Journal of Neurochemistry
Raven Press, Ltd ., New York
1994 International Society for Neurochemistry
Serotonin Binding Protein : Synthesis,
Secretion, and Recycling
*tHadassah Tamir, *Kuo-peing Liu, *Shu-chi Hsiung,
*Mella Adlersberg, and tMichael D . Gershon
*Division ol"Neuroscience, New York State Psychiatric Institute and (Department ql Anatomy and Cell Biology,
Columbia University, College of Physicians and Surgeons, New York, New York, U.S .A .
Abstract: Serotonin binding protein (SBP) is present in
all neurectodermally derived cells that store serotonin (5-
HT). Three forms of SBP have been detected (68, 56, and
45 kDa), and antibodies to SBP that interfere with the
binding of 5-HT react with each of these proteins . The
current experiments test two hypotheses : (a) that the 56-
and 45-kDa forms of SBP are produced by posttransla-
tional cleavage of a 68-kDa precursor molecule; and (b)
that 45-kDa SBP is a constituent of serotonergic secre-
tory vesicles . Pulse-chase experiments were carried out
using medullary thyroid carcinoma cells as a model.
These neurectodermally derived cells produce 5-HT and
all three forms of SBP. Following pulse labeling for 20
min with L-[s5S]methionine, the cells were incubated in
the presence of an excess of unlabeled L-methionine for
0, 30, 60, or 90 min at 37°C . Alternatively, the chase
was performed under conditions (20°C, inhibition of ATP
generation) that delay or stop transport of newly synthe-
sized proteins from the rough endoplasmic reticulum
through the Golgi apparatus. Following incubation, the
cells were washed and solubilized, and SBP was immu-
noprecipitated . Radioactive proteins in the immunopre-
cipitate were electrophoretically resolved and quantified .
Immediately after the pulse, each of the three forms of
SBP was found to be labeled with 35 S. The relative pro-
portions of 35 S-labeled 68-, 56-, and 45-kDa SBP re-
mained the same at each interval of chase. These propor-
tions were not changed when the chase was carried out
at 20°C or under conditions that blocked the biosynthesis
of ATP . These observations suggest that each form of
SBP is a primary product of translation, that the smaller
forms of SBP are not produced by cleavage from a larger
molecule, and that the size of the primary products of
translation is not altered by passage to the Golgi appara-
tus or a post-Golgi compartment. When secretion was
induced, 45-kDa SBP, but not 56- or 68-kDa SBP, was
released to the medium . When antibodies to 45-kDa SBP
were added to the medium at the time secretion was
induced, antibody binding sites appeared as patches on
the cell surfaces . Because of these sites, cells were lysed
when they were stimulated to secrete in the presence of
antibodies to 45-kDa SBP and guinea pig complement .
Antibody binding sites disappeared from cell surfaces
after 20 min, at which time antibodies to SBP were found
inside the cells. It is suggested that 45-kDa SBP is pack-
aged with 5-HT in secretory vesicles . Some 45-kDa SBP
97
is lost during secretion as a result of exocytosis ; however,
a fraction of the 45-kDa SBP remains bound to the luminal
surface of the membrane of secretory vesicles . This pro-
tein is exposed to the ambient medium as a consequence
of exocytosis, but is reinternalized when the vesicular
membrane is recaptured during vesicle recycling. Key
Words: Serotonin-Serotonin binding protein-Medul-
lary thyroid carcinoma-Pulse-chase labeling-Stimu-
lated release-Vesicle recycling.
J. Neurochem. 63, 97-107 (1994) .
Serotonin binding protein (SBP) is found in seroto-
nergic neurons of both the central and enteric nervous
systems (Gershon and Tamir, 1985 ; Tamir and Gers-
hon, 1990) and is also present in parafollicular cells
of the thyroid gland (Bernd et al ., 1981 ; Barasch et
al ., 1987, 1988) . These cells are all derived, ontogeneti-
cally, from neurectoderm . In contrast, SBP is not found
in other cells that store serotonin (5-HT), but are not
derived from neurectoderm, such as mast cells, plate-
lets, and enterochromaffin cells (Gershon and Tamir,
1985). SBP binds 5-HT with great specificity and high
affinity in the presence of Fe 2+ ions (KD, - 10 -y M;
K2 _ 10-7 M; Liu et al ., 1985). There is evidence
that SBP is present in synaptic and secretory vesicles
(Tamir and Gershon, 1979). In neurons, SBP is subject
to fast axonal transport, is concentrated at terminals,
and some at least is released by nerve stimulation (Jo-
nakait et al ., 1979 ; Gershon et al ., 1983) . In parafollicu-
lar cells, SBP immunoreactivity is found in secretory
Resubmitted manuscript received November 12, 1993 ; accepted
November 24, 1993 .
Address correspondence and reprint requests to Dr . H . Tamir at
Division of Neuroscience, New York State Psychiatric Institute, 722
West 168th Street, New York, NY 10032, U .S .A .
Abbreviations used : [Ca 2- ], , external calcium ; CCCP, carbonyl
cyanide in-chlorophenyl hydrazone ; 5-HT, serotonin ; MEM, mini-
mum essential medium ; MTC, medullary thyroid carcinoma ; PAGE,
polyacrylamide gel electrophoresis ; PBS, phosphate-buffered saline;
RER, rough endoplasmic reticulum ; SBP, serotonin binding protein ;
SDS, sodium dodecyl sulfate .
98 IL TAMIR ET AL .
vesicles (Barasch et al ., 1987; Tamir et al ., 1990) . Two
forms of SBP, 45 and 56 kDa, have been purified
from rat brain (Liu et al ., 1985) . Each of 12 different
monoclonal antibodies raised against these proteins re-
acts with both forms (Liu et al ., 1990b) ; however, some
polyclonal antisera have been raised, which are type
specific (Liu et al ., 1985) . When the 5-HT binding
sites on 45- or 56-kDa SBP are labeled with a photoaf-
finity probe and peptide maps are prepared, similar
patterns are obtained (Liu et al., 1990a). In addition,
anti-idiotypic antibodies that recognize 5-HT binding
proteins react with both 45- and 56-kDa SBP (Tamir
et al., 1991). These observations indicate that 45- and
56-kDa forms of SBP share sequence homology, par-
ticularly in domains that bind 5-HT; however, the
forms are not identical. More recently, a third 5-HT
binding protein (68 kDa) was identified that reacts with
each of the anti-SBP monoclonal antibodies and with
