Differential Association of the Pleckstrin Homology Domains of Phospholipases C-β1, C-β2, and C-δ1 with Lipid Bilayers and the βγ Subunits of Heterotrimeric G Proteins

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Abstract
Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes β, γ, and δ). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or βγ subunits of heterotrimeric GTP binding (G) proteins. Although the PH domains of PLC-δ and PLC-γ have been studied, the comparable domains of the β isotypes have not. Here, we have measured the affinities of the isolated PH domains of PLC-β1 and -β2 (PH-β1 and PH-β2, respectively) for lipid bilayers and G-βγ subunits. Like the intact enzymes, these PH domains bind to membrane surfaces composed of zwitterionic phosphatidylcholine with moderate affinity. Inclusion of the anionic lipid phosphatidylserine or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and inclusion of G-βγ subunits had little affect on their membrane affinity. In contrast, binding of PLC-δ1 or its PH domain was highly dependent on PI(4,5)P2. We also determined whether these domains laterally associate with G-βγ subunits bound to membrane surfaces using fluorescence resonance energy transfer. Affinities for G-βγ were in the following order: PH-β2 ≥ PH-β1 > PH-δ1; the affinities of the native enzyme were as follows: PLC-β2 ≫ PLC-δ1 > PLC-β1. Thus, the PH domain of PLC-β1 interacts with G-βγ in isolation, but not in the context of the native enzyme. By contrast, docking of the PH domain of PLC-β2 with G-βγ is comparable to that of the full-length protein and may play a key role in G-βγ recognition.