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Volume 271, Number 17, Issue of April 26, 1996 pp. 10023-10028
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth-associated Protein-43 (GAP-43) Facilitates Peptide Hormone Secretion in Mouse Anterior Pituitary AtT-20 Cells (*)

(Received for publication, October 11, 1995; and in revised form, January 19, 1996)

Chantal Gamby (1),  (2),  Martha C. Waage (1),  Richard G. Allen (3),  Lawrence Baizer (1)(§)

From the  (1)R. S. DOW Neurological Sciences Institute, Good Samaritan Hospital and Medical Center, (2)Department of Cell and Developmental Biology and (3)Center for Research on Occupational and Environmental Toxicology, Oregon Health Sciences University, Portland, Oregon 97209

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The neuronal growth-associated protein (GAP)-43 (neuromodulin, B-50, F1), which is concentrated in the growth cones of elongating axons during neuronal development and in nerve terminals in restricted regions of the adult nervous system, has been implicated in the release of neurotransmitter. To study the role of GAP-43 in evoked secretion, we transfected mouse anterior pituitary AtT-20 cells with the rat GAP-43 cDNA and derived stably transfected cell lines. Depolarization-mediated beta-endorphin secretion was greatly enhanced in the GAP-43-expressing AtT-20 cells without a significant change in Ca influx; in contrast, expression of GAP-43 did not alter corticotropin-releasing factor-evoked hormone secretion. The transfected cells also displayed a flattened morphology and extended processes when plated on laminin-coated substrates. These results suggest that AtT-20 cells are a useful model system for further investigations on the precise biological function(s) of GAP-43.

INTRODUCTION

Growth-associated protein (GAP)(^1)-43 (also known as B-50, F1, neuromodulin, P-57, and pp46) is a membrane-associated phosphoprotein expressed primarily in neurons (1, 2, 3, 4, 5, 6) . While its precise biological function remains to be determined, it is concentrated in the growth cone of developing neurons (7, 8) and expressed at elevated levels during periods of axonal growth and regeneration (for reviews, see (9, 10, 11, 12) ). Together with its association with the membrane skeleton(13, 14) , this suggests that GAP-43 may be involved in the membrane addition associated with axonal elongation.

GAP-43 is a major neuronal calmodulin-binding protein that displays higher affinity for calmodulin in the absence of Ca than in its presence(15, 16, 17, 18) . GAP-43 is also a prominent substrate of the calcium/phospholipid-dependent protein kinase(19, 20, 21, 22) ; phosphorylation of GAP-43 by calcium/phospholipid-dependent protein kinase decreases its affinity for calmodulin(16) . Additionally, GAP-43 has been shown to interact with and activate GTP-binding or ``G'' proteins(23, 24, 25, 26) . Thus, GAP-43 appears to be a common mediator of several second messenger pathways and is therefore in a position to modulate the rate, extent, or direction of axonal growth in response to external stimuli.

GAP-43 expression persists in regions of the mature nervous system that retain the potential for plasticity in response to neuronal activity (27, 28, 29, 30) . In the hippocampus, the correlation of calcium/phospholipid-dependent protein kinase-mediated phosphorylation of GAP-43 with long term potentiation (31, 32) suggests that GAP-43 may play a role in synaptic transmission. Support for this hypothesis has been provided by experiments demonstrating that in vitro phosphorylation of GAP-43 by calcium/phospholipid-dependent protein kinase is correlated with potassium-evoked neurotransmitter release(33) . Furthermore, introduction of antibodies which interfere with GAP-43 phosphorylation into permeabilized synaptosomes inhibits Ca-induced neurotransmitter release(34, 35, 36, 37, 38) . Finally, antisense RNA-mediated inhibition of GAP-43 in PC12 cells leads to a decreased release of dopamine in response to elevated potassium(39) . These investigations suggest that GAP-43 is necessary for exocytosis but additional experiments are required to further define the role of GAP-43 in this process.

