Interaction between pleckstrin homology domains and G protein βγ- subunits: Analyses of kinetic parameters by a biosensor-based method

Abstract

Pleckstrin homology (PH) domains, comprised of rather weakly conserved sequences of about 100 amino acid residues, are a protein motif found in many signaling and cytoskeletal proteins. PH domains have been shown to bind to the βγ subunits of heterotrimeric GTP-binding proteins (G(βγ)), but the affinity of PH domains for G(βγ) has not been quantitatively estimated in detail. To characterize the nature of the interaction between PH domains and G(βγ), its kinetic parameters were analyzed using a BIAcore(TM) instrument. All PH domains tested (PH domains of ras-specific guanine nucleotide exchange factor (ras-GRF), phospholipase (PLC) γ1, and Son of sevenless protein (Sos)) appeared to bind to G(β1γ2) with affinity constants K(D) of 0.108, 0.318, and 0.208 μM, respectively. The binding of PH domains to G(βγ) was inhibited by preincubation of G(βγ) with the GDP-bound but not the GTP- bound form of Gi(α). This study showed a high affinity interaction between PH domains and G(βγ), and suggests a potential role of PH domains in G(βγ)-mediated signal transduction in intact cells.