SNX19 Interacts with Caveolin-1 and Flotillin-1 to Regulate D1R Endocytosis and Signaling

Abstract

Background: Sorting nexin 19 (SNX19) is important in the localization and trafficking of the dopamine D(1) receptor (D(1)R) to lipid raft microdomains. However, the interaction between SNX19 and the lipid raft components caveolin-1 or flotillin-1 and, in particular, their roles in the cellular endocytosis and cell membrane trafficking of the D(1)R have not been determined. Methods: Caveolin-1 and flotillin-1 motifs were analyzed by in silico analysis; colocalization was observed by confocal immunofluorescence microscopy; protein-protein interaction was determined by co-immunoprecipitation. Results: In silico analysis revealed the presence of putative caveolin-1 and flotillin-1 binding motifs within SNX19. In mouse and human renal proximal tubule cells (RPTCs), SNX19 was localized mainly in lipid rafts. In mouse RPTCs transfected with wild-type (WT) Snx19, fenoldopam (FEN), a D(1)-like receptor agonist, increased the colocalization of SNX19 with caveolin-1 and flotillin-1. FEN also increased the co-immunoprecipitation of SNX19 with caveolin-1 and flotillin-1, effects that were prevented by SCH39166, a D(1)-like receptor antagonist. The FEN-mediated increase in the residence of SNX19 in lipid rafts and the colocalization of the D(1)R with caveolin-1 and flotilin-1 were attenuated by the deletion of a caveolin-1 (YHTVNRRYREF) (?Cav1) or a flotillin-1 (EEGPGTETETGLPVS) (?Flot1) binding motif. The FEN-mediated increase in intracellular cAMP production was also impaired by the deletion of either the flotillin-1 or caveolin-1 binding motif. Nocodazole, a microtubule depolymerization inhibitor, interfered with the FEN-mediated increase in the colocalization between SNX19 and D(1)R. Conclusion: SNX19 contains caveolin-1 and flotillin-1 binding motifs, which play an important role in D(1)R endocytosis and signaling.