(Received for publication, October 11, 1995; and in revised form, January 19, 1996)
From the
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 -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.
Growth-associated protein (GAP)()-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.
The original AtT-20 cells were transfected using
the LipofectAMINE reagent, according to the manufacturer's
instructions. Briefly, 20 10
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.
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
-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
-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
-endorphin in cultures of the original AtT-20 and the D16 cells.
Secretion experiments and quantitation of
-endorphin by
radioimmunoassay were performed as described under ``Experimental
Procedures.'' A, net CRF-evoked (CRF-evoked minus basal)
secretion of
-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
-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
-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.
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 -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).
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.
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 cells/plate on 35-mm
plates coated with laminin (10 µg/plate) and cultured for 3 days
before fixation. Scale bar, 50
µm.
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 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. 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) .
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.