HETEROTRIMERIC G-PROTEINS IN SYNAPTONEUROSOME MEMBRANES ARE CROSS-LINKED BY PARA-PHENYLENEDIMALEIMIDE, YIELDING STRUCTURES COMPARABLE IN SIZE TO CROSS-LINKED 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), G(s), G(i)) 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 G(s), G(o), and G(i) 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 G(s), G(i), 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.