Abstract 101: Association Of Snx19 With Flotillin-1 And Caveolin-1 In Its Lipid Raft Localization And Regulation Of D 1 R Endocytosis And Blood Pressure

Abstract

Sorting nexin 19 (SNX19), a member of the SNX-PXA-RGS-PXC subfamily of sorting nexins, plays a vital role in lipid raft microdomain localization, but its association with flotillin-1 and caveolin-1, two of the most important lipid raft components, and in particular its linkage to the endocytosis and trafficking of dopamine D1 receptor (D 1 R) have not been determined in the renal proximal tubule (RPT). In the present study, we found that SNX19 was localized with lipid rafts in human and mouse RPT cells (RPTCs), which regulated D 1 R endocytosis. In wild type (WT) mouse RPTCs, SNX19 co-localized with flotillin-1 and caveolin-1, which was increased by treatment with fenoldopam (Fen, 25 nM, 30 min, n=4), a D1-like receptor agonist but not by vehicle (Veh). The targeting of SNX19 in lipid rafts and its colocalization with flotillin-1 and caveolin-1 were attenuated by deletion of a flotillin binding domain ( EEGPGTETETGLPVS ) (ΔFlot1), a caveolin-1 binding domain ( YHTVNRRYREF ) (ΔCav1), or both domains (ΔFlot1/Cav) in mouse RPTCs. The increase in intracellular cAMP production caused by Fen was also impaired or eliminated by the deletion of either flotillin-1 or caveolin-1 or both binding domains (WT-Veh: 100±16.6%, n=3, WT-Fen: 195.3±10.6%, n=4; ΔFlot1-Veh: 93.1±11.8%, n=4, ΔFlot1-Fen: 144.8±9.4%, n=3; ΔCav1-Veh: 90.9±16.3%, n=3, ΔCav1-Fen: 106.9±11.7%, n=4; ΔFlot/Cav-Veh: 63.8±10.3%, n=4, ΔFlot/Cav-Fen: 86.5±8.1%, n=4). Fen (25 nM, 30 min, n=3) decreased the colocalization of SNX19 with microtubules without an effect on actin filaments, which was abrogated by nocodazole (10 μM, 1hr), a microtubule depolymerization inhibitor. Furthermore, the renal-selective silencing of SNX19 in C57BL/6 mice (n=3) by the renal subcapsular infusion of specific SNX19 siRNA decreased renal sodium excretion and increased systolic blood pressure (Mock siRNA: 96.8±0.88 mmHg, n=3; SNX19 siRNA: 117.7±5.77 mmHg, n=3). In conclusion, SNX19 contains both flotillin-1 and caveolin-1 binding domains and its binding with flotillin-1 and caveolin-1 are important in SNX19-mediated D 1 R signaling, renal sodium transport, and blood pressure regulation.