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

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Highlights

  • PIP3 theoretically binds to the C-terminal neurotensin binding site on sortilin.

  • PIP3 nanodiscs bind to sortilin.

  • diC4 PIP3 competes for neurotensin binding to sortilin.

  • Immunoreactive PIP3 is present on rat plasma VLDL.

  • A sortilin binding compound enhances neurotensin and PIP3 binding to sortilin.

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.

Introduction

Sortilin is a multi-ligand binding protein with numerous binding partners [1]. Sortilin binds neurotensin (NT) at two sites: the carboxy terminus (C-term) site 1, and the amino terminus (N-term) site 2 [2]. Sortilin also binds B100-containing very low density lipoprotein (VLDL) and LDL [3], [4]. We previously demonstrated that sortilin facilitates the secretion of VLDL-B100 by insulin-sensitive McArdle RH7777 (McA) cells [5]. We have further shown that insulin enhances B100 binding to sortilin in McA cells [6] preceding autophagic destruction [7]. Considering the association of sortilin with VLDL binding and with VLDL-B100 degradation in response to insulin, we explored the possibility for insulin-generated phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to interact directly with sortilin. We specifically tested the potential of PIP3 binding to NT defined binding sites on sortilin. Our results suggest a possible role for PIP3 present on VLDL to directly modulate lipoprotein-sortilin interactions.

Section snippets

Materials

Plasma from fasted Sprague Dawley rats was obtained from BioreclamationIVT, (Westbury, NY) and used to prepare rat lipoproteins. Bovine serum albumin (BSA) (A7285) was from Sigma-Aldrich Corp. (St. Louis, MO). The following materials and reagents were obtained from Echelon Biosciences, Inc. (St. Lake City, UT) including PIP3 PolyPIPosomes (Y-P039); dibutanoyl PIP3 (diC4-PIP3) (P-3904); dipalmitoyl PIP3 (diC16-PIP3) (P-3916); purified anti-PIP3 IgG (Z-P345b), and purified anti-PIP3 IgM (ZP345)

Sortilin is a PIP3 binding protein

Sortilin binding to NT has been characterized and involves N-term (site 2) and C-term (site 1) binding sites for binding to a single molecule of sortilin [2], [15]. A compound was identified with strong theoretical binding to site 2 (cpd984), and in biologic experiments cpd984 incubation increased VLDL-B100 secretion by McA cells [5]. In Fig. 1A, N-term NT is depicted in site 2 located across the central space of sortilin from PIP3 computationally docked into site 1 of hsortilin. The

Acknowledgements

We thank Dr. Stephen Sligar for his generous contribution of MSP1D1 protein and Dr. James Morrissey's laboratory in facilitating the preparation of PIP3 nanodiscs. This study was supported by grants from the National Institutes of Health DK100163 (JDS) and GM101132 (RAF).

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