From the
The NH
Endocrine cells and peptidergic neurons contain a regulated
secretory pathway, in addition to the ``default''
constitutive secretory pathway present in all cells
(1) .
Prohormones and proneuropeptides are intracellularly routed through the
endoplasmic reticulum and Golgi apparatus to the trans-Golgi network
(2, 3, 4, 5) . In the trans-Golgi
network they are actively sorted away from other proteins and packaged
into the granules of the regulated secretory pathway where they are
proteolytically processed to biologically active peptides that are
released from the cell upon stimulation
(6, 7) . The
nature of the sorting signal involved in directing prohormones and
proneuropeptides to these regulated secretory granules has not been
determined, because no consensus amino acid sequences have been found.
The adrenocorticotropin/endorphin prohormone pro-opiomelanocortin
(POMC)
In this study,
site-directed mutagenesis was used to identify the specific molecular
motif within N-POMC encoding this sorting signal. A unique 13-amino
acid amphipathic loop structure, stabilized by one disulfide bridge, at
the NH
In order to determine
whether other regions of NH
Previous studies from our laboratory have shown that the
first 26 amino acids of N-POMC contained information that was
sufficient to sort a reporter protein, chloramphenicol
acetyltransferase, to the regulated secretory pathway in AtT20 cells
(14) . In addition, we have shown that when 25 amino acids of
N-POMC (Cys
The participation of the disulfide bridges in sorting was also
tested by substituting a serine residue for cysteine at position 2,
position 8, or both, which effectively disrupted the disulfide bridges.
Disruption of the second disulfide bridge or both disulfide bridges
resulted in the missorting of POMC to the constitutive secretory
pathway. Mutant POMC secreted by the constitutive secretory pathway was
not processed. However, disruption of the first disulfide bridge had no
effect on sorting to secretory granules. Thus we conclude that sorting
of POMC to the regulated secretory pathway is dependent only on the
presence of the disulfide bridge closest to the amphipathic loop
(Cys
Roy et al. (22) have
reported similar results for two of the mutants described here, C2S,C8S
and C2S. However, our results differ concerning the fate of the C8S and
POMC-
First, for their secretion studies, Roy et al. (22) reported the result of only one experiment for the
POMC-
Second, the use by
Roy et al. (22) of the processing of POMC to
The requirement for the integrity of the disulfide bridge in
sorting proteins to the regulated secretory pathway has also been
demonstrated for chromogranin B
(31, 32) . Disruption of
the disulfide bridge by the addition of the thiol-reducing agent
dithiothreitol to PC12 cells caused rerouting of the endogenous
chromogranin B to the constitutive pathway. The constitutively secreted
chromogranin B, in the reduced state due to treatment with
dithiothreitol, lacked the loop structure formed by the disulfide
bridge (Fig. 1 A), implicating the involvement of this
structure as a sorting signal for the regulated secretory pathway.
A
sorting signal may also exist for prohormones without cysteine
residues, such as pro-somatostatin. In experiments in which the first
82 amino acids of the pro-region of pro-somatostatin were fused to 142
amino acids of
A comparison of the
NH
Misfolding of the natural conformation of proteins has been
associated with aggregation or retention and degradation in the early
stages of the secretory pathway
(35, 36) . Any of the
mutations ( i.e. disruption of the disulfide bridges, deletion
of the amphipathic loop structure, or the 78-amino acid deletion in
POMC) could potentially cause such misfolding. However, we found that
none of these mutations caused significant retention or degradation in
the endoplasmic reticulum, because large amounts of mutant POMC were
found to be secreted in a constitutive or regulated manner from these
cells.
How does the amphipathic heart-shaped loop of N-POMC act as a
sorting signal? The modeling results indicate that the acidic side
chains of Asp
In summary, we have provided evidence in this
paper for the first time that the regulated secretory pathway sorting
signal present in the NH
Stimulated secretion of ACTH
We thank Winnie Tam and Victor Hwang for excellent
technical assistance and Drs. Ana-Maria Bamberger and Theodore C.
