Alterations in D2 dopamine receptor internalization in the presence of the Na+/K+‐ATPase

Abstract

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.Support contributed by the NINDS intramural program.