In FY2009, we continued our investigation of the Na+, K+-ATPase (NKA, sodium pump) as an interacting protein for the D1 and D2 receptors. Using a co-immunoprecipitation assay for dopamine receptors (DARs) coupled with mass spectrometry (MS) sequencing, we previously identified the Na+/K+-ATPase (Na+ pump, NKA) as a member of both the D1 and D2 DAR signalplexes. Using fluorescent-labeled D2 DAR and NKAα1 constructs in confocal microscopy experiments, co-localization of the two proteins was observed at the plasma membrane. Dopamine stimulation of RFP-tagged D2 DAR-expressing cells resulted in marked DAR internalization twenty minutes after drug treatment. Interestingly, when GFP-tagged NKAα1 was co-expressed, the D2 DAR internalization twenty minutes after dopamine treatment was diminished. To achieve a more quantitative assessment of receptor expression, cell-surface binding assays were performed in the presence and absence of over-expressed NKA. These assays revealed a 45% decrease in cell-surface D2 DAR number twenty minutes after dopamine treatment, and a 30% decrease in receptor number when the NKA was also present. Longer dopamine incubations enhanced D2 DAR internalization, but co-expression of NKA prohibited complete internalization as compared to D2 DAR alone. Current studies are underway to determine the impact of the DAR-NKA complex on receptor recycling, and the role of NKA in lipid rafts on receptor activation and internalization. Our laboratory and others have previously shown that the D2 dopamine receptor (DAR) is internalized by agonist stimulation and either recycled back to the plasma membrane or sorted to lysosomes for degradation. However, the molecular components involved in these processes are only beginning to be characterized. In FY2009, we continued our investigation of sorting nexin-25 (SNX25) as a novel DAR interacting protein using co-immunoprecipitation-coupled mass spectrometry-based sequencing. Mammalian SNXs have been suggested to be involved in the internalization, intracellular trafficking, and endosomal recycling or sorting of membrane-bound cargo. In addition, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) has been found to play a distinct role in promoting the rapid recycling of internalized receptors back to the plasma membrane. In FY2009, we investigated the role of SNX25 and Hrs on D2 DAR internalization and recycling in HEK293T cells. Receptor internalization and recycling was initially assessed by quantifying cell surface receptors using intact cell 3H-sulpiride binding assays. Treatment with dopamine for 1 hr resulted in a 25% loss of cell surface receptor binding (internalization), which recovered to 93% of control after 1hr of dopamine removal (recycling). Over-expression of either SNX25 or Hrs had no effect on the total surface receptor expression, but decreased the amount of agonist-induced receptor internalization to 17% and 11%, respectively. Surprisingly, D2 DAR recycling was severely impaired in Hrs over-expressing cells. However, SNX25 over-expression resulted in only a slight decrease in receptor recycling. Taken together, over-expression of either SNX25 or Hrs perturbed both internalization and recycling of the D2 DAR. These data suggest that SNX25 and Hrs may play a role in D2 DAR trafficking through membrane compartments and that common machinery exists linking receptor internalization (endocytosis) and recycling. In FY 2009, we also continued our investigations of how protein kinase C regulation of the D1 receptor is regulated by alchohol. Alcohol abuse and alcoholism are of clinical and economic significance worldwide. The effectiveness of the current pharmacotherapies for alcoholism is limited and is partially due to lack of mechanistic data at the molecular level. Aberrant protein kinase C (PKC) signaling is associated with many diseases that include alcoholism and addiction. The PKC family of serine/threonine kinases is comprised of 12 isozymes that differ with respect to their structure, expression, and mechanisms of regulation. In many instances, only a subset of the PKC isozymes is associated with a specific disease state. We are particularly interested in the interplay between PKC and the D1 dopamine receptor in neuropsychiatric disorders such as alcohol abuse and alcoholism. We've recently found that PKC constitutively phosphorylates the D1 receptor and that this negatively regulates dopaminergic signaling. Moreover, we've shown that ethanol (EtOH) treatment decreases constitutive PKC phosphorylation of the D1 receptor with a concomitant potentiation of dopaminergic signaling. Importantly, EtOH was found to directly inhibit the lipid-activated enzymatic activities of PKCγand PKCδ, but only when they were isolated from the membrane fraction - a response that was not observed for other PKC isozymes, including α, β1 or ε. The molecular mechanisms underlying the EtOH-mediated inhibition of membrane-associated PKCγand PKCδkinase activities are at present unclear. We hypothesize that EtOH may regulate the interaction between the PKC isozyme and a membrane-associated interacting protein(s) and/or target the PKC interacting protein itself. We have mow employed a PKC isozyme-specific coimmunoprecipitation approach followed by 2-D gel electrophoresis and mass spectrometry-based sequencing to identify candidate PKC isozyme-specific interacting proteins. RanBP10, a known scaffolding protein, was identified in the proteomics screen involving PKCγ. Significantly, we confirmed the association of RanBP10 with both PKCγand PKCδusing coimmunoprecipitation techniques followed by Western Blotting. Since PKCγand PKCδboth phosphorylate the D1 receptor, we postulate that RanBP10 may function as a scaffolding molecule and may also associate with the D1 receptor. This notion was supported by the coimmunoprecipitation of RanBP10 with the D1 receptor. Interestingly, our preliminary functional data suggest that RanBP10 modulates D1 receptor expression and cAMP accumulation. In summary, we have identified a putative scaffolding molecule, RanBP10 that may be a critical component that regulates crosstalk between D1 receptor and PKC isozyme-specific signaling pathways.
