he low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of... more he low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated T protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR Ϫ / Ϫ MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK Ϫ cells. Untreated uPAR ϩ / ϩ MEFs demonstrated substantially increased Rac1 activation compared with uPAR Ϫ / Ϫ MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signalregulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked. *Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; GPI, glycosylphosphatidylinositol; LRF, receptor in the low density lipoprotein receptor gene family; LRP-1, low density lipoprotein receptorrelated protein; MEF, murine embryonic fibroblast; MLCK, myosin light chain kinase; PAI-1, plasminogen activator inhibitor-1; RAP, receptorassociated protein; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; VLDLr, VLDL receptor; VLK-p NA, H -D -Val-L -Leu-L -Lys-p -nitroanilide.
Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs through... more Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms 1-3 . ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses 4,5 ; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment 4-6 . However, the receptor(s) upstream of the ELMO/ Dock180/Rac module are still unknown. Here we identify brainspecific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region 7-9 and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells 10-13 , as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells.
he low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of... more he low density lipoprotein receptor-related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1-deficient MEFs demonstrated increased Rac1 activation compared with LRP-1expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated T protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR Ϫ / Ϫ MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK Ϫ cells. Untreated uPAR ϩ / ϩ MEFs demonstrated substantially increased Rac1 activation compared with uPAR Ϫ / Ϫ MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signalregulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked. *Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; GPI, glycosylphosphatidylinositol; LRF, receptor in the low density lipoprotein receptor gene family; LRP-1, low density lipoprotein receptorrelated protein; MEF, murine embryonic fibroblast; MLCK, myosin light chain kinase; PAI-1, plasminogen activator inhibitor-1; RAP, receptorassociated protein; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; VLDLr, VLDL receptor; VLK-p NA, H -D -Val-L -Leu-L -Lys-p -nitroanilide.
Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs through... more Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms 1-3 . ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses 4,5 ; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment 4-6 . However, the receptor(s) upstream of the ELMO/ Dock180/Rac module are still unknown. Here we identify brainspecific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region 7-9 and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells 10-13 , as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells.
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Papers by Zhong Ma