Growth cone transduction: G0 and GAP-43

STEPHEN M. STRITTMATTER, DARIO VALENZUELA, TIMOTHY VARTANIAN, YOSHIAKI SUDO, MAURICIO X. ZUBER and MARK C. FISHMAN Developm ental Biology Laboratory and Cardiovascular Research Center, D epartm ents o f N eurology and M edicine, M assachusetts General Hosipital and Harvard M edical School, Boston, M A , USA

Summary The neuronal growth cone plays a crucial role in forming the complex brain architecture achieved during development, and similar nerve terminal mechanisms may operate to modify synaptic struc­ ture during adulthood. The growth cone leads the elongating axon towards appropriate synaptic tar­ gets by altering motility in response to a variety of extracellular signals. Independently of extrinsic clues, neurons mainfest intrinsic control of their growth and form (Banker and Cowan, 1979). Hence, there must be intracellular proteins which control nerve cell shape, so-called ‘plasticity’ or ‘growth’ genes. GAP-43 may be such a molecule (Skene and Willard, 1981; Benowitz and Lewis, 1983). For example, GAP-43 is localized to the growth cone membrane (Meiri et al. 1986; Skene et al. 1986) and can enhance filopodial formation even in non­ neuronal cells (Zuber et al. 1989a). It includes a small region at the amino terminus for membrane associ­ ation and perhaps growth cone targeting (Zuber et al. 1989b, Liu et al. 1991). We have found that G0, a member of the G protein family that links receptors and second messengers, is the major non-cytoskeletal protein in the growth cone membrane (Strittmatter et al. 1990). Double staining immunohistochemistry for GAP-43 and G„ shows that the distributions of the two proteins are quite similar. Purified GAP-43 regulates the activity of purified G„ (Strittmatter et al. 1990), a surprising observation since GAP-43 is an intracellular protein. We have compared the mechanism of GAP-43 acti­ vation of G0 with that of G protein-linked receptors. GAP-43 resembles receptor activation in that both serve primarily to increase the rate of dissociation of bound GDP, with consequent increase in GTPyS binding and GTPase activity. Neither affects the intrinsic rate of hydrolysis of bound GTP by G„. They differ, however, in that pertussis toxin blocks inter­ action of the receptor with G0, but not that of GAP-43. Furthermore, whereas GAP-43 activates both iso­ lated «o subunits and a/Jy trimers, receptors require the presence of the fiy subunits. Thus like receptors, GAP-43 is a guanine nucleotide release protein, but of a novel class. The interactions between G0 and GAP-43 suggest that G0 plays a pivotal role in growth cone function, coordinating the effects of both extracellular signals and intracellular growth pro­ teins. Journal o f Cell Science, Supplement 15, 27-33 (1991) Printed in Great Britain © The Company o f Biologists Limited 1991

Key words: GAP-43, growth cone, GTP-binding proteins, signal transduction, G0.

Introduction The function of the central nervous system is absolutely dependent on the highly complex synaptic connections established during brain development. The ability of the extending neurite to identify and grow along appropriate pathways, and then to stop at synaptic targets, is mediated to a great extent by the axonal growth cone (reviewed by Lockerbie, 1987). The growth cone responds to extracellu­ lar signals such as soluble factors, matrix components and cell-bound adhesion molecules. Among the important soluble factors are neurotransmitters (Haydon et al. 1984; Lankford et al. 1988) and growth factors (Yankner and Shooter, 1982), both of which bind to specific transmem­ brane receptors. The matrix component with the most dramatic effect on growth cone motility is laminin in its various isoforms (Letourneau, 1975; Rogers et al. 1983; Carbonetto et al. 1983; Tomaselli et al. 1986). Its action is mediated primarily through the integrins (Turner et al. 1989; Bozyczko and Howitz, 1986; Horwitz et al. 1985; Hynes, 1987). Other matrix components which affect neurite outgrowth are fibronectin, tenascin, and type IV collagen (Grumet et al. 1985; Chiquet-Ehrismann et al. 1986; Rogers et al. 1983; Carbonetto et al. 1983). Cellbound adhesion molecules, such as NCAM, N-cadherin, LI, TAG-1 and F l l, also are important in controlling growth cone guidance (reviewed by Jessell, 1988). They function primarily via homophilic binding to like mol­ ecules on adjacent cells. It is clear that the growth cone has the capacity to transduce a wide variety of extracellular signals into altered motility and directed growth. In addition, there is evidence that neurons exhibit various intrinsic growth potentials (Banker and Cowen, 1979), which may reflect the regulated intracellular expression of certain genes (Skene and Willard, 1981; Benowitz and Lewis, 1983; Strittmatter and Fishman, 1991). GAP-43 is one such protein, with expression that is closely correlated with periods of active growth cone function. The levels of GAP-43 are dramatically increased within regenerating nerves after axonal injury (Benowitz and Lewis, 1983; Skene and Willard, 1981), and are higher during brain development than in the adult (Karas et al. 1987; Jacobsen et al. 1986). The expression pattern of GAP43 within the developing mouse brain closely reflects areas of active neurite outgrowth (Biffo et al. 1990). When cultured neurons are exposed to synaptic targets, the level of GAP-43 synthesis and axonal transport falls to 20 % of control levels (Baizer and Fishman, 1987). The correspon­ 27

dence of GAP-43 expression with active neurite outgrowth suggests that it may have a role in determining or modifying neurite extension.

GAP-43 structure and function GAP-43 is highly concentrated at the cytosolic face of the plasma membrane, especially in the growth cone (Meiri et al. 1986; Skene et al. 1986; Van Hooff et al. 1989). There is some data that GAP-43 directly regulates cell morphology. The expression of GAP-43 in non-neuronal cells enhances their propensity to form filopodia (Zuber et al. 1989a). This suggests that GAP-43 can interact with some generalized cell machinery to alter cell shape. In coordination with other growth cone proteins, it may modulate growth cone motility. Overexpression of GAP-43 in NGF-treated PC-12 cells does increase the extent of neurite outgrowth modestly (Yankner et al. 1990), although PC-12 strains with low levels of GAP-43 can extend neurites normally (Baetge and Hammang, 1991). What molecular interactions might underlie the func­ tion of GAP-43? The cDNA and the gene for GAP-43 have been cloned (Karns et al. 1987; Basi et al. 1987; Cimler et al. 1987; Grabczyk et al. 1990). The deduced sequence encodes a 226-amino acid protein with many charged residues and very little hydrophobicity. GAP-43 has been shown to bind to calmodulin in vitro (Alexander et al. 1988; Chapman et al. 1991). This interaction is strongest in the absence of calcium and at low ionic strength. The calmodulin binding site has been localized to amino acid residues 39-56 (Fig. 1). The region contains a serine residue at position 41, which is subject to phosphorylation

226

MEMBRANE TARGETING

CALMODULIN BINDING

Fig. 1. GAP-43 structure. A schem atic diagram illustrates the tw o dom ains in GAP-43 w ith identified functions. The aminoterm inal segm ent is responsible for mem brane binding and contributes to growth cone-targeting o f the m olecule (Zuber et al. 19896; Liu et al. 1991). The cysteines at position 3 and 4 are subject to palm itoylation (fatty acid (FA), Skene and Virag, 1989), w hich m ay be critical for m em brane association. This same region has a major role in stim ulating G0 (Strittm atter et al. 1990). The region betw een amino acid residues 3 9 -5 6 constitutes the calm odulin binding site (Alexander et al. 1988). The serine at position 41 can be phosphorylated by protein kinase C, decreasing the affinity of GAP-43 for calm odulin (Alexander et al. 1988; Chapman et al. 1991).

A

PEL B C

by protein kinase C (Coggins and Zwiers, 1989; Chapman et al. 1991). Phosphorylation of GAP-43 at this site inhibits calmodulin binding. The level of phosphorylation has also been correlated with the development of long term potentiation in vivo (Lovinger et al. 1985). Long term potentiation is a model for learning and memory, which may require synaptic remodelling. How calmodulin bind­ ing by GAP-43 might relate to growth cone motility is not well defined, but it has been suggested that GAP-43 serves as a ‘calmodulin sink’, regulating the level of free calmodulin (Alexander et al. 1988). There are a number of studies showing the importance of calcium levels in growth cone regulation (reviewed by Kater and Mills, 1991). Several other actions of GAP-43 have been described. Antibody to GAP-43 can alter the release of neurotrans­ mitter from isolated, permeabilized synaptosomes (Dekker et al. 1989), and the phosphorylated protein may inhibit phosphatidylinositol 4-phosphate (PIP) kinase activity (Van Dongen et al. 1985).

The amino terminus of GAP-43 and membrane binding We have studied how GAP-43 binds to the growth cone membrane, since this is a prerequisite for specific action at this site. GAP-43 behaves as an integral membrane protein, resisting extraction by high ionic strength, despite its lack of a domain containing hydrophobic amino acid residues (Skene and Virag, 1989; Karns et al. 1987). When expressed in non-neuronal cells, GAP-43 binds to the membrane (Zuber et al. 1989a). However, point mutations of GAP-43 at position 3 or 4 (the only two cysteines in the molecule) prevent membrane association in non-neuronal cells, demonstrating that the amino terminus is necessary for membrane association (Zuber et al. 19896, Fig. 1). The amino terminus is, in fact, sufficient for membrane binding. We showed this by expressing fusion proteins between the amino terminus of GAP-43 and chloramphenicol acetyl transferase (CAT). The first 10 amino acids of GAP-43 are sufficient to localize the normally cytosolic CAT to the membrane, even in non­ neuronal cells. This fusion protein behaves as an integral membrane protein, requiring detergent for extraction from the membrane and resisting solubilization with 1 m NaCl (Fig. 2). The amino terminus can undergo palmitoylation at positions 3 and 4 (Skene and Virag, 1989), and this may make the protein hydrophobic enough to partition into the membrane fraction. Alternatively, if the palmitates are added after membrane association, they might stabilize an interaction initiated by some other mechanism. The GAP-CAT fusion proteins also contain information for

SN D

A

B

C

D

M

C

Fig. 2. The amino term inus o f GAP-43 causes CAT to behave as an integral m em brane protein. A G AP(1_40)CAT fusion protein was expressed stably in P C -12 cells as described previously (Zuber et al. 19896). The mem brane fraction (M) and the cytosolic fraction (C) from these cells w ere analyzed for CAT im m unoreactivity on im m unoblots. Essentially all o f the fusion protein was found in the m em brane fraction. M em brane fractions were incubated w ith 1 % N onidet P40 (A), w ith 1 M N aCl (B), with 2 m M EGTA (C) or with 4m M CaCl 2 (D), and then centrifuged at 400 0 0 0 ^ for 30m in. The pellet (PEL) and supernatant (SN) were analyzed on im munoblots. N ote that detergent is required to extract the fusion protein from the membrane.

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S. M. Strittmatter et al.

distribution to certain sites within the membrane, as demonstrated by CAT immunohistochemistry after trans­ fection into PC-12 cells (Zuber et al. 19896). The chimeric proteins are localized to the same regions as native GAP43, including the growth cone. This suggests that this region of GAP-43 may interact with other proteins having restricted distributions in the plasma membrane. Such growth cone ‘receptors’ for intracellular GAP-43 have not yet been identified. Recent work by Storm and colleagues has shown that although the GAP-43 amino terminus can also localize a /3-gal fusion protein to the membrane, growth cone targeting does not occur for this protein in neurons (Liu et al. 1991). This may reflect a steric problem in targeting the larger /3-gal chimeric protein, or be specific to the small minority of neurons successfully transfected in these experiments, or it may be that other portions of GAP-43 are required for transport through the entire axonal length of nerve cells. The ability of the amino terminus of GAP-43 to target at least 50 % of the 13gal to the membrane in COS cells confirms this domain is largely responsible for membrane binding (Liu et al. 1991).

G0 is a major component of the growth cone membrane In order to provide a better understanding of the molecular events which underlie the growth cone’s ability to respond to this wide variety of extracellular and intracellular signals, we sought to identify some of the major proteins in the growth cone membrane. Fortunately, a subcellular

B

E

fractionation method for enriching in growth cone par­ ticles had been developed by Pfenninger et al. (1983). Analysis of growth cone membrane fractions reveals a remarkably simple protein composition (Simkowitz et al. 1989). Apart from actin and tubulin, there are two other major proteins detectable by SDS-PAGE. We employed a combination of electrophoresis, immunoblotting and par­ tial protein sequence to identify these two proteins as the a and b subunits of the GTP-binding protein, G0 (Strittmatter et al. 1990). Immunoblots of various fractions from this subcellular fractionation demonstrated enrichment of a0 in the growth cone membrane (Fig. 3). We also confirmed the growth cone localization by immunohistology, in both PC-12 cells and chick sympathetic neurons (Fig. 4). What might be the function of G„ in the growth cone? G0 is a member of the large family o f heterotrimeric GTPbinding proteins which link transmembrane receptors to intracellular second messenger systems (reviewed by Gilman, 1987). For example, G0 in the growth cone might respond to the neurotransmitter receptors for serotonin and dopamine, which are known to regulate potently growth cone motilty in certain neurons (Haydon et al. 1984; Lankford et al. 1988). Furthermore, the ability of pertussis toxin to abolish the effect of antibodies to LI and NCAM on intracellular second messengers suggests that a pertussis toxin-sensitive G protein, possibly G0, may be important in the action of the cell adhesion molecules (Schuch et al. 1989).

