Raft.1, A Monoclonal Antibody Raised Against the Raft Microdomain, Recognizes G-Protein β1 and 2, Which Assemble Near Nucleus After Shiga Toxin Binding to Human Renal Cell Line

Abstract

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 β subunits 1 and 2 (Gβ1 and 2). That Raft.1 recognized Gβ1 and 2 was further confirmed by the reactivity to recombinant Gβ1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gβ1 and 2. Because Gβ1 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.