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Journal of Biomolecular Screening
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Binding of a Pleckstrin Homology Domain Protein to Phosphoinositide in Membranes: A Miniaturized FRET-Based Assay for Drug Screening

Brian D. Hamman

Aurora Biosciences Corporation, San Diego, CA

Brian A. Pollok

Aurora Biosciences Corporation, San Diego, CA

Todd Bennett

Aurora Biosciences Corporation, San Diego, CA

Janet Allen

Institute de Recherche Jouveinal/Pfizer, Fresnes, France

Roger Heim

Aurora Biosciences Corporation, San Diego, CA

Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton’s tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP3) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP3, with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP3 at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP3 and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP3 in vesicles. This biochemical assay readily miniaturized to 1.8-µl reaction volumes and was validated in a 3456-well screening format.

Journal of Biomolecular Screening, Vol. 7, No. 1, 45-55 (2002)
DOI: 10.1177/108705710200700107
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