WO1993006851A1 - Mammalian gap-43 compositions and methods of use - Google Patents

Abstract

This invention relates to regulation of G protein functions and the related axonal growth. The invention further relates to the discovery that GAP-43 and palmitoylated GAP-43 and biologically active peptides derived therefrom function to modulate cell function. The present invention also is related to the clinical in vivo and in vitro diagnostic and therapeutic applications of GAP-43 and palmitoylated GAP-43 and their regulatory and membrane-targeting elements. Also disclosed is a method of palmitoylating the GAP-43 protein.

Description

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TΪT E OF THE INVENTION

MAMMALIAN GAP-43 COMPOSITIONS AND METHODS OF USE

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the fields of molecular genetics and neurology. More particularly, the invention relates to regulation of G protein functions and the related axonal growth. The invention further relates to the discovery that GAP-43 and palmitoylated GAP-43 and biologically active peptides derived therefrom function to modulate cell function. The present invention also is related to the clinical in vivo and in vitro diagnostic and therapeutic applications of GAP-43 and palmitoylated GAP-43 and their regulatory and membrane-targeting elements in, inter alia, neurological indications in animals including humans.

Description of the Background Art

A diverse group of post-translational modifications occurs in eukaryotic cells. Proteolytic processing, glycosylation, amino terminal myristoylation, phosphatidylinositol lipid linkage, isoprenylation, proline hydroxylation and carboxy terminal amidation are required to synthesize the active form of various proteins. Phosphorylation has a more dynamic role, with cycles of phosphorylation-dephosphorylation controlling the function of certain proteins. Palmitate is linked to cysteine residues via a thioester in a number of proteins including GAP-43, ank rin, major histocompatibility antigens, transferrin receptor, large T antigen SV40 virus, ros-like proteins, proteolipid protein of myelin, viral coat proteins, rhodopsin, and J₂-adrenergic receptor (Sefton et al,J. CelL Biol 104:1449- 1453 (1987); Schultz et aL, Annu. Rev. CelL BioL 4:611-647 (1988)). Where studied, this modification is dynamic, turning over with a much shorter t^ than the protein (Sefton et aL, J. CelL BioL 104:1449-1453 (1987); Schultz et aL, Annu. Rev. Cell BioL 4:611-647 (1988); Magee et aL, EMBOJ. 6:3353-3357 (1987); Staufenbiel et aL, Proc NaiL Acad. Sc USA £5:318-322 (1986); and Skene et al, J. Ce1L BioL 105:613-624 (1989))). GAP-43 is one of those proteins that is subject to palmitoylation

(Skene et al, J. CelL BioL 108:613-624 (1989)). It is a neuronal protein which is highly enriched in the tips of extending processes, a region termed the growth cone (Meiri et aL, Proc NatL Acad. Set USA 833537- 3541 (1986); Skene et aL, Science 253:783-785 (1986)). This localization, the protein's massive induction during nerve regeneration (Skene et aL, J. Cell BioL £9:96-103 (1981); Benowitz et al, J. NeurpscL 3:2153-2163 (1983)), and its increased gene expression during development (Karns et al, Science 236:597-600 (1987)) have suggested that GAP-43 may control growth cone motility (Skene, J.H.V.Annu. Rev. NeuroscL 12:127- 156 (1989)); Strittmatter et al, Nature 544:836-841 (1990)). Expression of GAP-43 in on-neuronal cells enhances filopodial formation (Zuberef al, Science 244:1193-1195 (1989)), and introduction of antibodies against GAP-43 into neurons blocks neurite outgrowth (Shea et al, J. NeuroscL 21:1685-1690 (1991)). At a molecular level, GAP-43 may function by altering the activity of G₀ (See U.S. Serial No. 07/683,455, filed April 10, 1991, which is incorporated in its entirety herein by reference). This heterotrimeric GTP binding protein is also enriched in the axonal growth cone, where is the major non-cytoskeletal protein. G₀ may transduce many of the extracellular signals which determine die extent and direction of axonal growth; some of these signals known to bind to G protein-linked receptors. Pharmacological agents which alter G protein activity have dramatic effects on neurite outgrowth in tissue culture (Vartanian et at, 1991). The ability of GAP-43 to stimulate GTPγS binding to G₀ may alter the response the growth cone G protein-based signal transduction system to extracellular morphogens. Whether some of the other properties GAP-43, such as calmodulin binding (Chapman et al, J. BioL Chem. 266:207-213 (1991)), protein kinase C substrate (Lovinger et al, Brain Res. 545:137-143 (1985)) and regulation of neurotransmitter release (Dekker et al, Nature 542:74-76 (1989)), are separate functions or are related to G protein coupling is not known. The C protein-interacting domain of GAP-43 has been localized to the amino terminal 25 amino acids (Strittmatter et al, Nature 544:836-841 (1990)).

More specifically, the disclosure in U.S. Serial No. 07/683,455 has demonstrated that the GAP-43 protein is encoded in three exons. The short (10 amino acid residues) amino-terminus exon was considered to encode a membrane-targeting peptide domain. Experiments in which large portions of the second GAP-43 exon were removed did not affect membrane binding of the remaining protein. Similarly, replacing the carboxy-terminus of GAP-43 had no effect on membrane binding. However, a synthetic GAP-43 gene lacking the initial four amino acids (MET LEU CYS CYS), and beginning at the MET of position five failed to bind to the membranes of neuronal or non-neuronal host cells, indicating that the first exon is responsible for this membrane-targeting function.

Pa nitoylation of GAP-43 occurs on the only two cysteine residues in the molecule, at positions 3 and 4 (Skene et aL, J. CelL Biol 108:613- 624 (1989)), within the region necessary for G protein stimulation. This modification probably contributes to the membrane binding of the otherwise hydrophilic GAP-43 protein (Skene et al, J. CelL BioL 108:613- 624 (1989); Zuber et al, Nature 542:345-348 (1989)).

The growth cone membrane can be considered a highly specialized device for the transduction of extracellular signals and intracellular growth programs into changes in cell shape (Strittmatter et al, Bioessays 25:127- 134 (1991)). By analyzing the components of the growth cone membrane, and their interaction, a better understanding of the pathways controlling cellular form may emerge.

One of the most prominent components of the neuronal growth cone membrane is the heterotrimeric GTP-binding protein, G_σ

Strittmatter et al, Nature 544:836-841 (1990)). It is known to couple transmembrane receptors for extracellular ligands to intracellular second messenger systems (Gilman, A.G. Annu, Rev. Biochem. 56:615-649

(1987)). Several neurotransmitters can cause growth cone collapse (Kater et al, J. NeuroscL 22:891-899 (1991)), and these effects are likefy to be transduced by G₀. Cell adhesion molecules have prominent effects on neurite outgrowth, and there is evidence that some of these may be mediated by a pertussis toxin-sensitive G protein, such as G₀ (Schuch et al, Neuron. 5:13-20 (1989)). There is also direct evidence that pharmacologic agents which alter G protein activity change the rate of neurite outgrowth (Vartanian et al, NeuroscL Abstr. 17:16 (1991)).

Activation of a pertussis toxin-sensitive G protein blocks neurite outgrowth, and inhibition promotes outgrowth. Thus, G₀ may have a . major role in transducing a number of the extracellular signals which alter axonal extension. Evidence for G protein transduction of other developmentally regulated cell orphogens comes from the Cta gene of

Drosophila (Parks et al, Cell 64:447-458 (1991)) and the frigid mutation in Dictyostelium (Deverotes, P. Science 245:1054-1058 (1989)).

Another growth cone membrane-enriched protein is GAP-43 (also called B-50, Fl, pp46, neuromodulin, reviewed by Skene, Skene, J.H.P. Annu. Rev. NeuroscL 22:127-156 (1989)). This protein was originally identified as an axonally transported molecule induced 10-100 fold during nerve regeneration (Skene et al, J. Cell Biol £9:96-103 (1981); Benowitz et al, J. NeuroscL 5:2153-2163 (1983)). The level of GAP-43 drops when neurons reach their synaptic targets (Bazier et al, J. NeuroscL 7:2305-2311 (1987)), and decreases as the brain matures (Karns et al, Science 236-597- 600 (1987)). The correlation of GAP-43 expression with periods of neurite extension has led to the proposal that it may affect growth cone motility, and there is some direct evidence that GAP-43 can alter cell shape. Expression of GAP-43 in non-neuronal cells transiently enhances the propensity to filopodial formation (Zuber et al, Science 244:1193-1195 (1989)). Over-expression of GAP-43 in pheochromocytoma cells increases NGF-induced neurite outgrowth (Yankner et al, Mol Brain Res. 739-44 (1990)), and intracellular anti-GAP-43 decreases neurite growth from neuroblastoma cells under certain conditions (Shea et al, J. NeuroscL 22:1685-1690 (1991)). However, GAP-43 is not necessary for outgrowth as suppression of GAP-43 in PC-I 2 cells with dexamethasone (Federoff et al, J. Biol Chem. 263:19290-19295 (1988)), or its constitutive absence from some PC-I 2 strains (Baetge et al, Neuron 6:21-30 (1991)), does not affect neurite outgrowth.

