Characterization of sorting nexin‐25, a D1 and D2 dopamine receptor interacting protein that regulates receptor expression and trafficking in HEK293 cells

Abstract

Dopamine receptors (DARs) do not exist as independent units within the synaptic membrane, but are part of large macromolecular complexes of interacting proteins. Our current studies employ co‐IP assays for DARs coupled with mass spectrometry‐based sequencing to identify interacting partners. One protein identified was sorting nexin‐25. Mammalian sorting nexins (SNXs) have been suggested to regulate intracellular trafficking, internalization, and endosomal recycling or sorting of membrane‐bound cargo. The physiological role of SNX25 is unknown, however, and multiple isoforms of SNX25 have been reported. Using RT‐PCR, we have found that the endogenous isoform in HEK293T cells is a 1004 residue protein containing two putative transmembrane‐spanning regions. When this SNX25 isoform is expressed as an RFP fusion protein, it is localized in distinct clusters near the plasma membrane. When SNX25 is over‐expressed with the DARs, an alteration in the cellular receptor distribution is seen using confocal microscopy. Radioligand binding and functional assays also show that the expression levels of both D1 and D2 DARs are altered. SNX25 over‐expression also appears to limit the internalization of D2 DARs after agonist treatment, but this had only a slight effect on receptor recycling. Overall, these data suggest that SNX25 regulates the trafficking of D1 and D2 DARs and is a novel member of the DAR signalplex.