MOLECULAR REQUIREMENTS FOR ATTACHMENT OF THE GLYCOSYLPHOSPHATIDYLINOSITOL ANCHOR TO THE HUMAN ALPHA FOLATE RECEPTOR

The alpha isoform of the folate receptor (FR) is a 38-KDa glycosylphos phatidylinositol (GPI) protein which mediates the internalization of f olates. The FR amino acid sequence has features typical of GPI-linked proteins, including the presence of a hydrophobic carboxyl-terminus, a hinge region, and a stretch of small and uncharged amino acids. Subst itution of predicted cleavage/attachment Ser(234) With arginine or thr eonine, or replacement of Gly(235) With proline by site-directed mutag enesis had no effect on GPI processing. In fact, CHO cells transfected with each of the three cDNA variants or with FR wild-type showed comp arable amounts of phosphatidylinositol-specific phospholipase C-resist ant FR in double-determinant radioimmunoassay. Western blot analysis o f total cell lysates from all transfectants consistently revealed the 38-KDa FR band. Deletion of residues 233-237 in the amino-terminal por tion of the FR cDNA constructs derived by a polymerase chain reaction strategy abrogated GPI processing, with only a small proportion of the FR remaining in the cytoplasm in four of the five clones tested. This finding suggests that FR residues 233-237 are essential in properly j uxtaposing the FR hydrophobic domain. Together, these data support the hypothesis that the postulated Ser(234) is not the only potential cle avage/attachment site of the alpha isoform of FR. J. Cell. Biochem. 72 :111-118, 1999. (C) 1999 Wiley-Liss, Inc.