Friedman for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal region of pro-opiomelanocortin
(POMC) is highly conserved across species, having two disulfide bridges
that cause the formation of an amphipathic hairpin loop structure
between the 2nd and 3rd cysteine residues (Cys
to
Cys
). The role that the NH
-terminal region of
pro-opiomelanocortin plays in acting as a molecular sorting signal for
the regulated secretory pathway was investigated by using site-directed
mutagenesis either to disrupt one or more of the disulfide bridges or
to delete the amphipathic loop entirely. When POMC was expressed in
Neuro-2a cells, ACTH immunoreactive material was localized in punctate
secretory granules in the cell body and along the neurites, with heavy
labeling at the tips. ACTH was secreted from these POMC-transfected
cells in a regulated manner. Disruption of both disulfide bridges or
the second disulfide bridge or removal of the amphipathic hairpin loop
resulted in constitutive secretion of the mutant POMC from the cells
and a lack of punctate secretory granule immunostaining within the
cells. We have modeled the NH
-terminal POMC Cys
to Cys
domain and have identified it as an
amphipathic loop containing four highly conserved hydrophobic and
acidic amino acid residues
(Asp
-Leu
-Glu
-Leu
).
Thus the sorting signal for POMC to the regulated secretory pathway
appears to be encoded by a specific conformational motif comprised of a
13-amino acid amphipathic loop structure stabilized by a disulfide
bridge, located at the NH
terminus of the molecule.
(
)
is representative of a class of
prohormones that is directed to the regulated secretory pathway in
neuroendocrine cells
(8, 9, 10, 11) .
Other members of this class of prohormones include pro-enkephalin,
pro-vasopressin, pro-dynorphin, and pro-oxytocin, all of which have in
common in their primary amino acid sequence one or more pairs of
cysteine residues that may form a disulfide bridge and thus a hairpin
loop structure in their NH
-terminal regions
(Fig. 1 A). For example, in POMC the first 26 amino acids
contain 4 cysteine residues that are highly conserved
(Fig. 1 A)
(12) and have been experimentally
shown to form a hairpin loop stabilized by two disulfide bridges
(Cys
/Cys
and Cys
/Cys
)
(13) (Fig. 1 B).
Figure 1:
A, alignment of the
NH-terminal regions showing homology between POMC,
pro-vasopressin, pro-oxytocin, pro-dynorphin, pro-enkephalin, and
chromogranin B. Dark shading indicates conserved cysteine
residues, and light shading indicates conserved acidic and
hydrophobic residues among the different prohormones (DLEL). The
heavy black outline indicates the Phe substitution for Cys in
Salmon II and Trout B POMC. B, line drawing representation of
the hairpin loop conformation and the disulfide bridges of
NH
-terminal POMC (13).
Recent work in our laboratory
(14) has demonstrated that the NH-terminal hairpin
loop consisting of 26 amino acids of POMC was sufficient for targeting
a non-native protein, chloramphenicol acetyltransferase, to the
regulated secretory pathway in AtT20 cells. However, the first 10 amino
acids of NH
-terminal POMC, which lacked the formation of
the hairpin loop, were insufficient for sorting chloramphenicol
acetyltransferase to the regulated secretory pathway. These data
suggested that residues 1-26 contained information necessary for
sorting POMC to the regulated secretory pathway.
terminus of POMC was identified as the signal
essential for the sorting of this prohormone into the regulated
secretory pathway of the mouse neuroendocrine cell line, Neuro-2a.
Site-directed Mutagenesis of Bovine POMC
Bovine
POMC cDNA (kindly donated by Dr. S. Nakanishi) was subcloned into the
cloning vector, dsM13p18
(15) . Using an Amersham mutagenesis
system (version 2, RPN 1523), substitution mutations were made by
annealing the ssM13-POMC to a 39-nucleotide probe complementary to
positions 72-111 in POMC. The probe contained glycine-to-cysteine
mutations in position 82, 101, or both, and after annealing was treated
with DNA polymerase (Klenow fragment) and T4-ligase to convert the
hybrid to dsM13-POMC. The non-mutant strand was nicked with
NciI, digested with exonuclease III, and polymerized by DNA
polymerase I and T4 ligase. The constructs were transfected into JM101
bacteria and plated. The M13-POMC phages were isolated, and the inserts
were sequenced. Wild type and mutated POMC cDNA were cut from the M13
vector by PstI and blunt ended by T4 polymerase, and
NheI linkers were ligated. The mixture was digested with
NheI and HincII, and the gel was purified. The POMC
cDNA was ligated to a NheI- and SmaI-digested pMSG
expression vector and transfected into HB101 Escherichia coli bacteria. Deletion mutations were created by restriction digestion
of the area to be deleted and blunt end ligation of the fragments. All
the POMC cDNA mutations were verified by sequencing. Bacteria
containing wild type and mutated POMC plasmids were grown in 500 ml of
LB broth and purified using Qiagen Maxi Prep kits (Chatsworth, CA).
