An evaluation of strategies available for the identification of GTP-binding proteins required in intracellular signalling pathways

Abstract

Strategies which can be used to elucidate the nature of a GTP-binding regulatory protein (G-protein) involved in an intracellular pathway of interest in the complex environment of the cell are described and evaluated. A desirable strategy is considered to be one in which the first stage indicates a requirement for one or more G-proteins, provides information on whether a monomeric, trimeric or other type of G-protein is involved, and gives some idea of the G-protein sub-class. In the second stage the specific G-protein involved is identified. Approaches available for investigations in the first stage include the use of analogues of GTP and GDP, AlF4-, inhibitors of G-protein isoprenylation, bacterial toxins which covalently modify G-proteins, and the introduction of a purified GDP dissociation inhibitor, GDP exchange and/or GTP-ase activating protein. Identification of the specific G-protein in the second stage can be achieved using anti G-protein antibodies, G-protein-or receptor-derived peptides, antisense G-protein RNA and over-expressed, constitutively-active or dominant-negative G-protein mutants. The correct interpretation of results obtained with GTP and GDP analogues and AlF4- in the first stage is complex and often difficult, and requires a thorough understanding of the functions and mechanisms of activation of G-proteins. Nevertheless, it is important to reach the correct conclusion at this stage since considerable time and expense are usually required for investigations in the second stage.