FINAL DISCUSSION AND FUTURE WORK

This thesis describes the identification o f a G TP-binding protein w hich m ay be involved in the regulation o f exocytosis. R acl, purified from bovine brain as a com plex w ith RhoG D I retards the o f loss o f responsiveness to stim ulation by Ca^^ and GTPyS (rundow n) o f perm eabilised mast cells. In contrast RhoGDI, applied alone under the same conditions accelerates the rundow n and this suggests that Rho fam ily proteins other then Rac may be involved in regulating mast cell secretion.

The case for Rac is supported by the finding that recom binant Rac2, expressed and purified from E .C oli, can also retard the run-dow n o f m ast cells stim ulated by the dual effectors, Ca^^ plus GTPyS. In addition, cells treated w ith this protein exhibit GTP-y-S-independent secretion. However, the recom binant protein is only capable o f producing these effects when “pre-activated” by GTPyS binding. This probably reflects the lack o f post-translational m odification know n to be required for translocation to the plasm a membrane (Abo et al., 1994; el B enna et al., 1994; Quirm et al., 1993; D idsbury et al., 1990; Heyworth et al., 1994) and interaction w ith som e exchange factors (GEFs) w hich catalyse activation (Ando et al., 1992; H eyw orth et al., 1993; Takai et al., 1993; Bokoch et al., 1994; Shirataki et al.,

1991; M izuno et al., 1991; Hori et al., 1991). The dom inant inhibitory m utant rT17N -Rac2 inhibits secretion induced by Ca^^ and GTPyS, and Rac has been show n to leak from the perm eabilised m ast cells under the conditions in w hich they lose responsiveness (Brown et al., 1997), lending further credence to the idea that Rac is an endogenous regulator o f secretion. However, the presence o f free GTPyS in addition to the preactivated rRac2 causes an enhancem ent o f the

secretory response and this must im plicate at least one other G TP-binding protein in the regulatory m echanism . This may not be a m em ber o f the Rho family, since an optim um concentration o f RhoGDI fails to inhibit m ast cell secretion

com pletely(M ariot et al., 1996).

One candidate m ust be Cdc42, since preactivated recom binant Cdc42H s can also retard the rate o f rundow n o f the secretory response and, like rRac2, it is also capable o f eliciting G TPyS-independent secretion. The dom inant inhibitory

m utant rT17N -Cdc42H s inhibits secretion induced by and GTPyS and with Cdc42 leaking from S L -0 perm eabilised mast cells it appears that this is another G TPase regulator o f secretion. Interestingly, the dose-response relationships for rRac2 and rCdc42H s in the stim ulation o f secretion from run-dow n m ast cells stim ulated w ith either C a“^ alone or by the dual effector system are different. rC dc42H s characteristically stim ulates secretion w hen applied over a m uch w ider range o f concentrations than rRac2.

W hen present at low concentrations rCdc42Hs enhances the extent o f secretion due to the presence o f a saturating concentration o f rR ac2.1 have concluded that while both G TPases can interact (under these conditions) w ith a com m on effector to induce secretion, rCdc42Hs can additionally interact w ith a second dow nstream effector w hich is not accessible to rRac2.

If Rac and Cdc42 are indeed the authentic regulators o f exocytosis ( G e) in mast cells, then this m ay provide a rationale for a num ber o f earlier observations. In particular, it m ay explain why the onset o f exocytosis from perm eabilised cells is delayed by the presence o f ATP (but not by A ppN H p) (Tatham and Gomperts,

1989). This w as originally taken as an indication o f a dephosphorylation step necessary for the induction o f exocytosis (see Introduction - “A TP”). It has already been m entioned that the com plexes o f R ho-G TPases w ith R hoG D I are m ore stable w hen the GDI is phosphorylated (see p ??), so it is possible that

dephosphorylation o f RhoG D I (which w ould allow Rac and/or Cdc42 to dissociate and becom e available for activation) constitutes a required step in the activation o f secretion. D ephosphorylation o f RhoG DI could also explain w hy the rapid loss o f sensitivity to stim ulation in cells deprived o f A TP can be restored by readdition o f A TP (H owell et al., 1989), and that after this, secretion can be stim ulated by alone (Churcher et al., 1990b). In the absence o f A TP, the com plexes m ay be sufficiently destabilised that the Rac and/or Cdc42 dissociate, bind to the plasm a m em brane and can thus activate secretion even in the absence o f a guanine nucleotide. This o f course, assumes that the proposal o f an equilibrium state (presented in Chapter 3 “D iscussion”) holds, and further that the critical condition for stim ulation is m em brane binding, not the identity o f the bound guanine

nucleotide. One m ust assum e, though, that in the absence o f a stim ulating guanine nucleotide the Rho proteins w ould be functioning less efficiently than norm al, and

