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Proteins are packaged into vesicles which proceed through the secretory pathway by budding from one compartment and fusing with the next until their destination is reached. Intracellular transport was found to be blocked by GTPyS (161) which indicated that G-proteins were involved in this process. Monomeric G-proteins have now been shown to be involved in almost every vesicular trafficking event (reviewed in (41)).

Whilst constitutive secretion is inhibited by GTPyS (162), the fusion of regulated secretory granules has been shown to be stimulated by guanine nucleotides in a variety of cells including neutrophils (8), mast cells (156), gastric chief cells (163), HL60 cells (139), adrenal chromaffin cells (164) and eosinophils (155). Stimulation of exocytosis by GTPyS occurs in the presence of MgATP and is independent of Ca^"^ (8,139,154,163); but in the absence of MgATP, Ca^^ is required (139,165,166). Enhanced release is observed if both Ca^^ and GTPyS are present (139,154,163). GTPyS-stimulated secretion can not be accounted for by the activation of known

signal-transducing G-proteins, which has led to the hypothesis that a novel G-protein referred to as Ge regulates exocytosis (165).

Until recently a role for heterotrimeric G-proteins in regulated secretion had not been demonstrated however evidence is now emerging that these proteins are required for regulated secretion.

Histamine release from mast cells can be stimulated by AIF4' which specifically

stimulates heterotrimeric G-proteins (86). Through the use of specific peptides and antibodies against G-protein a subunits it was shown that inhibition of Gccis (which is also involved in constitutive secretion (17,167)) resulted in the inhibition of histamine release. GcCis was localised to both the plasma membrane and Golgi membranes. Agents which disrupt the Golgi resulted in the delocalisation of the Golgi associated Go(i3, but no effect was observed on secretion and the plasma membrane locahsation remained intact. The authors therefore speculate that plasma membrane-bound G0(^3 regulates exocytosis whereas Golgi-bound Goti3 is not involved (168).

A requirement for Gi (the specific a subunit is as yet unknown) is also observed for insulin secretion from p-cells (169). Mastoparan, a known activator of heterotrimeric G-proteins (170) stimulates insulin secretion and causes increased GTPase activity on insulin secretory granules (ISGs). ISGs are enriched in Gi; and pertussis toxin treatment which inactivates Gi and Go inhibited secretion. It is therefore postulated that activation of Gi is required for the fusion of ISGs with the plasma membrane.

Alternatively, an inhibitory effect of heterotrimeric G-proteins has been observed in adrenal chromaffin cells (171). GAP-43 activates Go by increasing guanine nucleotide exchange. In the presence of GAP-43 the rate of GTPyS binding to granule membranes was increased whilst noradrenaline secretion was inhibited. Antibodies raised against Go reduced the inhibitory effect of GAP-43 indicating that secretion from chromaffin cells is under the regulation of Go.

Monomeric GTP-binding proteins play a well-established role in regulated exocytosis. The Rab family of small GTP-binding proteins regulate many transport steps in both the secretory and endocytic pathways. Only one member of the family -Rab3A- so far, has been shown to be involved in the control of regulated exocytosis.

The localisation of Rab3A specifically to cells containing regulated secretory pathways (172) provided the first indication that this protein maybe involved in regulated exocytosis. Further localisation studies have demonstrated that Rab3A associates with secretory granules in adrenal chromaffin cells (172) and synaptic vesicles in nerve terminals (173) thus emphasising a potential role for Rab3A in exocytosis.

Studies utilising a peptide which corresponds to the proposed effector domain of Rab3A have provided much of the evidence for the role of Rab3A. In the presence of this peptide, exocytotic release is stimulated in several cells including pancreatic p- cells (174), mast cells (175), adrenal chromaffin cells (176) and pituitary cells (174,177). Recent evidence from Stahl et al (178) has demonstrated that Rab3A which is associated with synaptic vesicles is predominantly in the GTP bound form. Stimuli which induce exocytosis results in GTP hydrolysis and therefore a shift to the GDP bound form. These observations are specific to Rab3A localised at the synaptic vesicle membrane with no observable changes in the cytosolic pool of Rab3A. The authors therefore propose that GTP hydrolysis by Rab3A is associated with vesicles docking or fusion.

Through the use of a photoactivated cross linking Rab3A peptide Olszewski et al (174) have provided evidence which suggests that Rab3A interacts specifically with two proteins (REEPl & 2) and that this interaction is perhaps important for the regulation of exocytosis in pancreatic p-cells. Both REEPl & 2 are predominantly membrane- associated in unstimulated cells but redistribute to the cytosol when release of insulin is maximal. It is postulated that these proteins and Rab3A may form an inhibitory complex which, once dissociated allows exocytosis to occur. A similar inhibitory role for Rab3A has been implicated in studies from adrenal chromaffin cells (176)

Recently, the involvement of Rho and Rac have been implicated in the regulation of secretion in mast cells (30). Constitutively-active mutants of these proteins enhanced secretion whilst specific inhibitors of endogenous Rac and Rho- N17Racl and C3 transferase respectively- inhibited secretion. Similar evidence was demonstrated for the regulation of exocytosis by Rho in RBL cells (179).

Rho and Rac are involved in the regulation of the actin cytoskeleton (24,25). It is proposed that the cytoskeleton forms a barrier to the docking of secretory vesicles and that localised disassembly of the cytoskeleton is required for exocytosis to occur. Preliminary results suggest that exocytosis from mast cells in response to Rho and Rac occurs in the absence of their observed effects on the actin cytoskeleton and vice versa

although the interdependence of these pathways need to be fully established (30).