The AtT-20/D16-16 (D16) cell line is a subclone (40) of the original AtT-20 mouse pituitary tumor cell line(41) . Both of these cell lines secrete pro-opiomelanocortin-derived peptide hormones(42, 43) . The D16 cells are more amenable to manipulation in culture and respond to a variety of secretagogues, including corticotrophin-releasing factor (CRF), noradrenaline, potassium, phorbol esters, and calcium ionophores (reviewed in (44) ) and have therefore been used widely to study secretion(45) . We have discovered that D16 cells express GAP-43 at high levels and display a robust secretory response to potassium-mediated membrane depolarization. In contrast, GAP-43 is undetectable in the original AtT-20 cells, and potassium evokes only a modest amount of hormone release. This correlation led us to investigate further the role of GAP-43 in neuropeptide secretion in the AtT-20 cell lines.

EXPERIMENTAL PROCEDURES

Plasmid Construction

The expression vector for rat GAP-43 was produced by ligating a 1.1-kilobase pair restriction fragment containing the entire rat GAP-43 coding sequence (5) into the pRc/RSV vector (Invitrogen) with the Rous sarcoma virus (RSV) promoter (46) driving expression of the cDNA. To generate the probe for the RNase protection assay a 300-base pair DraI/SacI restriction fragment derived from the rat GAP-43 expression vector (which includes a portion of 3`-untranslated region of the rat GAP-43 cDNA and some adjacent vector sequences) was ligated into pGEM-3Zf (Promega). This riboprobe will protect two fragments: the larger (approximately 270 nt) results from hybridization with the mRNA transcribed from the transfected rat GAP-43 cDNA, and the smaller fragment (approximately 240 nt) results from hybridization with the endogenous GAP-43 transcript.

Cell Culture and Transfections

All cell culture reagents were from Life Technologies, Inc. Monolayer cultures of D16 cells and transfected cell lines were maintained in 95% Opti-MEM I, 5% fetal bovine serum. The original AtT-20 cells were cultured in 85% Opti-MEM I, 10% equine serum, 5% fetal bovine serum; medium for routine culture of the transfected cells contained 200 µg/ml G418. All cell lines were incubated in humidified 95% air, 5% CO(2) at 37 °C. The AtT-20/D16 cells were generously provided by Dr. Lee Limbird, Department of Pharmacology, Vanderbilt University, and the original AtT-20 cells were obtained from the American Type Culture Collection (CCL89).

The original AtT-20 cells were transfected using the LipofectAMINE reagent, according to the manufacturer's instructions. Briefly, 20 times 10^6 cells were transfected with 15 µg of the GAP-43 expression plasmid and 60 µl of LipofectAMINE. After 6 h of incubation at 37 °C, the medium was removed and replaced by normal culture medium. After 72 h cells were split into selective medium containing 400 µg/ml G418. Clones were then isolated by limiting dilution and expanded in culture.

Intracellular Ca Measurements

Cells were spun and resuspended at 10^7 cell/ml in Krebs-Ringer-Hepes (KRH) buffer (125 mM NaCl, 4.8 mM KCl, 2.6 mM CaCl(2), 1.2 mM MgSO(4), 25 mM Hepes, 5.6 mM glucose, pH 7.4). Fura-2/acetoxymethylester (Fura-2/AM, Molecular Probes) was added at a final concentration of 1 µM, and loading was done for 30 min in the dark at room temperature. Unincorporated dye was removed by washing the cells once with KRH. Cells were resuspended at 0.5 times 10^6 cells/ml in KRH prewarmed at 37 °C and transferred in fluorimeter cuvette and incubated at 37 °C for 30 min, to allow complete de-esterification. Fura-2 fluorescence was measured using a LS50 luminescence spectrometer (Perkin-Elmer), and Ca concentrations were calculated as described by Grynkiewicz et al.(47) , using the Intracellular Biochemistry software package (Perkin-Elmer).

RNase Protection Analysis

Total cellular RNA was extracted using the acid-guanidinium-phenol-chloroform method(48) . Radiolabeled complementary RNA probes were generated in vitro with [alpha-P]UTP (DuPont NEN) as label. Five µg of total RNA from each sample were ethanol-precipitated and re-dissolved in 40 mM PIPES, pH 6.4, 1 mM EDTA, 0.4 M NaCl, 80% formamide with 10^5 cpm of labeled RNA probe. The RNAs were denatured by heating to 85 °C for 5 min, then hybridized at 48 °C overnight. RNase protection analysis was performed by standard methods (49) using digestion with RNase T1 (Life Technologies, Inc.) for 1 h at 30 °C. The resulting RNA fragments were resolved by electrophoresis in a 6% acrylamide, 8 M urea sequencing gel, which was dried and exposed to x-ray film (X-Omat AR, Eastman Kodak Co.), with an intensifying screen at -70 °C.

Immunoblot Analysis

Total cellular proteins were extracted with radioimmune precipitation buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS) containing 2 µg/ml aprotinin and quantified by the method of Bradford (50) using bovine serum albumin as a standard. Subcellular fractionation was performed by lysing cells in 20 mM Tris, pH 7.4, 2 mM EDTA, 1 mM EGTA and separating the particulate and soluble fractions by centrifugation at 100,000 times g. Proteins were separated by SDS-polyacrylamide gel electrophoresis on SDS-10% polyacrylamide minigels (Hoefer Scientific Instruments) and transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore). Protein blots were blocked with 5% non-fat milk in phosphate-buffered saline and incubated with either an anti-rat GAP-43 polyclonal antibody (30) or an anti-GAP-43 monoclonal antibody (clone GAP-7B10, Sigma,) followed by a peroxidase-conjugated secondary antibody (Sigma). Bound antibodies were detected by enhanced chemiluminescence (DuPont NEN) and exposure to x-ray film.

Secretion Studies

Cells were plated in six-well cluster dishes at an initial density of 2.5 times 10^5 cells/well and were used for experiments 6 days later. For incubations with CRF, the medium was replaced by prewarmed Opti-MEM I containing bovine serum albumin (2.5 mg/ml) and protease inhibitors (0.1 mg/ml trypsin inhibitor, 2 µg/ml aprotinin). After 30 min the medium was removed and replaced by fresh medium without (basal) or with 100 nM CRF (Sigma), and the cells were incubated an additional 30 min at 37 °C. For K stimulation, the cells were equilibrated for 15 min in KRH. The medium was then removed and the cells were incubated for 5 min either in KRH buffer (basal) or in KRH buffer containing 56 mM KCl (in which the NaCl concentration was decreased to maintain iso-osmolarity). After the incubation with either CRF or elevated potassium the medium was collected, centrifuged 3 min at 1,700 times g to remove dislodged cells and debris, and phenylmethylsulfonyl fluoride added to a final concentration of 2 mM. The cells were collected in phosphate-buffered saline, centrifuged, and protein was extracted from the cell pellets with radioimmune precipitation buffer containing protease inhibitors. Secreted and cellular hormones were measured by radioimmunoassay. Net secretion (CRF or K-stimulated minus basal) was expressed as the percent of total cellular stores of beta-endorphin released during the incubation period.

Radioimmunoassay

beta-Endorphin immunoassays were performed as described previously(51) , using an antiserum which is specific for beta-endorphin residues 15-26. Synthetic acetyl-beta-endorphin 1-27 was used as tracer and standard, and a 12-point standard curve was assayed with each group of samples. The unpaired ``t'' test was used to determine the statistical significance of the results.

RESULTS

Potassium-stimulated beta-Endorphin Secretion Is Correlated with GAP-43 Expression

Levels of GAP-43 in the original AtT-20 and D16 cell lines were determined by immunoblot analysis. As shown in Fig. 1, GAP-43 is undetectable in the original AtT-20 cells, but is expressed at high levels in the D16 cells. As previous investigations had suggested that GAP-43 might be involved in exocytosis(33, 34, 39) , we analyzed evoked hormone secretion in these two cell lines.

Figure 1: Immunoblot analysis reveals that GAP-43 is expressed at high levels in the D16 cells but is undetectable in the original AtT-20 cells. Proteins extracted from the original AtT-20 (odd numbered lanes) and D16 (even numbered lanes) cell lines were resolved by SDS-polyacrylamide gel electrophoresis, and immunoblot analysis was performed as described under ``Experimental Procedures,'' using a polyclonal anti-rat GAP-43 antibody. Lanes 1 and 2 contain 20 µg; lanes 3 and 4, 10 µg; and lanes 5 and 6, 5 µg of protein.


