AGONIST ACTIVATION PROMOTES DOPAMINE D3 RECEPTOR ENDOSSOMAL TRAFFICKING AND INTERACTION WITH GADD34: PP.13.475

Abstract

Objectives: During conditions of moderate sodium excess, the peripheral dopaminergic system regulates blood pressure and water and electrolyte balance by promoting natriuresis. Dopamine exerts its effects on dopamine receptors, including the dopamine D3 receptor. The human DRD3 gene at 3q13.3 is linked to essential hypertension. Drd3 knockout mice develop a renin-dependent form of hypertension and fail to excrete a sodium load. This study aimed to demonstrate the trafficking of agonist-activated DRD3 in human proximal tubule cells and to characterize its interaction with GADD34, which is the regulatory subunit of the serine/threonine-protein phosphatase 1 (PP1) holoenzyme that recruits the catalytic subunit of PP1 (PP1c) to promote dephosphorylation. We found that PD128907 (1 μM), a DRD3 selective agonist, promotes the internalization and trafficking of the receptor along the endosome network. Design and Methods: The interaction of DRD3 with GADD34, a novel interacting protein previously identified in a yeast two-hybrid screen, was confirmed through a His pull-down between heterologously expressed DRD3-His,myc and endogenous GADD34. To further characterize the spatial and temporal dynamics of the interaction, laser scanning confocal microscopy was performed. Results: Under basal conditions, DRD3 was mainly localized at the plasma membrane and partially at the cytoplasm of human proximal tubule cells, while GADD34 was scattered throughout the cytoplasm and the plasma membrane. Colocalization of DRD3 and GADD34 was observed at the plasma membrane. Stimulation with the PD128907 for 5–15 minutes resulted in the considerable internalization of both DRD3 and GADD34 and their co-localization at the perinuclear space. Thirty minutes after agonist treatment, both DRD3 and GADD34 recycled back to the surface membrane, where minimal colocalization was observed. Conclusions: Our results indicate that agonist activation promotes DRD3 endocytosis and endosomal trafficking and indicates an important role for GADD34 in DRD3 function in human proximal tubule cells.