The membrane phospholipid phosphatidylserine is exposed on the cell surface during apoptosis and acts as an eat-me signal in the phagocytosis of apoptotic cells in mammals and nematodes. However, whether this is also true in insects was unclear. When milk fat globule-epidermal growth factor 8, a phosphatidylserine-binding protein of mammals, was ectopically expressed in Drosophila, the level of phagocytosis was reduced, whereas this was not the case for the same protein lacking a domain responsible for the binding to phosphatidylserine. We found that the extracellular region of Draper, an engulfment receptor of Drosophila, binds to phosphatidylserine in an enzyme-linked immunosorbent assay-like solid-phase assay and in an assay for surface plasmon resonance. A portion of Draper containing domains EMI and NIM located close to the N-terminus was required for binding to phosphatidylserine, and a Draper protein lacking this region was not active in Drosophila. Finally, the level of tyrosine-phosphorylated Draper, indicative of the activation of Draper, in a hemocyte-derived cell line was increased after treatment with phosphatidylserine-containing liposome. These results indicated that phosphatidylserine serves as an eat-me signal in the phagocytic removal of apoptotic cells in Drosophila and that Draper is a phosphatidylserine-binding receptor for phagocytosis.

To identify molecules that play roles in the clearance of apoptotic cells by Drosophila phagocytes, we examined a series of monoclonal antibodies raised against larval hemocytes for effects on phagocytosis in vitro. One antibody that inhibited phagocytosis recognized terribly reduced optic lobes (Trol), a core protein of the perlecan-type proteoglycan, and the level of phagocytosis in embryos of a Trol-lacking fly line was lower than in a control line. The treatment of a hemocyte cell line with a recombinant Trol protein containing the amino acid sequence RGD augmented the phosphorylation of focal adhesion kinase, a hallmark of integrin activation. A loss of integrin ␤, one of the two ␤ subunits of Drosophila integrin, brought about a reduction in the level of apoptotic cell clearance in embryos. The presence of integrin ␤ at the surface of embryonic hemocytes was confirmed, and forced expression of integrin ␤ in hemocytes of an integrin ␤-lacking fly line recovered the defective phenotype of phagocytosis. Finally, the level of phagocytosis in a fly line that lacks both integrin ␤ and Draper, another receptor required for the phagocytosis of apoptotic cells, was lower than that in a fly line lacking either protein. We suggest that integrin ␤ serves as a phagocytosis receptor responsible for the clearance of apoptotic cells in Drosophila, independent of Draper.

Background: Drosophila integrin ␤ plays a role in the phagocytosis of apoptotic cells and bacteria, but its partner ␣-subunit remains to be identified. Results: Of 5 ␣-subunits, ␣PS3 was physically and functionally associated with ␤. Conclusion: ␣PS3/␤ serves as a receptor for phagocytosis in Drosophila. Significance: The heterodimeric structure of Drosophila integrin has been genetically and biochemically solved.

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