Preferred Sites of Glycosylphosphatidylinositol Modification in Folate Receptors and Constraints in the Primary Structure of the Hydrophobic Portion of the Signal

Abstract

The divergent carboxyl-terminal signal peptides for glycosylphosphatidylinositol (GPI) membrane anchor attachment in folate receptor (FR) types alpha and beta were characterized. All of the candidate amino acid residues for GPI modification were identified and tested by substituting individually and in combination with amino acids that cannot be modified by GPI. Thus the GPI modification in FR-alpha was decreased to 22% by mutation of Ser234 to Thr but unaltered by changing the other candidate, Gly235, to Met. However, the double mutant FR-alpha Ser234-Thr,Gly235-Met showed half of the GPI modification seen in FR-alpha Ser234-Thr. This result suggests that Ser234 is the preferred GPI modification site, while Gly235 is a minor, alternate GPI modification site. Similarly, in FR-beta, mutation of Asn230 to Gln decreased GPI modification to 32%, while mutation of the other candidate site, Gly237, to Met had no effect. However, mutation at both sites further reduced the GPI modification by a half. A five amino acid carboxyl-terminal deletion (FR-beta delta 5) caused no decrease in the extent of GPI modification. However, the same deletion in FR beta Asn230-Gln decreased the residual GPI modification by 66%. These results suggest that Asn230 is the preferred GPI modification site in FR-beta, while Gly235 offers a minor alternate modification site; consistent with this conclusion is the fact that modification at the downstream site is hindered by its proximity to the carboxyl terminus in FR-beta delta 5. Further, the suggestion that the hydrophobic portion of the GPI signal is a random sequence of neutral amino acids with overall moderate hydrophobicity was tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity

“…On the other hand, for a given environment, different C-terminal segments (native, Leu/Ala, and Val/Phe/Ala sequences) with the same predicted ⌬G app value show different efficiencies of GPI attachment. This result is especially noteworthy because sequence conservation among GPI-anchored proteins drops dramatically after the residue and it has proven difficult to identify any conserved sequence motifs in the C-terminal segment beyond the hydrophobicity constraint (41)(42)(43)(44).…”

Strategy for the treatment of acute myelogenous leukemia based on folate receptor β–targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid

“…18,19 The folate receptor (FR) is a promising target because of its narrow tissue specificity, its overexpression in malignant tissues, and its ability to bind and internalize folic acid conjugates. Of the 3 human folate receptor isoforms, [20][21][22] 2 (FR-␣ and FR-␤) are attached to the cell surface through a glycosyl-phosphatidylinositol (GPI) membrane anchor, 20,23 whereas the third (FR-␥) is constitutively secreted. 24 The expression of FR-␣ in normal tissues is restricted to certain epithelial cells, where it is inaccessible by the circulation.…”

mRNA Instability in the Nucleus Due to a Novel Open Reading Frame Element Is a Major Determinant of the Narrow Tissue Specificity of Folate Receptor α

“…The folate receptor (FR) types ␣ and ␤ are glycosyl-phosphatidylinositol-anchored cell surface glycoproteins (11,40). The expression of FR-␣ among normal tissues is restricted to the luminal surface of certain epithelial cells (37,38,39).…”

“…Accordingly, a wide variety of FR-␣-mediated diagnostic and therapeutic approaches are currently in various stages of preclinical and clinical testing (8, 9a, 12, 15, 17, 21, 25, 30, 36). FR-␤ bears approximately 70% amino acid sequence identity to FR-␣ (23,40) and is limited in its expression to cells in the myelomonocytic lineage (19,28,35).…”