Raft.1, a monoclonal antibody raised against the raft microdomain, recognizes G-protein beta1 and 2, which assemble near nucleus after shiga toxin binding to human renal cell line.
Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein beta subunits 1 and 2 (Gbeta1 and 2). That Raft.1 recognized Gbeta1 and 2 was further confirmed by the reactivity to recombinant Gbeta1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gbeta1 and 2. Because Gbeta1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.
Katagiri
Yohko U
YU
Department of Developmental Biology, National Research Institute for Child Health and Development, Tokyo, Japan.
Ohmi
Kazuhiro
K
Tang
Weiran
W
Takenouchi
Hisami
H
Taguchi
Tomoko
T
Kiyokawa
Nobutaka
N
Fujimoto
Junichiro
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D002292:Carcinoma, Renal Cell / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D020962:Heterotrimeric GTP-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007680:Kidney Neoplasms / Q000378:metabolism*; D008297:Male; D021962:Membrane Microdomains / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D051575:Proto-Oncogene Proteins c-yes; D051381:Rats; D011917:Rats, Inbred Lew; D022621:Shiga Toxin / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured / Q000378:metabolism; D019061:src-Family Kinases / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; C106354:flotillins; D022621:Shiga Toxin; D051575:Proto-Oncogene Proteins c-yes; C489834:YES1 protein, human; C496639:Yes1 protein, mouse; D019061:src-Family Kinases; D020962:Heterotrimeric GTP-Binding Proteins