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

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 <i>k</i>_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 <i>k</i>_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.