Neuron,

Vol. 6, 21-30,

January,

1991, Copyright

0 1991 by Cell Press

Neurite Outgrowth in PC12 Cells Deficient in GAP-43 E. Edward

Baetge*

and J. P. Hamman@

*Bristol-Myers Squibb Company CNS Molecular Biology Wallingford, Connecticut 06492 +Pathobiological Sciences School of Veterinary Medicine University of Wisconsin-Madison Madison, Wisconsin 53706

Summary The neuronal cell line PC12 undergoes a welldocumented morphological and biochemical differentiation when treated with NGF and other growth factors. A hallmark of this growth factor-mediated differentiation is the induction of the growth-associated protein, GAP43. Here we show that a PC1 2 cell line which is capable of NCF-, bFCF-, and CAMP-mediated neurite outgrowth is deficient in GAP-43 protein and full-length mRNA, as measured by immunocytochemistry, Western blot, Northern blot, and PCR analyses, respectively. We propose that the GAP-43 protein may not be essential for the initial extension and maintenance of neurites induced by these neuritogenic factors; rather, its role may lie predominantly in growth cone function and in the operation of the presynaptic terminal.

Introduction The growth-associated protein, GAP43, is believed to be intimately associated with the growth and regeneration of axonal processes (Skene and Willard, 1981a, 1981b; Skene, 1989). Indeed, it has been speculated based on a limited amino acid similarity with NF-L that GAP43 may be involved in the organization of cytoskeletal proteins during growth of axons (LaBate and Skene, 1989). Many investigations have demonstrated that GAP43 protein and mRNA levels increase rapidly just before axon extension in vivo (Kalil and Skene, 1986; Verhaagen et al., 1988; Hoffman, 1989) and in culture models of sympathetic ganglia explants (Meiri et al., 1988) and primary hippocampal (Coslin et al., 1988, 1990) and dorsal root ganglion (Woolf et al., 1990) neurons, as well as in PC12 cells treated with nerve growth factor (NGF; Van Hooff et al., 1986; Basi et al., 1987; Federoff et al., 1988; Costello et al., 1990). Histochemical and electron microscopic localizations of GAP43 show the protein to be closely associated with the inner membrane surface (Skene and Virag, 1989; Moss et al., 1990) and to be specifically concentrated at the tips of growing axons (Skene et al., 1986; Meiri et al., 1986,1988; Van Hooff et al., 1989; Van Lookeren Campagne et al., 1989). The aminoterminal IO amino acids are sufficient to direct GAP43 to the inner membrane surface and may allow for

specific sorting of the molecule to growth cone tips (Zuber et al., 1989). Some of the most intriguing and direct evidence supporting a role for CAP43 in mechanisms of process elongation comes from the expressing the entire coding region of the GAP43 cDNA in nonneuronal NIH-3T3 and COS cells. When transfected with the GAP43 expression vector, these cells elaborate a variety of filopodial extensions reminiscent of neurite processes in neurons (Zuber et al., 1989). In similar experiments Yankner et al. (1990) stably transfected PC12 cells with human GAP-43 and demonstrated that these cells extend neurites in a more rapid fashion in response to IO-fold lower concentrations of NGF. These experiments have supported the theory that GAP-43 is a key molecular player in the process of neurite extension. GAP-43 may also play a role in the adhesion of extending neurites to the extracellular matrix. Recently, GAP43 has been localized by immunocytochemistry in membrane patches that remained on culture surfaces after superior cervical ganglion neurons were dislodged (Meiri and Gordon-Weeks, 1990). In addition to its predicated role in axonal extension, many investigators have studied the protein as B-50, Fl, pp46, pp57, or neuromodulin (Andreason et al., 1983; Katz et al., 1985; Nelson and Routtenberg, 1985; Cimler et al., 1987; Snipes et al., 1987). These studies have demonstrated that GAP43 binds Ca2+/ calmodulin (Cimler et al., 1985; Alexander et al., 1988) and is a major substrate for protein kinase C, whose phosphorylation state is correlated with the process of long-term potentiation induction (Nelsonand Routtenberg, 1985). Phorbol ester application or tetanic stimulation of the perforant path hippocampal input results in long-lived postsynaptic long-term potentiation responses and presynaptic GAP-43 phosphorylation (Akers and Routtenberg, 1987; Routtenberg et al., 1985; Lovinger et al., 1985; Schrama et al., 1986; De Graan et al., 1988). In isolated synaptosomes and hippocampal slices, GAP43/B-50 is phosphorylated by protein kinase C in response to 4aminopyridine and K+-stimulated depolarization (Heemskerk et al., 1990) and appears to be directly involved in the Ca2+mediated excitation/secretion pathway of neurotransmitter release (Dekker et al., 1989,199O). Furthermore, GAP43 has been recently shown to regulateGTP binding to G, (Strittmatter et al., 1990). Activated G, may in turn serve to couple K+ (VanDongen et al., 1988) or Ca2+ (Ewald et al., 1989) channels in growth cones or synaptic terminals. It has been demonstrated that GAP-43and its mRNA are present in PC12 cells and that NGF treatment results in a marked increase in the levels of GAP-43 mRNA and protein (Van Hooff et al., 1986; Karns et al., 1987; Basi et al., 1987; Federoff et al., 1988; Costello et al., 1990). In this report we address the role of GAP43

Neuron 22

B

in neurite extension through the analysis of this processintheratclonalcell linePC12. WedescribeaPC12 cell line that is capable of rapid neurite outgrowth and a sustained morphological differentiation in the presence of NGF and other neurite-promoting factors and yet is highly deficient in protein and functional mRNA for GAP-43. Results

D

E

GAP-43 Protein Localization To determine whether NCF-responsive PC12 ceils always showelevated GAP-43 expression during neurite outgrowth, we examined two PC12 cell sublines, PC12(A) and PC12(B), grown under standard conditions (see Experimental Procedures). In the presence of 7S B-NGF at 50 ng/mI, neurites become apparent in both PC12 cell lines within 24 hr. By 48 hr, the cells flatten considerably and processes are present on more that 95% of the cells. After 72 hr in the presence of NGF, both PC12 cell lines were investigated for the presence of GAP-43 protein using the monoclonal antibody 91E12 (Goslin et al., 1988). The PC12(A) cells were intensely immnunoreactive for GAP-43 (Figure 1B)comparedwiththePC12(B)cells(Figure1D),which were devoid of immunoreactivity to the GAP-43 antibody. High-powered photomicrographs of growth cones from both PC12(A) and PC12(B) cells are depicted in Figures IE and IF, respectively. It is apparent that both cell types similarly manifest extensive filopodial extensions and swollen growth cone tips. Upon closer examination, there are a large number of microspike projections (Figure IF, arrows) emanating from PCl2(B) but not PC12(A) growth cone tips. The PC12(B) cells attained a highly differentiated appearance after treatment with NGF for 7 days (Figure IG), despite the fact that they were devoid of GAP-43 immunoreactivity even after treatment with NGF for 2 weeks (data not shown).

F

Figure 1. lmmunocytochemical Localization with the ABC-Vectastain Method (Band D) Bright-field photomicrographs immunostaining; (Aand C) corresponding

of GAP-43

of avidin-biotin phase-contrast

Protein GAP-43 photo-

micrographs. PC12(A) cells (B) are intenseiy immunoreactive for GAP-43 protein. They exhibit horseradish peroxidase reaction product in the cell bodies, especially at the neurite tips. Conversely, PC12(B) cells (D) do not exhibit immunoreactivity for GAP-43 protein under identical experimental conditions. (E and F) Higher magnification phase-contrast micrographs of PClZ(A) and PC12(B) growth cones taken from the same preparations as above, respectively. Although many of the PClZ(B) growth cones appear morphologically similar to those of the PClZ(A) cells, some possess numerous microspikes, as indicated by the arrows (F). These microspikes are a common feature of the NGFtreated PCl2(B) cells and are evident on growth cones that are photographed on live cells or on paraformaldehyde-fixed cells. Inaddition,themicrospikesareacommonfeatureofthePClZ(B) cells regardless of the growth substrate. Bar, 10 pm. (G) PC12(B) cells (live culture) plated on laminin substrate (Collaborative Research). This culture was treated with NCF (100 ngi ml)for7days (Nomarski DIC).The majority of PC12(B)cells elaborate multiple processes several times the diameter of the cell body. The PClZ(B) cells never exhibit immunoreactivity for GAP43above background underanyofthecultureconditionsorwith growth factor treatments. Bar, 5 pm.

PC12 Cells Deficient 23

in GAP-43

A

6

C

D

Figure teins

2. lmmunofluorescence

Localization

of the

NGF-R

Pro-

PClZ(A) (B) and PC12tB) (D) are intensely immunoreactive for the NGF-R; (A) and (C) show the corresponding phase-contrast photomicrographs. The NCF-R immunoreactivity is localized throughout the cell body as well as in the neurite processes of both cell lines. All of the micrographs are of the same exposure. Bar, 5 mm.

It has been previously reported that the GAP-43 monoclonal antibody 91E12 specifically recognizes soluble and membrane-bound GAP-43 and all isoforms resolvable by isoelectric focusing (Goslin et al., 1990). We further investigated the possibility that a GAP-43-like protein may exist in PC12(B)cells but lacks the epitope recognized by the monoclonal antibody 91E12. We repeated our immunocytochemical analysis using a polyclonal antibody to GAP-43 (L. I. Benowitz), and similarly, this antibodywas unableto detect GAP-43 protein by either avidin-biotin or fluorescence immunocytochemical methods in PClZ(B)cells, while PCl2(A) cells stained positively in both detection schemes (data not shown). In another set of experiments we determined whether both PC12 cell sublines expressed the NGF receptor (NCF-R). As shown in Figures 2B and 2D,

PC12 A I

1

p?Y

-

NGF-R was immunofluorescently labeled in both PC12 cell lines by a monoclonal antibody to PC12 NGF-R (192-IgG; Chandler et al., 1984). Both PC12(A) and PC12(B)cellswere intensely immunoreactivewith NGF-R antibody and appeared similar with respect to NCF-R levels and cellular distribution. To test further the immunocytochemical finding that PC12(B) ceils might be completely devoid of GAP43 protein, we analyzed GAP-43 protein content by Western blotting. PClZ(A) protein extracts were prepared from PC12(A) and PC12(B) cells that had been treated with NGF for 3 days, and Western blots were probed with the GAP-43 monoclonal antibody, 91E12. Serial IO-fold dilutions of the PC12(A) cell extract were compared with 30 PLgof PClZ(B) cell extract. As shown in Figure 3, GAP-43 protein is readily apparent in as little as 0.3 kg of total cell extract. In contrast, GAP-43 protein was barely detectable in 30 pg of identically prepared PC12(B) extract and is approximately 200- to 500-fold less abundant than in PC12(A) cells. To determine whether PC12(B) cells exhibited other properties characteristic of “normal” PC12 cells, we examined PC12(A) and PC12(B) cells for the expression of a variety of neuronal markers consistently found in these cells. The results of these experiments are expressed in Table 1. Both cell lines were immunoreactive for the catecholaminergic enzyme tyrosine hydroxylase(INCSTAR), twoof the neurofilament molecular subunits, NF-L and NF-M (V. Lee), and the microtubule-associated protein MAP5 (IWM-3C5; L. I. Binder). MAP5 is induced in PC12 cells by NGF and is present in cell bodies, axons, and dendrites of embryonic neurons (Riederer et al., 1986; Brugg and Matus, 1988). Additionally, both cell lines were completely negative when reacted with the monoclonal antibody to MAP2 (V. Lee), which is expressed primarily in dendritic processesofadult neurons. It has been reported for embryonic day 19 (E19) primary rat hippocampal cultures that GAP-43 is concentrated in axonal growth cones and axonal processes, which are negative for MAP2 (Goslin et al., 1988, 1990). PC12 cells also extend neurites after application of basic fibroblast growth factor (bFGF; Togari et al., 1983). This growth factor is presumed to exert effects similar to those of NGF on the differentiation of PC12 cells (Rydel and Greene, 1987). In addition, dibutyrl

GAP-43 Table 1. lmmunocytochemical Marker Proteins in PC12(A)

Figure 3. Western Blot of Protein Extracts from PC12(B) Cells Using GAP-43 Monoclonal Antibody

PClZ(A) 91E12

and

Lanes marked PC12 A are serial IO-fold dilutions of total protein extract isolated from cells treated with NGF (100 rig/ml) for3 days. The lane labeled PC12 B, contains 30 ug of identically prepared PClL(B) cell protein extract. PClP(B) cells contain approximately 200 to 500fold less protein compared with the serially diluted PClZ(A) cell extract. Molecular weight markers are indicated on the left side of the figure: bovine serum albumin 69,000, ovalbumin 46,000, and carbonic anhydrase 30,000.

and

Comparison of Neuronal PC12(B) Cells

Marker

PC12(A)

PC12(B)

Tyrosine hydroxylase Neurofilament-L Neurofilament-M MAP2 MAP5

+ + +

+ + +

+

+

(+) indicates positive immunoreactivity with the specified protein antibody. (-) indicates no immunoreactivitv.

marker

Neuron 24

A. GAP-43-m 1.4Kb

cyclic adenosine monophosphate (dbcAMP) treatment results in PC12 process extension but through mechanisms believed to be nonoverlapping with those of NGF and bFGF (Heidemann et al., 1985). We investigated both PC12 sublines for the presence of GAP-43 protein immunoreactivity after the addition of bFGF or dbcAMP. Both cell lines extended neurites equally in response to these effector molecules. Following these treatments, we were again unable to detect GAP-43 protein in PC12(B) cells, whereas PC12(A) cells were always positive. When comparing the neuror;al characteristics of both PC12 ceil lines, we conclude that PC12(B) cells are identical to other PC12 cell lines with respect to expression of the neuronal markers examined above, with the exception of CAP43. In addition, it is interesting to note that the PC12(B) cells are easily dislodged from the culture surface by a gentle tap, whereas PC12(A) cells remain tightly adherent and can be removed only by vigorous washing in Ca2+- and Mg2’-free buffer.

