Although many glycosylphosphatidylinositol (GPI)‐anchored proteins have been observed as soluble forms, the mechanisms by which they are released from the cell surface have not been demonstrated. We show here that a cell‐associated GPI‐specific phospholipase D (GPI‐PLD) releases the GPI‐anchored, complement regulatory protein decay‐accelerating factor (DAF) from HeLa cells, as well as the basic fibroblast growth factor‐binding heparan sulfate proteoglycan from bone marrow stromal cells. DAF found in the HeLa cell culture supernatants contained both [3H]ethanolamine and [3H]inositol, but not [3H]palmitic acid, whereas the soluble heparan sulfate proteoglycan present in bone marrow stromal cell culture supernatants contained [3H]ethanolamine. 125I‐labeled GPI‐DAF incorporated into the plasma membranes of these two cell types was released in a soluble form lacking the fatty acid GPI‐anchor component. GPI‐PLD activity was detected in lysates of both HeLa and bone marrow stromal cells. Treatment of HeLa cells with 1,10‐phenanthroline, an inhibitor of GPI‐PLD, reduced the release of [3H]ethanolamine‐DAF by 70%. The hydrolysis of these GPI‐anchored molecules is likely to be mediated by an endogenous GPI‐PLD because [3H]ethanolamine DAF is constitutively released from HeLa cells maintained in serum‐free medium. Furthermore, using PCR, a GPI‐PLD mRNA has been identified in cDNA libraries prepared from both cell types. These studies are the first demonstration of the physiologically relevant release of GPI‐anchored proteins from cells by a GPI‐PLD.
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