I w ould certainly expect that the stim ulation o f secretion by the Ca"^-only stimulus is not sim ply due to Rac and/or Cdc42, but reflects the activity o f m ore than one com ponent. In particular, the requirem ent for ATP in the stim ulation o f secretion under these conditions indicates that there is another A TP-dependent step in the stim ulation o f secretion, the nature o f w hich is unknow n at present. However, Rac/Cdc42 activation may be rate-limiting. W hen m ast cells are treated with okadaic acid (phosphatase inhibitor) or PMA before perm eabilisation in the absence o f A TP and then restored by ‘"add-back” o f A TP an activating guanine nucleotide becom es an absolute requirem ent once again (H ow ell et al., 1989; Churcher et al., 1990b). This is consistent with my proposal since it w ould be expected that w hilst RhoGDI remains in a phosphorylated condition forming stable com plexes with Rac and Cdc42, activation o f secretion can only occur in the conventional manner, requiring the presence o f an activating guanine

nucleotide. However, as already mentioned, w hilst the activation and deactivation o f Rac/Cdc42 may provide a logical explanation for these observations, there is as yet no evidence for the involvem ent o f a phosphatase in these events, and it is also unlikely that the observations are due solely to Rac/Cdc42. Other, as yet unknown m echanism s m ay occur alongside Rac and Cdc42 activation in the stim ulation o f secretion from m ast cells. Indeed, this seems likely, since Rac/Cdc42 activation due to dephosphorylation o f RhoGDI does not provide an explanation for the finding that A TP induces onset delays even in the absence o f Mg^^, w hich itself im plies that a dephosphorylation does not constitute an essential step in the pathw ay leading to exocytosis in mast cells (Lillie et al., 1991).

A t present, there are no clues as to the mechanism s w hereby Rac and Cdc42 exert their stim ulatory effects on secretion. As already m entioned (Chapters 4 and 5), candidates for the com m on effector for these GTPases include the

CRTB-containing proteins (Burbelo et al., 1995), such as PA K (M anser et al., 1994; M anser et al., 1995; Teo et al., 1995; Prigmore et al., 1995), PI-3kinase (Tolias et al., 1995; Zheng et al., 1994a), IQ G A Pl and IQ GA P2 (Hart et al., 1996; Brill et al., 1996), w hilst candidates for the Cdc42-only effector include ACK (M anser et al., 1993) and W ASp (Symons et al., 1996). So far, o f course, there is no evidence for the involvem ent o f any o f these proteins in the regulation o f secretion from any cell type. As already mentioned, there is evidence for the involvem ent o f PfP-5kinase in the regulation o f secretion (H ay et al., 1995), and

this protein has been shown to interact w ith Rac (Tolias et ai., 1995). Cdc42 interacts only w eakly with this enzyme (Tolias et al., 1995) m aking it an unlikely candidate for the shared effector. However, it may be that w hen high

concentrations o f activated rCdc42Hs are provided in the absence o f activated rRac2 during the rundow n assay, the rCdc42Hs will stim ulate PIP-5kinase in addition to its specific effector sufficiently to induce secretion (Figure 5.2a). Even if this is the case, it is unlikely that Cdc42 interacts w ith PIP-5kinase in vivo, and if so, there w ould be no physiological “shared effector” so that under norm al conditions, Cdc42 and Rac would each have their own effectors (assum ing that the Rac effector is indeed PIP-5kinase).

It is clear that the next m ajor step forward must be to identify the dow nstream effectors o f these GTPases in the regulation o f exocytosis. This could be achieved by either affinity chromatography, using activated rRac2 or rCdc42H s as the ligand, or by a yeast two hybrid method. In addition, because a Ca^^-binding exchange factor for Cdc42, Cdc24, has been identified in yeast (Ohya et al., 1986; M iyam oto et al., 1987; Zheng et al., 1994b), it will be interesting to investigate the Ca^^-dependence o f the secretory response to Cdc42 in m ast cells (as outlined in the discussion to Chapter 5) since a sim ilar factor m ay be responsible, at least in part, for the Ca^^-dependence o f secretion. However, post-translational

m odification is required for interaction o f Rho proteins w ith som e exchange factors (A ndo et al., 1992; Heyworth et al., 1993; Takai et al., 1993; B okoch et al., 1994; Shirataki et al., 1991; M izuno et al., 1991; Hori et al., 1991), so a source o f m odified protein w ould be required.

M y w ork has unearthed tw o possible candidate G TPases, Rac and Cdc42, for G e,

the G TP-binding protein that mediates the exocytotic process in m ast cells. Since these tw o proteins, w hich until recently were never even vaguely perceived to be involved, are turning out to be regulators o f many diverse cellular processes, this begs the question: are these proteins situated well upstream o f the exocytotic fusion machinery? In addition, are there really, as im plied by the shift in

sensitivity to rCdc42H s in the presence o f GTPyS (Figure 5.2a) w hich w as found not to be due to Rac2 (Figure 5.6), further GTPases involved? I f so, are they directly involved in the fusion process, as hypothesised for R ab3? A s previously discussed (Chapter 5, “D iscussion”), candidate regulators o f m ast cell secretion

w hich may m ediate the enhanced sensitivity to rCdc42H s include Rho (Price et ai., 1995), Gi3 (A ridor et al., 1993) and Py subunits (Pinxteren et al., 1997).

However, it appears that there is m ore than one, maybe m ore than two, GTPases involved in the regulation o f secretion, at least in m ast cells, and it rem ains to be seen exactly w hich these are, and how they carry out their duties. The GTPases continue to surprise us by their versatility, and I am sure that the surprises w ill not cease now. As more data become available, so the signalling pathways regulated by these proteins become more and m ore complex. Clearly, for those interested in dissecting these pathways, a m am m oth task lies ahead.

B ib lio g ra p h y

Abo, A., Pick, E., Hall, A., Totty, N., Teahan, C.G.. and Segal, A.W. (1991). Activation o f the NADPH oxidase involves the small GTP-binding protein pli'"'''. Nature 353, 668-670.

Abo, A., Webb, M.R., Grogan, A., and Segal, A.W. (1994). Activation o f NADPH oxidase involves the dissociation o f p 2 r “‘" from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its

translocation to the plasma membrane. Biochem. J. 298. 585-591.

Adamson, P., Marshall, C.J., Hall, A., and Tilbrook, P.A. (1992). Post-translational modifications o f p21rho proteins. J. Biol. Chem. 267, 20033-20038.

Ahnert-Hilger, G. and Weller, U. (1993). Comparison o f the intracellular effects o f Clostridial neurotoxins on exocytosis from Streptolysin 0-permeabilized rat pheochromocytoma (PC 12) and bovine adrenal chromaffin cells. Neurosci. 53, 547-552.

Alder, J., Lu, B., Valtorta, P., Greengard, P., and Poo, M. (1992a). Calcium-Dependent Transmitter Secretion Reconstituted in Xenopus Oocytes: Requirement for Synaptophysin. Science 257, 657-661. Alder, J., Xie, Z.P., Valtorta, F., Greengard, P., and Poo, M. (1992b). Antibodies to synaptophysin interfere with transmitter secretion at neuromuscular synapses. Neuron 9, 759-768.

All, H., Richardson, R.M., Tomhave, E.D., DuBose, R.A., Haribabu, B., and Snyderman, R. (1994). Regulation o f stably transfected platelet activating factor receptor in RBL-2H3 cells. Role o f multiple G proteins and receptor phosphorylation. J. Biol. Chem. 269, 24557-24563.

All, H., Choi, O.H., Fraundorfer, P.F., Yamada, K., Gonzaga, H.M., and Beaven, M.A. (1996). Sustained activation o f phospholipase D via adenosine A3 receptors is associated with enhancement o f antigen- and Ca(2+)-ionophore-induced secretion in a rat mast cell line. J. Pharmacol. Exp. Ther. 276, 837-845. Ali, S.M., Geisow, M.J., and Burgoyne, R.D. (1989). A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells. Nature 340, 313-315.

Ali, S.M. and Burgoyne, R.D. (1990). The stimulatory effect o f calpactin (annexin II) on calcium- dependent exocytosis in chromaffin cells: requirement for both the N-terminal and core domains o f p36 and ATP. Cell Signal. 2, 265-276.

Aim, P.E. and Bloom, G.D. (1982). Cyclic nucleotide involvement in histamine release from mast cells - a réévaluation. Life Sci. 30, 213-218.

Ando, S., Kaibuchi, K., Sasaki, T., Hiraoka, K., Nishiyama, T., Mizuno, T., Asada, M., Nunoi, H., Matsuda, L, Matsuura, Y., and et al (1992). Post-translational processing o f rac p21s is important both for their interaction with the GDP/GTP exchange proteins and for their activation o f NADPH oxidase. J. Biol. Chem. 267, 25709-25713.

Araki, S., Kikuchi, A., Hata, Y., Isomura, M., and Takai, Y. (1990). Regulation o f reversible binding o f smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. J. Biol. Chem. 265, 13007-13015.

Aridor, M., Traub, L.M., and Sagi-Eisenberg, R. (1990). Exocytosis in mast cells by basic secretagogues: Evidence for direct activation o f GTP-binding proteins. J. Cell Biol. Il l , 909-917.

Aridor, M., Rajmilevich, G., Beaven, M.A., and Sagi-Eisenberg, R. (1993). Activation o f exocytosis by the heterotrimeric G protein G|3. Science 262, 1569-1572.

Bader, M., Sontag, J., Thierse, D., and Aunis, D. (1989). A Reassessment o f Guanine Nucleotide Effects on Catecholamine Secretion from Permeabilized Adrenal Chromaffin Cells. J. Biol. Chem. 264,16426-

16434.

Bagrodia, S., Derijard, B., Davis, R.J., and Cerione, R.A. (1995). Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation. J. Biol. Chem. 270, 27995- 27998.

Banerjee, A., Barry, V.A., DasGupta, B.R., and Martin, T.F.J. (1996a). TV-Ethylmaleimide-sensitive Factor Acts at a Prefusion ATP-dependent Step in Ca'^-activated Exocytosis. J. Biol. Chem. 271, 20223- 20226.

Banerjee, A., Kowalchyk, J.A., DasGupta, B.R., and Martin, T.F.J. (1996b). SNAP-25 Is Required for a Late Postdocking Step in Ca“^-dependent Exocytosis. J. Biol. Chem. 271, 20227-20230.

Banin, S., Gan, T., Katz, D.R., Waterfield, M.D., Brickell, P.M., and Gout, I. (1996). Wiskott-Aldrich syndrome protein (WASp) is a partner for c-Src family protein-tyrosine kinases. Curr. Biol. 6, 981-988.

Bar-Sagi, D. and Gomperts, B.D. (1988). Stimulation of exocytotic degranulation by microinjection o f the

ras oncogenic protein into rat mast cells. Oncogene i . 463-469.

Barfod, E.T., Zheng, Y., Kuang, W.J., Hart, M.J., Evans, T., Cerione, R.A., and Ashkenazi, A. (1993). Cloning and expression o f a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain. J. Biol. Chem. 268, 26059-26062.

Barker, S.A., Caldwell, K.K., Hall, A., Martinez, A.M., Pfeiffer, J.R., Oliver, J.M., and Wilson, B.S. (1995). Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset o f responses induced by FcÎRl cross-linking. Mol. Biol. Cell 6, 1145-1158.

Barrowman, M.M., Cockcroft, S., and Gomperts, B.D. (1986). Two roles for guanine nucleotides in the stimulus secretion sequence o f neutrophils. Nature 319, 504-507.

Beaven, M.A., Moore, J.P., Smith, G.A., Hesketh, T.R., and Metcalfe, J.C. (1984a). The calcium signal and phosphatidylinositol breakdown in 2H3 cells. J. Biol. Chem. 259, 7137-7142.

Beaven, M.A., Rogers, J., Moore, J.P., Hesketh, T.R., Smith, G.A., and Metcalfe, J.C. (1984b). The mechanism o f the calcium signal and correlation with histamine release in 2H3 cells. J. Biol. Chem. 259,

7129-7136.

Beaven, M.A. and Kassessinoff, T. (1997). Role o f phospholipases, protein kinases and calcium in FcÎRl- induced secretion. In IgE Receptor (FcÎRl) Function in Mast cells and basophils. M.M. Hamawy, ed. (R G Landes Co), pp. 55-73.

Bender, L., Lo, H.S., Lee, H., Kokojan, V., Peterson, V., and Bender, A. (1996). Associations among PH and SH3 domain-containing proteins and Rho-type GTPases in Yeast. J. Cell Biol. 133, 879-894. Benhamou, M., Gutkind, J.S., Robbins, K.C., and Siraganian, R.P. (1990). Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release. Proc. Nat. Acad. Sci. (USA).

87, 5327-5330.

Bennett, J.P., Cockcroft, S., and Gomperts, B.D. ( 1981 ). Rat mast cells permeabilised with ATP secrete histamine in response to calcium ions buffered in the micromolar range. J. Physiol. (Lond) 317, 335-345. Bennett, M.K., Calakos, N., and Scheller, R.H. (1992). Syntaxin: A Synaptic Protein Implicated in Docking o f Synaptic Vesicles at Presynaptic Active Zones. Science 257, 255-259.

Beranger, F., Cadwallader, K., Porfiri, E., Powers, S., Evans, T., de Gunzburg, J., and Hancock, J.F. ( 1994). Determination of structural requirements for the interaction o f Rab6 with RabGDl and Rab

geranylgeranyltransferase. J. Biol. Chem. 269, 13637-13643.

Bird, G.S. and Putney, J.W. (1993). Inhibition o f thapsigargin-induced calcium entry by microinjected guanine nucleotide analogues. Evidence for the involvement o f a small G-protein in capacitative calcium entry. J. Biol. Chem. 268, 21486-21488.

Bimbaumer, L., Abramowitz, J., and Brown, A.M. (1990). Receptor-effector coupling by G proteins. Biochim. Biophys. Acta 1031, 163-224.