Initial experiments with cultures of D16 cells demonstrated that 56 mM K-evoked and 100 nM CRF-evoked beta-endorphin secretion were linear for at least 5 and 30 min, respectively (data not shown). Net CRF-stimulated secretion (stimulated minus basal) from the D16 cells averaged 6.9% of the total cellular stores in 30 min and produced an identical secretory response in the original AtT-20 (Fig. 2A). All of the essential components of the secretory machinery are thus present and functional in both cell lines. In contrast to the results with CRF, potassium depolarization resulted in a marked stimulation of beta-endorphin secretion from the D16 cells (17.7% of total cellular stores in 5 min), but produced a dramatically lower amount of secretion from the original AtT-20 (net 1.6% of total cellular stores; Fig. 2B). We have verified by spectrofluorimetric analysis with the dye Fura2-AM that depolarization-mediated calcium influx is similar in these two cell lines (Fig. 3). These results prompted us to ask whether expression of GAP-43 in the original AtT-20 cells would restore potassium-evoked secretion.

Figure 2: CRF- and potassium-evoked secretion of beta-endorphin in cultures of the original AtT-20 and the D16 cells. Secretion experiments and quantitation of beta-endorphin by radioimmunoassay were performed as described under ``Experimental Procedures.'' A, net CRF-evoked (CRF-evoked minus basal) secretion of beta-endorphin from the two cell lines. Cultures were incubated for 30 min at 37 °C in either control medium (basal) or in medium containing 100 nM CRF. Basal release was on the average 4.6% ± 2.2 (D16) and 13.5% ± 6.0 (AtT-20). B, net potassium-evoked (potassium-evoked minus basal) secretion of beta-endorphin from the two cell lines. Cultures were incubated for 5 min at 37 °C in either KRH buffer (basal) or in buffer containing 56 mM KCl. Basal release was on the average 3.7% ± 1.7 (D16) and 6.9% ± 4.1 (AtT-20). The values shown here represent the mean of three separate determinations, each carried out in triplicate. Error bars indicate the standard deviation of the mean. *, p < 0.05 significantly different from the net beta-endorphin release from the original AtT-20 cells.


Figure 3: K-evoked influx of calcium in AtT-20 cells. Cells were loaded with the fluorescent dye Fura-2/AM. At the time indicated by the arrow, KCl was added to the cells to a final concentration of 56 mM and intracellular calcium was measured as described under ``Experimental Procedures.'' A, intracellular calcium concentration in D16 cells. B, intracellular calcium concentration in the original AtT-20 cells. The graphs shown are representative of at least three independent determinations.


Transfection of GAP-43 in the Original AtT-20

The original AtT-20 cells were transfected with a plasmid in which expression of the rat GAP-43 cDNA was driven by the RSV promoter and permanently transfected cells were selected with G418. RNase protection analysis with a radiolabeled antisense RNA GAP-43 probe demonstrates that five cell lines, designated AtT-20:rGAP-43 #1, AtT-20:rGAP-43 G8D, AtT-20:rGAP-43 G4G, AtT-20:rGAP-43 K3F, and AtT-20:rGAP-43 H5E, transcribe GAP-43 RNA from both the transfected GAP-43 cDNA and the endogenous gene (Fig. 4A, lanes 8-12). Four other G418-resistant cell lines that were obtained by transfecting the backbone plasmid pRc/RSV into AtT-20 cells and were designated as AtT-20:pRc/RSV BB1, AtT-20:pRc/RSV CC1, AtT-20:pRc/RSV DD1, and AtT-20:pRc/RSV DD2 (lanes 4-7) express only the endogenous GAP-43 mRNA, which is also readily detectable in the parental AtT-20 (lane 3) and D16 cell lines (lane 2).

