Guanine nucleotide-dependent translocation of RhoA from cytosol to high affinity membrane binding sites in human erythrocytes.
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.
Boukharov
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AA
Department of Biomedical Research, St. Elizabeth's Medical Center of Boston, Boston, MA 02135, USA.
Cohen
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CM
D036002:ADP Ribose Transferases; D000906:Antibodies; D001665:Binding Sites; D001905:Botulinum Toxins; D003600:Cytosol / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000097:blood*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
D000906:Antibodies; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D008274:Magnesium