Functional Analysis of RhoX, a New Branch of the Rho Family

Johns Hopkins University, Baltimore MD

Investigators

Abstract

The Rho family of intracellular signaling proteins mediates a variety of critical cellular responses to external stimuli. Specifically the family members Rho, Rac and Cdc42 are activated in response to receptor tyrosine kinases and G-protein coupled receptors, among others, and can have rapid effects on the organization of the actin cytoskeleton as well as delayed effects on transcription. Rho family GTPases have been shown to be essential for cell polarity in yeast, as well as for cytokinesis, epithelial sheet invagination, cell migration and axon pathfinding in more complex organisms. Preliminary studies have identified a novel, evolutionarily conserved branch of the Rho family which has been tentatively named RhoX. cDNAs encoding members of this family are found in fruit flies, mice, rats and humans. Drosophila appears to have one family member, whereas there are at least two highly related human genes. All of the encoded proteins share characteristics that are otherwise unique amongst Rho family GTPases. In addition to an N-terminal Rho homology domain, these proteins contain 200-400 additional residues at the C-terminus. Characteristically, the C-terminus contains a proline rich segment reminiscent of SH3 binding domains, followed by one or more BTB oligomerization motifs. The remaining sequences share slight homology with members of the Kelch family of actin binding proteins. The Drosophila and human RhoX mRNAs are expressed widely, perhaps ubiquitously in embryos. In preliminary studies the Drosophila gene has been mapped and a P-element insertion mutation has been identified. The mutation is homozygous lethal, which confirms the functional importance of this new branch of the Rho family. Homozygous mutant clones in the adult Drosophila eye exhibit a rough eye phenotype, due to loss of photoreceptor cells and ommatidial fusions, that resembles EGF receptor (EFGR) pathway mutants. Specific aims of this project are to determine whether RhoX proteins bind and hydrolyze GTP and to test the hypothesis that the BTB domains mediate dimerization or interactions with other BTB domain proteins such as Kelch. Additionally, experiments will test whether the C-terminal domain is responsible for the subcellular localization of RhoX. In collaboration with Alan Hall, fibroblasts that express mammalian RhoX proteins will be characterized. The RhoX loss-of-function mutations in Drosophila will be used to study the in vivo functions of this protein. Through this combined use of biochemistry, cell biology and genetics, these studies are likely to reveal the fundamental roles of this new and essential member of the Rho family.