Determination of interaction sites on the small G protein RhoA for phospholipase D.
Phospholipase 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-Ras 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 II and internal regions are responsible for the differential activation of PLD by RhoA and Cdc42Hs.
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Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
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Fleming
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Exton
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JH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000378:metabolism*; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein
D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein