Ethanol Regulation of D1 Dopamine Receptor Signaling is Mediated by Protein Kinase C in an Isozyme-Specific Manner

Abstract

Ethanol consumption potentiates dopaminergic signaling that is partially mediated by the D 1 dopamine receptor; however, the mechanism(s) underlying ethanol-dependent modulation of D 1 signaling is unclear. We now show that ethanol treatment of D 1 receptor-expressing cells decreases D 1 receptor phosphorylation and concurrently potentiates dopamine-stimulated cAMP accumulation. Protein kinase C (PKC) inhibitors mimic the effects of ethanol on D 1 receptor phosphorylation and dopamine-stimulated cAMP levels in a manner that is non-additive with ethanol treatment. Ethanol was also found to modulate specific PKC activities as demonstrated using in vitro kinase assays where ethanol treatment attenuated the activities of lipid-stimulated PKCg and PKCd in membrane fractions, but did not affect the activities of PKCa, PKCb 1 , or PKCe. Importantly, ethanol treatment potentiated D 1 receptormediated DARPP-32 phosphorylation in rat striatal slices, supporting the notion that ethanol enhances D 1 receptor signaling in vivo. These findings suggest that ethanol inhibits the activities of specific PKC isozymes, resulting in decreased D 1 receptor phosphorylation and enhanced dopaminergic signaling.

Identification of RanBP 9/10 as Interacting Partners for Protein Kinase C (PKC) γ/δ and the D1Dopamine Receptor: Regulation of PKC-Mediated Receptor Phosphorylation

“…Moreover, we found that EtOH decreases constitutive PKC phosphorylation of the D 1 receptor with a concomitant potentiation of dopaminergic signaling (Rex et al, 2008). It is noteworthy that EtOH was found to directly inhibit the enzymatic activities of PKC␥ and PKC␦, but only when they were isolated from the plasma membrane fraction, an effect that was not observed for other PKC isozymes that were tested (Rex et al, 2008). The molecular mechanisms underlying the EtOH-mediated inhibition of membrane-associated PKC␥ and PKC␦ kinase activities and how they target the D 1 receptor are at present unclear.…”

“…We determined recently that PKC constitutively phosphorylates the D 1 receptor and that this negatively regulates dopaminergic signaling (Rex et al, 2008). Moreover, we found that EtOH decreases constitutive PKC phosphorylation of the D 1 receptor with a concomitant potentiation of dopaminergic signaling (Rex et al, 2008). It is noteworthy that EtOH was found to directly inhibit the enzymatic activities of PKC␥ and PKC␦, but only when they were isolated from the plasma membrane fraction, an effect that was not observed for other PKC isozymes that were tested (Rex et al, 2008).…”

“…In contrast to GRKs and PKA, very little is known about the regulation of the D 1 receptor by PKC phosphorylation. We determined recently that PKC constitutively phosphorylates the D 1 receptor and that this negatively regulates dopaminergic signaling (Rex et al, 2008). Moreover, we found that EtOH decreases constitutive PKC phosphorylation of the D 1 receptor with a concomitant potentiation of dopaminergic signaling (Rex et al, 2008).…”

“…Each pellet was resuspended with 70 l of PBS. Kinase assays were performed using a PKC Assay Kit (Millipore, Billerica, MA) as described previously (Rex et al, 2008). In brief, the kinase activity in 10 l of each sample was assayed by measuring the transfer of 32 P i from [␥-32 P]ATP to a specific substrate peptide (Millipore).…”

Protein Kinase D1-Dependent Phosphorylation of Dopamine D1 Receptor Regulates Cocaine-Induced Behavioral Responses

“…PKA can phosphorylate T268 in the third intracellular loop and T380 in the carboxyl terminus of D1R, which regulates either the rate of desensitization or intracellular trafficking of the receptor once internalized (Jiang and Sibley, 1999;Mason et al, 2002). Constitutive or heterologous PKC (including PKCa, bI, g, d, and e) phosphorylates D1R and dampens DA activation of the receptor, thus attenuating D1R-mediated signaling (Gardner et al, 2001;Rankin and Sibley, 2010;Rex et al, 2008;Rex et al, 2010). Here we provided both in vitro and in vivo evidence for the involvement of PKD1 in D1R phosphorylation.…”