Characterization of D 1 and D 2 dopamine receptor interactions with the Na + /K + ‐ATPase

Abstract

It is evident that dopamine receptors (DARs) do not exist as singular independent units within the synaptic membrane, but rather are part of a large macromolecular complex of interacting proteins. These interacting proteins may be transmembrane or cytosolic, and can play a variety of roles in receptor function. We have employed a co-immunoprecipitation assay for DARs (from mouse brain and transfected cell lines), coupled with mass spectrometry (MS) sequencing to identify these interacting partners. Interacting proteins identified through these experiments include the α1 subunit of sodium-potassium ATPase (NKA α1). The NKA is a transmembrane protein consisting of both an α and β subunit, with α1 being the predominant α isoform. Studies indicate that the α subunit is primarily responsible for the transport of Na+ and K+ ions across the plasma membrane. Co-immunoprecipitation of DARs followed by Western analysis has confirmed specific D1 and D2 DAR interactions with NKA α1. To determine the impact of NKA on the DARs, biological assays were conducted in the presence of enhanced levels of NKA. Over-expression of NKA DNA yields a dramatic decrease in total D2 DAR expression, but has little impact on the expression of D1 DAR; a functional consequence of this diminished receptor level is also evident. These preliminary data indicate that NKA interactions are distinct across DAR isoforms. Experiments are underway to determine the impact of DAR expression on NKA function and activity, and to ascertain whether the observed effects are mediated by direct or indirect interactions. Support Contributed By: NIH and NIGMS PRAT Program