sensitization of Cocaine Effects at the Dopamine Transporter

“…Therefore, it is possible that changes in the interaction of the DAT protein with downstream effectors responsible for these post-translational modifications are altered following self-administration in a way that changes the ability of ligands to bind to the DAT and inhibit the clearance of DA. In addition, the DAT has been shown to form oligomer complexes (Hastrup et al, 2003) and be modulated by receptors such as the D2 autoreceptor (Chen et al, 2013), which are two additional mechanisms that could have a role in the observed changes in cocaine potency.…”