Phosphatidylserine receptors: role of CD36 in binding of anionic phospholipid vesicles to monocytic cells.

Exposure of phosphatidylserine (PtdSer) has been implicated in the recognition and phagocytosis of senescent and apoptotic cells, and CD36 has been proposed as one receptor protein that recognizes PtdSer and other anionic phospholipids. We investigated the binding of phospholipid vesicles to the monocytic leukemia cell lines THP-1 and J774A.1 with a flow cytometric assay; vesicles contained 50 mol% PtdSer, phosphatidylinositol (PtdIns), or phosphatidylglycerol (PtdGro), with the balance being phosphatidylcholine. Specific, high affinity binding was observed for vesicles containing PtdSer, PtdIns, or PtdGro. Specificity of the assay was confirmed by control experiments with erythrocytes, which showed minimal vesicle binding, and with annexin V, which blocked the binding of PtdSer, PtdGro, and PtdIns vesicles to the THP-1 cells. However, O-phospho-L-serine (to 1 mM) had no effect on the binding of PtdSer vesicles, indicating that high affinity binding requires a surface containing multiple phosphoserine groups rather than a single molecule. A monoclonal antibody to CD36 blocked up to 60% of the specific binding of PtdSer vesicles but had minimal to no effect on the binding of PtdGro or PtdIns vesicles. This antibody also selectively inhibited the phagocytosis of PtdSer-containing vesicles as measured by fluorescence microscopy, indicating that CD36 is functionally significant for phagocytosis of this vesicle type. In addition, collagen and thrombospondin, two other putative ligands of CD36, were unable to inhibit the binding of PtdSer vesicles. We conclude that CD36 is the primary protein responsible for the high affinity binding of PtdSer vesicles to these monocyte-like cells. In addition, CD36 appears to be specific for PtdSer among anionic phospholipids, and non-phospholipid ligands of CD36 do not share binding sites with PtdSer on CD36.