w

B. GAP-43 1.4Kb

+

CVCLOPHILIN1.1 Kb

C. GAP431.4Kb

CYCLOPHILIN“,RNA 1.1 Kb

Figure 4. Northern Analysis Tissues and the Rat-Derived

of GAP-43 mRNA PC12 Cell Lines

Expression

in Rat

Total cellular RNA was isolated by the lithium chloride precipitation method (Cathala et al., 1983), electrophoretically separated on formaldehyde-agarose gels, and transferred to nitrocellulose (15 hg of total RNA per lane unless otherwise stated). For (A), a double-stranded oligonucleotide probe complementary to the amino terminus of the rat GAP-43 cDNA sequence was used (see Experimental Procedures). For (6) and (C), a full coding length rat cDNA probe was cloned and labeled as described in Experimental Procedures. (A) El9 BRAIN = El9 rat brain; El9 LIVER = El9 rat liver; El9 KIDNEY = El9 rat kidney; ADULT BRAIN = 5 pg of adult rat brain poly(A)+ RNA. (B)ANAlVE = PClZ(A), untreated; B NAIVE = PC12(B), untreated; A NCF = PC12(A), 50 rig/ml NGF, 72 hr; B NCF = PClZ(B), 50 ng/ ml NGF, 72 hr; A FGF = PC12(A), 5 ngiml bFGF, 72 hr; B FCF = PC12(B), 5 ngiml bFGF, 72 hr; A dbcAMP = PClZ(A), 1 mM dbcAMP, 24 hr; B dbcAMP = PClL(B), 1 mM dbcAMP 24 hr. (C) poly(A)’ mRNA isolated from PClZ(A) and PClZ(B) cells treated with NCF (50 q/ml) for 72 hr. 4 pg PC12A = 4 pg; 2 wg PCIZA = 2 pg; 1 pg PC12A = 1 Kg; 0.5 pg PCIZA = 0.5 hg; 0.25 pg PC12A = 0.25 pg; 10 vg PCIZB = 10 pg. Panels labeled CYCLOPHILIN immediately below the GAP-43 blots correspond to the same blot reprobed with the lB15 rat cyclophilin cDNA clone (Danielson et al., 1988). The rat cDNA 1815 includes the complete coding sequence of cyclophilin and selves as a useful probe for total mRNA loading and transfer on Northern gels.

GAP-43 mRNA Expression Having determined that the PCl2(B) cell line is highly deficient in GAP-43 protein, as detected by immunocytochemistryand Western blotting, we further investigated GAP-43 expression at the mRNA level. In Figure 4A, the GAP-43 mRNA is readily detected in 15 pg of total RNA from El9 rat brain and 5 wg of poly(A)’ mRNAfrom adult rat brain. El9 rat liver and kidney are completely negative. In Figure4B wecompare PCl2(A) and PC12(B) cells for GAP-43 mRNA expression after treating with NGF or bFGF for 3 days or with dbcAMP for 24 hr. It is clear that GAP-43 mRNA cannot be detected in the PC12(B) cell lineafter any of these manipulations even though the PCl2(B) cells respond by extending neurites in a fashion similar to the GAP-43containing PC12(A) cells (see Figure 1). The cyclophilin mRNAcontent of the same blot is shown below for comparison of mRNA loading and transfer. GAP-43 mRNAexpression levels in response to added growth factors are normalized to cyclophilin content, as shown in Table 2. The PC12(A) cells show a IO-fold induction of GAP-43 mRNA in response to NGF and a 2-fold stimulation after treatment with bFGF. Toenhanceourabilitytodetect low IevelsofGAP-43 message, we isolated poly(A)+ mRNA from both PC12 cell lines treated with NCF for 3 days. In Figure 4C, a gradient of decreasing amounts of poly(A)’ mRNA from PC12(A) cells was compared with IO pg of poly(A)’ mRNA from PC12(B) cells. The cyclophilin mRNA content of the same blot is shown below for comparison. The expected 1.4 kb GAP-43 mRNA was abundantly clear in as little as 0.25 pg of poly(A)’ mRNA generated from NGF-treated PC12(A) cells. In comparison, a diffuse mRNA band measuringonlyl.l-1.2 kb in sizewas visible in the lane loaded with IO pg of NCF-treated PC72(B) poly(A)+ mRNA. This aberrant mRNA species is approximately 200-300 nucleotides shorter than the full-length 1.4 kb GAP-43 message. Quantitation of this mRNA species by normalization tothecyclophilin

PC12 Cells Deficient 25

Table

in GAP-43

2. Radioanalytical

Quantitation

of GAP-43

and

Cyclophilin

mRNA

Levels

Net cpma GAP-43 CON PClZ(A) PClLfB)

73 0

Cyclophilin

GAP-43

mRNA

lnductiot+

NGF

FGF

CAMP

CON

NGF

FCF

CAMP

CON

NGF

FCF

CAMP

551 0

163 0

78 0

628 347

460 638

671 1130

748 618

1 0

IO 0

2 0

1 0

a Net cpm were calculated using an AMSIS radioanalytical imaging system in which the radioactivity in each probe per mRNA hybridization signal was outlined and quantitated as net cpm after subtraction of a representative sample background area on the imaged blot. b Net GAP-43 mRNA induction was calculated by dividing the specific GAP-43 message cpm by the specific cyclophilin message cpm for each lane. The number obtained from the control treated PC12(A) cells was arbitrarily assigned a value of 1.

hybridization this smaller equivalent

signal showed the signal intensity of band to be 3800-fold less than that of an amount of PC12(A) poly(A)+ mRNA.

Polymerase Chain Reaction mRNA Analysis To characterize the smaller PClZ(B) mRNA species further, poly(A)’ mRNA from PC12(A) and PC12(B) cells was reverse transcribed using random primers and amplified with oligonucleotides flanking the entire coding region of GAP-43 using the polymerase chain reaction (PCR). To our surprise, the predicted 708 bp GAP-43 coding sequencewas amplified from both cell lines (Figure 5A). Both of these 708 bp PCR products were cloned, restriction mapped, and completely sequenced. The PCR-amplified DNA sequences from both PC12(A) and PC12(B) cells were identical in both cases to the rat cDNA sequence previously published (data not shown; Basi et al., 1987). However, when PC12(B) poly(A)+ mRNAwas reverse-transcribed using oligo(dt) as a primer and then PCR-amplified using the sametwooligonucleotides(Figure5B),amuch smaller but detectable amount of the 708 bp GAP-43 DNA fragment was obtained. In contrast, when PCR analysis was performed using random-primed poly(A)+ mRNA and a 3’ PCR oligonucleotide complementary to the last 24 nucleotides of the 3’untranslated region of GAP-43 mRNA, theexpected 1207 bp DNAfragment was amplified only from PC12(A) cells (Figure 5C). These data indicate that the PC12(B) cells are synthesizing a truncated form of GAP-43 mRNA in very low abundance. Wearecurrentlyattemptingtodetermine the exact structure of the truncated GAP-43 mRNA by PCR cloning and sequencing. Discussion Since its introduction, the rat clonal cell line PC12 has been widely employed as a model of a neural crestderived precursor of the adrenomedullary lineage (Greene and Tischler, 1976). In the presence of NGF, PC12 cells elaborate extensive neurites and through a differentiation cascade assume morphological and biochemical properties analogous to sympathetic neurons (Greene and Shooter, 1980; Guroff, 1985). PC12 cells are highly mutable in culture. Many of these mutant PC12 cells show altered growth charac-

teristics, includingfailureto respond normallyto NGF or failure to express the high-affinity NGF-R (Bothwell et al., 1980; Green et al., 1986). The PC12(B) cells described here have a robust NGF response and stain intensely for NGF-R using the monoclonal antibody that specifically recognizes the low-affinity receptor on PC12 cells (Chandler et al., 1984). In addition, these cells show tyrosine hydroxylase, NF-L, NF-M, and MAP5 expression patterns similar to those in PClZ(A) cells. Furthermore, PC12(B) cells show an induction of neurites with NGF, bFGF, and dbcAMP similar to that seen in GAP-43-containing PC12(A) cells. Recently, CHO cells were stably transfected with a GAP-43 expression vector. These altered cell lines produced unusually long and numerous GAP-43immunoreactive filopodial processes (Zuber et al., 1989). Based on these experiments, the authors suggested that in neurons, GAP-43 may be responsible for the regulation of cell membrane structure at the tip of the extending growth cone. In a related set of experiments, stable transfection of an expression vector containing the human cDNA for GAP-43 into PC12 cells imparted these cells with a more rapid initial neurite outgrowth response and a IO-fold increased sensitivity to NGF (Yankner et al., 1990). Since the PC12(B) cells are capable of initiating neurite extension in response to neuritogenic factors in a fashion analogous to the PC12(A) cells, we suggest that normal levels of GAP-43 may not be required for this process. Furthermore, normal levels of GAP-43 expression do not appear to be required for the continued outgrowth of neurites in PC12(B) cells, as GAP-43 can be detected only by Western blotting at levels 200- to 500-fold lower than those normally found in PClZ(A) cells. Nonetheless, when compared with the elevated levels of GAP-43 protein normally found in NGFtreated PC12 cultures or with GAP-43 protein levels expressed during development and regeneration of axonal processes in vivo, the PC12(B) cells appear extremely deficient in this protein. Our Northern blot analysis indicates that a GAP-43 gene is transcribed in PC12(B) cells, but results in the production of a diffuse, possibly degraded, GAP-43 mRNA. The PC12(B) GAP-43 mRNA is truncated by approximately 200-300 nucleotides and is less abundant by more than 3 orders of magnitude when compared

26

RANDOM PRIMER I I

RANDOM PRIMER II/

:B% 6535174% 394.

-706

OLIGO tit

CYCLOPHILIN

GAP-43 bp

GAP.43 706 bP65jr

234-

CYCLOPHILIN -556 bp

c.

RANDOM PRIMER

-2167 bp -1766 bp GAP.43

I

-1230 bp -1033 bp -653 bp -517 bp

Figure

5. cDNA

PCR Analysis

of PC12(A)

and PClZ(B)

Poly(A)+

mRNA

For each lane, 200 ng of poly(A)+ mRNA was reverse-transcribed and PCR-amplified as described in Experimental Procedures. CAP-1 and CAP-2 oligonucleotides were used to synthesize the 708 bp product and CAP-l, and CAP-5 oligonucleotides were used to synthesize the 1207 bp product (see Experimental Procedures). Two oligonucleotides complementary to the rat cyclophiiin gene were used as PCR controls (556 bp product) for monitoring the intactness of both mRNA preparations. Random primer or oligo(dt) primer labels indicate which primer was employed to prime the mRNA in the reverse transcriptase reaction. Standard size markers are pBR322 cut with Haelil and pBR328 cut with Bgll and Hinfl (Boehringer Mannheim). (A) PC12(A) and PC12(B) mRNA can program the synthesis of the 708 bp full coding sequence of rat CAP-43. (B) The 708 bp PCR product can easily be detected from random- or oligo(dt)-primed PClZ(A) mRNA. As in (A), random-primed PC12(B) mRNA can program the synthesis of the 708 bp product, but oligo(dt)-primed RNA is much less efficient. The 556 bp cyclophilin control is readily produced from randomor oligo(dt)-primed mRNA. (C)Only PClZ(A) mRNA is capable of programming the synthesis of the 1207 bp PCR product encompassed by the CAP-1 and GAP-5 oligonucleotides.

with identically prepared mRNA from PC12(A) cells. PCR analysis points to an alteration in the 3’ untranslated region or poly(A) tail of the PC12(B) mRNA, due perhaps to the lack of an appropriate polyadenylation signal sequence and therefore resulting in little or no poly(A) tail addition. It bears mentioning that the 3 untranslated region of the rat GAP-43 mRNA contains astretch of 12consecutiveadenines starting at nucleotide 156 downstream from the TCA stop codon. In addition, starting 70 nucleotides 5’ of the end of the rat GAP-43 mRNA, there is a stretch of 17 adenines interrupted by only 2 thymidine residues (Basi et al., 1987). These internal poly(A) tracts may serve as sufficient adenine runs for purification of the message by oligo(dt) chromatography and as weak oligo(dt) priming sites in our cDNA PCR reactions. This could ex-

plain our abiiity to purify a small quantity of this truncated message by oligo(dt) chromatography and our subsequent ability to reverse-transcribe a limited amount of GAP-43 mRNA with oligo(dt), thus allowing PCR amplification of the 708 bp DNA fragment. Combined with our Northern blot results showing a diffuse mRNA band approximately 200-300 nucleotides smaller (see Figure 4C), we would predict that the PC12(B) cells are producing a truncated GAP-43 mRNA. The truncated mRNA may lack a functional polyadenylation consensus signal sequence (AAUAAA), precluding functional polyadenylation. This may predispose the PC12(B) GAP-43 mRNA to more rapid nuclear or cytoplasmic degradation; thus, preventing functional translation and expression of GAP-43 protein. As recently reviewed by Jackson and Standart, (1990), lack

PC12 Cells Deficient 27

in GAP-43

of a poly(A) tail has also been demonstrated to be associated with decreased (re)initiation of mRNA translation; therefore, even if a small amount of poly(A)GAP-43 mRNA is present in PC12(B) cells, this mRNA may be severely compromised in its ability to be efficiently translated into protein. Final proof of our findings awaits the cloning and structural characterization of the 3’end of this abnormal GAP-43 mRNA molecule. GAP-43 is expressed at high levels during development of the peripheral and central nervous systems and is highly concentrated in neuronal growth cones (Skene et al., 1986; Meiri et al., 1988). In the adult nervous system, GAP-43 expression is up-regulated during peripheral nerve regeneration and selectively maintained in populations of CNS neurons presumed to undergo synaptic remodeling (Neve et al., 1987; Akers and Routtenberg, 1987; Rosenthal et al., 1987; Nget al., 1988; De la Monte et al., 1989). The results presented above suggest that normal levels of GAP-43 expression are not required for growth factor-mediated neurite outgrowth in PC12 cells in vitro and raises the question of whether this may also be true of neurons in vivo. Nonetheless, because PC12 cells constitute a transformed cell line that is highly mutable in culture, it is not possible to state unequivocally that the mechanisms controlling neurite outgrowth in PC12 cells are identical to those found in vivo. Our findings indicate that these GAP-43-deficient PCl2(B) cells will initiate neurite outgrowth in a fashion identical to that exhibited by GAP-43-expressing PC12(A) cells. When compared with GAP-43-containing PC12(A) cells, however, the PClZ(B) cells are less adherent. These observations may further support a role for GAP-43 in the process of growth cone-substrate adhesion (Meiri and Gordon-Weeks, 1990). At this juncture, it is interesting to speculate that if GAP43 is involved in growth cone-substrateadhesion processes, the over-expression of GAP-43 in COS cells or PC12 cells might increase the number of filopodial extensions observed simply by making them more adherent and less transitory. In mature synaptic endings, there is now strong evidence supporting a role for GAP-43-facilitated, protein kinase C-dependent synaptic vesicle fusion and transmitter release (Dekker et al., 1989). In addition to these findings, GAP-43 has recently been demonstrated to bind to G, and stimulate GTP binding to this G protein (Strittmatter et al., 1990). In light of our discovery of this CAP4Zdeficient PClZ(B) cell line, we believe that the major role of GAP-43 may lie primarily in synaptic function and not in the neurite extension process per se. These functions may be directly linked to presynaptic processes involved in the generation of long-term potentiation and synaptic remodeling (Akers and Routtenberg, 1987; Rosenthal et al., 1987; Ng et al., 1988; Neve et al., 1988; Benowitz et al., 1988, 1989). It has been reported that PC12 cells are capable of forming functional cholinergic synapses in vitro

(Schubert et al., 1977; Ruzzier et al., 1988). A further test of the role of GAP-43 in growth cone or synaptic function may reside in assessing the ability of the PC12(B) cells to locate and to form functional synapses with target cells in vitro. In addition, in vitro mutagenesis of the GAP-43 gene may allow structure-function studies to’ be performed in the GAP-43-deficient PC12(B) cells. Experimental

Procedures

Culture Methods PC12(A) is an NGF-responsive clonal cell line isolated by E. Schweitzer. PClP(B) cells were originally purchased from the American Type Culture Collection in 1987 under the name PC12 cells. Growth medium was Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% fetal bovine serum (Hyclone), 5% supplemented calf serum (Hyclone), L-glutamine (CIBCO), pen/strep (Sigma), and 25 mM HEPES (GIBCO). Both cell lines were grown in an atmosphere of 9.5% CO1 at 37°C. The growth medium was changed twice weekly, and cells treated with NGF (Sigma) received the growth factor every 2 days. In addition, some cells were treated with bFGF (twice daily) and 1 mM dbcAMP (Sigma) (once) for 24 hr. lmmunocytochemistry Cells from both PC12 lines were plated on poly-L-lysinetreated glass coverslips and grown under standard conditions as de scribed. For NGF-R immunocytochemistry, the cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, washed with the same buffer, permeabilized with 1% Triton X-100, washed and immersed in normal goat serum, and incubated in 192-IgG MAb at a I:500 dilution for 1 hr. Following the primary antiserum, the cells were washed with phosphate buffer and incubated in biotinylated goat anti-mouse followed by streptavidin-FITC(bothfromVectorLabs).ForCAP-43immunocytochemistry, the coverslips were fixed with 4% paraformaldehyde for 20 min. Following fixation, the cells were washed in the same phosphate buffer, permeabilized with 100% ethanol, washed again with buffer, immersed in normal goat serum, and then incubated in monoclonal antibody 91E12 at a I:5000 dilution for 1 hr. The cells were then washed in phosphate buffer and incubated in biotinylated goat anti-mouse IgC followed by avidinhorseradish peroxidase for ABC immunocytochemistry. After immunocytochemistry, the coverslips were mounted on glass microscope slides, viewed, and photographed by either phasecontrast, bright-field, or fluorescence optics. Western Blot Analysis PClZ(A) and PClZ(B) cells were grown under standard culture conditions as described. Both naive and NGF-treated (3 days; 100 nglml NCF) PC12 cells were loosened with and suspended in Ca2+- and Mg2+-free Hanks’ balanced salt solution (GIBCO), pelleted by centrifugation, and resuspended in a lysis buffer consisting 10 mM potassium phosphate (pH 7.4), 1% Triton X-100, 1 PM PMSF, 1 mM benzamidine, 2 mM EDTA, 2 mM ECTA, and 20 pg/ml of trypsin inhibitor, antipain, pepstatin A, and leupeptin (all purchased from Sigma). After 1 hr at 4OC in the lysis buffer, the samples were centrifuged, the supernatants were adjusted to 1 x SDS sample buffer, and the protein content was determined by the Bio-Rad (Bradford) system. The protein samples were boiled for IO min, and the indicated amounts were loaded into each well and subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose, washed in TBS (0.1 M Tris [pH 7.61, 150 mM NaCI), and blocked with 5% lowfat powdered milk in TBS (TBS-milk). The nitrocellulose membranes were incubated overnight at 4OC with the monoclonal antibody 91E12 to CAP-43, diluted 1:10,000 in TBS-milk, and washed threetimes (IO min each) in TBS. GAP-43 immunore activitywas detected using a mouse IgG standard Vectastain ABC kit with diaminobenzidine-nickel as the chromagen.

Neuron 28

GAP-43 cDNA Clone isolation Two 27-mer oligonucleotides complementary to the first 15 amino acids of the published rat cDNA sequence (Basi et al., 1987) were heated to 65Yfor 15 min and slowly cooled to room temperature at a final concentration of 2 pmol/ml in 500 mM NaCI, 50 mM M&b, 50 mM Tris (pH 8.0). Annealed oligomers were labeled in a 10 PI reaction containing 2 ~1 of oligomer hybridization mix, 50 PCi of [QP]dCTP, 300 pmol each of unlabeled dATP, dCTP, TTP, 1 mglml BSA, 1 mM dithiothreitol, and 2 U of Klenow polymerase and incubated at room temperature for 20 min. The double-stranded, 45 bp probe described above was employed to screen a rat brain kgtll library (Clonetech Inc.), resulting in the isolation of four independent cDNA clones. Two clones, designated GAP-2 (835 bp) and GAP-4-4 (1087 bp], were characterized by DNA sequence analysis. GAP-Z spans the region beginning 21 bp 5’ of the translation start site and extends 154 bp 3’ of the translation stop codon (data not shown). GAP-4-4 begins 9 residues 5’ of the ATC start and ends at residue 1130, using the numbering system of Basi et al. (1987). The purified cDNA insert was labeled with [32P]dCTP as described below. mRNA Isolation and Northern Blot Analysis Total cellular RNA was isolated bythe lithium chloride precipitation method of Cathala et al. (1983), and the poly(A)’ fraction was isolated by two purification rounds over oligofdt) spin columns (Pharmacia). For Northern blot analysis, 15 Kg aliquots of total RNA or 0.25-10 pg amounts of poly(A)+ RNA were resolved by electrophoresis through a 0.66 M formaldehyde, 1.1% agarose gel and capillary-transferred with 20x SSC to nitrocellulose membrane. The full coding sequence GAP-2 or GAP-4-4 clones were labeled with [a-=P]dCTP to >109 cpm per pg of DNA using Klenow enzyme and a random hexanucleotide kit (Boehringer Mannheim). Blots were stripped and reprobed for cyclophilin transcripts using a full coding sequence complementary to the published rat cDNA sequence (Basi et al., 1987). The blots were hybridized using 3 x IO6 cpmimi labeled probe in 50% formamide, 3x SSC, 50 mM Na,P04, 100 pgiml sonicated herring sperm DNA, 10% dextran sulfate at 45OC overnight. Blots were washed to a final stringency of 68OC in 0.2x SSC, 0.5% SDS for 1 hr. Washed membranes were exposed at -7O’C to Kodak X-Omat AR film with intensifying screens for 1-6 days. cDNA PCR Analysis Poly(A)’ mRNA isolated from PC12(A) and PClZ(B) cells was reverse-transcribed and PCR-amplified as described by Kawasaki and Wang (1989). To a final volume of 20 ~1 of 1 x PCR buffer (as supplied in the Perkin-Elmer Gene Amp kit), the following were added: dNTPs at a final concentration of 1 mM each, 20 U of RNAasin (Boehringer Mannheim), 100 pmol of random hexamer or oligo(dt)15, 200 ng of poly(A)’ mRNA, and 300 U of MULV reverse transcriptase (Bethesda Research Laboratories). The mRNA was heated at VOT for 5 min in HZ0 and quickly cooled on ice prior to addition of the reaction mix. The reaction mix was incubated for 10 min at room temperature and then for 60 min at 42’C. The reaction was stopped by heating at 95OC for 10 min followed by quickly chilling on ice. The heat-treated 20 ~1 reverse transcriptase reaction was diluted to a final volume of 100 ~1 with 80 ~1 of 1 x PCR buffer containing 50-100 pmol of each upstream and downstream primer and 1.25 U of Taq polymerase (Perkin-Elmer Cetus). The reaction was overlaid with 100 PI of light mineral oil and cycled for 30 rounds of amplification in a Perkin-Elmer Cetus thermal cycler at 95°C for 1 min, 55OC for 2 min, 72OC for 2 min, and ending with 72OC, for 7 min. The samples were extracted once with Tris-EDTA-saturated chloroform and analyzed on 1% CTC, 2% Nusieve-agarose composite gels in Tris acetate buffer. The GAP-43 PCR primers are as follows: The upstream primer GAP-1 (S-ATCCTGTGCTCTATCACAAGAACC-3’) begins at the ATG of the published rat cDNAand extends24 bases 3’; the downstream GAP-2 oligonucleotide (5’GGCAACGTGGAAAGCCGTTTCTTAAAGT-3’) is complementary to the first 28 residues past the TGA stop codon; and the GAP-5 primer (S-ACGCTAATTGGCACATTTG-3’) is complementary to the terminal 23 residues of the rat

cDNA sequence. The combination of GAP-l and GAP-2 primers results in a PCR product of 708 bp covering the entire coding region of GAP-43. The combination of CAP-I and GAP-5 primers spans theentirecoding and 3’untranslated region for rat GAP-43, resulting in a 1207 bp PCR product. Acknowledgments The authors thank P. Claude for NGF-R 192-IgG monoclonal antibody, L. Binder for the IWM-3G5 MAP5 antibody, V. Lee for NF-L, NF-M, and MAP2 antibodies, D. Schreyer for the GAP-43 91E12 monoclonal antibody,and L. I. Benowitzforthe sheep polyclonal antibody to GAP-43. We also thank A. Messing, K. Kalil, K. Felsenstein, and T. Martin for comments on the manuscript; T. Martin, M. Bachinsky, and E. Schweitzer for providing cell lines; C. Sampson for DNA sequencing; and B. Olwin for the recombinant human bFGF. This work was supported in part by National Institutes for Health grant 22475 and the Milwaukee Foundation grants to A. Messing. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenf’ in accordance with 18 USC Section 1734 soley to indicate this fact. Received

August

6, 1990; revised

November

14, 199C.

References Akers, R. F., and Routtenberg,A. (1987). Calcium-promoted translocation of protein kinase C to synaptic membranes: relation to the phosphorylation of an endogenous substrate (protein Fl) involved in synaptic plasticity. J. Neurosci. 7, 3976-3983. Alexander, K. A., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988). Identification and characterization of the calmodulin binding domain of neuromodulin, a neurospecific calmodulin-binding protein. J. Biol. Chem. 263, 75467549. Andreason, T. Jo, Luetje, C. W., Heidman, W., and Storm, (1983). Purification of a novel calmodulin binding protein bovine cerebral cortex. Biochemistry 22, 4615-4618.

D. R. from

Basi, G. S., Jacobson, R. D., Virag, I., Schilling, J., and Skene, J. H. P. (1987). Primary structureand transcriptional regulation of GAP-43, a protein associated with nerve growth. Cell 49,785-791. Benowitz, L. I., Apostolides, P. J., Perrone-Bizzozero, N. I., Finklestein, S. P., and Zweirs, H. (1988). Anatomical distribution of thegrowth-associated proteinsGAP-43/B-50in theadult rat brain. J. Neurosci. 8, 339-352. Benowitz, L. I., Perrone-Bizzozero, N. I., Finklestein, S. P., and Bird, E. D. (1989). Localization of the growth-associated phosphoprotein CAP-43 (B-50, Fl) in the human cerebral cortex. J. Neurosci. 9, 990-995. Bothwell, M. A., Schechter, A. L., and Vaugn, K. M. (1980). Clonal variants of PC12 pheochromocytoma cells with altered response to nerve growth factor. Cell 27, 857-866. Brugg, B., and Matus, A. (1988). PC12 cells express microtubule-associated proteins during nerve growth induced neurite outgrowth. J. Cell Biol. 107, 643-650. Cathala, G., Sovouret, I. F., Mendez, Martial, J. A., and Baxter, J. D. (1983). intact translationally active ribonucleic

juvenile factor-

B., West, B. J.., Karin, M., A method for isolation of acid. DNA 2, 329-335.