Figure 4: Expression of GAP-43 in the transfected AtT-20 cells. Original AtT-20 were transfected with the expression plasmid for rat GAP-43 and stably transformed cell lines were selected as described under ``Experimental Procedures.'' A, RNase protection analysis of GAP-43 RNA expression in the original AtT-20 cells, D16 cells, and several transfected cells lines. Lane 1, intact GAP-43 (300 nt) and beta-actin (245 nt) riboprobes. The GAP-43 riboprobe includes some pGEM 3Zf sequences so the protected fragments are shorter. This riboprobe will protect two fragments: the larger (approximately 270 nt, exogenous) results from hybridization with the mRNA transcribed from the transfected rat GAP-43 cDNA, and the smaller fragment (approximately 240 nt, endogenous) results from hybridization with the endogenous GAP-43 transcript; lane 2, D16; lane 3, original AtT-20; lane 4, AtT-20:pRc/RSV BB1; lane 5, AtT-20:pRc/RSV CC1; lane 6, AtT-20:pRc/RSV DD1; lane 7, AtT-20:pRc/RSV DD2; lane 8, AtT-20:rGAP-43 #1; lane 9, AtT-20:rGAP-43 G8D; lane 10, AtT-20:rGAP-43 G4G; lane 11, AtT-20:rGAP-43 K3F; lane 12, AtT-20:rGAP-43 H5E; lane 13, control yeast tRNA. B, immunoblot analysis of GAP-43 expression in the original AtT-20 cells, D16 cells, and the transfected AtT-20 cell lines. 20 µg of protein were resolved on a 10% SDS-polyacrylamide gel and immunoblot analysis was performed as described previously with a monoclonal anti-GAP-43 antibody. Lane 1, D16; lane 2, original AtT-20; lane 3, AtT-20:pRc/RSV BB1; lane 4, AtT-20:pRc/RSV CC1; lane 5, AtT-20:pRc/RSV DD1; lane 6, AtT-20:pRc/RSV DD2; lane 7, AtT-20:rGAP-43 #1; lane 8, AtT-20:rGAP-43 G8D; lane 9, AtT-20:rGAP-43 G4G; lane 10, AtT-20:rGAP-43 K3F; lane 11, AtT-20:rGAP-43 H5E.


The expression of GAP-43 protein in these cell lines was then analyzed by immunoblot (Fig. 4B). As demonstrated in Fig. 1, the D16 cells (lane 1) produce high amounts of GAP-43, but the protein is undetectable in the original AtT-20 (lane 2). The AtT-20:rGAP-43 #1, G8D, G4G, K3F, and H5E cell lines, which transcribe GAP-43 RNA from the transfected cDNA, also produce significant amount of GAP-43 protein (lanes 7-11). In contrast, this polypeptide is undetectable in the control cell lines AtT-20:pRc/RSV BB1, CC1, DD1, and DD2, which express only the endogenous GAP-43 mRNA (lanes 3-6).

In neurons, GAP-43 is mostly associated with the plasma membrane(7, 8, 10, 13, 52, 53) and when the GAP-43 cDNA is transfected into non-neuronal cells the protein shows a similar subcellular distribution (54, 55, 56, 57) . Subcellular fractionation indicated that GAP-43 localizes to the particulate fraction in the transfected AtT-20 and the D16 cell lines as well (data not shown).

Expression of GAP-43 in the Original AtT-20 Cells Restores Potassium-evoked Secretion

The transfected AtT-20 cell lines were stimulated for 5 min with 56 mM KCl and secretion of beta-endorphin was measured as described. These studies revealed that beta-endorphin secretion in the GAP-43-expressing AtT-20:rGAP-43 cell lines was markedly stimulated (on the average 7.5% of total cellular stores in 5 min). In contrast, secretion in the control AtT-20:pRc/RSV cell lines (2.4% of total cellular stores in 5 min) was not significantly different from that in the parental AtT-20 cell line (Table 1). Potassium-evoked Ca influx in the transfected cell lines (measured by spectrofluorimetric analysis using the dye Fura-2/AM) was similar to that in the parental AtT-20 and D16 cells (Fig. 5). CRF-evoked hormone secretion in the transfected cells expressing GAP-43 was not significantly increased (data not shown).