The molecular details of GAP-43 action are not clear. The highly acidic, hydrophilic protein has a small region at the amino terminus which is responsible for the membrane attachment of the protein, and probably its growth cone localization (Zuber et al, Nature 542:345-348 (1989)). The cysteines at position three and four can be palmitoylated in vivo, and this presumably contributes to the membrane association of the molecule (Skene et al, J. Cell Biol 108:613-624 (1989)). Several investigators have proposed a role for GAP-43 in transduction, since it inhibits phosphatidylinositol 4-phosphate kinase (Oestricher et al, J. Neurochem. 42:331-340 (1983)), contains a cahnodulin binding site (residues 39-56, Chapman, 1991), is a substrate for protein kinase C (serine 41, Coggins et al, J. Neurochem. 55:1895-1901 (1989)), and may contribute to depolarization-induced neurotransmitter release (Dekker et al, Nature 542:74-76 (1989)).

SUMMARY OF THE INVENTION

Recognizing the potential importance of GAP-43 in mammalian CNS function and disease, the present inventors have succeeded in finding that palmitqylation of GAP-43 amino terminal peptides, or GAP-43 protein prevents interaction with G₀. Thus, palmitoylation is a novel type of dynamic regulatory protein modification for GAP-43. Inasmuch as the ability to modulate GAP-43 expression may be of great therapeutic utility in treating mammals, and particularly humans, suffering from damage to, or from disease or dysfunction of, the central or peripheral nervous system, the significance of these discoveries will be readily apparent GAP-43 stimulation of G. suggests a novel type of intracellular regulation for G proteins, which have been thought of as responding only to the concentrations of extracellular ligands for transmembrane receptors. The present inventors therefore, developed an assay for GAP- 43 effects on cell shape, and then made various alterations in GAP-43 and G proteins to test their interaction. This assay shows that GAP-43 effects on cell spreading in non-neuronal cells are caused by changes in G protein transduction, and that GAP-43 may uncouple some receptors from G proteins.

In view of the findings presented herein, the present invention provides for palmitoylated mammalian GAP-43, or a functional derivative thereof. Also provided by the present invention is a method of inactivating G protein activity comprising administering an effective amount of palmitoylated GAP-43.

In another aspect of the present invention, a pharmaceutical composition comprising an effective amount of palmitoylated GAP-43 and a pharmaceutically acceptable carrier is provided.

Additionally, the present invention provides for a peptide comprising an amino acid sequence selected from the group consisting of I. MET LEU CYS CYS MET ARG ARG THR LYS GLN; H. MET LEU CYS CYS MET ARG ARG THR LYS; m. MET LEU CYS CYS MET ARG ARG THR;

IV. MET LEU CYS CYS MET ARG ARG;

V. MET LEU CYS CYS MET ARG; VL MET LEU CYS CYS MET, VH. MET LEU CYS CYS; and

Viπ. functional derivatives thereof, wherein at least one of the CYS components is palmitoylated.

This amino acid sequence is capable of being effectively incorporated into a pharmaceutical composition. In a further aspect of the present invention, a method of palmitoylating GAP-43 protein is provided. The method comprises contacting the GAP-43 protein with CoA-plamitate. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood by reference to the Description of the Preferred Embodiments when taken together with fheattached drawings, which are described as follows:

Figure 1. GAP-43 amino terminal peptides stimulate G-.

Peptides of the indicated length (left) with the same sequence as GAP-43 protein were added at 100 uM to G₀, and GTPγS binding was measured. The level of increase in GTPγS binding, as compared to no addition, is indicated at right Note that the 1-6 through 1-25 peptides stimulate binding of GTPγS to GQ, but that the 1-10 peptide with threonines in place of cysteines and the 11-25 peptide do not effect G₀. The results are the average of 3-6 separate determinations, which varied by less than 20%.

Figures 2 and 2B. Palmitoylated peptides are less effective in stimulating G„

(Figure 2A) GAP-43 amino terminal peptides of 25 amino acid residues without palmitate (■) or with pahnitate linked to cysteines at position 3 (•), position 4 (^), or position 3 and 4 (♦) were added 1 G₀- GTPγS binding assay. Note that over the range of peptide concentrations indicated, the nonpalmitoylated peptide augments GTPγS binding, while the singly palmitoylated peptides are less stimulatory and the dipalmitoylated peptide has no effect An average of three experiments with similar results is shown. In Figure 2B, the binding of GTPγS to G₀ is shown as a function of timing with the no addition (■), 100 μM nonpalmitoylated ϊ-25 peptide (•) or 100 μM dipalmitoylated 1-25 peptide (- ). Note that the nonpalmitoylated peptide increases the initial rate of binding to G₀ by 55%, but that the dipalmitoylated form has no effect One example from 3 with similar results is illustrated.

Figure 3. Palmitoylated GAP-43 peptides do not stimulate vesicle- incorporated G₀.

G₀ was incorporated into phosphatidylcholine vesicles and then assayed for GTPγS binding in the presence of the indicated 1-25 amino terminal GAP-43 peptides at 100 μM. Note that the nonpalmitoylated peptide stimulated GTPγS binding to vesicle-incorporated GQ, but the dipalmitoylated peptide had no significant effect on binding to either preparation.

Figure 4. Palmitoylation of GAP-43 non-enzvmatically.

GAP-43 incubated with (^wC)CoA-palmitate as described in Procedures, a then boiled in 2% SDS and electrophoresed through a 10% polyaciylamide gel. The presence of GAP-43 was detected by Commassie Blue staining in fraction 8. Note that the same fraction contains a peak of radioactivity (■). When GAP-43 was omitted from the sample all radioactivity was at the dye front (A). If the sample was incubated with 1 M neutral hydroxylamine prior to electrophoresis, then no peak of radioactivity was detected (•).

Figure 5. Separation of palmitoylated from non-palmitoylated GAP-43

After incubation with (¹ C)CoA-palmitate, a purified GAP-43 preparation was applied to a phenyl-sepharose resin and eluted decreasing KC1 concentrations. Non-palmitoylated GAP-43 (1 mg total protein, ■■■) does not bind to this column. The palmitate-modified GAP-43 (3 mg total protein, ) is separated into two fractions by this procedure.

The protein which elutes in the void volume contains no (^MC)pa_mitate co-migrating with GAP-43 on SDS-PAGE, but the second peak has a molar ratio of protein to palmitate of 1 to 1.5 when analyzed as in Figure 4. The region indicated as "Pal-GAP" was used in further experiments as palmitoylated GAP-43.

Figures 6A and 6B. Palmitoylation blocks GAP-43 stimulation of G O, i

GAP-43 (■) and palmitoylated GAP-43 (•) protein were added to a G< GTPγS binding assay at the indicated concentrations. Note that GAP-43 stimulates binding to 230% of control levels, but that the palmitoylated protein produces binding levels which are only 140% of control values. The data are averaged from 5 separate experiments with similar results.

Figure 7. Hvdroxalamine restores the activity of palmitoylated GAP-43

The level of GTPγS binding to G₀ in the presence of different μM GAP-43 protein preparations is shown. The palmitoylated protein is only 35% as effective in stimulating binding as nonpalmitoylated GAP-43. When both preparations are treated with 1 M hydroxalmamine to cleave thioester bonds, the previously palmitoylated protein is nearly as active as the nonpalmitoylated sample. The data from one experiment of 3 with similar results shown. Figure 8. Palmitoylation as a dynamic regulator of the GAP-43/G

A schematic model illustrates how palmitovlation might prevent GAP-43 from stimulating the activation of the a subunit of G₀. The palmitoylated form of GAP-43 is shown as more tightly adherent to the membrane. The interaction of nonpalmitoylated GAP-43 with G₀ causes the release of bound GDP, and the binding of GTP. Then, the activated subunit can alter the activity of various effector systems.

Figures 9A-E. Decreased spreading of A431 cells transfected with GAP-43

Four control cell lines transfected with pDOJ (a,b,c,d) exhibit a more flattened, spread phenotype as compared to four GAP-43-expressing A431 cell lines (e,f,g,h). These cells were fixed 2 h after plating. The scale bar is 50 μ.

Figure 10. Quantitation of decreased spreading in GAP-43 transfectants.

Control or GAP-43-expressing CHO cell lines (a) or A431 lines

(b,c) were assessed for cell spreading. The data are shown for n lines with standard errors. The spreading assay was on laminin-coated glass (a,b) or poly-I_-lysine-coated glass (c).

Figures 11A and 11B. Immunoblot detection of GAP-43 in A431 transfectants.

A431 cell lines transfected with a GAP-43 expression vector (a,b,c,d,e), with both GAP-43 and a_a vectors (f,g,h,i), or with control plasmid (j,k) were analyzed for GAP-43 expression. Note that control cells do not express GAP-43 but that the transfected do so. Figures 12A-C. GAP-43 does not alter adhesion of A431 cells.

The adhesion of metabolically labelled GAP-43 and control A431 lines was determined as in Experimental Procedures. The percent of radioactivity adherent to plates is plotted with standard errors for n separate cell lines.

Figure 13A-C. GAP-43 mutants alter COS cell spreading.

COS-7 cells were transfected with expression vectors encoding GAP-43 (a, b), GAP-43 with cys³ and cys⁴ mutated to thr (c), GAP-43 with cys⁴ mutated to thr (d), GAP-43 with phe⁴² changed to ala (e), or a fusion protein of GAP-43(1 -40) followed by CAT (f). Cells expressing the transfected DNAs are visualized by immunofluorescence for GAP-43 (a,b,c,d,e) or CAT (f). Note that the GAP-43 cells have smaller areas than those with the amino terminal mutations. The phe⁴² mutation and the fusion protein produce small areas just like the intact GAP-43 transfectants. The scale bar is 50 μ.