F

§li® mm

8§;

rn&UiI '■ Tm I■ iP k 'd >' S'v W$$mjj§ -

Fig. 3. Enrichm ent o f a0 im m unoreactivity in the growth cone mem brane. N eonatal rat brain was fractionated to prepare grow th cone mem branes (Simkowitz et al. 1989), and then analyzed for a0. Twenty ng o f protein from the homogenate (A), the low speed supernatant (B), the low speed pellet (C) and the high speed supernatant (D), and ten fig o f protein from the grow th cone particle fraction (E) and the growth cone mem brane fraction (F) w ere analyzed by im munoblots. The G0 lane is a control w ith 2 ,ug o f purified bovine brain G0. G0 is enriched in the growth cone particles, and especially in the growth cone mem brane. Filled squares, position o f M r markers o f 116 000, 84 000, 58 000, 48 500, 36 500 and 26 600 from top to bottom. Bar, position o f a0.

..

ft. V

:

-

A ■, ;

Fig. 4. Localization o f a0 to the grow th cones o f chick sym pathetic neurons. The sym pathetic chain was rem oved from ten day old chick embryos, and dissociated cells were plated on a lam inin-coated surface for 3 h. The cells were fixed and stained for a0 im m unoreactivity as previously described for PC-12 cells. The grow th cone in the m iddle o f the figure is enriched in a0. Bar, 15 f/m.

Growth cone transduction: G0 and GAP-43

29

Interaction of G0 and GAP-43 G0 and GAP-43 have indistinguishable distributions at the light microscopic level in NGF-treated PC-12 cells. Both are highly concentrated in the tips of many neuritic processes (Fig. 5), whereas other processes appear to lack both (not shown). This high degree of concordance between G0 and GAP-43 immunoreactivity led us to explore the possibility that GAP-43 might interact with G0. The activation of G0 can be monitored by its guanine nucleotide-binding properties (Gilman 1987). In the basal state, G0 contains tightly bound GDP. When activated by receptor-ligand complexes, the rate of GDP release is increased, and GTP then binds. The GTP-bound a subunit activates appropriate second messenger systems. GTP hydrolysis terminates activation. GAP-43 alters the guanine nucleotide-binding characteristics of G0, by increasing its level of GTPyS binding (Fig. 6, Strittmatter et al. 1990). The increase in GTPyS binding cannot be

GAP-43 (log M) Fig. 6. GAP-43 stim ulates GTPyS binding o f G0. [35S]-GTPyS binding (■ ) to a fixed concentration o f G0 at the indicated concentrations o f GAP-43 was m easured as described previously (Strittm atter et al. 1990).

T able 1. Comparison o f GAP-43- and G protein-linked receptors Similarities Increase GDP release Increase GTPyS binding Increase steady state GTPase Unchanged GTPase &cat Small region of homology

Differences Pertussis toxin blockade /3y subunit requirement Phospholipid dependence Cellular location Overall structure

The rate of release o f [3H]GDP, the initial rate of [35S]GTPyS, the rate of steady state GTPase and the turnover number for bound GTP were determined as described previously (Linder et al. 1990). Each rate was determined in the presence and the absence o f 1 ,um GAP-43. The small region o f homology between GAP-43 and receptors has been noted previously (Strittmatter et al. 1990). GTPyS binding to pertussis toxintreated G0 is stimulated to the same degree as untreated G0 by GAP-43. GAP-43 stimulates GTPyS binding to isolated aQ as effectively as to a jiy trimer. Reconstitution of Gc into phosphatidylcholine vesicles (Cerione et al. 1984) did not change the ability of GAP-43 to stimulate GTPyS binding. The response o f receptor/G protein coupling to these experimental conditions has been documented (Gilman, 1987).

Fig. 5. Co-localization o f a0 and GAP-43 in PC-12 cells. PC-12 cells were treated with NGF, fixed and stained for a0 as described previously (Strittm atter et al. 1990) using a fluorescein-labelled, anti-rabbit IgG antibody (lower panel). The same section was also stained for GAP-43 using a mouse monoclonal anti-GAP-43 and Texas Red-labelled, anti-mouse IgG antibody (upper panel). Both proteins are enriched in the growth cone region o f the tw o neurites shown. Bar, 15,um.

