Ex vivo identification of protein–protein interactions involving the dopamine transporter

The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT–protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca2+/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein–protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein–protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT.

Research highlights
► Description of a method to immunoprecipitate DAT and identify potential DAT protein–protein interactions ex vivo by Western blot and LC/MS/MS.
► DAT can be immunoprecipitated from striatal synaptosomes and numerous DAT co-immunoprecipitating proteins are detected by silver-stain.
► CaMKII, PKC, and PICK1, co-immunoprecipitate with DAT.
► DAT, CaMKII, and PKC are identified from the immunoprecipitate by LC/MS/MS.