Chandler, C. E., Parsons, L. M., Hosang, M., and Shooter, E. M. (1984). A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells. J. Biol. Chem. 259, 68826889. Cimler, B. M.,Andreasen,T.J.,Andreasen, K. I., and Storm, D. R. (1985). P-57 is a neural specific calmodulin-binding protein. J. Biol. Chem. 260,10784-10788. Cimler, B. M., Giebelhaus, D. H., Wakim, B. T., Storm, D. R., and Moon, R. T. (1987). Characterization of murine cDNAs encoding P-57, a neural-specific calmodulin-binding protein. J. Biol. Chem. 262, 12158-12163.

PC12 Cells Deficient 29

in GAP-43

Costello, B., Meymandi, A., and Freeman, J. A. (1990). Factors influencing GAP-43 gene expression in PC12 pheochromocytoma cells. J. Neurosci. 70, 1398-1406. Danielson, P. E., Forss-Petter, S., Brow, M.A., Calavetta, lass, J., Milner, R. J., and Sutcliffe, J. G. (1988). A cDNA the rat mRNA encoding cyclophilin. DNA 7, 261-267.

L., Dougclone of

De Graan, P. N. E., Heemskerk, F. M. J., Dekker, L. V., Melchers, B. P. C., Cianotti, C., and Schrama, L. H. (1988). Phorbol esters induce long- and short-term enhancement of B-50/CAP43 phosphorylation in rat hippocampal slices. Neurosci. Res. Commun. 3, 175-182. Dekker, L. V., De Graan, P. N. E., Oestreicher, D. H. G., and Gispen, W. H. (1989). Inhibition release by an+%odies to B-50 (GAP-43). Nature Dekker, L. V., Cispen, W. H. of the protein synaptosomes.

A. B., Versteeg, of noradrenaline 342, 74-76.

De Craan, P. N. E., De Wit, M., Hens, J. J. H., and (1990). Depolarization-induced phosphorylation kinase C substrate B-50 (CAP-43) in rat cortical J. Neurochem. 54, 1645-1652.

De la Monte, S. M., Federoff, H. J., Ng, S.-C., Crabczyk, E., and Fishman, M. C. (1989). GAP-43 gene expression during development: persistence in a distinctive set of neurons in the mature central nervous system. Dev. Brain Res. 46, 161-168. Ewald, D.A., Pang, I.-H., Sternweis, P. C., and Miller, R. J. (1989). Differential C protein-mediated coupling of neurotransmitter receptors to Ca2+ channels in rat dorsal root ganglion neurons in vitro. Neuron 2, 1185-1193. Federoff, H. J., Crabczyk, E., and Fishman, M. C. (1988). Dual regulation of GAP-43 gene expression by nerve growth factor and glucocorticoids. J. Biol. Chem. 263, 19290-19295. Goslin, K., Schreyer, D. J., Skene, J. H. P., and Banker, Development of neuronal polarity: GAP-43 distinguishes from dendritic growth cones. Nature 336, 672-674. Goslin, K., Schreyer, D. J., Skene, J. H. P., and Changes in the distribution of GAP-43 during of neuronal polarity. J. Neurosci. 70, 588-602.

C. (1988). axonal

Banker, C. (1990). the development

Green, S. H., Rydel, R. E., Connolly, J. L., and Greene, L. A. (1986). PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor. J. Cell Biol. 702, 830-843. Greene, L. A., and Shooter, E. M. (1980). The nerve growth factor: biochemistry, synthesis and mechanism of action. Annu. Rev. Neurosci. 3, 353-402. Greene, L. A., and Tischler, A. S. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nervegrowth factor. Proc. Natl. Acad. Sci. USA 73, 2424-2428. Guroff, C. (1985). PC12 cells as a model of neuronal differentiation. In Cell Culture in the Neurosciences, J. E. Bottenstein and G. Sato, eds. (New York: Plenum Publishing Corp.), pp. 245-272. Heemskerk, F. M. J., Schrama, L. H., Gianotti, C., Spierenburg, H., Versteeg, D. H. C., De Craan, P. N. E., and Gispen, W. H. (1990). CAminopyridine stimulates B-50 (GAP-43) phosphorylation and [‘Hlnoradrenaline release in rat hippocampal slices. J. Neurochem. 54, 863-869. Heidemann, S. R., Joshi, H. C., Schechter, A., Fletcher, J. R., and Bothwell, M. (1985). Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells. J. Cell Biol. 700, 916-927. Hoffman, P. N. (1989). Expression of GAP-43, a rapidly transported growth-associated protein, and class II beta tubulin, a slowly transported cytoskeletal protein, are coordinated in regenerating neurons. J. Neurosci. 9, 893-897. Jackson, R. J., and Standart, N. (1990). 3’ untranslated region control mRNA

Do the poly(A) tail and the translation? Cell 62,15-24.

Kalil, K., and Skene, J. H. P. (1986). Elevated synthesis of an axonally transported protein correlates with axon outgrowth in normal and injured pyramidal tracts. J. Neurosci. 6, 2563-2570. Karns, L. R., Ng, S.-C., Freeman, J. A., and Fishman, M. C. (1987). Cloning of complementary DNA for CAP-43, a neuronal growth-

related

protein.

Science

236, 597-600.

Katz, F., Ellis, L., and Pfenninger, isolated from fetal rat brain. phosphorylation. J. Neurosci. Kawasaki, E. S., and Wang, sion In PCR Techniques, Press), pp. 89-97.

K. H. (1985). Nervegrowth III. Calcium-dependent 5, 1402-1411.

A. M. (1989). Detection H. A. Erlich, ed. (New

cones protein

of gene expresYork: Stockton

LaBate, M. E., and Skene, J. H. P. (1989). Selective conservation of GAP-43 structure in vertebrate evolution. Neuron 3, 299-310. Lovinger, D. M., Akers, R. F., Nelson, R. B., Barnes, C. A., McNaughton, B. L., and Routtenberg, A. (1985). A selective increase in phosphorylation of protein Fl, a protein kinase C substrate, directly related to 3 day growth of long-term synaptic enhancement. Brain Res. 343, 137-143. Meiri, K. F., and Gordon-Weeks, P. R. (1990). GAP-43 in growth cones is associated with areas of membrane that are tightly bound to substrate and is a component of a membrane skeleton subcellular fraction. J. Neurosci. 70, 256-266. Meiri, K. F., Pfenninger, K. H., and Willard, M. (1986). Crowthassociated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones. Proc. Natl. Acad. Sci. USA 83, 3537-3541. Meiri, K. F., Willard, M., and Johnson, M. I. (1988). Distribution and phosphorylation of the growth-associated protein GAP-43 in regenerating sympathetic neurons in culture. J. Neurosci. 8, 2571-2581. Moss, D.J., Fernyhough, P., Chapman, K., Baizer, L., Bray, D., and Allsopp, T. (1990). Chicken growth-associated protein GAP-43 is tightly bound to the actin-rich neuronal membrane skeleton. J. Neurochem. 54, 729-736. Nelson, R. B., and protein Fl (47 kDa, to neural plasticity.