some of the polyclonal anti-SBP sera. The 45- and 56-
kDa forms of SBP appear to be highly restricted to 5-
HT-containing cells ; however, the 68-kDa protein is
also found in neurons that are not known to be seroto-
nergic (Barasch et al., 1987; Kirchgessner et al., 1988) .
The relationships of the three forms of SBP to one
another are not clear. It is possible that a larger precur-
sor protein is synthesized first and then cleaved after
translation to yield smaller products . The 68- and/or
56-kDa proteins could thus be precursors of 45-kDa
SBP . Alternatively, each of these proteins may be syn-
thesized independently . There is evidence that 56- and
45-kDa SBP may be located in different intracellular
compartments (Gershon et al ., 1983). For example,
newly taken up [3H]5-HT forms a complex in situ
mainly with 45-kDa SBP; moreover, 45-kDa SBP is
the form that predominates in the spinal cord, which
contains serotonergic axons and synapses, whereas 56-
kDa SBP predominates in the region of the raphe nu-
clei, which is rich in serotonergic nerve cell bodies
(Gershon et al., 1983) . During ontogeny, 56-kDa SBP
appears before 45-kDa SBP, but the acquisition of the
45-kDa protein is coincident with the formation of
serotonergic synapses (Liu et al ., 1987; Lauder et al.,
1988; lvgy-May et al., 1994) .
The current study was undertaken to determine
whether 45-kDa SBP is synthesized independently or
is derived posttranslationally from the larger forms of
SBP ; the hypothesis that 45-kDa SBP is packaged with
5-HT in secretory vesicles was also tested. Parafollicu-
lar cells and medullary thyroid carcinoma (MTC) cells,
which are derived from them, were investigated as
model systems . MTC cells have been characterized
extensively and shown to be useful models for the
study of mechanisms relevant to serotonergic neurons
(Tamir et al ., 1989, 1990, 1992) . These cells produce
all three forms of SBP, and they synthesize, store,
secrete, and take up 5-HT. A pulse-chase technique
was used to follow the intracellular processing and
transport of each of the forms of SBP. MTC cells were
metabolically labeled with [ 35 S]methionine and then
J. Neurochem., Vol. 63, No . 1, 1994
chased for various periods of time in the presence of
unlabeled methionine. After each period of chase, the
amount of radioactivity incorporated into the various
forms of SBP was ascertained . In addition, parafollicu-
lar and MTC cells were stimulated to secrete to deter-
mine whether any of the three forms of SBP are se-
creted. After evidence was obtained that 45-kDa SBP
is secreted with 5-HT, studies were done to determine
whether some 45-kDa SBP might also be retained on
the plasma membrane of cells and recycled. The data
suggest that 45-, 56-, and 68-kDa SBP are translated
independently and that only the 45-kDa SBP is se-
creted ; however, a portion of the vesicular store of
45-kDa SBP remains membrane bound and can be
recaptured by endocytosis .
MATERIALS AND METHODS
Cells
Sheep thyroid glands were dissociated with trypsin and
chromatographically purified as described previously (Bernd
et al., 1981). Essentially, dissociated cells were exposed to
thyroid stimulating hormone and passed through columns of
thyroglobulin coupled to Sepharose beads . This method
takes advantage of the fact that stimulated thyroid follicular
cells attempt to phagocytize thyroglobulin, but parafollicular
cells do not. The follicular cells are thus retained on the
columns while parafollicular cells pass through. MTC cells
were obtained from Dr . Barry Nelkin (Johns Hopkins Uni-
versity, Baltimore, MD, U.S.A.) .
Cell culture, radiolabeling, and SBP extraction
MTC cells were propagated in vitro as described pre-
viously (Tamir et al., 1989) . The amounts of total SBP in
MTC cells were quantified using an enzyme-linked immuno-
sorbent assay, carried out as described previously (Liu et al.,
1985). Cultures were metabolically labeled within 3 days
after reaching confluence (approximately 1 week in culture).
For metabolic labeling of SBP, confluent cultures (5 X 10`'
cells) were grown in low-methionine media and labeled with
[ 35 S]methionine (50 pCi/ml) for 20 min . Chase periods were
initiated by removing the labeling medium and briefly wash-
ing the nnonolayers twice with cold phosphate-buffered sa-
line (PBS) before adding medium containing 50 Ng/ml of
nonradioactive i.-methionine . Cells were chased in this me-
dium for 30, 60, or 90 min . At the end of the chase period,
cells were placed on ice, rinsed three times with cold PBS,
and lysed with 400 pl of lysis buffer 11% Triton X-100,
0.5% sodium deoxycholate, and 0.1% sodium dodecyl sul-
fate (SDS)] in PBS containing proteolytic enzyme inhibitors:
phenylmethyl sulfonyl fluoride (1 mM; Sigma, St. Louis,
MO, U.S.A.) and leupeptin, pepstatin, antipain, and aprotinin
all at 10 Ng/ml (Sigma) . SBP in cell lysates was immunopre-
cipitated as described below . In some experiments, the chase
was performed in glucose-free media that contained the H+
ionophore, carbonyl cyanide m-chlorophenyl hydrazone
(CCCP ; 20 pM; Sigma), or at 20°C. The lower temperature
is known to delay or stop transport of newly synthesized
protein from the rough endoplasmic reticulum (RER) to the
Golgi apparatus (Burgess and Kelly, 1987) . Because this
process is ATP dependent, it is also inhibited by CCCP.
SDS-polyacrylamide gel electrophoresis (PAGE) was car-
ried out as described by Laemmli (1970) on 8.5% acrylamide
 SEROTONIN BINDING PROTEIN
slab gels . Equal amounts of protein were layered in each
lane . The positions of labeled bands were visualized by fluo-
rography . Gels were fixed, dried, affixed to Kodak XAR-2
film, and exposed at -70°C. The resulting fluorograms were
scanned densitometrically (using a UMax 600 scanner) and
analyzed using the NIH image program (version 1 .49) on a
Macintosh Quadra 800 computer . Peaks were identified on
the scans and the proportion of the radiolabeled proteins that
migrated with the three forms of SBP was determined by
measuring the area under each peak . A portion of each gel
was electrophoretically transferred to nitrocellulose . Lanes
in these blots were either stained with Amido Black IOB
(Bio-Rad, Richmond, CA, U .S .A .) or used for the immunolo-
calization of SBP. The blots were treated for 1 h with 2 .5%
nonfat dry milk (Carnation) in PBS (pH 7 .5) containing
0.05% Tween-20, rinsed, and immunostained with mono-
clonal antibodies that recognize all three forms of SBP (Liu
et al ., 1990b) .
Effect of CCCP on transport of proteins from the
RER through the Golgi complex
To confirm that CCCP prevents transport of newly synthe-
sized proteins from RER to Golgi, the effect of CCCP on
the conversion of the high-mannose residues of asparagine-
linked glycoproteins to complex oligosaccharides was inves-
tigated. Newly synthesized glycoproteins were pulse labeled
with ['H]mannose and chased in the presence or absence of
CCCP. High-mannose sugars are added to asparagine resi-
dues in the RER and then trimmed and processed further to
complex oligosaccharides in the Golgi apparatus (Kornfeld
and Kornfeld, 1985 ; Kornfeld, 1987). The [3H]mannose la-
bel, therefore, is added only in the RER and cannot render
a complex oligosaccharide radioactive unless the labeled gly-
coprotein moves into the Golgi apparatus, which is where
the terminal sugars of complex oligosaccharides are added.
Wheat germ agglutinin binds to complex oligosaccharides,
but not to high-mannose sugars ; wheat germ agglutinin bind-
ing, therefore, was utilized to isolate glycoproteins with com-
plex oligosaccharides . MTC cells (6 x 106) were incubated
for 30 min at 37°C with 50 kCi/ml of [3H]mannose (15 Ci/
mmol ; American Radiolabel Co ., St. Louis, MO, U.S .A .) .
Cells were then rinsed with PBS and chased for 90 and 180
min with or without CCCP (20 yM). Cells were washed
again with PBS, collected by centrifugation, and lysed (30
min at 4°C) in buffer (25 mM HEPES, pH 7.0 ; 1 % Triton
X-100; 0.2% deoxycholate ; and the proteolytic enzyme in-
hibitors mentioned above) . Cell debris was removed by cen-
trifugation (10 min; 12,000 g) . The lysate (200 Fig of protein)
was then incubated with wheat germ agglutinin coupled to
agarose beads (Sigma) for 17 h at 4°C. The beads were
collected by centrifugation and washed with 25 mM HEPES
buffer, and the radioactivity of the glycoproteins bound to
the beads was measured by liquid scintillation.
Effect of phospholipase C on the apparent
molecular weight of the three forms of SBP
Posttranslational modification of a single protein could
give rise to products of different size, for example, by adding
carbohydrate or lipid moieties . SBP is not glycosylated (Ger-
shon and Tamir, 1985) . To determine whether one or more
forms of SBP is covalently attached to phosphatidylinositol,
a crude preparation that contained all three forms of SBP
(500 N,g) in 0.02 M Tris-HCI, pH 7.0, containing 0.1 M NaCl
and 0.0025 MMgCl2, was treated with phospholipase C (200
p,,g ; 13 U/mg ; Sigma) . Following exposure to the enzyme (60
99
min at 37°C), the treated proteins were separated by 10%
SDS-PAGE, transferred to nitrocellulose sheets, and stained
with Coomassie Blue R-250 (Sigma) as described previously
(Tamir et al ., 1989).
Measurement of [35 S]SBP release from
metabolically labeled MTC cells
MTC cells were labeled with 135 S]methionine by incuba-
tion overnight as described above. Medium was aspirated
and the monolayers were rinsed twice with Ca 21 -free mini-
mum essential medium (MEM) . Cells were then incubated
at 37°C in Hanks salt solution containing 2 or 5 mM Ca`+.
The medium was collected after 30 min and the radioactivity
was determined . The harvested cells were washed exten-
sively and lysed . [35 S] SBP was immunoprecipitated as de-
scribed below and fractionated by SDS-PACE .
Immunochemistry
Immunoprecipitation was used to isolate 35 S-labeled SBP.
Cell lysates were passed five times through a 19-gauge nee-
dle and centrifuged at 12,000 g for 10 min at 4°C. The
supernatant fluid was stored at -70°C until analyzed . For
the precipitation of labeled SBP released into the media,
aspirated media were centrifuged (12,000 g) and the superna-
tant fluids were concentrated to a volume of 1 ml (using a
Centricon microconcentrator; Amicon, Danvers, MA,
U.S .A .) . The solutions were then incubated at 4°C overnight
with a monoclonal antibody that recognizes all three forms
of SBP (44-10 ; Liu et al ., 1990b) . The resulting immune
complexes were collected by precipitation with secondary
antibodies (rabbit anti-mouse IgM; 2 h at 4°C) . Precipitates
were washed twice with lysis buffer. The final pellets were
suspended in SDS sample buffer, resolved by SDS-PAGE,
and quantified .
Immunocytochemistry
SBP immunoreactivity was located as described pre-
viously (Tamir et al ., 1989) . In brief, cells were fixed with
4% formaldehyde and permeabilized with 0.2% Triton X-
100 (Sigma). Polyclonal antibodies to 45-kDa SBP were
applied and sites of immunoreactivity were visualized using
goat anti-rabbit IgG coupled to rabbit horseradish peroxidase
(Kirkegaard and Perry, Gaithersburg, MD, U .S .A .) . Peroxi-
dase activity was visualized with 3,3'-diaminobenzidine and
H2O, . In control experiments, nonimmune serum was substi-
tuted for primary antibodies .
Retention and membrane binding of SBP
Isolated parafollicular cells or MTC cells were plated onto
glass cover slips coated with poly-tAysine (12.5 Ng/ml ;
Sigma) and cultured overnight in MEM supplemented with
10% fetal bovine senun (Tamir et al ., 1990) . Cells were
stimulated to secrete by the addition of 5 .0 mM Ca 2+ or
thyroid stimulating hormone (10.0 mU/ml; NIH) . To deter-
mine whether 45-kDa SBP appears on the plasma membrane
of secreting cells, secretion-dependent uptake of antibodies
to 45-kDa SBP was investigated . Secretogogues were added
to cells simultaneously with partially purified antibodies to
45-kDa SBP (y-globulin fraction) and incubated for 20 min
at 37 °C. As a control, secretogogues were added with preim-
mune serum and incubated similarly. The stimulated cells
were washed once with PBS and allowed to incubate for an
additional 30 min at 37°C . The cells were then washed three
times with PBS, fixed with 4% formaldehyde in PBS for 20
