Disease relevance of RHOA

• Gb (b for botulinum) is an Mr 21,000-22,000 guanine nucleotide binding protein which is specifically ADP-ribosylated by an ADP-ribosyltransferase produced by types C1 and D Clostridium botulinum (Morii, N., Sekine, A., Ohashi, Y., Nakao, K., Imura, H., Fujiwara, M., and Narumiya, S. (1988) J. Biol. Chem. 263, 12420-12426) [1].
• Extensive digestion of rhoA p21 with Achromobacter protease I yielded a C-terminal peptide, Ser-Gly-Cys190, that lacked the three C-terminal amino acids predicted from the cDNA but was geranylgeranylated and carboxyl methylated at the cysteine residue [2].
• To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli [3].

High impact information on RHOA

• The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins) [4].
• Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo [4].
• This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein [3].
• There are also marked similarities of sequence in regions of the G proteins, elongation factors, and the ras p21 gene products that are believed to be involved in guanine nucleotide binding and GTP hydrolysis [5].
• We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol [6].

Chemical compound and disease context of RHOA

• Clostridium botulinum C3 transferase (C3) was used as a specific inhibitor of rho p21 [7].

Biological context of RHOA

• Substrate for botulinum ADP-ribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product [1].
• These results suggest that rhoA p21 is first geranylgeranylated followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the exposed cysteine residue [2].
• A 1.5 kilobase cDNA coding for the complete amino acid sequence of Gb, the substrate for ADP-ribosyltransferase in C1 and D botulinum toxins from bovine adrenal gland, has been isolated from a cDNA library of bovine adrenal gland [8].
• In the present study, we investigated the function(s) of these post-translational modifications of the C-terminal region of rhoA p21 by use of the rhoA p21s purified from bovine aortic smooth muscle and rhoA p21-overexpressing Escherichia coli since the bacterial protein was not modified with a geranylgeranyl moiety [9].
• These results suggest that the post-translational modifications of the C-terminal region of bovine rhoA p21, most presumably the geranylgeranylation, which are absent in bacterial rhoA p21, play important roles in its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GAP [9].

Anatomical context of RHOA

• Bovine brain cytosol geranylgeranylated the bacterial rhoA p21 having the three C-terminal amino acids predicted from the cDNA but not the protein lacking the three C-terminal amino acids [2].
• The posttranslationally modified C-terminal structure of bovine aortic smooth muscle rhoA p21 [2].
• Bovine brain membranes methylated the synthetic C-terminal peptide with 10 amino acids of rhoA p21 which was geranylgeranylated at its C-terminal cysteine residue but not the peptide which was not geranylgeranylated [2].
• cDNA cloning of Gb, the substrate for botulinum ADP-ribosyltransferase from bovine adrenal gland and its identification as a rho gene product [8].
• The regulation of endothelial cell motility by p21 ras [10].

Associations of RHOA with chemical compounds

• Consistently, Raney nickel treatment of rhoA p21 released a geranylgeranyl moiety as estimated by gas chromatography/mass spectrometry [2].
• On the other hand, rho p21 was previously shown to be ADP-ribosylated by bacterial ADP-ribosyltransferases, named C3 and EDIN, at Asn41 in the putative effector region of rho p21 [11].
• The Mr values of smg p21 GAP1 and -2 are estimated to be 250-400 x 10(3) and 80-100 x 10(3) by gel filtration and sucrose density gradient ultracentrifugation, respectively [12].
• In the present studies, we resolved the bovine aortic smg p21 fraction into two distinct G protein fractions on hydroxyapatite column chromatography and purified them separately to near homogeneity (22K G1 and -2) [13].
• Bovine rhoA p21 bound to plasma membranes and phosphatidylserine-linked Affigel, but bacterial rhoA p21 did not bind to them [9].

Analytical, diagnostic and therapeutic context of RHOA

• Purified Gb was digested with various proteases, and the proteolytic fragments were separated by high performance liquid chromatography [1].
• Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins [14].
• 21K G and the bovine brain rhoA gene product (rhoA p21) were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography and migrated at the same positions on two-dimensional gel electrophoresis [15].
• Microinjection of oncogenic ras-p21 protein (0.12 pmol/50 nl) induced maturation in 79% of oocytes, beginning at 6 hours and reaching completion by 12 hours [16].

References