title: Structural determinants required for the interaction between Rho GTPase and the GTPase-activating domain of p190.
abstract: The Rho family small GTP-binding proteins are subjected to regulation by Rho GTPase-activating proteins (GAPs) in the course of transmitting diverse intracellular signals. To understand the mechanism of GAP-catalyzed GTP hydrolysis of Rho GTPases, we have studied the interaction between RhoA and p190, the RasGAP binding phosphoprotein which has been implicated as a Rho-specific GAP, by delineating the structural determinants of RhoA and p190 GAP domain (p190GD) that are involved in their functional coupling. Besides the conserved residues Tyr34, Thr37, and Phe39 in the switch I region of RhoA which are required for p190GD interaction, chimeras made between RhoA and Cdc42, a close relative of RhoA with which p190GD interacts 50-fold less efficiently, revealed that residues outside the switch I and neighboring regions of RhoA, residues 85-122 in particular, contain the major p190GD-specifying determinant(s). Mutation of the unique Asp90 of RhoA in this region mostly abolished p190GD stimulation, whereas the corresponding reverse mutation of Cdc42 (S88D) was able to respond to p190GD-catalysis similarly as RhoA. Further kinetic analysis of these mutants provided evidence that Asp90 of RhoA contributes primarily to the specific binding interaction with p190GD. On the other hand, two charged residues of p190GD, Arg1283 and Lys1321, which are located in the putative G-protein binding helix pocket of GAP domain, were found to be involved in different aspects of interaction with RhoA. The R1283L mutant of p190GD lost GAP activity but retained the ability to bind to RhoA, while K1321A failed to stimulate and to bind to RhoA. These results indicate that residue Asp90 constitutes the second GAP-interactive site in RhoA which is mostly responsible for conferring p190GD-specificity, and suggest that the role of p190GD in the GTPase reaction of RhoA is in part to supply active site residue Arg1283 for efficient catalysis.
authors.affiliation: Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA.
authors.forename: R
authors.initials: R
authors.lastname: Li
authors.forename: B
authors.initials: B
authors.lastname: Zhang
authors.forename: Y
authors.initials: Y
authors.lastname: Zheng
mesh_terms: D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein
chemical_list: C074560:ARHGAP35 protein, human; C513043:ARHGAP5 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein