The hydrophobic domains in the carboxyl-terminal signal for GPI modification and in the amino-terminal leader peptide have similar structural requirements

Abstract

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 characterized by a moderately hydrophobic domain downstream from the cleavage/modification site. The essential features of this domain were characterized using a truncated version of folate receptor (FR) type β (FR-βΔ5) in which its five carboxyl-terminal amino acid residues were deleted without affecting the efficiency of GPI modification. The amino acids at various positions in the hydrophobic domain were systematically altered and the extent of GPI modification of the recombinant proteins was determined by measuring [3H]folic acid binding at the cell surface, by Western blot analysis and from the sensitivity of the 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 decreases with increasing hydrophilicity. Further, the hydrophobic domain is characterized by a hydrophobicity profile and not merely a minimum overall hydrophobicity. Thus, a leucine-rich core hydrophobic segment of six to eight amino acid residues is more sensitive to relatively small hydrophilic substitutions compared to its flanking regions and such mutations could be compensated by a hydrophobic substitution elsewhere within this core segment. Such a hydrophobicity profile is characteristic of the amino-terminal leader peptide. When the entire hydrophobic domain of the leader peptide of FR-β (12 amino acid residues) was substituted with the hydrophobic domain of the GPI signal (13 amino acids), it was possible to obtain expression of FR-β on the cell surface. In this construct, point mutations in the core hydrophobic segment and in the flanking regions within the substituting peptide produced a similar pattern of effects on the cell surface receptor expression compared to the corresponding mutations in the GPI signal of FR-β. The results suggest that common principles may govern interactions of the hydrophobic domains of the GPI signal and the leader peptide with the endoplasmic reticulum.