Heterotrimeric G proteins in synaptoneurosome membranes are crosslinked by p-phenylenedimaleimide, yielding structures comparable in size to crosslinked tubulin and F-actin.
We have treated rat brain synaptoneurosomes with the crosslinking agent N,N'-1,4-phenylenedimaleimide under conditions that cause extensive crosslinking of tubulin, F-actin, and the alpha and beta subunits of three major types of heterotrimeric GTP-binding regulatory proteins (G(o), Gs, Gi) present in brain membranes. The major crosslinked products are coeluted from Bio-Gel sizing columns as very large structures that do not penetrate stacking gels during SDS/PAGE. The alpha subunits but not the beta subunits of Gs, G(o) and Gi also yield crosslinked products of intermediate sizes. None of the products are as small as the heterotrimeric G proteins extracted from brain by cholate or Lubrol. However, the large and intermediate crosslinked structures are strikingly similar to the large, polydisperse structures of the alpha subunits of Gs, Gi, and G(o) extracted from synaptoneurosomes by the detergent octyl glucoside, which have sedimentation properties of multimeric proteins. Several ways in which multimeric forms of G proteins can explain the dynamic and pleiotropic actions of hormones and GTP on signal-transducing systems are discussed.
Coulter
S
S
Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Rodbell
M
M
D000199:Actins / Q000737:chemistry; D000818:Animals; D001923:Brain Chemistry; D003432:Cross-Linking Reagents; D019204:GTP-Binding Proteins / Q000737:chemistry*; D046911:Macromolecular Substances; D008301:Maleimides / Q000737:chemistry; D051381:Rats; D013574:Synaptosomes / Q000648:ultrastructure*; D014404:Tubulin / Q000737:chemistry
D000199:Actins; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008301:Maleimides; D014404:Tubulin; C011328:N,N'-4-phenylenedimaleimide; D019204:GTP-Binding Proteins