Cell Culture and Generation of Stable Neuro-2a Cell Lines
Expressing Mutant POMC for Immunocytochemistry
Stable Neuro-2a
cell lines were made by using Lipofectin (Life Technologies, Inc.) to
transfect the plasmids containing wild type or mutated POMC and were
selected using the hypoxanthine-guanine phosphoribosyltransferase
selection marker. Ten different clones for each mutant were screened
for expression of the mutant POMC, after which three final clones were
selected for the experiments. The cells were grown on
poly-
L-lysine-coated coverslips, and expression of the
POMC/mutant POMC was stimulated with 100 n
M dexamethasone.
After 24 h the cells were fixed in 2% paraformaldehyde, permeabilized
with 0.1% Triton X-100, blocked with 10% goat serum in
phosphate-buffered saline, and incubated for 16 h with antibody to
ACTH(DP4)
(16) at 1:2500 dilution. The
cells were washed with phosphate-buffered saline and stained using a
goat anti-rabbit serum conjugated with rhodamine (Boehringer Mannheim).
Wheat germ agglutinin conjugated to fluorescein (1:250 dilution) was
used for staining the Golgi apparatus. The labeled cells were
photographed using a Nikon Optiphot fluorescent microscope.
Stimulated Secretion with
K
Neuro-2a cells
were grown in 60-mm plates and transfected with 20 µg of POMC or
mutated POMC using Lipofectin (Life Technologies, Inc.). A comparison
between stably and transiently transfected cells indicated that similar
amounts of POMC or mutant POMC were produced per well of 10/Ca
cells. Subsequently, transient transfections were used for the
secretion experiments. After 48 h of stimulation with 100 n
M dexamethasone in complete media, the transfected cells were
incubated for 3 h in a basal release medium (Medium 1) containing 25
m
M Hepes, 125 m
M NaCl, 4.8 m
M KCl, 1.2
m
M KH
PO
, 5.6 m
M dextrose, 1
m
M CaCl
, 0.5 m
M MgCl
, and
0.1% bovine serum albumin at pH 7.4. After 3 h the medium was collected
and one-half of the cells received Medium 1, while the other half
received a stimulated release medium (Medium 2) identical to Medium 1
except for the substitution of 51 m
M K
and
5.4 m
M Ca
. Both media contained 200
kallikrein-inactivating units/ml aprotinin and 1 m
M 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (ICN
Biochemicals, Aurora, OH). After 3 h of incubation in Medium 2, the
medium was collected, extracted on a Sep-Pak C
, and
fractionated by HPLC. The system used for HPLC was a Beckman 421 system
and a Bio-Rad HiPore RP-304 column (4.6
250 mm). Solvent A was
0.1% trifluoroacetic acid, and solvent B was 80% acetonitrile in 0.1%
trifluoroacetic acid. Samples were injected as 100-µl volumes and
separated using a linear gradient from 10 to 20% solvent B in 10 min
and from 20 to 70% solvent B in 60 min at a flow rate of 1 ml/min. 1-ml
samples were collected and analyzed for ACTH
by
radioimmunoassay using antibody DP6, which is specific for
ACTH
but also cross-reacts with
ACTH
, ACTH
, and
POMC
(17) . The values represent the mean ± S.E. for
three to four experiments. The percentage of total released ACTH
was calculated by dividing the ACTH
in Medium 2 by
the total ACTH
( i.e. ACTH
in Medium 1
+ Medium 2 + the cell extract).
Molecular Modeling of N-POMC
The peptide structure
was built with disulfide bridges between Cys/Cys
and Cys
/Cys
and was subjected to 250 ps
of molecular dynamics at 600 K. At each picosecond, the structure was
captured and extensively minimized using a
``conjugate-gradient'' algorithm. All calculations were
performed using the Discover and Insight programs (Biosys, Palo Alto,
CA). The backbone of the loop from Cys
to Cys
is shown as an oval ribbon with the side chains shown as shaded
sticks.
