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Lipid Raft Residency is Required for Renal Dopamine D 1 Receptor Activity
Author(s) -
Tiu Andrew Chua,
Asico Laureano D,
Zheng Xiaoxu,
Jose Pedro A,
Villar Van Anthony M
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1026.15
Subject(s) - lipid raft , g protein coupled receptor , palmitoylation , chemistry , caveolae , caveolin , receptor , raft , biochemistry , cysteine , microbiology and biotechnology , signal transduction , biology , organic chemistry , copolymer , enzyme , polymer
Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol and sphingolipids that serve as a platform for an effective G protein‐coupled receptor (GPCR) signaling. The renal dopamine D 1 receptor (D 1 R) partitions to the LR in human renal proximal tubule cells and interacts with the LR protein marker caveolin‐1. We tested the hypothesis that the LR is crucial for the organization and signal transduction of the renal D 1 R. Palmitoylation, a process that involves the formation of thioesters between palmitate and cysteine residues which then serve as membrane anchors, promotes LR partitioning of a few GPCRs. The human D 1 R has palmitoylation sites at two cysteine residues at positions 347 & 351. Thus, we mutated and converted the cysteine residues to alanine (347C>A & 351C>A) and evaluated their distribution and activity in human renal proximal tubule cells. Through biotinylation and sucrose gradient ultracentrifugation experiments, we found that the mutant D 1 R 347A and 351A still targeted to the plasma membrane but failed to localize in the LR, unlike the endogenous wild‐type D 1 R which partitioned to lipid and non‐lipid raft microdomains, its normal plasma membrane distribution. A di‐leucine mutant (344L>G & 345L>A), used as a control, was observed to mis‐ target the plasma membrane. The plasma membrane distribution of the mutant and endogenous wild‐type D 1 R was confirmed via confocal microscopy. The cAMP response to the D 1 R/D 5 R agonist fenoldopam was blunted in D 1 R 347A and 351A mutants, reflecting the requirement for LR residency for normal renal D 1 R function. We next evaluated the importance of LR in the kidneys of C57Bl/6J mice by infusing methyl‐β‐cyclodextrin (β‐MCD; a LR disruptor by depletion of LR cholesterol) subcapsularly for 7 days into the kidneys of C57Bl/6J mice; methyl‐ α‐cyclodextrin (α‐MCD) and vehicle were used as negative controls. We found that chronic β‐MCD treatment increased the systolic blood pressure (118±12 mm Hg, measured under anesthesia), similar to that observed in Drd1 knockout mice. Treatment with α‐MCD and vehicle did not change the blood pressure (98±1 mm Hg and 98±1 mm Hg, respectively) of C57Bl/6J mice. Confocal microscopic examination of the chronically treated kidneys revealed the disappearance of the LR without affecting the brush border architecture, and the redistribution of the D 1 R and its regulatory proteins (e.g., GRK4 and SNX19) from the brush border to the cytoplasm, indicating the importance of the LR in the proper deployment and activity of renal D 1 R. Our data underline the crucial role of D 1 R residency in LR membrane microdomains for its full activity. Support or Funding Information The work was funded by grants from the US National Institutes of Health, P01HL074940, P01HL068686, R01HL092196, R37HL023081, R01DK039308, and DK090918.