Figure 14. Ouantitation of spreading by COS cells expressing different GAP-43 mutations.

The percent of spreading byimmunofiuorescent cells was measured after transfections with the indicated DNA molecules. The error bars indicate the standard error from 3-5 separate transfections.

Figure 15. Immunoblot detection of a_^ in transfected A431 cell lines.

A431 cell lines transfected with expression vectors for _Q (a,b,c,d), for both a₀ and GAP-43 (e), or for neither protein (f,g,h) were analyzed for ₀ expression by immunoblot Note that those cells with the a₀ vector produce ₀ protein but control cells do not

Figure 16. GAP-43 increases spreading of _r₀-A431 cells.

A431 cell lines transfected with control plasmid (a,b), α₀ expression vector (c,d), or with both ₀ and GAP-43 vectors (e,f) were assayed for cells spreading. Note that the area of the a₀ transfectants is less than control, and that the doubly transfected cells are larger than the ₀- transfected cells. The scale bar is 50 μ.

Figure 17. Ouantitation of GAP-43 effect on the spreading of α--A43 1

The spreading of cell lines of the type shown in Fig. 16 was measured as in Experimental Procedures. The percentage of spread cells for n cell lines of each type is illustrated wit standard errors.

Figure 18. Pertussis toxin and mastoparan block GAP-43 action on A431 spreading.

A control cell line (a,c,e) and a GAP-43 expressing line (b,d,f) were assayed for spreading in the presence of no additions (a,b), pertussis toxin

(c,d), or mastoparan (e,f). Whereas the GAP-43 cells are smaller without additions, they are indistinguishable from control cells in the presence of either drug. The scale bar is 50 μ. Figures 19A and 19B. Ouantitation of the effect of mastoparan and pertussis toxin on A431 spreading.

Six control lines (Con) and six GAP-43 lines (GAP) were plated without additions, with pertussis toxin or with mastoparan, and the degree of cell spreading measured. The mean percentage of spread cells with standard error is shown in Figure 19 A. The ratio of spreading in control cells to that in GAP-43 cells is shown in Figure 19B.

Figure 20. GAP-43 blocks isoproterenol stimulation of cAMP levels.

Control and GAP-43-expressing A431 cells were assayed for cAMP level in the presence of no drugs (Basal), 100 nM isoproterenol (Iso) or

100 nM isoproterenol with 10 uM propranolo (Pro) . The mean values with standard errors for determinations from n separate cell lines is shown.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following description, reference will be made to various methodologies known to those of skill in the art of molecular genetics and neurology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full. Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J.D. et al, Molecular Biology of the Gene, Volumes I and π, The Benjamin/Cunuαings Publishing Company, Inc., publisher, Menlo Park, CA (1987); Darnell, J.E. et al, Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, B.M., Genes II, John Wiley & Sons, publishers, New York, N.Y. (1985); Old, R.W., et al, Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2d edition, University of California Press, publisher, Berkeley, CA (1981); and Maniatis, T., et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, NY (1982).

By "cloning" is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.

By "cDNA" is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Thus a "cDNA clone" means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.

By "cDNA library" is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire genome of an organism. Such a cDNA library may be prepared by methods known to those of skill, and described, for example, in Maniatis et aL, Molecular Cloning: A Laboratory Manual supra. Generally, RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene. Preferred for the purposes of the present invention are mammalian, and particularly human, cell lines.

By "vector" is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. Thus, by "DNA expression vector" is meant any autonomous element capable of replicating in a host independently of the hosf s chromosome, after additional sequences of DNA have been incorporated into the autonomous element's genome. Such DNA expression vectors include bacterial plasmids and phages. Preferred for the purposes of the present invention is the lambda gtll expression vector.

By "substantially pure" is meant any antigen of the present invention, or any gene encoding any such antigen, which is essentially free of other antigens or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature. By "functional derivative" is meant the "fragments,"

"variants," "analogs," or "chemical derivatives" of a molecule. A "fragment" of a molecule, such as any of the cDNA sequences of the present inven- tion, is meant to refer to any nucleotide subset of the molecule. A

"variant" of such molecule is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof. An "analog" of a molecule is meant to refer to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.

A molecule is said to be "substantially similar" to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesir¬ able side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Penn. (1980).

Similarly, a "functional derivative" of a gene of any of the antigens of the present invention is meant to include "fragments," "variants," or "analogues" of the gene, which may be "substantially similar" in nucleotide sequence, and which encode a molecule possessing similar activity. A DNA sequence encoding GAP-43 or its functional derivatives, or the membrane-targeting peptide or functional derivatives thereof, may be recombined with vector DNA in accordance with conventional techni¬ ques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., et al, supra, and are well known in the art

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box; capping sequence, CAAT sequence, and the like.

If desired, the non-coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and pofyadenylation. Thus, by retaining the 3'-region naturally contiguous to the DNA sequence coding for the protein, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.

Two DNA sequences (such as a promoter region sequence and a GAP-43 encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the GAP-43 gene sequence, or (3) interfere with the ability of the GAP-43 gene sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.

Thus, to express the protein, transcriptional and translational signals recognized by an appropriate host are necessary.

The present invention encompasses the expression of the GAP-43 protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells (as described in U.S. Serial No. 07/683,455, filed April 10,

1991, which is incorporated herein by reference in its entirety) and subsequent palmitøylization of the protein.

The GAP-43 encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the GAP-43 protein may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotropic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co- transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol Cel BioL 5:280 (1983).

In another embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host Any of a wide variety-of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector, the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. Preferred prokaiyotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, •π-VX. Such plasmids are, for example, disclosed by Maniatis, T., et al. In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). Bacillus plasmids include pC194, ρC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pUlOl (Kendall, KJ., et aL, J. Bacteriol 269:4177-4183 (1987)), and streptomyces bacteriophages such as φ C31 (Chater, K.F., et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp.45-54). Pseudomonas plasmids are reviewed by John, J.F., et al (Rev. Infect Dis. 8:693-704 (1986)), and Izald, K. (Ipn. J. Bac- terioL 55:729-742 (1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2- micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D., et al, Miami Wntr. Symp. 29:265-274 (1982); Broach,

J.R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY, p.445-470 (1981); Broach, J.R., Cell 28:203-204 (1982); Bollon, D.P., et al, J. Clin. HematoL Oncol 20:39-48 (1980); Maniatis, T., In: Cell

Biology: A Comprehensive Treatise, Vol 3, Gene Expression, Academic

Press, NY, pp. 563-608 (1980)). Once the vector or DNA sequence containing the constructs) has been prepared for expression, the vector or DNA constructs) may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoefhyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al, Science 240(4858):153S (1988)), etc.

After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the GAP-43 protein, or in the production of a fragment of this protein. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromo- deoxyuracil to neuroblastoma cells or the like).

The expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like. However, GAP-43 protein is normally depalmitoylated using these general purification techniques. Therefore, the protein must be palmitoylated after purification.

The present inventors have provided a technique for palmitoylating GAP-43 protein wherein the protein is incubated with CoA-plamitate with a mild acid solution. Such a mild solution is achieved by incubating the components at a pH of less than 7. Preferabty the pH will be above 5 to avoid denaturation of the protein, the overall conditions being apparent to those of ordinary skill in the art It is also preferred that the mild acid be a mild organic acid such as a carboxylic acid, preferably acetic acid. Using this^' technique, the membrane targeting peptide of GAP-43 can be palmitoylated. In addition, any active part of palmitoylated GAP-43 can be incorporated into a pharmaceutical composition.

By "membrane-targeting peptide," then, is meant any amino acid sequence as follows:

METLEUCYS CYSMETARGARGTHRLYSGLN or a functional derivative thereof, which, when attached at or near the amino-terminus end of a desired protein or peptide, will effect the direction of said protein or peptide to the cell membrane.

The membrane-targeting peptide may be attached to a desired protein or peptide by well known methods, including but not limited to direct synthesis by manual or, preferably, automated methods. An alternate preferred method by which the membrane-targeting peptide of the invention may be attached to the desired protein or peptide involves modifying the gene encoding the desired protein or peptide, so that the expressed gene product will include the membrane-targeting peptide at its amino-teπninus end. This may be accomplished by well-known methods, including but not limited to blunt-ended or sticky-ended ligation methods as described herein.

The membrane-targeting peptide is capable of being palmitoylated using the technique of this invention. Specifically provided for in this invention is a protein which includes an amino acid sequence selected from the group consisting of:

L MET LEU CYS CYS MET ARG ARG THR LYS GLN; IL MET LEU CYS CYS MET ARG ARG THR LYS; HL MET LEU CYS CYS MET ARG ARG THR;

IV. MET LEU CYS CYS MET ARG ARG;

V. MET LEU CYS CYS MET ARG; VL MET LEU CYS CYS MET; VIL MET LEU CYS CYS; and VHL ^• functional derivatives thereof, wherein at least one of the CYS components is palmitoylated.

The present inventors have undertaken experiments designed to elucidate the regulatory mechanisms which control G protein activity by the palmitoylization and de-palmitoylization of GAP-43. Regulation of G protein activity offers a convenient and effective manner in which mammals, including humans, suffering from damaged, diseased or dysfunctioning central or peripheral nervous tissue, may be therapeutically treated. Further, methods of modulating structural remodeling in normal central or peripheral nervous tissue in mammals, including humans, according to the present invention, will be a significant aid to those of skill in further elucidating the mechanisms of neuron structure and function.

The preclinical and clinical therapeutic use of the present invention in the treatment of neurological disease or disorders will be best accomplished by those of skill, employing accepted principles of diagnosis and treatment Such principles are known in the art, and are set forth, for example, in Petersdorf, R.G. et al., eds., Harrison's Principles of Internal Medicine, 10th Edition, McGraw-Hill, publisher, New York, N.Y. (1983), especially at Part 6, Section 11 of that work, entitled "Disorders of the Central Nervous System."

The compositions of the present invention, or their functional derivatives, are well suited for the preparation of pharmaceutical compositions. The pharmaceutical compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended to be so limited.

The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. For example, administration maybe byparenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In addition to the pharmacologically active compounds, the new pharmaceutical preparations may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.001 to about 99 percent, preferably from about 0.01 to about 95 percent of active compound(s), together with the excipient

The dose ranges for the administration of the compositions of the present invention are those large enough to produce the desired effect, whereby, for example, the neoplastic tissue is reduced or eliminated or ameliorated. The doses should not be so large as to cause adverse side effects, such as unwanted cross reactions anaphalactic reactions and the like. Generally, the dosage will vaiy with the age, condition, sex and extent of the disease in the patient Counterindication, if any, immune tolerance and other variables will also affect the proper dosage. The anti¬ bodies can be administered parenterally by injection or by gradual profusion over time. The antibodies of the present invention also can be administered intravenously, intraparenteraUy, intramuscularly or subcutaneously. The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lycψhilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl -starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetyl- cellulose phthalate or hydrσxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.

Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or sus¬ pended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, stabilizers may be added. Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable foπnulatio__;forparenteralad___istration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipo- philic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

The manner and method of carrying out the present invention may be more fully understood by those of skill by reference to ^"the following examples, which examples are not intended in any manner to limit the scope of the present invention or of the claims directed thereto. EXAMPLE 1

INTERACTION BETWEEN PALMΠΌYLATED GAP-43

AND G PROTEIN

A_ EXPERIMENTAL PROCEDURES

Materials

(3⁵S)GTPγS (1000 Ci/mmol) and CoA-(¹ C)palmitate (60 mCi mmol) were obtained from New England Nuclear. All peptide except the 1-6 peptide were prepared as described previously at Howard Hughes Medical Institute laboratory (Strittmatter et al, Nature 544:836-841 (1990)). The 1-6 peptide and a second synthesis of the 1-10 peptide were a generous gift of Shigeo Ogino of Sumitomo Chemical Co. Sequences were verified by amino-acid sequencing. After synthesis, all peptide solutions contained 1 to 1 0 mM dithiothrietol. The stability of peptides was monitored by reverse phase HPLC on a C resin in 0.1% TFA with a 10-60% acetonitrile gradient

Purification of GAP-43 and GO

Bovine brain G₀ was purified as described previously (Neer et al, J. Biol Chem. 259:14222,14229 (1984)). GAP-43 was purified from the brains of 10-day old rats by a modified method (Zwiers et al, J. Neurochem. 44:1083-1090 (1985); Strittmatter et al, J. Biol Chem. in press (1991)). This purification extracts GAP-43 from membranes with 0.1 N NaOH, that any endogenous palmitate linked by thioesters is cleaved from GAP-43. This preparation is not palmitoylated. Synthesis of Palmitoylated Peptides

To prepare the dipalmitoylated 1-25 peptide, the met-leu-cys-cys (1-4) peptide was synthesized and palmitate was linked to both cysteine thiols by treatment with glutaraldehyde. The palmitoylated amino terminus was then coupled to the 5-25 GAP-43 peptide. The structure of the final dipalmitoylated product was confirmed by mass spectrometry. The singly palmitoylated peptides were synthesized by coupling palmitoylated 1-3 peptide to 4-25 peptic or coupling the 1-3 peptide to the palmitoylated 4-25 peptide.

Palmifoyiation of GAP-43

Purified GAP-43 protein (3 mg, 80 μM) was incubated with CoA- (¹ C)paImitate (1.6 mM, 5 mCi/mmol) for 14 h at 370 in IOC mM Na- Acetate, pH 6. Inclusion of Lubrol PX (1%), or dithiothietc (1 mM) decreased the incorporation into GAP-43. To assess the degree of palmitoylation, the preparation was boiled in 2% SDS, without p- mercaptoethanol, and electrophoresed through a polyaciylamide gel. The gel was stained with Coomassie Blue to detect GAP-43 protein, and then slices of the gel were incubated with 30% H202 for 16 h at 650. Radioactivity was determined by liquid scintillation spectrophotometry, and protein concentration GAP-43 by BCA protein reagent (Pierce). Molar stochiometries calculated on the basis of a Mr of 24,000 for GAP- 43, and the specific activity of the CoA-(¹⁴C)palmitate. Separation of palmitoylated from nonpalmitoylated GAP-43

After palmitoylation of GAP-43, the sample (10-200 μg of protein) was dialyzed versus 1.5 M KC1, 10 mM Tris -HO, pH 7 and then applied to a 0.4 ml column of Phenyl-Speharose (Pharmacia). Nonpalmitoylated GAP-43 was not retained under these conditions. After elution with 4 ml of 50 mM Tris-HCl, 1.5 M KC1, 7.5, and 4 ml of 50 mM Tris-HCl, 1.0 M KO, pH 7.5, palmitoylated GAP-43 was eluted with 20 mM Tris-HCl, pH 7.5, without KC1. The total recovery of protein was 40-75% of that applied to the column.

Hydroxalamine treatment of palmitoylated GAP-43.

To demonstrate the thioester nature of the bond linking palmitate to GAP-43, preparations were diluted into an equal volume of 2 M hydroxalamine, 2 mM DTT, and then incubated at 370 for 14 The sample was then dialyzed against 20 mM Tris-HCl, 1 mM DTT, 7.5 to remove the hydroxalamine prior to addition to GTPγS binding assays or before column chromatography.

GTPvS binding to Go

The binding of (³⁵S)GTPγS to G₀ was determined as described previously. For kinetic studies, assays included 20 nM G_Q, 50 nM (^GTPγS, 0.1% Lubrol PX, 5 mM MgCl₂ mM EDTA, 1 mM DTT, 50 mM Na Hepes, pH 7.5 and any GAP-43 additions. After incubation for 1-10 min at 200, the sample was filtered over nitrocellulose, and bound radioactivity was measured. For standard assays, lower concentrations of the reagents were used, 1 nM G₀ and 2 nM GTPyS, and 100 μg/ml bovine serum albumin was included in the assay. In assays of vesicle incorporated G₀, Lubrol PX was omitted from the assay buffer. In assays of palmitoylated GAP-43 protein, DTT was omitted from the control and experimental tubes to promote stability of the palmitate thioester.

Incorporation of G₀ into posphatidylcholine vesicles

Vesicle incorporation of G₀ was by the method of Cerione et al, Biochemistry 25:4519-4525 (1984), and was confirmed as described previously.

Ε_ RESULTS

GAP-43 amino terminal peptides stimulate GTPγS binding to G_σ.

The activity of G₀ can be assessed by quantitating its guanine nucleotide binding characteristics. Both GAP-43 and a 1-25 amino terminal peptide have been shown to stimulate GDP release and hence GTPγS binding to G_Q (Strittmatter et al, Nature 544:836-841 (1990); and Strittmatter et aL, J. BioL Chem. in press (1991)). To better localize this stimulatory domain within GAP-43, a several amino terminal peptides from 1-6 through 1-25 were tested for their effect on GTPγS binding G-. The 1-6, 1-10, 1-15, 1-20 and 1-25 peptides all stimulate GTPγS binding as effectively as GAP-43 protein itself (Fig. 1). This localizes the stimulatory region of GAP-43 to its first six amino acid residues. A peptide of residues 11-25 does not alter GTPγS binding to G₀, so this region can not be as important Within the amino terminus, the cysteine residues at position 3 and 4 are required for activity, since a 1-10 peptide with threonines in pla of the cysteines is inactive (Strittmatter et al, Nature 544:836-841 (1990)) and Fig. 1). The presence of this activity in such a short stretch of GAP-43 increases the likelihood that adding palmitates to two of these amino acid residues would alter the effect of GAP-43 on G_G.

Previous data showing an inhibitory effect of the 1-10 peptide (Strittmatter et al, Nature 544:836-841 (1990)) was likely due to oxidation of cysteine thiols at position 3 and 4 of this peptide. In the present study all peptides were maintained in solutions with 1 mM DTT. Reverse phase HPLC analysis demonstrates that the peptides are stable for greater than two weeks under these conditions (including two new syntheses of 1-10 peptide), but that the previous preparation of the 1-10 peptide with inhibitory activity elutes the HPLC column at a lower acetonitrile concentration, consistent with oxidation of the thiol groups. A change in motility due to oxidation of thiols supports the data for the 1-10 threonine-substituted peptide which indicates that the cysteine residues are important for G₀ stimulation.