Routtenberg, A. (1985). Characterization of 4.5 pi): a kinase C substrate directly related Exp. Neurol. 89, 213-224.

Neve, R. L., Perrone-Bizzozero, N. I., Finklestein, S., Zwiers, H., Bird, E., Kurnit, D. M., and Benowitz, L. I. (1987). The neuronal growth-associated protein GAP-43 (B-50, Fl): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNAs. Mol. Brain Res. 2, 177-183. Neve, R. L., Finch, E. A., Bird, E. D., and Benowitz, L. I. (1988). Growth-associated protein GAP-43 is expressed selectively in associative regions of the adult human brain. Proc. Natl. Acad. Sci. USA 85, 3638-3642. Ng, S.-C., de la Monte, S. M., Conboy, Fishman, M. C. (1988). Cloning of human tion and ischemic resurgence. Neuron

C. L., Karns, L. R., and GAP-43: growth associa7, 133-139.

Riederer, B., Cohen, R., and Matus, A. (1986). MAP5: a novel brain microtubule-associated protein under strong developmental regulation. J. Neurocytol. 75, 763-775. Rosenthal, A., Chan, S. Y., Henzel, W., Haskell, C., Kuang, W.-J., Chen, E., Wilcox, J. N., Ullrich, A., Goeddel, D. V., and Routtenberg, A. (1987). Primary structure and mRNA localization of protein Fl, a growth-related protein kinase C substrate associated with synaptic plasticity. EMBO J. 6, 364-3646. Routtenberg, A., Lovinger, D., and Steward, O., (1985). Selective increase in the phosphorylation of a 47 kD protein (Fl) directly related to long-term potentiation. Behav. Neural Biol. 43, 3-11. Ruzzier, F., Lee, S., Dryden, W. F., and Miledi, R. (1988). In vitro reinnervation of adult rat muscle fibers by foreign neurons and transformed chromaffin PC12 cells. Proc. R. Sot. (Lond.) B 234, l-9. Rydel, R. E., and Greene, growth factors promote differentiation in cultures

L. A. (1987). Acidic and basic fibroblast stable neurite outgrowth and neuronal of PC12 cells. J. Neurosci. 7,3639-3653.

Schrama, L. H., De Graan, P. N. E., Wadman, Silva, F. H., and Gispen, W. H. (1986). Long-term Caminopyridine-induced changes in protein

W. J., Lopes da potentiation and and lipid phos-

phorylation 257.

in the

hippocampal

slice.

Prog.

Brain

Res. 69, 245-

Schubert, D., Heinemann, S., and Kidokoro, Y. (1977). Cholinergic metabolism and synapse formation by a rat nerve cell line. Proc. Natl. Acad. Sci. USA 74, 2579-2583. Skene, J. H. P. (1989). Axonal Rev. Neurosci. 72, 127-156.

growth-associated

proteins.

Annu.

Skene, I. H. P., and Virag, I. (1989). Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J. Cell Biol. 108, 613-624. Skene, J. H. P., and Willard, M. (1981a). Changes in axonaily transported proteins during axon regeneration in toad retinal ganglion cells. J. Cell Biol. 89, 86-95. Skene, proteins ripheral

J. H. P., and Willard, M. (1981b). Axonally transported associated with axon growth in rabbit central and penervous systems. J. Cell Biol. 96, 96-103.

Skene, J. H. P., Jacobson, Norden, J. J., and Freeman, nerve growth (CAP-43) is membranes. Science 233,

R. D., Snipes, G. J., McGuire, C. B., J. A. (1986). A protein induced during a major component of growth cone 783-786.

Snipes, G. J., Chan, S. Y., McCuire, C. B., Costello, B. R., Norden, J. J., Freeman, J. A., and Routtenberg, A. (1987). Evidence for the coidentification of GAP-43, a growth-associated protein, and F1, a plasticity-associated protein. J. Neurosci. 7, 4066-4075. Strittmatter, S. M., Valenzuela, D., Kennedy,T. E., Neer, Fishman, M. C. (1990). G, is a major growth cone protein to regulation by GAP-43. Nature 344, 836-841.

E. J., and subject

Togari, A., Baker, D., Dickens, C., and Guroff, C. (1983). The neurite-promoting effect of fibroblast growth factor on PC12 cells. Biochem. Biophys. Res. Commun. 774, 1189-1193. VanDongen, A. M. J., Codina, J., Olate, J., Mattera, R., Joho, R., Birnbaumer, L., and Brown, A. M. (1988). Newly identified brain potassium channels gated by the guanine nucleotide binding protein C,. Science 242,1433-1437. Van Hooff, C. 0. M., De Graan, P. N. E., Boonstra, J., Oestreicher, A. B., Schmidt-Michels, M. H., and Gispen, W. H. (1986). Nerve growth factor enhances the level of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells. Biochem. Biophys. Res. Commun. 739, 644651. Van Hooff, C. 0. M., Holthius, J. C. M., Oestreicher,A. B., Boonstra, J., De Craan, P. N. E., and Cispen, W. H. (1989). Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells. J. Cell Biol. 708, 1115-1125. Van Lookeren Campagne, M., Oestreicher, A. B., Van Bergen en Henegouwen, P. M. P., and Cispen, W. H. (1989). Ultrastructural immunocytochemical localization of B-50/CAP-43, a protein kinase C substrate, in isolated presynaptic nerve terminals and neuronal growth cones. J. Neurocytol. 18, 479-489. Verhaagen, J., Oestreicher, A. B., Edwards, P. M., Veldman, H., Jennekens, F. G. I, and Gispen, W. H. (1988). Light- and electronmicroscopical study of phosphoprotein B-50 following denervation and reinnervation of the rat soleus muscle. J. Neurosci. 8, 1759-1766. Woolf, C. J., Reynolds, M. L., Molander, C., O’Brien, C., Lindsay, R. M., and Benowitz, L. I. (1990). The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury. Neuroscience 34, 465-478. Yankner, B. A., Benowitz, L. I., Villa-Komaroff, L., and Neve, R. L. (1990). Transfection of PC12 cells with the human GAP-43 gene: effects on neurite outgrowth and regeneration. Mol. Brain Res. 7, 39-44. Zuber, M. X., Goodman, D. W., Karns, L. R., and Fishman, M. C. (1989). The neuronal growth-associated protein GAP-43 induces filopodia in non-neuronal cells. Science 244, 1193-1195. Zuber, M. X., Strittmatter, S. M., and Fishman, M. C. (1990). A membrane-targeting signal in the amino terminus of the neuronal protein GAP-43. Nature 347, 345-348.