min at room temperature, and permeabilized with 0.2% Tri-
ton X-100. A biotinytated goat anti-rabbit antibody (1 :200 ;
J. Neurohem ., Vol . 63, No. l, 1994
 10 0 H. TAMIR ET AL .
.Jackson Labs ., West Grove, PA, U.S .A .) and avidin-fluores-
cein isothiocyanate (1 :500 ; Jackson Labs .) were used to visu-
alize the internalized antibodies to SBP.
The possibility that secretion causes SBP to appear on
cell surfaces was also investigated by determining whether
cells lyse when they are made to secrete in the presence of
antibody to SBP and complement . MTC cells were stimu-
lated to secrete by the addition of elevated external calcium
([Ca'- ' 1, ; 5 mM) or cholera toxin (10 pg/ml; List Biological
Laboratories Inc., Campbell ; CA, U.S .A .) for 2 h at 37°C . In
the experimental preparations, the secretogogue was added
simultaneously with antibodies to SBP (1 :200) and/or guinea
pig complement (1 :100 ; GIBCO, Grand Island, NY, U.S .A .)
in a medium free of fetal bovine serum. Cells were washed
and harvested for assay of SBP by ELISA and for the mea-
surement of 5-HT by HPLC . The concentrations of 5-HT
and 5-hydroxyindoleacetic acid were determined using
HPLC with electrochemical detection as described pre-
viously (Tamir et al ., 1990). The ability of complement to
kill cells was investigated by means of the fluorescent live/
dead viability test described below. In additional experi-
ments, MTC cells were subjected to repeated rounds of se-
cretogogue-dependent complement-mediated lysis, to pro-
duce an SBP-free variant cell line . When this was done, the
experiments were carried out as described above; however,
the cells were not harvested following treatment with com-
plement. Instead, they were washed, replated, and allowed
to grow to confluency .
Live/dead viability test
MTC cells were grown in glass petri dishes and stimulated
to secrete by the addition of elevated [Ca" 1, (5 mM) or
cholera toxin (10 Ng/ml) for 2 h at 37°C . In the experimental
preparations, the secretogogues were added simultaneously
with antibodies to SBP and/or guinea pig complement as
described above. To assess the viability of the cells, 2 pM
calcein acetoxymethyl ester and 3 .75 uM ethidium homodi-
mer (Bioprobes, Eugene, OR, U .S .A .) were added to each
dish . The cells were incubated at room temperature for 15
min and observed at a magnification of x40 using an in-
verted fluorescence microscope with excitation and emission
wavelengths of 456-490 and 515-565 nm, respectively .
Live cells generate an intense green fluorescence . Dead cells
are distinguished by the red fluorescent staining of nucleic
acids (Moore et al ., 1990) . Three control experiments were
carried out, in which complement, the secretogogue, or anti-
bodies to SBP were individually omitted. At least 100 cells
were counted in each dish .
RESULTS
The 68-, 56-, and 45-kDa SBP are each primary
products of translation
The observation that there are three SBPs of differ-
ent size led to the hypothesis that there may be a pre-
cursor-product relationship between the higher molec-
ular mass (68 and 56 kDa) and the lower molecular
mass (45 kDa) species (Liu et al ., 1990a,b) . Pulse-
chase experiments were carried out to test this hypoth-
esis . Confluent cultures of MTC cells were pulse la-
beled with ["S]methionine for 20 min and chased for
up to 90 min in the presence of an excess of nonradio-
active methionine . SBPs were immunoprecipitated
with antibodies that react with all three isoforms and
J. Nrurachem_ Val . 63, No . 1, 1994
FIG. 1 . The relative proportions of 45-, 56-, and 68-kDa SBP
do not change as a result of posttranslational processing or
intracellular transport. The results of pulse-chase experiments
are illustrated. MTC cells were incubated in the presence of
[35S]
methionine for 20 min and chased for 30-90 min in a me-
dium containing an excess of nonradioactive methionine . Radio-
labeled SBP was immunoprecipitated and resolved by 8.5%
SDS-PAGE . A: Fluorograph showing the intensity of radioactivity
migrating as 45-, 56-, or 68-kDa SBP. The positions of the three
forms of SBP are indicated . Each lane corresponds to a different
time in chase medium in the absence or presence of CCCP . B:
Density of the bands was measured and plotted as a function
of time in the absence (-) or presence (+) of CCCP (20 N".
The relative proportions of each of the three forms of SBP were
unchanged as a function of time in chase medium . When added
after the pulse-labeling period, CCCP did not alter the relative
proportions of 45-, 56-, or 68-kDa SBP. The error bars represent
standard errors of the mean of three independent experiments .
fractionated by SDS-PAGE (Fig . IA) . No change was
detected in the proportions of 35 S-labeled 68-, 56-, or
45-kDa SBP as a function of time in chase media (Fig .
I B) . Neither a time-dependent increase in 45-kDa SBP
nor a time-dependent decrease in either 68- or 56-kDa
SBP was observed . These data suggest that neither of
the higher molecular weight species of SBP is post-
translationally converted to the lower molecular weight
molecules. To examine further the possible role of in-
tracellular transport through the vacuolar apparatus in
the processing of SBPs, transport was interrupted with
CCCP. CCCP was added to the chase and not to the
pulse medium, so as not to interfere with the synthesis
of '-5S-labeled SBP. CCCP is an ionophore that uncou-
ples oxidative phosphorylation and thus depletes ATP,
which is required for transport of newly synthesized
secretory proteins from the RER to the Golgi apparatus
(Burgess and Kelly, 1987). The addition of CCCP did
not alter the proportions of 68-, 56-, or 45-kDa SBP
at 30, 60, or 90 min of chase (Fig . 1 B) . A control
experiment was carried out to determine how effec-
tively CCCP interferes with transport of newly synthe-
sized protein from RER to the Golgi apparatus . [;H]-
Mannose was used to pulse label newly synthesized
glycoproteins. The effect of adding CCCP to the chase
medium on the Golgi-dependent conversion of high-
mannose to complex oligosaccharides (Kornfeld and
Kornfeld, 1985 ; Kornfeld, 1987) was then determined .
 SEROTONIN BINDING PROTEIN

FIG. 3. Phospholipase C does not alter

the apparent molecular weight of the
three forms of SBP. A crude preparation
that contained all three forms of SBP
(500 pg) was incubated with phospholi-
pase C (200 pg ; 13 LI/mg) for 60 min at
37°C . The treated proteins were sepa-
rated by 10% SIDS-PAGE . Lane A repre-
sents molecular weight standards; lane
B represents control proteins ; lane C
represents proteins following incubation
with phospholipase C.

FIG. 2. MTC cells were pulse labeled with [3H]mannose for 30

min. Glycoproteins containing complex oligosaccharides were
affinity isolated by binding to wheat germ agglutinin (WGA) beads
at 0, 90, and 180 min of chase in the absence (-) or presence
(+) of CCCP . To correct for transport of protein from the RER with ["S]methionine for 17 h . The labeled cells were
to the Golgi apparatus during the pulse, the mean radioactivity washed and stimulated to secrete by increasing the
found to be bound to the WGA beads at time 0 was subtracted
from that at each time of chase. Data were then normalized as concentration of Calf in the medium ([Ca-+ [,) to 5 .0
a ratio of bound to total radioactivity. The bars show the standard mM . [AS S]SBP was immunoprecipitated from the me-
errors of the mean of three measurements . The rise in complex dium and fractionated by PAGE . Both MTC cells and
oligosaccharides at 90 (p < 0.001) and 180 min (p < 0.005) in their parent parafollicular cells are stimulated to secrete
the absence of CCCP is significant. None of the values obtained
in the presence of CCCP differs significantly from the value at by increasing [Ca2' ]e (Hirsch and Munson, 1969 ;
time 0; however, at 90 and 180 min, the radioactivity of the Tamir et al ., 1990). Under basal conditions (2 .0 mM
complex oligosaccharides is significantly greater in the absence Ca"), 45-kDa, but not 56- or 68-kDa, SBP was found
than in the presence of CCCP (p < 0.001). CCCP prevents con- to be present in the medium ; however, a clear increase
version of high-mannose sugars to complex oligosaccharides .
in the concentration of 45-kDa SBP in the medium was
elicited by increased [Ca2+ ] ~ (Fig . 4A) . No significant
CCCP, at the same concentration used in the pulse- release of 56- or 68-kDa SBP was observed . In contrast
chase experiments with ["S]methionine, totally to the medium, increased [Ca2+ ], caused the concentra-
blocked the conversion of high-mannose to complex tion of 45-kDa SBP within the MTC cells to decrease
oligosaccharides (Fig . 2) . It thus can be concluded that
CCCP prevents transport from RER to Golgi. An addi-
tional experiment was carried out by incubating cells
at 20°C during the chase period . This treatment, like
exposure to CCCP, should interfere with passage of
newly synthesized secretory proteins from the RER to
the Golgi apparatus. Again, no change was seen in the
proportions of AS S-labeled 68-, 56-, or 45-kDa SBP
after 30, 60, or 90 min of chase (not illustrated) . These
observations indicate that passage through the Golgi
apparatus or a post-Golgi compartment is not required
to produce any of these SBP isoforms .
In addition to being altered by cleavage, the size of
the primary product of translation potentially could
be altered by glycosylation or covalent attachment of
phosphatidylinositol via a glycan linkage. None of the FIG. 4. Increased [Ca"], stimulates MTC cells to secrete 45-
three forms of SBP was glycosylated ; however, a mix- kDa SBP. MTC cells were metabolically labeled with [35 S]-
ture of the three proteins was incubated with phospho- methionine for 17 h. Cells were then stimulated to secrete by
adding Ca' to the medium to bring the [Ca2 ]e to 5 .0 mM. After
lipase C to determine whether any were attached to 30 min, the medium was collected and the cells were homoge-
phosphatidylinositol. Following exposure to the en- nized. Radiolabeled SBP was immunoprecipitated separately
zyme, the apparent size of each of the three forms of from the medium (A) and the cell homogenate (B). The three
SBP remained unchanged as judged by SDS-PAGE forms of SBP were then resolved by 8.5% SDS-PAGE (equal
amounts of protein were added to each lane). The 45-kDa form,
(Fig . 3) . but not the 56- or 68-kDa form, of SBP was found in the medium,
Secretion of 45-kDa SBP is evoked by a both in the presence of increased [Ca'`], and under resting con-
ditions; however, in the presence of increased [Ca"],, the
secretogogue amount of 45-kDa SBP released to the medium was increased.
To determine which, if any, of the isoforms of SBP The 56-kDa form of SBP could be detected in cellular homoge-
are secreted, MTC cells were metabolically labeled nates, but not in the medium .

