Arabidopsis Phospholipase Dα1 Interacts with the Heterotrimeric G-protein α-Subunit through a Motif Analogous to the DRY Motif in G-protein-coupled Receptors

Abstract

Phospholipase D (PLD) and heterotrimeric G-protein both play important, diverse roles in cellular regulation and signal transduction. Here we have determined the physical interaction between plant PLD and the only canonical α-subunit (Gα) of the G-protein in Arabidopsis thaliana and the molecular basis for the interaction. PLDα1 expressed in either Escherichia coli or Arabidopsis was co-precipitated with Gα. PLDα1 contains a sequence motif analogous to the Gα-interacting DRY motif normally conserved in G-protein-coupled receptors. Mutation of the central Lys residue PLDK564A of this motif abolished the PLDα1-Gα binding, whereas mutation of the two flanking residues PLDE563A and PLDF565A decreased the binding. Addition of Gα to PLDα1 inhibited PLDα1 activity, whereas the PLDK564A mutation that disrupted the Gα-PLDα1 binding abolished the inhibition. GTP relieved the Gα inhibition of PLDα1 activity and also inhibited the binding between PLDα1 and Gα. Meanwhile, the PLDα1-Gα interaction stimulated the intrinsic GTPase activity of Gα. Therefore, these results have demonstrated the direct binding between Gα and PLDα1, identified the DRY motif on PLDα1 as the site for the interaction, and indicated that the interaction modulates reciprocally the activities of PLDα1 and Gα.