RESUMO
The Rac GTPases are currently being subjected to intensive study due to their involvement in a wide array of cellular phenomena. Many studies of Rac function have relied upon the use of relatively uncharacterized Rac dominant active, dominant negative, and effector domain mutants on the basis of the analogy to Ras structure. We have generated and purified such Rac2 mutants and characterized their guanine nucleotide binding properties in vitro. The Rac2 G12V and Q61L activating mutations were shown to hydrolyze bound GTP very slowly and were unresponsive to p190 Rac GTPase-activating protein. Distinct differences in the kinetics of nucleotide binding to individual mutant proteins were observed, accounting for the behavior of these proteins in biological assays. The structural features required for the responsiveness of Rac2 to the guanine nucleotide exchange protein smgGDS were examined. We show that guanine nucleotide exchange by smgGDS is dependent upon intact switch 1 and switch 2 regions in Rac2. Functional interactions between the switch 1 and switch 2 regions and the G12V mutation of Rac2 are described. These data form the basis for rational use of Rac mutants in biological studies.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Nucleotídeos de Guanina/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Proteínas rac de Ligação ao GTP , Proteínas rap de Ligação ao GTPRESUMO
The Rac GTP-binding proteins regulate the actin cytoskeleton and the superoxide-forming NADPH oxidase of phagocytic leukocytes. These functions of Rac are determined by the GTP/GDP state of the protein, which can be modulated by GTPase-activating proteins (GAPs). The interaction of Ras with both downstream signaling targets and GAPs is mediated via an "effector" domain (amino acids 30-40). We demonstrate that the effector domain of Rac2 is required for both NADPH oxidase activation and actin assembly, but that mutations in this region do not decrease the responsiveness of Rac to GAPs. In contrast, mutations of residues 12 (Gly-->Val) or 61 (Gln-->Leu) inhibit both intrinsic- and GAP-stimulated GTP hydrolysis by Rac2. A double mutation in which both the effector domain and Q61L were modified restored NADPH oxidase activation and membrane ruffling, while the equivalent effector domain and G12V double mutation did not. The Rac2 Q61L mutant had an increased "affinity" for NADPH oxidase activation and for GAP binding as compared to the wild type or G12V proteins. These experiments suggest that Rac contains at least two "effector" interaction sites, and that changes in binding interactions at one of these sites may influence the function of the other.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , NADH NADPH Oxirredutases/metabolismo , Proteínas/metabolismo , Sequência de Bases , Ligação Competitiva , Primers do DNA/química , Ativação Enzimática , Proteínas Ativadoras de GTPase , Técnicas In Vitro , Dados de Sequência Molecular , NADPH Oxidases , Proteínas Recombinantes , Relação Estrutura-Atividade , Proteínas rac de Ligação ao GTP , Proteínas Ativadoras de ras GTPaseRESUMO
Recurrent ulceration following transmetatarsal amputation commonly results from hypertrophic bone formation or equinus deformity. In the current study, 31 diabetic patients underwent 33 Achilles tendon procedures for recurrent ulcerations at the distal stump of their transmetatarsal amputation. Primary healing was achieved in 21 procedures (64%) and secondary healing in 9 procedures (27%) for an overall healing rate of 91%. Two procedures failed to resolve the original ulceration (6%). The average follow-up examination was 27 months. The authors conclude that Achilles tendon procedures are an effective means of managing ulcerations in transmetatarsal amputation feet exhibiting an equinus deformity.