J. Neutoc'lwin . . Vol. 63, No . 1, IVV4

/02 H . TA MIR ET AL .
(Fig . 4B) . These observations suggest that 45-kDa SBP
is secreted when MTC cells are stimulated with in-
creased [Ca 2 ' 1 ' .
A fraction of the secreted SBP remains bound to
the plasma membrane
The fact that 45-kDa SBP is secreted implies that
the protein is present in secretory vesicles . MTC cells
secrete 5-HT as well as SBP (Tamir et al ., 1990) . Fol-
lowing secretion, recycled vesicles can be restocked
with 5-HT, which is synthesized in the cytosol and
can be transported into secretory vesicles . No such
mechanism is available for a protein, such as SBP . The
hypothesis was thus tested that a portion of the SBP
ordinarily present in vesicles is bound to the inner face
of the vesicular membrane, remains bound to the cell
surface following exocytosis, and is recaptured with
the vesicular membrane by endocytosis during the re-
covery period . Antibodies to SBP were added to cul-
tures of living cells as a probe to detect the appearance
of surface-bound SBP . When nonstimulated MTC cells
were exposed to antibodies to SBP, no immunoreactiv-
ity was detected (Fig . 5A) . Because the cells were not
permeabilized, antibodies would bind to the cells only
if immunoreactive material were present on the cell
surface. Secretion was evoked by raising the (Ca-+1,
to 5 mM. Under these conditions, MTC cells became
intensely SBP immunoreactive (Fig . 5B) . The appear-
ance of the cell surface SBP immunoreactivity was
punctate . As there was no detectable immunoreactivity
on the cell surfaces when the cells were stimulated
in the presence of preimmune serum (Fig . 5D), the
secretion-induced immunostaining of the cell surface
was specific . The stimulated cells were allowed to re-
cover for 20 min . After this period, there was a pro-
nounced decrease in the immunoreactivity of the sur-
face (not illustrated) . Cells were fixed and permeabil-
ized at this stage . The permeabilized cells were
exposed to secondary antibodies to determine whether
the cells had internalized antibodies to SBP during the
stimulation and subsequent rest periods . Immunoreac-
tivity was observed (Fig . 5C) . These experiments were
repeated with primary parafollicular cells isolated from
sheep thyroid glands to determine whether the secre-
tion-induced appearance of cell surface SBP immuno-
reactivity also occurred in a nontumor cell of the type
from which MTC cells are derived . Relatively little
SBP immunoreactivity was found on the surfaces of
resting parafollicular cells . Stimulation with increased
[Ca" 1, (5 .0 mM), however, caused immunoactive SBP
to appear on the plasma membrane . When either MTC
cells or parafollicular cells were stimulated with in-
creased [Ca 2 ~]~ in the presence of an unrelated anti-
body, the cell surface-immunoreactive material did not
appear (not illustrated) . These observations are consis-
tent with the idea that membrane-bound SBP becomes
externalized as a result of secretion and is reinterna-
lized during the postsecretion recovery period .
I. New . . hem
., Vol. 63, No. l, 1994
MTC cells lyse when they are stimulated to
secrete in the presence of antibodies to SBP plus
complement
If, as the experiments described above suggest, anti-
bodies bind to SBP that appears on cell surfaces as a
result of secretion, then MTC cells should be subject
to lysis when they are stimulated to secrete in the
presence of antibodies to SBP upon addition of com-
plement. This prediction was tested . The live/dead via-
bility test was used as a probe to detect cell lysis . As
cells lyse in the presence of ethidium homodimer, they
become permeable and intensely orange-red fluores-
cent . Resting MTC cells were found to be stable in
vitro when they were exposed to antibodies to SBP
alone, to complement alone, or to the complement plus
antibodies to SBP (Fig . 6A) . Secretion was induced
with increased [Ca'-+1, (5 .0 rnM) or cholera toxin (10
h, g/ml) . Cholera toxin was used for these experiments
because it has been shown previously to stimulate
MTC cells potently and rapidly to secrete (Tamir et
al ., 1990) . Both secretogogues led within 15 min to
the appearance of lysed cells (Fig . 6B and C) . When
cholera toxin was employed as the secretogogue, 15
± 5% of the total number of cells was lysed . In con-
trast, no lysis was detected when either secretogogue
was applied in the absence of antibodies to SBP and
complement (not illustrated) .
MTC cells were exposed to repeated cycles of se-
cretogogue stimulation in the presence of antibodies
to 45-kDa SBP and complement. Following each cycle
of complement-mediated lysis, the surviving cells were
permitted to regrow to confluency . After 10 such cy-
cles, immunocytochemical examination revealed that
the resulting cells (Fig . 7A) contained much less SBP
immunoreactivity than did control cells that had not
been subjected to cycles of immunolysis (Fig . 7B) .
Enzyme-linked immunosorbent assay was used to esti-
mate the degree to which the SBP content of MTC
cells surviving after repeated cycles of immunolysis
had been reduced ; the 45-kDa SBP content of the ex-
perimental cells was 48 - 8% of controls . The 5-
HT concentration in the MTC cells remaining after
repeated cycles of complement-mediated lysis was 50
+ 10% of controls . It is concluded that subjection of
MTC cells to repeated cycles of lysis secondary to
stimulation to secrete in the presence of antibodies to
SBP and complement selects a variant line that is both
SBP and 5-HT deficient .
DISCUSSION
The present study was undertaken to test whether the
smaller forms of SBP are derived by posttranslational
proteolysis from a larger precursor and to test the hy-
pothesis that 45-kDa SBP is secreted . In view of the
known similarities among the proteins, the data ob-
tained were surprising because they suggested that
none of the three forms of SBP is derived from i
larger precursor molecule. The relative proportions of'
 SEROTONIN BINDING PROTEIN 10 3

FIG . 5 . The 45-kDa form of SBP

immunoreactivity appears on the
surface of stimulated MTC cells . A :
Control cells incubated in the pres-
ence of 2 .0 mM Ca" and antibod-
ies to SBP . Little surface labeling is
detected . B : Secretion was evoked
in the presence of antibodies to
SBP by bringing the [Ca21]e to 5 .0
mM . Antibodies to SBP bound to
the cells . C : Cells that were treated
as in B were washed and incubated
for an additional 30 min . When fixed
and permeabilized, the cells con-
tained internalized rabbit immuno-
globulin (antibodies to SBP) . D:
Cells treated identically as in C, but
stimulated in the presence of preim-
mune sera contained no detectable
internalized rabbit immunoglobulin .
The markers represent 10 pm .