Palmitoylated peptides do not stimulate GTPvS binding to G₀.

To test whether palmitoylation of the amino terminal cysteines of GAP-43 alters interaction with G₀, peptides were prepared with palmitate stably attached to residue 3, residue 4, residues 3 and 4 through a glutaraldehyde linkage. Their structure was confirmed by mass spectrometry. The thioester linkage present in palmitoylated protein was not employed since it would probably be too labile to permit isolation of the modified peptide. When mixed with G-, the singly palmitoylated peptides have a small stimulatory effect on GTPγS binding, about 40% as much as nonpalmitoylated 1-25 peptide or GAP-43 protein (Fig. 2A). The dipalmitoylated peptide has no detectable stimulatory activity, at concentrations well above those which saturate the nonpalmitoylated 1-25 peptide 's effect (Fig. 2A). The equilibrium GTPγS binding assay in these preliminary studies is dependent or kinetics of guanine nucleotide binding as well as the thermal inactivation of G_Q (Ferguson et al, J. Biol Chem. 262:7393-7399 (1986)). To insure that the two peptides have different effects on the GTPγS binding characteristics of GQ, the initial association rate for GTPγS was determined at higher concentrations of G_σ and GTPγS at higher concentrations of G_σ and GTPγS, conditions in which thermal inactivation is insignificant The dipalmitoylated peptide is inactive in this assay, but the nonpalmitoylated peptide, like GAP-43 protein, stimulates the binding of GTPγS to G₀ (Fig. 2B). The palmitoylated peptides should be more hydrophobic than those without fatty acid, and might therefore function more effectively in a lipid bilayer than in the detergent solution of the standard assay. Previously, it was shown that GAP-43 protein is equally effective in stimulating G₀ in detergent solution and in lipid vesicles (Strittmatter et al, Bioessays 25:127-134 (1991)). Phosphatidylcholine vesicle incorporated G_Q is stimulated by the nonpalmitoylated 1-25 peptide, but not by the dipalmitoylated peptide (Fig. 3). Thus, palmitoylation blocks the action of the amino terminal peptides on G₀ irrespective of the assay conditions.

Palmitoylation of GAP-43 protein

To establish the relevance of the peptide data to intact GAP-43 protein, palmitoylation of intact GAP- 43 protein in the naturally occurring thioester linkage was tested for blocking activation of G₀. Although the majority of GAP-43 is palmitoylated in vivo, purification results in a protein with free thiols presumably due to hydrolysis of thioester bonds during alkali extraction of GAP-43 from membrane fractions (Strittmatter et al, J. Biol Chem. in press (1991)). Since rhodopsin (O'Brien et al, I. Biol Chem. 262:5210-5215 (1987)), viral glycoproteins (Schmidt et al, Biochem. Soc Meetings 27:625-626 (1989)) and the proteolipid protein of myelin (Bizzozero et al, J. Biol Chem. 262:13550-13357 (1987) incorporate palmitate when incubated with CoA- palmitate in vitro, we employed the same method for GAP-43. After GAP-43 and CoA-(¹⁴C)palmitate in a 1:20 molar ratio are incubated for 14 h at 37°C and boiled in the presence of SDS, a fraction of the radioactivity comigrates with GAP-43 protein during polyacryaln gel electrophoresis (Fig. 4). The stoichiometry of GAP-43 protein palmitate is 1.7 to 1, indicating that some but not all of the GAP is palmitoylated in this reaction. The highest levels of palmitoylation were achieved in the absence of detergent and DTT. To confirm that (^l4C)palmitate is attached to GAP-43 by a thioester, the preparation was incubated with 1 M neutral hydroxylamine for 14 h at 37°C. After this treatment, all radioactivity migrates on SDS-PAGE at the dye front and not with GAP- 43 (Fig. 4), as expected for hydrolysis of a thioester bond this mild treatment

In order to test the palmitoylated GAP-43 for G₀ stimulation, it is necessary to separate the different forms of GAP-43. Hydrophobic interaction chromatography on phenyl-sepharose allowed resolution of palmitoylated from nonpalmitoylated GAP (Fig. 5). In the presence of 1.5 M KC1, untreated GAP-43 does not bind to this resin, but about half of the modified GAP-43 preparation does so. By lowering the ionic strength, a second fraction of GAP-43 protein is eluted. SDS-PAGE analysis of (¹⁴C) radioactivity show that nonpalmitoylated GAP-43 is in the first fraction, and palmitoylated GAP-43 in the lower ionic strength eluate. The phenyl-sepharose eluted preparation has a molar stoichiometry protein to palmitate of 1 to 15. Thus, this method produces GAP-43 that is both monopalmitoylated and dipalmitoylated. Treatment of the sample with hydroxylamine prior to column chromatography converts all of the GAP-43 to the void volume, nonpalmitoylated form, as expected for thioester-linked palmitoyl groups. Palmitoylated GAP-43 does not stimulate G

With palmitoylated and nonpalmitoylated GAP-43 protein, interaction of GAP-43, as provided for by this invention, with G₀ could be tested. This modified form of GAP-43 has little stimulatory effect on GTPγS binding to G_Q, even when present at concentrations above those which saturate the GAP-43 effect (Figure 6). As shown for the palmitoylated peptides in GTPγS association experiments (Fig. 2B), the inactivity of the palmitoylated protein due to a loss of effect on the guanine nucleotide binding characteristics of G_σ. If palmitoylation is to have a dynamic role in controlling GAP-43/G₀ iαteraction, then inactivation by palmitate must be reversible. As shown above (Fig. 4 and 5), treatment with neutral hydroxylamine cleaves palmitate from GAP-43. Hydroxylamine treatment of the inactive palmitoylated GAP-43 protein also restores its ability to stimulate GTPγS binding to G_σ (Fig. 7).

C DISCUSSION

Cysteine thiol groups appear to be important for a productive interaction between GAP-43 and G_G. When these residues are a by substitution with threonine, GAP-43 peptides are no longer active. Addition of palmitate to these cysteine residues in GAP-43 peptides and in GAP-43 protein also blocks the stimulation of G_G by GAP-43. Thus, the interaction between these two proteins can be controlled by the level of palmitoylation of GAP-43. Since significant amounts of both nonpalmitolyated and palmitoylated GAP-43 exist w vivo, and since GAP- 43 palmitate turns over rapidly witiiin growth cone preparations (Skene et al, J. Cell Biol 108:613-624 (1989)), it is plausible that the level of palmitoylation varies within a range that would alter the level of G protein activation.

The level of GAP-43 palmitoylation is also correlated with subcellular distribution of GAP-43 (Skene et aL, J. Cell BioL 108:613-624 (1989)). The amino terminal domain which contains the two palmitoylation sites is necessary and sufficient for membrane association (Zuber et al, Nature 542:345-348 (1989); Skene et al, J. Cell BioL 108:613-624 (1989)). Membrane-associated GAP-43 is predominantly palmitoylated even though the palmitate turns over rapidly in membrane fractions, and cytosolic GAP-43 contains little palmitate in metabolic labelling studies (Skene et al, J. Cell Biol 108:613-624 (1989)). Thus, soluble GAP-43 may interact with GQ, whereas the membrane associated form may be more important in creating the growth cone localization of the protein and in serving as a reservoir of inactive protein at the distal tip of neurites (Fig. 8).

The effect of palmitoylation on the GAP-43/G₀ system provides the first example of a protein-protein interaction which altered by palmitoylation. The palmitoyl groups described in other proteins may have a similar regulatory role. Most directly related GAP-43 are the G protein-coupled seven transmembrane domain receptors. The agonist- bound form of these receptors stimulates GDP release from specific G proteins in order to transduce extracellular signals into changes in intracellular second messengers (Gilman, A.G. Annu, Rev. Biochem. 56:615-649 (1987)). Two proteins of this family are known to be palmitoylated, rhodopsin (O'Brien et al, /. BioL Chem. 262:5210-5215 (1987)) and the 3₂-adrenergic receptor (O'Dowd et al, J. Biol Chem. 264:7564-7569 (1989)). The site of palmitoylation is one or two cysteine residues within the cytoplasmic carboxyl terminal tail. These palmitoylated cysteine residues and a few surrounding residues are conserved between many of the sequence in this gene family (O'Dowd et al, J. BioL Chem. 264:7564-7569 (1989)), and show a short stretch homology with GAP-43 (Strittmatter et al, Nature 544:836-841 (1990)).

