Determination of interaction sites on the small G protein RhoA for phospholipase D

Phespholipase D (PLD) has been identified as a target of small G proteins of the Rho family. The present study was directed at defining the interaction sites of RhoA with rat brain PLD in vitro using chimeric proteins between RhoA and Ha-Has or Cdc42Hs and point mutations. The switch I region of RhoA, which is the common effector domain of Ras-like G proteins, was a crucial interaction site for PLD. Mutations in conserved amino acids (Tyr³⁴, Thr³⁷, Phe³⁹) totally abolished PLD activation, while mutations in Val³⁸ or Tyr⁴² caused partial loss. Two additional sites were responsible for the differential PLD activation ability between RhoA and Cdc42Hs. Changing Asp⁷⁶ in the switch II region of RhoA to the corresponding amino acid in Cdc42Hs led to partial loss of PLD activation. A chimeric protein with the N-terminal third of Cdc42Hs changed to RhoA showed enhanced PLD activation. Analysis of other Rho/Ha-Ras chimeric proteins and mutations indicated that Gln⁵² adjacent to the switch II region is responsible for this gain of function. In conclusion, the present study shows that conserved amino acids in the switch I region of RhoA are major PLD interaction sites and that residues in the switch H and internal regions are responsible for the differential activation of PLD by RhoA and Cdc42Hs.