Dataset Information

Guanine nucleotide-dependent translocation of RhoA from cytosol to high affinity membrane binding sites in human erythrocytes.

ABSTRACT: The translocation of the small GTP-binding protein Rho from the cytosolic to membrane-bound form is an early step in many cellular signal-transduction events, but little is known regarding the mechanism of Rho association with the plasma membrane. We have used membranes from human erythrocytes to uncover a novel class of integral membrane components involved in the Rho-membrane association. Membranes of human erythrocytes contain several proteins of the Ras superfamily. Using specific antibodies and C3 exoenzyme of Clostridium botulinum we have identified one of them as RhoA. This protein was detected in both cytosol and membrane fractions of hypotonically lysed erythrocytes. We found that cytosolic Rho bound specifically to the cytoplasmic surface of the erythrocyte membrane and that the translocation of Rho to the membrane was absolutely dependent on the prior incubation of the cytosol with guanosine 5'--gamma-thio-triphosphate (1-50 microM) at low Mg2+ concentration. Rho binding sites could not be extracted from the membrane using conditions that extracted all other peripheral proteins and were unaffected by heat treatment and protease digestion. Rho binding was saturable, with a Kd in the range 1-5.0 nM, and the number of binding sites was estimated to be approx. (1-2) x 10(3) sites per cell. This is the first report of Rho binding to integral membrane components. The identity of these components may reveal novel aspects of the mechanism by which Rho exerts its multiple biochemical effects.

SUBMITTER: Boukharov AA

PROVIDER: S-EPMC1219287 | biostudies-other | 1998 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundThe Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains.ResultsHere we show that RhoA is a substrate for ARHGEF10. In both G1/S and M phases, ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa cells. Furthermore, RNA interference-based knockdown of ARHGEF10 resulted in multipolar spindle formation in M phase. Each spindle pole seems to contain a centrosome consisting of two centrioles and the pericentriolar material. Downregulation of RhoA elicited similar phenotypes, and aberrant mitotic spindle formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively activated RhoA. Multinucleated cells were not increased upon ARHGEF10 knockdown in contrast to treatment with Y-27632, a specific pharmacological inhibitor for the RhoA effector kinase ROCK, which induced not only multipolar spindle formation, but also multinucleation. Therefore, unregulated centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10 knockdown-dependent multipolar spindle formation. We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10. Knockdown of KIF3B again caused multipolar spindle phenotypes. The supernumerary centrosome phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of ARHGEF10, RhoA or KIF3B was abrogated by RNA interference.ConclusionCollectively, our results suggest that a novel RhoA-dependent signaling pathway under the control of ARHGEF10 has a pivotal role in the regulation of the cell division cycle. This pathway is not involved in the regulation of cytokinesis, but instead may regulate centrosome duplication. The kinesin-like motor protein KIF3B may modulate the ARHGEF10-RhoA pathway through the binding to ARHGEF10.

Dataset Information

Guanine nucleotide-dependent translocation of RhoA from cytosol to high affinity membrane binding sites in human erythrocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets