The Pleckstrin Homology Domain of PLCδ1 Exhibits Complex Dissociation Properties at the Inner Leaflet of Plasma Membrane Sheets

Abstract

Using total internal reflection fluorescence microscopy, we followed the dissociation of GFP-tagged pleckstrin homology (PH) domains of AKT and PLCδ1 from the plasma membranes of rapidly unroofed cells. We found that the AKT-PH-GFP and PLCδ1-PH-GFP dissociation kinetics can be distinguished by their effective k_off values of 0.39 ± 0.05 and 0.56 ± 0.16 s^-1, respectively. Furthermore, we identified substantial rebinding events in measurements of PLCδ1-PH-GFP dissociation kinetics. By applying inositol triphosphate (IP₃) to samples during the unroofing process, we measured a much larger k_off of 1.54 ± 0.42 s^-1 for PLCδ1-PH-GFP, indicating that rebinding events are significantly suppressed through competitive action by IP₃ for the same PH domain binding site as phosphatidylinositol 4,5-bisphosphate (PIP₂). We discuss the complex character of our PLCδ1-PH-GFP fluorescence decays in the context of membrane receptor and ligand theory to address the question of how free PIP₂ levels modulate the interaction between membrane-associated proteins and the plasma membrane.

Keywords: PIP2; PLCδ1; pleckstrin homology domain; rebinding; second messenger lipids; unroofing.