Figure 5: K-evoked influx of calcium in transfected AtT-20 cells. Cells were loaded with the fluorescent dye Fura-2/AM. At the time indicated by the arrow, KCl was added to the cells to a final concentration of 56 mM, and intracellular calcium was measured as described under ``Experimental Procedures.'' A, intracellular calcium concentration in AtT-20:pRc/RSV CC1 cells. B, intracellular calcium concentration in AtT-20:rGAP-43 #1 cells. The graphs shown are representative of K-evoked Ca influx in AtT-20:pRc/RSV and AtT-20:rGAP-43 cell lines. At least three independent determinations were performed for each cell line.


GAP-43 Induces Morphological Changes in AtT-20 Cells

As GAP-43 has been shown to induce process outgrowth when transfected into non-neuronal cells(54, 58, 59, 60, 61, 62) , we investigated if transfection of GAP-43 would also cause morphological changes in AtT-20 cells. The D16 cells grow as a monolayer, flatten, and extend processes when plated at low density (Fig. 6A). In contrast, the original AtT-20 cells normally grow in suspension but will attach to laminin-coated culture substrates while retaining a rounded morphology (Fig. 6B). The transfected cell lines also grow in suspension under routine culture conditions. However, when seeded on laminin-coated plates, about 30% of the transfected cells expressing GAP-43 flattened and extended processes (Fig. 6C). These morphological changes were not observed for the control AtT-20:pRc/RSV CC1 cell line cultured in the same conditions (Fig. 6D).

Figure 6: Transfection and expression of GAP-43 into AtT-20 cells induces morphological changes. D16 cells (A), original AtT-20 cells (B), AtT-20:rGAP-43 #1 cells (C), or AtT-20:pRc/RSV CC1 control cells (D) were plated at an initial density of 10^5 cells/plate on 35-mm plates coated with laminin (10 µg/plate) and cultured for 3 days before fixation. Scale bar, 50 µm.


DISCUSSION

We report here that transfection of GAP-43 into mouse anterior pituitary AtT-20 cells dramatically augments depolarization-mediated hormone secretion without a change in calcium influx. Additionally, induced expression of GAP-43 results in morphological alterations that include process outgrowth from the cells.

GAP-43 Promotes Depolarization-mediated Hormone Secretion in AtT-20 Cells

Expression of GAP-43 is not detectable in either the anterior lobe cells of the pituitary (63) or in the original AtT-20 cell line, which was derived from a mouse anterior pituitary tumor. In marked contrast, GAP-43 expression is robust in the AtT-20/D16 cell line that was subcloned from the original AtT-20. Our initial studies indicated that the secretory response to elevated extracellular potassium was well correlated with the expression of GAP-43 in the two cell lines. The possibility that this differential response to membrane depolarization might be due to some difference between these cells other than GAP-43 expression is ruled out by the demonstration that transfection of GAP-43 into the original AtT-20 cells restores potassium-evoked secretion.

GAP-43 augments potassium depolarization-mediated hormone release without a significant effect on CRF-induced secretion. This differential effect may be due to the fact that these two secretagogues appear to act via different biochemical mechanisms, which have been characterized extensively in the AtT-20/D16 cells. CRF, the normal secretagogue for anterior pituitary corticotrophs, binds to cell surface receptors coupled to adenylate cyclase through G(s). The ensuing increase in cellular cyclic AMP levels activates cAMP-dependent protein kinase which phosphorylates calcium channels(64) . This effect occurs within minutes after the addition of CRF to the cells. Potassium depolarization produces a much larger and more rapid calcium mobilization through voltage-gated channels(65) , without a change in intracellular cAMP. GAP-43 may serve to facilitate transmission of the biochemical signal that is initiated by the rapid transient depolarization-induced influx of calcium.

GAP-43 has been characterized previously as a calmodulin-binding protein with the unusual property of binding calmodulin under low calcium conditions and releasing it in response to elevations in calcium(16, 17, 18, 66) . This property has led to the hypothesis that GAP-43 may serve to sequester calmodulin at the inner face of the plasma membrane to permit the rapid activation of calmodulin-dependent processes(66) , including CaM kinase II, which has been implicated in neurotransmitter release(67, 68) . The fact that the AtT-20 cells that do not express GAP-43 are nevertheless capable of calcium-dependent secretion of hormone in response to CRF suggests that the action of GAP-43 in the secretory process is likely to be indirect.