This cysteine residue is required for efficient coupling of the β₂- adrenergic receptor with Gj, but it is not known whether the free thiol versus palmitoylated form of receptor have different capacities for G protein coupling. The frequently reported observation that reconstitution of purified receptor with G protein is enhanced by incubation with DTT (Florio et al, J. Biol Chem. 264:3909-3915 (1989)) could be due to the cleavage of palmitate thioesters. It will be necessary to isolate receptor molecules in their palmitoylated a nonpalmitoylated forms and then reconstitute these preparations with G proteins in order to determine whether palmitoylation has similar effects on G protein coupling for receptors and for GAP-4. The ability to palmitoylate rhodopsin in vitro (O'Brien et aL, J. BioL Chem. 262:5210-5215 (1987)) by the same method as used for GAP-43, suggests that this may possible for other receptors. The fatty acylation of the group of small G proteins related to the ras gene product has received much interest recently. All of these proteins undergo farnesylation of a cysteine residue close to the carboxyl terminus (Hancock et al, Cell 57:1167-1177 (1989)). This is a post- translational modification which persists for the life of the protein and is necessary for normal function of the protein. In addition, there are several members of this family which undergo palmitoylation on nearby cysteine residues (Magee et al, EMBO J. 63353-3357 (1987); Hancock et al, Cell 57:1167-1177 (1989)). The significance of the later modification not yet clear, although a dynamic regulatory role has been suggested (Magee et al, EMBOJ. 6 353-3357 (1987); Hancock et al, Cell 57:1167-1177 (1989)). Mutations of oncogenic ras genes by serine substitution at the palmitoylated cysteines produce products with less transforming activity and lower avidity for the plasma membrane (Hancock et al, Cell 57:1167-1177 (1989)). However, the relative activity of the free thiol versus the palmitoylated cyste form of ras proteins remains unclear. The ability to produce lard, amounts of these proteins and then palmitqylate and depalmitoy them in vitro, as done here for GAP-43, should allow direct assessment of palmitoylation on various functions of the small G proteins.

If palmitoylation is a general type of dynamic protein modification, then there may be a highly specific machinery to control the level of palmitoylation. The mechanisms by which proteins are palmitoylated and depalmitoylated within cells have not been elucidated as yet (Berger et aL, /. Biol Chem. 259:7245-7252 (1984)). The activated nonpalmitoylated GAP-43 places particular emphasis on the depalmitoylation process. There is no obvious amino acid similarly when all known palmitoylated sequences are compared (Sefton et al, J. CelL BioL 204:1449-1453 (1987)), even though there is some homology between GAP-43 and the G protein- linked receptors (Strittmatter et al, Nature 544:836-841 (1990)). Therefore, no obvious mechanism exists whereby a hypothetical protein palmitoyl transferase could recognize the correct palmitoylation sites. Nonetheless, the possibility that palmitoylation has a dynamic role in controlling various cellular processes suggests that further study is warranted.

The rabies virus is also known to be palmitoylated and has been shown to block opiate receptor-mediated signal transmission, a system that is G protein linked. However, it is not known whether systems besides GAP-43/G. can use palmitoylated or unpalmitoylated proteins in such a way that might change the protein-protein functions. EXAMPLE 2 MODULATION OF CELL SHAPE BY GAP-43

A_ EXPERIMENTAL PROCEDURES

GAP-43 and a_a expression vectors

The synthesis of all but two of the plasmids has been described previously. In brief, the GAP-43 plasmids were all derived from CDM-8 and contain an SV40 origin of replication, and the rat GAP-43 cDNA sequence under the control of a CMV promoter (Zuber et al, Science 244:1193-1195 (1989); Zuber et al, Nature 542:345-348 (1989)). The point mutations of ser⁴¹to ala, and of phe⁴² to ala were created from the GAP- 43 vector by oligonucleotide-directed mutagenesis, and were confirmed by DNA sequencing. The CAT expression vectors were constructed in the same fashion. The _Q expression vector (pDOG) contains the rat α₀ sequence under the control of the MMLV-LTR promoter and possesses a neomycin expression cassette. The pDOJ vector is identical but lacks the ₀ sequence.

Cell culture and transfection.

A431 epithelial cells were maintained in DMEM, 7.5% fetal bovine serum. DNA transfections were^"by the calcium phosphate procedure, and contained pDOG, or equal amounts of pDOG and a GAP-43 expression plasmid, or pDOJ and a 5 fold excess of a GAP-43 expression vector. Stable transfectants were selected in 400 μg ml G418 and then screened for protein expression by immune-blotting. After selection clones were maintained without G41 8. Those clones with the highest levels of expression were used in subsequent experiments. The control and GAP-43-expressing CHO cell lines have been described previously (Zuber et al, Science 244:1193-1195 (1989)).

COS-7 cells were transfected with equal amounts of different DNA by the DEAE-dextran method. Forty hours after transfection, cells were analyzed in the spreading assay.

lmmunoblots.

Twenty μg of total protein from various A431 lines was separated by SDS-PAGE, and then transferred to nitrocellulose and stained for GAP- 43 or a₀ as described previously (Strittmatter et al, Nature 544:836-841 (1990).

Analysis of cell spreading.

Cultures of CHO, A431 or COS-7 cells were trypsinized at 30-50% confluency with 0.25% trypsin for 5 min at 370 The cells were then diluted into at least 20 volumes of serum-containing medium and plated onto precoated glass or plastic surfaces. Glass slides were precoated with laminin at 100 μg ml in PBS for 1 h, or with poly-L-fysine at 50 mg ml in PBS for 1 h. Routine experiments employed laminin. After 70 min (COS cells) or 120 min (CHO and A431 cells) at 37° in 5% CO_j, the medium was aspirated and the cells were fixed. CHO and A431 cells were fixed with 2% glutaraldehyde in PBS, and stained with 0.05% Coomassie Blue in 50% methanol, 5% acetic acid. COS-7 cells were fixed with 3.7% formaldehyde in PBS and then incubated for 1 h in 5% normal goat serum, 0.1% Triton X-l 00, PBS with 1 :1 000 rabbit anti-GAP-43 serum, or with 1 μg/ml anti-CAT antibody. Bound antibody was detected by fluorescence after incubating with fluorescein-labelled goat anti-rabbit IgG. The size of the stained cells was determined from micrographs of randomly chosen fields by an observer unaware of the identity of the samples. For 75 consecutive cells, the maximal and minimal diameter passing through the nucleus was recorded. The trends in the data were identical regardless of whether maximal or minimal data were analyzed, but all data in this paper is based on the minimal diameter. Cells were classified as "spread" if the minimal diameter exceeded 15 μm. Each cell line or transient expression assay was examined on at least three separate days.

Adhesion of A431 cell lines.

Twenty-four hours prior to the attachment assay, A431 cultures at

20-30% confluency were fed with medium containing 1 μCi ml of ( ⁵S)methionine. To initiate the assay cells were washed twice with PBS, incubated for 5 min with 0.25% trypsin and then washed again with growth medium and plated on laminin coated plastic at a density that would yield approximatefy 20% confluency if all cells attached. After 45 min at 370, the plates were washed three times for 30 sec with PBS, adherent cells were released in 2% Triton X-l 00, and radioactivity was determined by liquid scintillation spectrophotometry. The 45 min incubation produces attachment of about a third of the cells. By 90 min, 70-80% of the total radioactivity incorporated into cells was adherent

Growth rate of A431 cell lines.

Different cell lines were plated at 5-10% of confluency, and then trypsinized at 24 h intervals over the next five days and counted with a hemocytometer. The exponential rate of increase between 10 and 50% confluency was used to determine the doubling time. Pertussis toxin and mastoparan treatment

A431 cell lines were pretreated with or without 200 ng ml pertussis toxin in growth medium for 2 h prior to the tiypsinization described above. When replated on glass slides for the analysis of spreading, 100 uM mastoparan or 200 ng ml pertussis was present

Cyclic AMP levels in A431 cells.

A431 cell lines were grown to approximatefy 40% confluency on tissue culture grade plastic. Two hours prior to the assay, the cells were fed with DMEM and no serum. To initiate cAMP accumulation cells were refed with 500 μM IBMX, 20 mM Na Hepes, Hanks balanced salt solution, pH 7.4, 0 or 100 nM isoproterenol, and 0 or 10 μM propranolol. After 20 min at 23°, the cells were fixed with 2 vol of ethanol, the material was centrifuged and the supernatant was evaporated, to dryness. The cAMP content was determined by a radioimmunoassay method per the manufacturer's procedure (New England Nuclear). Cyclic AMP levels were standardized for protein content analyzed from duplicate wells by the BCA method (Pierce).

B_ RESULTS

GAP-43 blocks cell spreading in non-neuronal cells.

To examine the mechanism of GAP-43 action, we needed to develop an assay system for GAP-43's effect on cell shape. Previously, we had observed that expression of GAP-43 in nonneuronal cell lines enhances the propensity to form filopodia (Zuber et al, Science 244:1193-1195 (1989)). The advantages of this system are that an aspect of cell morpholσgy can be examined under conditions with no GAP-43, normal GAP-43 or mutant GAP-43 protein. However, this assay has some drawbacks in that the effect is quite time-dependent, requiring analysis of cells within a 15-30 minute time frame after plating. The filopodia are seen only at 100X magnification. Fixation must be complete and rapid or the phenomenon is not observed. Finally, some cells which were tested, such as A431 epithelial cells, produce many filopodia without GAP-43, so that any change is very difficult to judge.

Given these limitations, GAP-43 transfected nonneuronal cell lines were examined for other changes in cell morphology. The most easily reproduced alteration in GAP-43-expressing cell lines is a decrease in spreading. GAP-43 cell lines and control lines were examined two hours after tiypsinization, replating and fixation. At low magnification, a clear decrease in cell area for the GAP-43 lines is noted (Figures 9A-E). This is most conveniently quantitated by measuring the cell diameters. In a number of independent cell lines from both CHO and A431 strains, GAP- 43 produces a 60-75% decrease in the number of cells which obtain an arbitraiy diameter within two hours after plating (Figure 10). This difference is most apparent 1.5 to 4 h after plating, but is still detectable 24 h after plating. The expression of GAP-43 protein was confirmed by immunoblots (Figure 11).

