Probing phosphoinositide and isoprenoid binding proteins by photoaffinity labeling

Phosphoinositides act as second messengers and are important regulators of protein functions such as in membrane vesicle trafficking and cytoskeleton rearrangements. The availability of phosphoinositide affinity probes has greatly facilitated the characterization of a series of phosphoinositide binding proteins (PIBPs). The total synthesis of diacyl and head group-modified phosphatidylinositol (4) phosphate is described herein. Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polyphosphate analogs. The N-terminal half and the C-terminal half of gelsolin showed synergy in the binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. In addition to the previously identified PtdIns(4,5)P2 binding sites in the N-terminal half of gelsolin, a new bind side was identified in the C-terminal half by mapping of the photocovently modified peptide fragments. The binding of human AKAP79 with phosphoinositides was also examined. The binding of PtdIns(4,5)P2 has been limited to the first 153 amino acid residues, a membrane targeting region of AKAP79. In addition to the binding to the membrane targeting region, PtdIns(4,5)P3 has also been shown to bind to the C-terminal regions of AKAP79. The interaction of PtdIns(4,5)P2 and PtdIns(4,5)P3 with AKAP79 suggests that phosphoinositides may have certain regulatory roles in the targeting process of protein kinase A. The identification of the Phosphoinositide binding sites in gelsolin and AKAP79 may provide sites where competing ligands may be targeted for possible therapeutic uses. A diazo-derivatized juvenile hormone analog was used in the discovery of a putative isoprenoid binding protein p50. This 50 kDa protein was purified from fetal bovine serum directed by photoaffinity labeling with [3H] eposybishomofarnesyl diazoacetate (EBDA), a juvenile hormone I analog. Further studies identified this protein as alpha1 acid glycoprotein (AGP). Both human and ovine AGP were shown to be strongly labeled by [3H]EBDA. Competitive displacement experiments have determined that AGP has potent binding to isoprenoids preferably with three or more isoprene units. The putative isoprenoid binding protein is being proposed to be involved the protection of neighboring cells from apoptosis.