Strong, albeit indirect, evidence suggests that a GTP-binding (G) protein(s) can act directly on the secretory machinery by a post-second messenger mechanism. The type and function of this putative G(e) (exocytosis) protein were investigated in streptolysin-O-permeabilized rat basophilic leukemia (RBL) cells. The exocytotic response to calcium was first characterized both morphologically and biochemically using the release of preloaded [3H]serotonin as an index of exocytosis. Calcium-induced secretion (EC50 about 3 μM) in RBL cells requires ATP (EC50 about 2.5 mM) and is modulated by pH, the optimal value being 7.2. Another requirement for calcium-induced secretion is an activated G protein, since inactivators of G proteins such as GDPβS (EC50 about 800 μM) inhibit the secretagogue effect of 10 μM free calcium. Conversely, GTPγS (EC50 about 1 μM) and other nonhydrolyzable analogs of GTP, which keep G proteins in a permanently active conformation, potentiate the effect of calcium. GTPγS alone is without effect. The effect of GTPγS on exocytosis is apparently not mediated by known second messengers, suggesting that a G(e) protein is involved. Electron microscopic images show that in resting cells, secretory granules are clustered in the perinuclear area, whereas they become scattered upon calcium stimulation. A paradoxical effect of GTPγS is observed when applied during permeabilization; under these conditions, in fact, the nucleotide inhibits the subsequent secretory response to calcium. The scattering of granules is also inhibited. This effect of GTPγS is counteracted by coadministration of GTP. These responses to guanine nucleotides are typical of vectorially acting G proteins involved in protein synthesis and in intracellular vesicle transport. Taken together, the data presented suggest that calcium-dependent release requires a vectorially acting G protein controlling the movement of secretory granules. This and alternative models are discussed.
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1991|
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