Cell spreading is a complex phenomenon which can be altered by a number of factors, including cell density, stage of the cell cycle, substrate coating, and soluble factors. In all of the experiments described here, cell were at the same density in control and experimental cultures, both during tiypsinization and replating, so that cell density differences cannot account for the decreased spreading of GAP-43 cells. The cell cycle stage is unlikely to be altered by GAP-43 expression, since the doubling time for control cultures is 23 ± 1 h (SE, 6 separate cell lines), and that for GAP- 43 cells is 22 ±: 2 h (SE, 6 separate cell lines). Several different substrates were tested for the spreading assay, with little change in the results from routine assays on laminin-coated glass. Spreading was more rapid and extensive on either poly-L-fysine (Figure 10) or tissue culture plastic (not shown), but the GAP-43 cell lines still spread less than control cells. All spreading assays were conducted in serum-containing medium, so it is possible that components of the serum could be contributing to the substratum, or directly influencing these cells.

Ultimately, changes in spreading are likely to involve alterations in adhesion to the substratum and/or cytoskeletal configuration. By simply counting the number of adherent cells 30-90 min after plating, no obvious difference between the GAP-43 and control lines were noted. To quantitate the adhesion of these cell lines, cells were labelled with (³ _)methionine, replated for various times, washed, and then the radioactivity adherent to the plate was counted. At 45 min after plating, no difference is detectable in adhesion of the GAP-43 and control cell lines (Figure 12A). Thus, GAP-43's effects on spreading are likely due to some other mechanism.

The amino terminus of GAP-43 causes a decrease in cell spreading.

Transient expression of a number of GAP-43 mutations in COS-7 cells facilitated the analysis of how GAP-43 might decrease cell spreading. Cells expressing GAP-43 or a control protein, chloramphenicol acetyltransferase (CAT), were detected by indirect immunofluorescence of replated cells 40 h post-transfection. As in CHO and A431 cells, expression of GAP-43 decreases the number of cells which reach a certain diameter by about 50% (Figure 13A).

A series of GAP-43 mutations suggest that the amino terminus is critical for this decrease in cell spreading. Point mutations at residue 3, residue 4, or residues 3 and 4 abrogate the GAP-43 effect on COS cell spreading, causing cells to spread as fully as control CAT cells. The mid- portion of the molecule, including the cahnodulin binding domain (39-56), is not necessary for GAP-43 action since a large internal deletion (40-189) does not prevent the decrease in cell spreading by GAP-43. Similarly, a point mutation of phenylalanine 42, which renders the protein incapable of binding cahnodulin, does not alter its effect on cell spreading. A point mutation of the protein kinase C phosphorylation site (serine 41) also has no effect on GAP-43 activity in this assay. Although phosphorylation and cahnodulin are not required for GAP-43 action on cell spreading, they might modulate GAP-43's effect To demonstrate that the amino terminus alone can change cell shape we tested a chimeric protein which contains the first 40 amino acids of GAP-43 fiised to CAT. This truncated GAP- 43 sequence decreases spreading to the same extent as the intact GAP-43 molecule. Therefore, the amino terminus of GAP-43 is sufficient for its modulation of cell shape in this assay. This is the same region of GAP-43 which possesses the property of stimulating GTPgS binding to G₀ in vitro. The localization of both activities to the same domain of GAP-43 supports the hypothesis that the protein's action on cellular form is due to an interaction with G proteins.

The effect of GAP-43 is reversed by the presence of

In the non-neuronal cells described above there is no ₀ detectable on immunoblots (Figure 15), so GAP-43 would have to be causing these effects by interacting with another G protein. This is plausible since studies with purified proteins show that GAP-43 can stimulate GTP binding to α_a as efficiently as to α₀, altiiough α, is not stimulated. If the major growth cone membrane G protein, G„, were present GAP-43 might have a different action on these nonneuronal cells. To examine a possible interaction of GAP-43 with G₀ in A431 cells, we created cell lines stably transfected with ₀ alone, GAP-43 alone, or both proteins. The expression of one or both proteins after transfection is detectable by immunoblotting, and the levels are qualitatively the same in singly and doubly transfected cells (Figures 11A and 15). The ₀- transfected A431's exhibited 40% less spreading than control cell lines (Figures 16 and 17). Thus, both GAP-43 and G_Q are capable of altering pathways which impinge on the determination of cell shape. The α₀ transfectants might spread less due to a direct coupling of ₀ to a second messenger system in these cells, or the activity of an endogenous α- subunit might be altered by a different ratio of total a to βγ subunits, or ratio of a subunits to receptor. When both GAP-43 and α₀ are expressed in A431 cells, spreading is close to control levels (Figures 16 and 17). The doubly transfected cell lines are larger than either group of the singly transfected cell lines. Thus, GAP-43 enhances spreading in α₀-A431 cells. Because the action of GAP-43 is reversed when the G protein compliment of the cells is changed, these data argue that GAP-43 does indeed interact with a G protein transduction system which controls spreading in these cells.

G protein drugs alter spreading and block the GAP-43 effect

If GAP-43 action on spreading is modulated through G proteins, directly stimulating or inhibiting G proteins should alter GAP-43's effect Since GAP-43 acts on purified _Λ and a₀, but not a_t agents were tested which have a similar selectivity. Pertussis toxin catalyzes the ADP- ribosylation of Gj and GQ, but not G„ and in so doing blocks receptor- stimulated GTP binding (Gilman, A.G. Annu, Rev. Biochem. 56:615-649 (1987)). Mastoparan increases GTP binding to G; and G₀, and less so to G, (Higashijima et al, J. BioL Chem 265:141176-14186 (1990)). In control A431 cells, mastoparan increases spreading by 30% of control values, and pertussis toxin blocks nearly all spreading (Figures 18 and 19A-B). The mastoparan effect is selective for G proteins under these conditions because pertussis toxin pretreatment prevented mastoparan from increasing spreading. Since pertussis toxin decreases spreading, there must be some endogenous stimulation of a receptor linked to a toxin- sensitive G protein that enhances the spreading phenomenon. This could be due to unliganded receptor (Parker et al, J. Biol Chem. 266:519-527 (1991)), or to ligand in the growth medium or to autocrine stimulation of A431 cells (Stoppelli et al, CeU 45:675-684 (1986)). As for the α_{0 t}ransfections, these results show that G proteins, as well as GAP-43, are capable of altering cell morphology.

The addition of these agents to the GAP-43 cell lines caused their spreading to resemble control cells (Figures 18 and 19A-B). Mastoparan increased spreading from 15 to 45%, and pertussis toxin decreased spreading slightly. The ratio of spreading in the control lines to the GAP- 43 lines decreased from 2.3 1 without additions, to 1.1 : 1 with mastoparan, and 1.1 : 1 with pertussis toxin (Figure 19B). The ability of G protein activation or inhibition to abrogate the GAP-43 effect is expected if GAP-43 acts by altering G protein transduction, and supports the conclusion from the a GP&43 doubly-transfected cells. Taken alone, the results with pertussis toxin and mastoparan are also possible if a G protein cascade is a parallel and overriding pathway to that affected by GAP-43. However, the later hypothesis would not explain the results in the GAS-43 cells.

These data have several other implications if GAP-43 does indeed interact with G proteins to control cell shape. GAP-43 cannot act downstream of the site of mastoparan and pertussis toxin in a G protein transduction system, otherwise these agents would not block its effect GAP-43 must either act on G proteins directly or G protein-receptor coupling. Furthermore, GAP-43 affects A431 cells in the same way as pertussis toxin, and in the opposite manner from mastoparan. Since GAP-43 and these agents act on the same G proteins in vitro, these data suggest that GAP-43 may uncouple receptors from G proteins which alter A431 cell shape.

GAP-43 blocks <3_?-adrenergic stimulation of cAMP.

To directly test GAP-43 action on one receptor coupling event, cAMP levels were measured after the addition of isoproterenol. The β_z- adrenergic receptor is known to couple to G, in A431 cells and stimulate adenylate cyclase (Guillet et al, Proc Natl Acad. ScL USA £2:1781-1784 (1985)). In control cells, cAMP levels increase 15-fold in the presence of isopreterenol, and this is completely blocked by the receptor antagonist propranolol (Figure 20). The basal cAMP level in GAP-43 cell lines is the same as in control lines, but the levels rise only 5-fold with the addition of isoproterenol. Thus, GAP-43 does result in partial uncoupling of the ₂-adrenergic receptor from G, in A431 cells. This provides direct evidence that GAP-43 can alter a G protein transduction cascade. As mentioned above, GAP-43 does not stimulate GTP binding to purified recombinant α_t. Therefore, the uncoupling of G, in A431 could have several explanations. It may be that the recombinant protein is in some manner inactivated with regard to GAP-43, even though it can be reconstituted with receptors. Alternatively, GAP-43 might interact with a_s and prevent receptor coupling, but not have the mild stimulatory effect on GTP binding seen with a_Λ and α₀. Thirdly, interaction of GAP-43 with G; in A431 cells might release more free β γ subunit and this in turn prevents α_s from having the same effect on adenylate cyclase as in control cells. Regardless of which mechanism is present in A431 cells, GAP-43 can uncouple this response. C DISCUSSION

GAP-43 interacts with G proteins.

