THE HYDROPHOBIC DOMAINS IN THE CARBOXYL-TERMINAL SIGNAL FOR GPI MODIFICATION AND IN THE AMINO-TERMINAL LEADER PEPTIDE HAVE SIMILAR STRUCTURAL REQUIREMENTS

Proteins having a glycosyl-phosphatidylinositol (GPI) membrane anchor are synthesized with a carboxyl-terminal signal that is cleaved in the endoplasmic reticulum prior to GPI modification. The signal is charac terized by a moderately hydrophobic domain downstream from the cleavag e/modification site. The essential features of this domain were charac terized using a truncated version of folate receptor (FR) type beta (F R-beta Delta 5) in which its five carboxyl-terminal amino acid residue s were deleted without affecting the efficiency of GPI modification. T he amino acids at Various positions in the hydrophobic domain were sys tematically altered and the extent of GPI modification of the recombin ant proteins was determined by measuring [H-3]folic acid binding at th e cell surface, by Western blot analysis and from the sensitivity of t he proteins to phosphatidylinositol-specific phospholipase C (PI-PLC). The results indicate that a threshold level of hydrophobicity exists at a single position below which the efficiency of GPI modification de creases with increasing hydrophilicity. Further, the hydrophobic domai n is characterized by a hydrophobicity profile and not merely a minimu m overall hydrophobicity. Thus, a leucine-rich core hydrophobic segmen t of six to eight amino acid residues is more sensitive to relatively small hydrophilic substitutions compared to its flanking regions and s uch mutations could be compensated by a hydrophobic substitution elsew here within this core segment. Such a hydrophobicity profile is charac teristic of the amino-terminal Leader peptide. When the entire hydroph obic domain of the leader peptide of FR-beta (12 amino acid residues) was substituted with the hydrophobic domain of the GPI signal (13 amin o acids), it was possible to obtain expression of FR-beta on the cell surface. In this construct, point mutations in the core hydrophobic se gment and in the flanking regions within the substituting peptide prod uced a similar pattern of effects on the cell surface receptor express ion compared to the corresponding mutations in the GPI signal of FR-be ta. The results suggest that common principles may govern interactions of the hydrophobic domains of the GPI signal and the leader peptide w ith the endoplasmic reticulum. (C) 1998 Academic Press Limited.