Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors.
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 alpha-subunit (Galpha) of the G-protein in Arabidopsis thaliana and the molecular basis for the interaction. PLDalpha1 expressed in either Escherichia coli or Arabidopsis was co-precipitated with Galpha. PLDalpha1 contains a sequence motif analogous to the G alpha-interacting DRY motif normally conserved in G-protein-coupled receptors. Mutation of the central Lys residue PLD(K564A) of this motif abolished the PLDalpha1-Galpha binding, whereas mutation of the two flanking residues PLD(E563A) and PLD(F565A) decreased the binding. Addition of Galpha to PLDalpha1 inhibited PLDalpha1 activity, whereas the PLD(K564A) mutation that disrupted the Galpha-PLDalpha1 binding abolished the inhibition. GTP relieved the Galpha inhibition of PLDalpha1 activity and also inhibited the binding between PLDalpha1 and Galpha. Meanwhile, the PLDalpha1-Galpha interaction stimulated the intrinsic GTPase activity of Galpha. Therefore, these results have demonstrated the direct binding between Galpha and PLDalpha1, identified the DRY motif on PLDalpha1 as the site for the interaction, and indicated that the interaction modulates reciprocally the activities of PLDalpha1 and Galpha.
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Jian
J
Zhao
Xuemin
X
Wang
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry*; D001665:Binding Sites; D020558:GTP Phosphohydrolases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry*; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*
D029681:Arabidopsis Proteins; D044385:GTP-Binding Protein alpha Subunits; D043562:Receptors, G-Protein-Coupled; C582894:PLDA1 protein, Arabidopsis; D010739:Phospholipase D; D020558:GTP Phosphohydrolases; D020962:Heterotrimeric GTP-Binding Proteins