RanBPM protein acts as a negative regulator of BLT2 receptor to attenuate BLT2-mediated cell motility.
BLT2, a low affinity receptor for leukotriene B4 (LTB4), is a member of the G protein-coupled receptor family and is involved in many signal transduction pathways associated with various cellular phenotypes, including chemotactic motility. However, the regulatory mechanism for BLT2 has not yet been demonstrated. To understand the regulatory mechanism of BLT2, we screened and identified the proteins that bind to BLT2. Using a yeast two-hybrid assay with the BLT2 C-terminal domain as bait, we found that RanBPM, a previously proposed scaffold protein, interacts with BLT2. We demonstrated the specific interaction between BLT2 and RanBPM by GST pulldown assay and co-immunoprecipitation assay. To elucidate the biological function of the RanBPM-BLT2 interaction, we evaluated the effects of RanBPM overexpression or knockdown. We found that BLT2-mediated motility was severely attenuated by RanBPM overexpression and that knockdown of endogenous RanBPM by shRNA strongly promoted BLT2-mediated motility, suggesting a negative regulatory function of RanBPM toward BLT2. Furthermore, we observed that the addition of BLT2 ligands caused the dissociation of BLT2 and RanBPM, thus releasing the negative regulatory effect of RanBPM. Finally, we propose that Akt-induced BLT2 phosphorylation at residue Thr(355), which occurs after the addition of BLT2 ligands, is a potential mechanism by which BLT2 dissociates from RanBPM, resulting in stimulation of BLT2 signaling. Taken together, our results suggest that RanBPM acts as a negative regulator of BLT2 signaling to attenuate BLT2-mediated cell motility.
From the School of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul 136-701.
Jun-Dong
JD
Wei
Joo-Young
JY
Kim
Ae-Kyoung
AK
Kim
Sung Key
SK
Jang
Jae-Hong
JH
Kim
D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002633:Chemotaxis; D006224:Cricetinae; D003412:Cricetulus; D003598:Cytoskeletal Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation; D015289:Leukotrienes / Q000378:metabolism; D008024:Ligands; D009687:Nuclear Proteins / Q000502:physiology*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D018102:Receptors, Leukotriene B4 / Q000502:physiology*; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; C485878:LTB4R2 protein, human; D015289:Leukotrienes; D008024:Ligands; D009687:Nuclear Proteins; C116005:Ran binding protein 9; D017382:Reactive Oxygen Species; D043562:Receptors, G-Protein-Coupled; D018102:Receptors, Leukotriene B4; D013912:Threonine
BLT2; Chemotaxis; G Protein-coupled Receptors (GPCR); GPCR; Leukotriene; RanBPM; Reactive Oxygen Species; Reactive Oxygen Species (ROS); Scaffold Proteins