LETTERS BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module Daeho Park 1,2 , Annie-Carole Tosello-Trampont 2 , Michael R. Elliott 2 , Mingjian Lu 3 {, Lisa B. Haney 2 , Zhong Ma 2 , Alexander L. Klibanov 4 , James W. Mandell 5 & Kodi S. Ravichandran 2,3 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 sig- nalling 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 brain- specific 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 recog- nition 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 engulf- ment of apoptotic cells. Last, decreased BAI1 expression or inter- ference 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. Previous studies revealed two ‘functional’ regions within ELMO1 and its Caenorhabditis elegans homologue CED-12 during phagocyt- osis 5,14–17 . The amino-terminal 558 amino-acid residues (N-term) were necessary for targeting of the ELMO–Dock180 complex to the membrane 14,17 , whereas the carboxy-terminal 196 residues (C-term) were necessary for binding Dock180 and for optimal Rac activa- tion 15,16 . Because the receptor(s) upstream of ELMO1 during engulf- ment were not known, we performed a yeast two-hybrid screen, with N-term as bait. After screening more than 1.1 3 10 7 colonies from a mouse embryo library, followed by several subscreens for specificity, we identified a single membrane protein, BAI1. BAI1 belongs to subgroup VII of the adhesion-type G-protein- coupled receptor (GPCR) family 7–9 , with extended extracellular ter- mini containing multiple domains and motifs that are thought to function in cell–cell or cell–matrix interactions 9 . BAI1 (1,584 residues) has an 943-residue extracellular region, a seven-transmembrane ‘heptahelical body’ and a 392-residue cytoplasmic tail 7,8 (Fig. 1a). bai1 was initially cloned as a p53-regulated message in the brain 7 and received its name because an extracellular fragment inhibited neovascularization in experimental angiogenesis 7 . However, no physiological ligands for BAI1 have been reported. The BAI1 fragment isolated in the two-hybrid screen (residues 1431–1582) was part of its cytoplasmic tail. Yeast transformants expressing BAI1 1431–1582 and either N-term or full-length ELMO1 (but not C-term) were able to grow under selective conditions (Fig. 1b). Binding of N-term to the cytoplasmic tail of BAI1 or to full-length BAI1 was also confirmed in mammalian cells (Supplementary Fig. 2, and data not shown). Publicly available gene expression databases indicate the expres- sion of bai1 outside the brain, and microarray analyses also reported bai1 expression in primary human monocytes and macrophages 18 . We detected bai1 mRNA and BAI1 protein at different levels in macrophage cell lines (J774 and RAW264.7) and primary tissues such as bone marrow and spleen (Fig. 1c and Supplementary Fig. 1). As reported previously 7,8 , endogenous BAI1 migrated at the predicted 1 Department of Cell Biology, 2 Carter Immunology Center, 3 Department of Microbiology, 4 Cardiovascular Division and 5 Department of Pathology, University of Virginia, Charlottesville, Virginia 22908, USA. {Present address: Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. a mBAI1 clone(1431–1582) d BAI1 Erk2 250 } c IP IP IgG E1 kDa kDa kDa IP Flag Flag–BAI1 – + + ELMO1 – – + ELMO1 Flag ELMO1 TCL BAI1 TCL ELMO1 BAI1 e Full ELMO1 N-term ELMO1 C-term ELMO1 ELMO1 mBAI1 N-term mBAI1 C-term mBAI1 – mBAI1 pGBT pVP16 Selective Non-selective b PEC Astro Spln BM J774 BAI1 150 37 250 150 75 75 250 150 25 75 1 727 727 1 558 532 Figure 1 | Identification of BAI1 as an ELMO1-interacting protein. a, Diagram of BAI1. RGD, integrin-binding motif; HomR, hormone receptor; GPS, GPCR proteolytic site; 7TMR, seven-transmembrane receptor. b, A BAI1 clone was tested for interaction with the indicated ELMO1 constructs for growth on selective plates at tenfold dilutions. c, Immunoblotting for BAI1 expression shows a 160–170-kDa lower band (often a doublet) and a 220-kDa upper band 7,8 . BM, bone marrow; Spln, spleen; Astro, astrocytes; PEC, peritoneal exudate cells. d, Flag–BAI1 was transfected into LR73 cells, and simultaneous precipitation of endogenous ELMO1 (or transfected ELMO1; lane 3) was determined by anti-ELMO1 immunoblotting. IP, immunoprecipitation. TCL, total cell lysates. e, Mouse brain lysates were immunoprecipitated with ELMO1 antibody and simultaneous precipitation of endogenous BAI1 (arrowheads), assessed by immunoblotting. E1, ELMO1. doi:10.1038/nature06329 1 Nature ©2007 Publishing Group