This increase was maintained on shPLSCR1-transduced THP-1 cells treated with PMA (Fig 4C). in phagosomes, our results reveal a specific role for induced PLSCR1 expression in the modulation of the phagocytic process in differentiated macrophages. Introduction Phospholipid scramblase 1 (PLSCR1) is a member of a protein family referenced as phospholipid scramblases that are conserved in all eukaryotic organisms. In human, the scramblase family is constituted of four known homologues named PLSCR1, 2, 3 and 4 [1]. As the most studied member of the scramblase family, Elaidic acid the 37 kD ubiquitous PLSCR1 protein has been described as a type-II transmembrane protein comprised of a short 9 amino acid (aa)-long C-terminal extracellular domain (aa 310C318), a single transmembrane helix (aa 291C309) and a long intracytoplasmic N-terminal domain of 290 aa (aa 1C290), containing a cysteine-rich palmitoylation motif (C184CCPCC189) that could stabilize PLSCR1 anchoring in biological membranes [2C4]. PLSCR1 mutants with substitutions in this palmitoylation motif have been shown to localize in the nucleus where PLSCR1 can also carry out biological functions, such as transcriptional activity [5]. The main function ascribed to PLSCR1 has been related to its potential involvement in bidirectional and nonspecific movements of phospholipids between the inner and outer leaflets of the plasma membrane in TSPAN9 response to intracellular calcium mobilization [6C8]. Scrambling of membrane phospholipids then leads to the cell surface exposure of phosphatidylserine (PS), a critical signal for biological processes such as cell activation, coagulation, apoptosis and secretion [9,10]. However, this specific role of PLSCR1 in regulating phospholipid movements within the plasma membrane has Elaidic acid been recently challenged in several experimental systems (for Elaidic acid reviews, [2,9]). While the exact involvement of PLSCR1 in the translocation of membrane phospholipids remains controversial, increasing evidence now indicates that this transmembrane protein could also be involved in cell signaling processes at the plasma membrane. Indeed, PLSCR1 is found in lipid rafts where it has been shown to interact directly with several plasma membrane receptors, including the epidermal growth factor receptor, the high-affinity IgE receptor Fc?RI and the CD4 T-cell receptor [11C14]. In T lymphocytes, we have shown that both PLSCR1 and PLSCR4 are cellular receptors for the secretory leucocyte protease inhibitor (SLPI) and interact with CD4 at the plasma membrane [14]. In addition, PLSCR1 can also associate with cellular tyrosine kinases containing Src-homology 3 (SH3) domains, such as c-Abl [15] and Syk [16], and Src family kinases including Src and Lyn [13,16]. Association of PLSCR1 with these kinases is probably related to the multiple SH3-binding proline-rich motifs found in the long cytoplasmic domain of PLSCR1 (for review, [2]). However, the exact contributions of these interactions to specific functions of PLSCR1 are still poorly understood. To further characterize these functions, PLSCR1 expression was first examined in CD4-positive myeloid and lymphoid cells, and PLSCR1 levels were found to be higher in monocytic cells than in T lymphocytes. We next analyzed the expression and potential functions of PLSCR1 in the professional phagocytic myeloid cells, monocytes and macrophages. We found that the level of PLSCR1 was markedly increased during differentiation of primary monocytes to macrophages, and more interestingly, PLSCR1 specifically modulated phagocytosis in differentiated macrophages. Materials and Methods Cell culture and differentiation Adherent HeLa cells were grown in Dulbecco minimal essential medium supplemented with 10% fetal calf serum (FCS), 100 IU of penicillin/ml, and 100 g of streptomycin/ml (Invitrogen). Human THP-1 monocytic and HPB-ALL T lymphoid cells have been already described [17]. THP-1 and HPB-ALL non-adherent cells were cultured in RPMI 1640 medium with Glutamax-1 (Invitrogen) supplemented with 10 mM HEPES, 10% FCS, 100 IU of penicillin/ml, and 0.1 mg streptomycin/ml (complete medium). For differentiation in macrophages, THP-1 cells were treated in complete medium, supplemented with 1 M phorbol 12-myristate 13-acetate (PMA) (Sigma) alone or in combination with ionomycin where indicated, for the indicated time periods. Peripheral.