Abstract Body: Background: Cholesterol homeostasis is tightly regulated by LXR-RXR heterodimer complex. However, it’s not fully clear if any specific lipid can modulate the activity of LXR-RXR complex. We found that cholesterol loading increases nuclear PIP2. One intriguing question is how increased PIP2 relays cholesterol status to LXR/RXR complex.

Hypothesis: PIP2 has a specifically high critical micelle concentration (~10 µM) and inverted conical shape, allowing PIP2 to translocate/desorb from membrane bilayer and interact with RXR.

Goal: To determine the interaction between PIP2 and RXR and mechanism for PIP2 desorption/translocation from lipid bilayer.
Methods and Results: We identified a conserved putative PIP2 binding motif in human RXRA (aa:185-196). Using Rosetta-fold and PONDER prediction, RXR-PIP2 binding domain (RXR-PBD) comprising aa 111-228 was found to be highly disordered, forming a putative binding surface for anionic molecules. Microscale thermophoresis showed binding of DOPC:PIP2 with Alexa488-RXR-PBD (Kd-19.6±1.5µM, N=3, mean±S.E) vs no binding with DOPC. Surface Plasmon Resonance (SPR) using immobilized biotinylated-PIP2 and RXR-PBD showed binding with Kd of 1.65±0.3 µM (N=3 mean±S.E). Fluorescence resonance energy transfer (FRET) using Alexa 488-RXR-PBD and RhodPE-PIP2 pair showed robust binding, while RXR with mutations in PIP2 binding region showed markedly reduced binding (N=3, mean±S.E). STEDyCON microscopy, using giant unilamellar vesicles (GUV) doped with PIP2 showed strong binding of RXR-PBD-WT, while RXR-PBD-mutant showed no binding (N=30), confirms interaction between RXR and PIP2. Increased PIP2 levels and exogenous PIP2 addition in DOPC-GUVs drive nanotube formation. Fluorescence recovery after photobleaching (FRAP) revealed modulation of lipid mobility—DOPC:5% PIP2 showed increased diffusion (30.5 ± 0.24 µm2/s) vs DOPC (18.7±0.35 µm2/s), whereas exogenous PIP2 reduces diffusion (17.9 ± 4.5 µm2/s) compared with control DOPC (29.5 ± 4.2 µm2/s). Atomic force microscopy of giant plasma membrane vesicles (GPMVs) from HepG2 cells treated with PIP2 showed significantly increased Young’s modulus and membrane tension while reduced tether radius (n>30) vs GPMVs treated with PC.
Conclusion: Nuclear PIP2 binds RXR via a novel and conserved PIP2-binding domain. Mechanistically, PIP2 lowers tether radius, increases membrane tension and stiffness promotes high-curvature nanotube formation, and PIP2 desorption. The desorbed PIP2 interacts with RXR.