Inhibition by anti-RCC1 monoclonal antibodies of RCC1-stimulated guanine nucleotide exchange on Ran GTPase.
Nine monoclonal antibodies to RCC1, the guanine nucleotide exchange factor on Ran GTPase, were obtained using recombinant RCC1 as the antigen. Epitopes of three monoclonal antibodies, which did not inhibit RCC1 function, were localized in the N-terminus outside the RCC1 repeat, while epitopes of the other 6 monoclonal antibodies were localized within the RCC1 repeat. Three of the latter 6 monoclonal antibodies, 2B6, 6C3, and 8D9, inhibited RCC1-stimulated nucleotide release. Two of them, 2B6 and 6C3, recognized the same amino acid residues in the N-terminus of the second RCC1 repeat, Tyr89, Ser90, Phe91, and Gly92, of which one, Gly92, is conserved in Saccharomyces cerevisiae and mutated in an rcc1(-) strain, mtr1-2. The monoclonal antibody 8D9 recognized two amino acid residues, Arg320 and Ala321, downstream of Gly319 in the N-terminus of the 6th RCC1 repeat, which corresponds to Gly92 in the second RCC1 repeat. The monoclonal antibodies which inhibited RCC1 function bound to RCC1 in homogeneous solution and stained cellular RCC1. We propose that the N-terminus of the RCC1 repeat is exposed at the surface of RCC1 on the coated plate or in fixed cells, and is involved in the RCC1-stimulated nucleotide exchange on the Ran GTPase.
Azuma
Y
Y
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Hachiya
T
T
Nishimoto
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000494:pharmacology*; D001665:Binding Sites; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology*; D018604:Epitope Mapping; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D013268:Stimulation, Chemical; D020931:ran GTP-Binding Protein
D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D009687:Nuclear Proteins; C062521:RCC1 protein, human; C499302:Rcc1 protein, mouse; D020558:GTP Phosphohydrolases; D020931:ran GTP-Binding Protein