The hypothesis that GAP-43 interacts with G proteins is now supported by a number of lines of evidence. Both proteins are highly concentrated in the growth cone membrane (Strittmatter et al, Nature 544:836-841 (1990)). Purified GAP-43 protein acts as a guanine nucleotide release protein for G_Q (Strittmatter et al, Nature 544:836-841 (1990)). The domain of GAP-43 which stimulates GTPγS binding to G₀ shares sequence homology with G protein-linked receptors (Strittmatter et al, Nature 544:836-841 (1990)). This same amino terminal domain is the region of GAP-43 which decreases cell spreading in non-neuronal cells. Both GAP-43 and G proteins modulate neurite outgrowth, and the spreading of nonneuronal cells. The action of GAP-43 is altered when the G protein complement of a cell is changed. Pharmacologic agents which directly activate or inhibit G proteins block the GAP-43's effect on cell spreading. GAP-43 expression partially prevents 3₂-adrenergic receptor coupling. Taken together, these data strongly suggest that GAP- 43 does act in concert with G proteins to determine growth cone motility. This confirms the hypothesis that Go responds to an intracellular protein as well as to extracellular ligands for transmembrane receptors.

GAP-43 may uncouple G proteins from receptors.

What is the molecular consequence of GAP-43 and G protein interaction? When the two purified proteins are mixed GAP-43 produces a weak guanine nucleotide release action (Strittmatter et al, Nature 544:836-841 (1990). Whereas receptors stimulate GTP binding by up to

20-fold, GAP-43 does so only 2-fold. It is possible that this discrepancy is due to some deviation of the GAP-43 system from optimal reconstitution conditions, but it is also possible that GAP-43 does not produce as major a change in GTP binding as do receptors. Since GAP- 43 shares some sequence homology with receptors, it might compete with receptors for a site on G proteins. GAP-43 could then block receptor activation. Thus, the mild stimulation observed in vitro which reflect primarily the association of the two proteins and the net effect of GAP-43 in vivo would be opposite.

Several lines of evidence suggest that GAP-43 acts as a G protein uncoupler. By catalyzing the ADP-ribosylation of G proteins, pertussis toxin prevents receptor stimulation of G proteins (Gilman, A.G. Annu, Rev. Biochem. 56:615-649 (1987)). In two instances, GAP-43 has the same effect as pertussis toxin. A431 cell spreading is inhibited by both GAP-43 and the toxin. Neurite outgrowth from PC-12 cells is increased by excess GAP-43 (Yankner et al, Mol Brain Res. 7:39-44 (1990)) and from neuroblastoma cells is decreased by GAP-43 antibodies (Shea et al, J. NeuroscL 22:1685-1690 (1991)). Similarly, pertussis toxin stimulates neurite elaboration from sympathetic neurons (Vartanian et al, NeuroscL Abstr. 17:16 (1991)). Furthermore, GAP-43 blocks 8₂-adrenergic stimulation of cAMP levels in A431 cells. Thus, the net effect of GAP-43 may be to render cellular G proteins less sensitive to ligands in the extracellular space.

GAP-43 action in the growth cone membrane.

How could GAP-43-mediated uncoupling of G proteins from receptors alter growth cone motility and axonal extension? Several neurotransmitters which bind to G protein-linked receptors rapidly collapse growth cone structure and halt axonal extension (Kater et al, J.

NeuroscL 22:891-899 (1991)). If GAP-43 makes G proteins less responsive to low level stimulation of these receptors, then the growth of axons would be promoted. In this model, GAP-43 would act primarily to allow growth of the axon, by preventing the influence of inhibitory signals. G₀ would then have a central role in coordinating growth signals from both within and without the growth cone.

Several mechanisms exist which could alter the efficacy of GAP-43 action. Although the mutational studies show that cahnodulin binding and phosphorylation are not absolutely required for GAP-43 control of cell spreading, one or both might regulate the potency of GAP-43 as an upcoupler of G₀ transduction. The importance of the amino terminal domain for GAP-43 action raises the possibility that palmitoylation of this region (Skene et al, J. CelL BioL 20£:613 _24 (1989)) could have a dynamic regulatory role in GAP-43 function.

Recent data are confirming the existence and identity of growth cone collapse molecules, although the mechanisms by which they halt growth remains unknown (Strittmatter et al, Bioessays 25:127-134 (1991)). The prominence of G_c in the growth cone and its ability to halt growth when activated, raises the possibility that some of these "stop" ligands might bind to G protein-linked receptors, a possibility subject to experimental verification.

G proteins and cell shape

In both growth cone motility and in the spreading of nonneuronal cells, G proteins have major effects on cellular form. G proteins might act through one or more of several second messenger systems known to be responsive to G protein activation. The major growth cone protein, Go, has been linked to the activity of phospholipase C, phospholipase A calcium channels, and potassium channels (Freissmuth et al, FASEB J. 5:2125-2131 (1989)). The influence of calcium levels on growth cone motility is well documented (Kater et al, J. NeuroscL 22:891-899 (1991)), and there is evidence that protein kinase C and hence phospholipase C activity also influences neurite outgrowth (Bixby, J.L. Neuron. 5:287-297 (1989)). The spreading of some non-neuronal cells has been linked to the activation of G protein-linked receptors and their second messengers (Petty et al, J. Cell Physiol 138:247-256 (1989)). The possibility that G proteins may directly alter cytoskeletal confirmation has received some attention. The βy subunits fractionate with the cytoskeleton (Carlson et al., 1986). Chemotactic peptides for human neutrophils alter F-actin polymerization by a G protein-mediated pathway which may not require second messenger systems (Bengtsson et al, Proc Natl Acad. ScL USA £7:2921-2925 (1990)).

This analysis of the major growth cone proteins, GAP-43 and G_Q, places G protein transduction in a pivotal role for determining cellular morphology. At least for the neuronal growth cone, G protein control of form responds to both intracellular and extracellular growth signals. The importance of G proteins in specifying developmental morphogenesis is also emphasized by mutant organisms of Drosophila and Dictyostelium (Deverotes, P. Science 245:1054-1058 (1989); Parks et al, Cell 64:447-458 (1991)). Further work on the shape of many cells may be promoted by focusing on G protein transduction.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters of composition and conditions without departing from the spirit or scope of the invention or of any embodiment thereof. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Scrittmatter, Stephen M Voshiak , Sudo Beck-Sic inger, Annette Valenzuela, Dario Fishman, Mark C

(ii) TITLE OF INVENTION: MAMMALIAN GAP-43 COMPOSITIONS AND METHODS OF USE

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Sterne, Kessler, Goldstein & Fox

(B) STREET: 1225 Connecticut Avenue, NW

(C) CITY: Washington

(D) STATE: DC

(E) COUNTRY: USA

(F) ZIP: 20036

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/774,094

(B) FILING DATE: 11-0CT-1991

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Hopkins-Kilma, Susanne

(B) REGISTRATION NUMBER: 33,247

(C) REFERENCE/DOCKET NUMBER: 0609.3450000

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202)833-7533

(B) TELEFAX: (202)833-8716

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: PEPTIDE

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

Met Leu Cys Cys Met Arg Arg Thr Lys Gin 1 5 10

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Leu Cys Cys Met Arg Arg Thr Lys 1 ^' 5

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: PEPTIDE

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Leu Cys Cys Met Arg Arg Thr 1 5

(2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: PEPTIDE

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

Met Leu Cys Cys Met Arg Arg

1 5

(2) INFORMATION FOR SEQ ID N0:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: PEPTIDE

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Leu Cys Cys Met Arg 1 5

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: PEPTIDE

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Leu Cys Cys Met 1 5 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: PEPTIDE

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Leu Cys Cys

1

Claims

-5.5 -WHAT IS CLAIMED IS:

1. Palmitoylated mammalian GAP-43, or a functional derivative thereof.

2. The composition of claim 1, wherein said mammalian GAP-43 is palmitoylated rat GAP-43.

3. The composition of claim 1, wherein said mammalian GAP-43 is palmitoylated human GAP-43.

4. A method of inactivating G protein activity comprising administering an effective amount of palmitoylated GAP-43.

5. The method of claim 4, wherein the G protein activity is G₀ protein activity.

6. A pharmaceutical composition comprising an effective amount of palmitoylated GAP-43 and a pharmaceutically acceptable carrier.

7. A peptide comprising an amino acid sequence selected from the group consisting of

I. MET LEU CYS CYS MET ARG ARG THR LYS GLN; π. MET LEU CYS CYS MET ARG ARG THR LYS; m. MET LEU CYS CYS MET ARG ARG THR;

IV. MET LEU CYS CYS MET ARG ARG;

V. MET LEU CYS CYS MET ARG;

VI. MET LEU CYS CYS MET; VH. MET LEU CYS CYS; and Viπ. functional derivatives thereof, wherein at least one of the CYS components is palmitoylated.

8. A pharmaceutical composition comprising an effective amount of the peptide of claim 7 and a pharmaceutically acceptable carrier.

9. A method of palmitoylating GAP-43 protein comprising contacting the GAP-43 protein with CoA-plamitate.

10. The method of claim 9, wherein the CoA-palmitate is in an acid solution.

11. The method of claim 10, wherein the acid is acetic acid.