PROTEOLYSIS OF THE CARBOXYL-TERMINAL GPI SIGNAL INDEPENDENT OF GPI MODIFICATION AS A MECHANISM FOR SELECTIVE PROTEIN SECRETION

Variable amounts of soluble forms of a variety of glycosyl-phosphatidy linositol (GPI)-anchored proteins occur extracellularly, but the molec ular mechanisms governing their release are not entirely clear. When t he GPI-anchored folate receptor (FR) type beta was expressed transient ly in human 293 fibroblasts, there was a roughly equal distribution of [H-3]folic acid binding protein between the cell surface and the medi um after 24 h over a wide range of expression levels of FR-beta. The d ifference in apparent molecular masses between the soluble FR-beta and the PI-PLC-treated membrane protein indicated that the former was not released from the membrane by the action of phospholipase. Brefeldin A inhibited the release of soluble FR-beta from both the transfected 2 93 cells and stable recombinant CHO (CHO-FR-beta) cells while pre-exis ting levels of cell surface FR were unaltered suggesting the absence o f a precursor-product relationship between the membrane-associated FR- beta and the soluble protein in the medium. [S-35]Cysteine pulse-chase analysis was consistent with this finding. Interchanging of carboxyl- terminal peptides between FR-beta and FR-alpha revealed that the natur e of the processed signal for GPI modification was responsible for the quantitative membrane anchoring of FR-alpha and the production of sol uble FR-beta. When total cell lysates were analyzed by Western blot, a diffuse band of apparent 41 kDa and three additional sharp bands of a pparent 35, 33, and 29.3 kDa were seen. The 41 kDa band was identified as the PI-PLC sensitive cell surface receptor. Several mutant constru cts of FR-beta, in which the carboxyl-terminal signal for GPI modifica tion was either disrupted or deleted only gave the three lower bands. The three sharp bands from the wild-type and the mutant forms of FR-be ta were identified as nonglycosylated (29.3 kDa) or glycosylated polyp eptides in which the carboxyl-terminal peptide was at least partially proteolyzed without GPI modification. All of the mutations in the GPI signal resulted in the recovery of [H-3]folic acid binding protein in the media which, similar to the wild-type FR recovered from the media, were converted to the 29.3 kDa band by N-glycanase. The results from this study indicate that a carboxyl-terminal peptide in FR-beta is eff iciently proteolyzed intracellularly by a pathway that is independent of GPI signal recognition resulting in proper protein folding and secr etion. Such carboxyl-terminal sequences could represent a simple adapt ation for proteins whose physiologic functions reside both at the cell surface and in extracellular fluids, allowing their selective and tis sue-specific release.