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S. M. Strittmatter et al.

explained by changes in G0 thermal stability, as GAP-43 has no effect on this parameter (not shown). This effect is saturable, with an EC50 of about 300 nM GAP-43 (Fig. 6). The concentrations of Gc and GAP-43 in vivo probably exceed this level. GAP-43 is the first potential intracellular regulator of G proteins. Therefore, we compared the mechanism of G0 activation by GAP-43 and receptors (Table 1). GAP-43 is clearly a guanine nucleotide release protein (GNRP), like receptors. GAP-43 nearly doubles the rate of release of bound GDP. Since GDP release is the rate limiting step in GTP binding, GAP-43 also doubles the initial rate of GTPyS binding to G0 and the steady state GTPase activity of G0. Also like receptors, the intrinsic kcat of G0 for the hydrolysis of bound GTP is not altered by GAP-43. Since, in all of these regards, the GAP-43 effect resembles that of the G protein-linked receptors (Gilman 1987), it is clear that GAP-43 is a GNRP, and that it does not have activity as a GTPase-activating protein (GAP). In several other respects, however, GAP-43 action on G0 can be distinguished from that of receptors (Table 1). Pertussis toxin treatment of G0 prevents its regulation by

receptors, but not by GAP-43. GAP-43 can double the rate of GTPyS binding and GTPase of isolated recombinant aQ, while receptors require /3y in addition to a subunits for productive interactions. GAP-43 is equally effective at stimulating detergent soluble- and vesicle-incorporated G0, whereas receptors stimulate G proteins only when the two are incorporated into phospholipid vesicles. Thus, by these mechanistic studies GAP-43 is a GNRP, but is clearly distinguishable from those previously studied. We have begun to define the domains of GAP-43 that contribute to its stimulation of G0. The amino terminus of GAP-43 is known to be critical for association of GAP-43 with the plasma membrane and for growth cone localiz­ ation. Residues 39-56 include a calmodulin-binding site,

and there is a phosphorylation site for protein kinase C at serine 41 (Fig. 1, Alexander et al. 1988; Chapman et al. 1991; Coggins and Zwiers, 1989). A synthetic peptide corresponding to the calmodulin binding site has no effect on G0 activity (Strittmatter et al. 1990), and 40 [im calmodulin does not alter the GAP-43/G0 interaction (not shown). This suggests that the calmodulin-binding domain of GAP-43 is not important for G protein interaction. In contrast, a peptide composed of the first 25 amino acids o f GAP-43 does stimulate GTPyS binding to G0 (Strittmatter et al. 1990). Although the amino terminal peptide requires a twenty to hundred-fold higher concen­ tration than does intact GAP-43 for G0 activation, the

o ut •cams: GCM] in

1. CYTOSKELETON 2 . MEMBRANE DYNAMI CS 3. OTHER

1 GROWTH CONE : : m o tility Fig. 7. A m odel for growth cone transduction. G0 is a major protein in the grow th cone m em brane (GCM ), and can be regulated by receptors (R) o f the seven transm em brane dom ain fam ily (Gilm an, 1987), and by GAP-43 (Strittm atter et al. 1990). The receptors respond to extracellular concentrations o f ligands such as neurotransm itters. GAP-43 levels m ay function as an intracellular growth signal (Strittm atter et al. 1991). A fter hom ophilic binding to like m olecules on adjacent cells, cell adhesion m olecules o f the im m unoglobulin superfam ily m ay regulate G proteins (Schuch et al. 1989), as well as the cytoskeleton. Activated G0 could both stim ulate phospholipase C (PLC) (M oriarty et al. 1990) and regulate ion channels (Dunlap et al. 1987; H escheler et al. 1987; Van D ongen et al. 1988). PLC activation produces diacylglycerol (DAG) and inositol triphosphate (IP3), the form er activating protein kinase C (PK C), and the later increasing intracellular free calcium ([Ca2+li). PKC activation leads to phosphorylation o f various substrates, including GAP-43 (Chan et al. 1986; Chapman et al. 1991; Coggins and Zwiers, 1989), and can modulate grow th cone m otility (Bixby, 1989). In a possible feedback loop, phospho-GAP-43 inhibits the form ation o f phosphatidylinositol 4,5-bisphosphate (PIP2), the substrate for PLC (Van Dongen et al. 1985; Van H ooff et al. 1986). Dephospho-GAP-43 binds to free calm odulin, perhaps localizing or m odulating the action o f [Ca2+]j (Alexander et al. 1988). The level o f [Ca2+]i could be regulated by G0 both through IP3 and through direct action on Ca2+ and K + channels. Calcium ions have a profound effect on grow th cone m otility (K ater and M ills, 1991). Ca2+/ca lm od u lin dependent kinase (CaM kinase) m ay contribute to the action o f [Ca2+li. W hether calcium ’s effect on neuronal shape is m ediated via cytoskeletal changes (Yam ada and W essells, 1973; Sm ith, 1988; Bam burg et al. 1986), alteration in mem brane dynam ics (Lockerbie et al. 1991; Cheng and Reese, 1987) or som e other m echanism is unclear. PIP, phosphatidylinositol 4-phosphate.

