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Engagement of S1P1-degradative mechanisms leads to vascular leak in mice
Myat Lin Oo, … , David K. Han, Timothy Hla
Myat Lin Oo, … , David K. Han, Timothy Hla
Published May 9, 2011
Citation Information: J Clin Invest. 2011;121(6):2290-2300. https://doi.org/10.1172/JCI45403.
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Research Article Cell biology

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

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Abstract

GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P1) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.

Authors

Myat Lin Oo, Sung-Hee Chang, Shobha Thangada, Ming-Tao Wu, Karim Rezaul, Victoria Blaho, Sun-Il Hwang, David K. Han, Timothy Hla

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Figure 4

WWP2-dependent C-terminal polyubiquitinylation and degradation of S1P1 after FTY720P treatment.

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WWP2-dependent C-terminal polyubiquitinylation and degradation of S1P1 a...
(A) S1P1-GFP, S1P1-Δ1-GFP, or S1P1-S5A-GFP cells were transfected with pcDNA3 or pcDNA3-WWP2, lysed, and subjected to IP with anti-GFP IgG and IB with ubiquitin antibody. The same membrane was stripped and reprobed with GFP antibody to examine receptor levels, and expression of WWP2 was determined. (B) Cells were treated with 100 nM FTY720P for the indicated times, and expression of S1P1, WWP2, and β-actin were examined by IB analysis. (C) HEK293 cells expressing S1P1-GFP were stably transduced with lentiviral shRNA for WWP2 (sh-WWP2) and treated with 100 nM FTY720P for the indicated times. Expression of S1P1, WWP2, and β-actin were examined by IB analysis. sh-Cont, control shRNA. (D) HUVECs were stably transduced with lentiviral particles of pCDH-WWP2 or pCDH-copGFP and treated with 100 nM FTY720P for the indicated times, after which IB analysis was done. (E) Endogenous WWP2 in HUVECs was silenced by transduction of GIPZ lentiviral shRNAmir for WWP2 or control, and expression of S1P1, WWP2, and β-actin was determined by IB analysis. The experiment was repeated at least 3 times with similar results.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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