radioactive 45-, 56-, and 68-kDa SBP did not change
as a function of time in chase medium following their
biosynthesis during a pulse-labeling incubation in the
presence of [ 35S]methionine . If 68-kDa SBP or another,
still larger, molecule had been a precursor of the
smaller forms of SBP, then the proportional radioactiv-
ity of the smaller molecules would have been expected
to increase during the chase period, whereas that of
the larger molecules would have been expected to de-
crease . Instead, the relative proportions of each of the
three forms of SBP remained relatively constant
throughout a 90-min incubation in chase medium .
These data suggest that each of the three isoforms of
SBP is probably a primary product of translation . The
possibility that a large and still unidentified molecule
is cleaved to yield each of the three isoforms of SBP
in less than 20 min, the time allotted for pulse labeling
in the presence of [ 35 S]methionine, has not been ex-
cluded ; however, the data imply that 68-kDa SBP does
not give rise to 56- or 45-kDa SBP, and that 56-kDa
SBP is not a precursor of the 45-kDa protein . These
conclusions were supported by observations of the ef-
fects of addition of CCCP to the chase medium and
by subjecting pulse-labeled cells to a chase at 20°C.

1 . Neuio,hem ., Vu/ . hß. No . 1 . 1994

 104 H. TAMIR ET AL.

FIG . 6 . MTC cells lyse when they

are stimulated to secrete in the
presence of antibodies to 45-kDa
SBP plus complement . A: MTC
cells were incubated in the pres-
ence of both antibodies to SBP
and complement . The [Ca' - ], was
2 .0 mM . No lysis (orange fluores-
cence) can be detected . B : Cells
were incubated as in A, in the
presence of antibodies to SBP
and complement ; however, in-
creased [Ca2 '] e (5 .0 mM) was
used to stimulate secretion . Or-
ange cells indicate that a portion
of the cells was lysed . C : MTC
cells were again incubated in the
presence of antibodies to SBP
and complement ; however, chol-
era toxin (10 ug/ml) was used to
evoke secretion . Orange cells
again were observed, indicating
that a subset was lysed under
these conditions . The markers
represent 15 pm .