Growth cone transduction: G0 and GAP-43

31

effect on G0 is two-fold and saturable, as it is for the protein. These findings demonstrate the importance of this domain in G protein interaction. We compared this domain of GAP-43 with the portions of G protein-linked receptors necessary for coupling (Stritt­ matter et al. 1990). Despite the fact that the two proteins have disparate overall structures, a small region of homology exists between GAP-43 and receptors. This stretch, at the GAP-43 amino terminus, contains the sequence nonpolar-nonpolar-cys-cys-x-basic-basic, with the cysteines subject to palmitoylation (Skene and Virag, 1989, O’Dowd et al. 1989).

membrane receptors and GAP-43 places it at a central position in the growth cone control apparatus (Fig. 7). Extracellular signals could bind to transmembrane recep­ tors and then be transduced into second messanger changes by G„. In addition, G0 might respond to intracellu­ lar gene programs for growth, as signaled by increased GAP-43 synthesis. Thus both extracellular and intracellu­ lar signals to the growth cone would converge on, and be integrated by, G0. Whether such G0 activation enhances or retards neurite extension is under study.

References Discussion

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The evidence that G0 is a major component of the growth cone membrane suggests a critical role for this signaltransducing protein in coordinating growth cone function. Fig. 7 is a model incorporating notions from several investigators as to how the growth cone might be regulated, one that places heavy emphasis upon G0, GAP43 and the calcium ion. Although the signals are not known with certainty, growth cone G0 might respond to the neurotransmitter receptors and possibly to some celladhesion molecules. The potential role of G0 in transduc­ ing a number of other extracellular signals has not been investigated. There is evidence in other systems that G0 can link receptors to several effector systems: Ca2+ channels (Holz et al. 1986; Heschler et al. 1987), phospholi­ pase C (Moriarty et al. 1990), K + channels (Von Dongen et al. 1988) and phospholipase A2 (Bloch et al. 1989). Since the first three of these effectors can alter intracellular Ca2+ levels, and since the level of Ca2+ is a major regulator of growth cone activity (Kater and Mills, 1991), many G0-linked signals may regulate growth cone dy­ namics via the Ca2+ ion. Alternatively, G0 activation of phospholipase C may lead to diacylglycerol-mediated protein kinase C phosphorylation of various substrates in the growth cone. Protein kinase C activation has been implicated in laminin stimulation of neurite outgrowth (Bixby, 1989). Whether the final mediator in these pathways is the cytoskeleton (Yamada and Wessells, 1973; Smith, 1988; Bamburg et al. 1986), membrane cycling (Lockerbie et al. 1991; Cheng and Reese, 1987) or some other mechanism remains to be seen. G0 responds to both transmembrane receptors (Gilman, 1987) and the intracellular protein GAP-43 (Strittmatter et al. 1990). GAP-43 has GNRP activity as do receptors, and the two proteins share a small region of sequence homology. However, the mechanism of action of GAP-43 can be distinguished from that of receptors in several ways. This defines GAP-43 to be in a new class of GNRP. The differences between GAP-43 and receptors suggest that GAP-43 interacts with different or additional region of ft'o- GAP-43 also appears to affect a different domain than do ¡3y subunits because there is no competition or synergism between GAP-43 and fly. This raises the possibility that GAP-43 might interact directly with either the guanine nucleotide-binding domain or the effector domain. If GAP-43 acted at the effector domain, its primary role might be as downstream responder to activated Gc rather than a positive upstream regulator of G0-like receptors. However, since it enhances GDP release, and therefore interacts with the GDP-bound form, it is more likely an upstream positive regulator of G0. The possibility that G0 might respond to both trans­ 32

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