Our results are consistent with those of Gispen and colleagues, who have demonstrated that introduction of GAP-43 antibodies into permeabilized synaptosomes inhibits calcium-dependent GAP-43 phosphorylation and neurotransmitter release(34, 35, 37, 38) . Similarly, Ivins et al. (39) showed that antisense RNA-mediated inhibition of GAP-43 expression in PC12 pheochromocytoma cells significantly diminishes depolarization-mediated catecholamine secretion. Possible mechanisms for the action of GAP-43 in secretion have been addressed in additional studies with synaptosomes using antibodies directed specifically against the amino terminus of GAP-43, which have provided further evidence for a role of calmodulin in this process(38) . Furthermore, introduction of GAP-43 peptides into permeabilized chromaffin cells has been shown to modulate Ca-regulated exocytosis via interactions with GTP-binding proteins(69) . In contrast to these previous studies, most of which have relied upon the inhibition of GAP-43 function, our investigations have demonstrated a robust and readily quantifiable positive effect that results from the stable expression of GAP-43 in a well characterized cell line. This suggests that AtT-20 cells will be a useful model system for future studies of the precise molecular mechanisms of GAP-43 action and may help to resolve some of the unresolved issues that remain from previous investigations(35) .

GAP-43 Produces Morphological Changes in AtT-20 Cells

Forced expression of GAP-43 also caused the original AtT-20 cells to flatten and extend processes on laminin-coated culture substrata. These processes are clearly present for several days in culture and are thus significantly more stable than the transient processes induced by GAP-43 in COS and CHO cells(54, 61, 62) . The stability of this response in our studies suggests again that AtT-20 cells will be a useful model system for future investigations of the mechanism of GAP-43 action.

The morphological alterations noted here may relate to the postulated role of GAP-43 in axonal growth and regeneration, in which context this polypeptide was first identified(70, 71) . A multitude of subsequent investigations has sought convincing evidence for a role of GAP-43 in process extension (reviewed in Refs. 10, 52, and 72); the results of these studies have been somewhat equivocal. For example, although the expression of GAP-43 is highly correlated with axonal growth in a variety of experimental systems(7, 52, 73, 74, 75, 76, 77, 78, 79, 80) , the protein is absent from the dendrites of hippocampal pyramidal neurons that still extend prominently in culture(81) . Furthermore, a line of PC12 pheochromocytoma cells that lacks GAP-43 can elongate long branching processes in response to nerve growth factor(82) . Phosphatidylcholine-mediated introduction of GAP-43 antibodies into NB2a/d1 neuroblastoma cells in culture does, however, prevent the initial phase of neurite outgrowth in response to cyclic AMP(83) .

This variability in the requirement for GAP-43 may result from the fact that the precise function of this protein is dependent upon the cell type in which it is expressed. Alternatively, the role of GAP-43 in axonal growth may be indirect, as suggested by recent studies on the effects of disruption of the GAP-43 gene in the mouse embryo(84) . The effects of GAP-43 on cellular morphology noted in the current study represent a positive effect of the induced expression of the protein and again indicate the utility of AtT-20 cells for studies of the mechanism of action of this protein, which we are now pursuing.

FOOTNOTES

*
This work was supported by National Institutes of Health Grant NS26806 and National Science Foundation Grant IBN-9409721. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: R. S. DOW Neurological Sciences Institute, Good Samaritan Hospital and Medical Center, 1120 N.W. 20th Ave., Portland, OR 97209. Tel.: 503-413-7950; Fax: 503-413-7229; baizerl{at}ohsu.edu.

(^1)
The abbreviations used are: GAP, growth-associated protein; CRF, corticotropin-releasing factor; RSV, Rous sarcoma virus; nt, nucleotide(s); PIPES, 1,4-piperazinediethanesulfonic acid.

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