1. Neuroehern., Vol . 63, No . 1, 1994

 SEROTONIN BINDING PROTEIN
FIG. 7 . Repeated cycles of secre-
tion-dependent, complement-me-
diated lysis in the presence of anti-
bodies to SBP selects an SBP-de-
ficient variant MTC cell line. A :
Immunoreactivity of 45-kDa SBP
demonstrated in MTC cells derived
from progenitors that were exposed
10 times to cycles of stimulation
with cholera toxin (10 pg/ml) in the
presence of antibodies to SBP and
complement . B : Immunoreactivity
of 45-kDa SBP demonstrated in
control MTC cells . The surviving
cells were permitted to grow to con-
fluence between cycles . SBP im-
munoreactivity decreased markedly
in the MTC cell variant line selected
by repeated cycles of secretion-de-
pendent, complement-mediated ly-
sis in the presence of antibodies to
SBP . Markers represent 30 pm .
Each of these perturbations is known to interfere with
the transfer of newly synthesized protein from the RER
to the Golgi apparatus . The effectiveness of CCCP in
this regard was verified by ascertaining that it com-
pletely blocked the conversion of high-mannose sugars
to complex oligosaccharides, a step that depends on
transport from RER to Golgi (Kornfeld and Kornfeld,
1985 ; Kornfeld, 1987) . Neither CCCP nor incubation
at 20°C altered the proportions of radioactive 68-, 56-,
or 45-kDa SBP found in the cells . These data suggest
that the size of newly synthesized SBP is not altered
as a result of processing in a compartment reached as
a consequence of intracellular transport through the
vacuolar apparatus of the cells . Finally, incubation of
68-, 56-, or 45-kDa SBP with phospholipase C failed
to alter the apparent molecular weight of any of the
proteins ; furthermore, none of the forms of SBP was
glycosylated . It can be concluded that the three iso-
forms of the SBP are products of different genes, or
that they are synthesized as the result of alternative
splicing of mRNA transcribed from a single gene. In
considering these observations, it should be recalled
that secretion by parafollicular and MTC cells is regu-
lated and that no secretogogue was added during any
of the three pulse-chase experiments . The proportions
of the three forms of SBP were thus not altered by
secretion, as they would have been unless all three
were secreted in equal amounts .
Studies of the regulated secretion of the three iso-
forms of SBP provided evidence that only 45-kDa SBP
is secreted . Exposure of metabolically labeled MTC
cells to increased [Ca"],, which is known to induce
the cells to secrete 5-HT (Tamir et al ., 1990), caused
them to secrete 35 S-labeled 45-kDa SBP as well . In
105
contrast, no secretion of 35 S- labeled 56- or 68-kDa SBP
could be detected under these conditions . These data
suggest that 45-kDa SBP is probably synthesized in
the RER of MTC cells and transported via the secretory
pathway of intracellular transport to be packaged in
secretory vesicles . These results are compatible with
prior electron microscopic immunocytochemical ob-
servations that the immunoreactivities of 45-kDa SBP
and 5-HT are found in the same secretory vesicles in
both MTC cells and parafollicular cells (Barasch et al.,
1987 ; Tamir et al., 1990) . The failure of increased
[Ca21], to cause the cells to secrete 56- or 68-kDa
SBP may be due to a difference in the intracellular
cornpartmentation of the three isoforms of SBP. In
contrast to 45-kDa SBP, it seems likely that the 56-
and 68-kDa isoforms are probably not packaged in
5-HT-containing secretory vesicles and thus are not
released by stimuli that induce the secretion of 5-HT .
Whether they enter the cisternal space at all remains
to be determined. It has been shown previously that
SBP is secreted from electrically stimulated enteric
neurons and that this release is Ca`+ dependent (Jona-
kait et al ., 1979) . Enteric neurons, moreover, contain
only 45-kDa SBP [the larger isoforms cannot be de-
tected in the bowel (Gershon et al., 1983)] ; therefore,
it is likely that in serotonergic neurons, as well as in
the MTC paraneurons, 45-kDa SBP is a component of
synaptic vesicles that is secreted together with 5-HT
as a result of exocytosis . This idea would explain the
observation that 45-kDa SBP and not 56-kDa SBP is
concentrated in the terminals of serotonergic neurons
in the brain, and explain why 45-kDa SBP is subject
to fast axonal transport (Gershon et al ., 1983) .
Costorage of 45-kDa SBP with 5-HT in the same vesi-
J. Neurochern ., Vol . 03, Nu. 1, 1994
106 H. 7AMIR ET AL.
cles of serotonergic neurons would also account for the
observation that newly taken up [3H]5-HT is rapidly
complexed with 45-kDa SBP in the CNS in situ .
The secretion of a large protein, such as SBP, from
a neuron raises a question that does not arise with
respect to a small-molecule neurotransmitter, such as
5-HT . This question is how synaptic vesicles or their
membranes can be recycled . Synaptic vesicle recycling
has not been studied in serotonergic neurons, but it is
known to occur in other systems (see Südhof and Jahn,
1991 ; Kelly, 1993), including the motor nerve terminal
at the neuromuscular junction, where it has been inves-
tigated extensively (Heuser and Reese, 1981 ; Koening
and Ikeda, 1989) . Vesicle recycling thus would be ex-
pected to occur in serotonergic cells as well . Recycling
involves the recapture by endocytosis of the synaptic
vesicle membrane that has fused with the plasma mem-
brane of the nerve terminal during exocytosis. The
retrieved membrane is then reutilized to form new syn-
aptic vesicles that have to be restocked with a neuro-
transmitter. Integral membrane proteins that are com-
ponents of the synaptic vesicle membrane are retrieved
by endocytosis together with the membrane and thus
do not have to be resynthesized or added to the re-
formed vesicles. The neurotransmitter 5-HT is synthe-
sized in the cytosol, but is transported into vesicles by
a reserpine-sensitive carrier protein that recently has
been cloned (Liu et al., 1992). How a soluble protein
of the vesicular interior could be added to a recycled
vesicle is not at all clear. Two possibilities can be
envisioned. Because proteins enter the vacuolar space
of cells cotranslationally, 45-kDa SBP cannot, like 5-
HT, be transported through the vesicular membrane .
Addition of newly synthesized 45-kDa SBP would re-
quire recycling vesicles to fuse with virgin vesicles.
As there are no ribosomes or RER in nerve terminals,
such a mechanism would necessitate the transport of
vesicles containing 45-kDa SBP from the cell body.
An alternative mechanism involving the recycling of
45-kDa SBP along with the vesicular membrane would
make the system more efficient . The 45-kDa SBP origi-
nally present in 5-HT-storing secretory vesicles could
continue to be utilized if a fraction of that protein were
retained at the time of exocytosis . Retention of 45-kDa
SBP could be accomplished if a fraction of the total
present in vesicles were to be bound to the luminal
face of the vesicular membrane. If this binding occurs,
then patches of 45-kDa SBP would be expected to
appear on the cell surface when cells are stimulated
to secrete. This exteriorized 45-kDa SBP would be
reinternalized when the vesicular membrane is re-
trieved by endocytosis . The process would result in a
progressive decline in the quantity of vesicular 45-kDa
SBP as vesicles recycle repeatedly ; however, vesicles
have a finite lifetime and there is probably a limit to
the number of times recycling can occur.
Experiments were carried out to test the hypothesis
that 45-kDa SBP is recycled, as well as released, when
parafollicular cells or MTC cells are stimulated to se-
J. Neurocheni_ Vol. 63, No. l, 1994
crete. The predicted secretion-dependent appearance
of 45-kDa SBP was detected by stimulating cells to
secrete in the presence of antibodies to the protein. The
fact that 45-kDa SBP was bound to the cell surfaces of
secreting cells was demonstrated by both immunocyto-
chemistry and complement-mediated lysis. Patches of
45-kDa SBP immunoreactivity appeared as a result of
stimulation, and the cells were lysed when they were
stimulated in the presence of antibodies to 45-kDa SBP
and complement. Furthermore, when time was allotted
for endocytosis to occur after the cells were stimulated
to secrete, the antibodies to SBP that were present in
the medium were taken up by the cells . These observa-
tions are compatible with the idea that the secretory
vesicles of MTC and parafollicular cells, like synaptic
vesicles, recycle and that a fraction of the vesicular
content of 45-kDa SBP is retained as a result of binding
to the luminal face of the vesicular membrane. Re-
maining to be demonstrated is the proportion of 45-
kDa SBP secreted versus that retained, and the mecha-
nism of 45-kDa SBP binding . In several other systems
also, components of synaptic vesicles are known to be
partially released and partially retained during exo-
cytosis . These molecules include dopamine /3-hydrox-
ylase (Viveros et al., 1968; De Potter et al ., 1969;
Sabban et al., 1987), carboxypeptidase E (Fricker et
al., 1990), the prehormone convertases, PC] and PC2
(Kirchmair et al., 1992), and chromogranins A and B
(Settleman et al ., 1985 ; Pimplikar and Huttner, 1992).
MTC cells normally are heterogeneous with respect
to their content of 45-kDa SBP. It follows, therefore,
that subjection of MTC cells to repeated cycles of
secretion-dependent, complement-mediated lysis can
be used to select a variant cell line that is 45-kDa SBP-
deficient . The technique of repeated cycles of activity-
dependent, complement-mediated lysis previously has
been used successfully to obtain a synaptotagmin-
deficient variant cell line (Kasai et al., 1992) . Synapto-
tagmin is a vesicular membrane protein . MTC cell
variants deficient in 45-kDa SBP were obtained by
repeating cycles of' secretion-dependent, complement-
mediated lysis. These variants were found to have
lower than control levels of 5-HT. This observation
suggests that 45-kDa SBP may be important in the
vesicular storage of 5-HT.
Acknowledgment : This study was supported by NIMH
grant 37575, NS grant 12969, and DK grant 19743 .
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