Phosphatidylinositol (3,4,5)-trisphosphate binds to sortilin and competes with neurotensin: Implications for very low density lipoprotein binding

Abstract

Sortilin is a multi-ligand sorting receptor that interacts with B100-containing VLDL and LDL as well as other ligands including neurotensin (NT). The current study investigates the hypothesis that phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated downstream of insulin action can directly bind to sortilin. NT binds to sortilin at a well characterized site via its carboxy terminus (C-term). Using a crystal structure of human sortilin (hsortilin), PIP3 is predicted to bind at this C-term site. Binding of PIP3 to hsortilin is demonstrated using surface plasmon resonance (SPR) flowing PIP3 nanodiscs over immobilized hsortilin. Studies were performed using SPR where dibutanoyl PIP3 is shown to compete with NT for sortilin binding. Rat VLDL and LDL were evaluated for PIP3 content immunologically using monoclonal antibodies directed against PIP3. Rat plasma VLDL contained three times more immunoreactive PIP3 than LDL per µg of protein. Because VLDL contains additional ligands that bind sortilin, to distinguish specific PIP3 binding, we used PIP3 liposomes. Liposome floatation assays were used to demonstrate PIP3 liposome binding to sortilin. Using SPR and immobilized hsortilin, the C-term NT tetrapeptide (P-Y-I-L) is shown to bind to hsortilin. A compound (cpd984) was identified with strong theoretical binding to the site on sortilin involved in NT N-terminal binding. When cpd984 is co-incubated with the tetrapeptide, the affinity of binding to sortilin is increased. Similarly, the affinity of PIP3 liposome binding increased in the presence of cpd984. Overall, results demonstrate that sortilin is a PIP3 binding protein with binding likely to occur at the C-term NT binding site. The presence of multiple ligands on B100-containing lipoproteins, VLDL and LDL, raises the interesting possibility for increased interaction with sortilin based on the presence of PIP3.

Non-Canonical Binding of a Small Molecule to Sortilin Alters Cellular Trafficking of ApoB and PCSK9 in Liver Derived Cells

“…1C). Results are consistent with our previous study showing that cpd984 bound Sortilin in the presence of C-terminal NT 17 .…”

“…We speculate this to be the result of increased ligands for Sortilin on the VLDL surface other than ApoB-100, e.g. PI(3,4,5)P 3 or ApoE, in contrast to decreased alternative ligands for LDL other than ApoB-100 17,40 .…”

“…Sortilin is a multiligand receptor that can interact with the various ligands found on VLDL including B100 14 , ApoE 15,16 , and PI(3,4,5)P 3 17 . Circulating VLDL content of PI(3,4,5)P 3 is enriched as compared to HDL and LDL 17 . The paradox for the ability of Sortilin to both increase or decrease VLDL secretion could relate to the relative concentration and position of ligands on the VLDL surface.…”

“…Having concluded previously that PI(3,4,5)P 3 binds to Site-1 17 , we tested in vitro binding using SPR to determine the effect of cpd541 and cpd984 on Sortilin binding of PI(3,4,5)P 3 . First, we found that diC8-PI(3,4,5)P 3 bound with high affinity to crosslinked Sortilin with a K D of 4.2 µM ± 380 nM ( Fig.…”

“…The system was equilibrated for 20 ns restraining the Cα atoms of the protein (1.0 kcal/mol/Å 2 ) to allow for solvation. This was followed by a production run of 50 ns without restraints for 4 poses taken from ensemble docking, 3 poses with the highest affinity pose for Site-1 of Sortilin run in duplicate for each, and the pose for Site-2 that represented the predicted pose as shown previously 17 .…”