Expression of POMC in Neuro-2a Cells
When
full-length POMC was expressed in Neuro-2a cells
(18, 19, 20, 21) , ACTHwas
immunocytochemically localized to punctate secretory granules in both
the main cell body and along the neurites with very heavy punctate
labeling at the tips of the neurites, which suggested accumulation in
this region (Fig. 2 A). Wheat germ agglutinin-fluorescein
isothiocyanate staining was used to show the Golgi apparatus in these
cells (Fig. 2 A`). Immunoelectron microscopy confirmed
that these granules were similar in size to ACTH
-containing
granules described by others in the Neuro-2a cell line
(22, 23) .
(
)
Analysis of the
ACTH
products showed processing of POMC to
ACTH
and ACTH
(Fig. 3). These
ACTH products were secreted into the medium in a stimulated manner with
high K
/Ca
(Table I). These results
showed that POMC was sorted to the regulated secretory pathway in
Neuro-2a cells, thus making this a useful cell line in which to study
prohormone sorting.
Figure 2:
Immunocytochemical localization of mutant
and wild type POMC in stably transfected Neuro-2a cells. A,
wild type POMC; A`, Golgi stain; B, C2S,C8S;
B`, Golgi stain; C, C2S; D, C8S; E,
Cysto Cys
loop deletion; F, 78-amino
acid deletion. Thin arrows indicate punctate secretory
granules. Triangular arrowheads indicate neurite tips.
Large arrows in A` and B` indicate Golgi
staining, and the open triangular arrowheads in B indicate ACTH
. The data for each mutant shown are
representative of
100 cells photographed from at least 20 different
experiments. Variability in the staining of the stably transfected
cells expressing POMC or POMC mutants may be due to a decrease or loss
of expression in some cells after multiple
passages.
Effect of the Disruption of the Disulfide Bridges of POMC
on Sorting
Because the Cys residues are highly conserved across
species
(12) and are present in several prohormones (Fig.
1 A), we first determined whether the two disulfide bridges in
N-POMC (Cys/Cys
and
Cys
/Cys
) were involved in the sorting signal
motif. Substitution mutations were made to disrupt either one or both
of the disulfide bridges (Fig. 4). Disruption of both disulfide bridges
(C2S,C8S) resulted in a perinuclear ACTH immunostaining pattern,
suggesting localization within the Golgi (Fig. 2 B). Wheat germ
staining for the Golgi confirmed this co-localization
(Fig. 2 B`). Substantial amounts of ACTH
,
primarily as unprocessed POMC (Fig. 5), were released from these cells
in a non-stimulated manner, characteristic of constitutive secretion
(). Disruption of the first disulfide bridge (Fig. 4,
C2S) resulted in punctate secretory granule ACTH
immunostaining at the tips and along the length of the neurites,
similar to that of cells transfected with wild type POMC
(Fig. 2 C). Stimulated secretion of ACTH
(ACTH
and ACTH
) from
these cells was found (). Disruption of the second
disulfide bridge (Fig. 4, C8S) resulted in the
immunostaining of large (500-2000 nm) cytoplasmic vesicular-like
structures containing ACTH
(Fig. 2 D).
Stimulated secretion was not observed, but constitutive secretion of
ACTH
as unprocessed POMC (data not shown) was found (Table
I). These data suggest not only that at least one disulfide bridge is
necessary for sorting but also that the position of this disulfide
bridge (Cys
/Cys
) was critical for sorting.
Figure 4:
Deletion mutations of POMC were created by
deleting 78 amino acids from Cys to Arg
or
by deleting the hairpin loop region from Cys
to
Cys
. Substitution mutations were created by substituting
serine for cysteine at position 2, position 8, or
both.
Analysis of Sorting Information within the Hairpin
Loop and the Lys
The hairpin loop region was further analyzed as a
sorting signal by making a deletion mutant to remove the 13 amino acids
forming the loop (Cto Arg
Domain of
POMC
QDLTTESNLLAC
) (Fig. 4,
Cys8-Cys20 Loop Deletion). The Cys
to Cys
loop deletion-mutated POMC protein was localized to a perinuclear
region characteristic of localization in the Golgi (Fig. 2 E).
There was only constitutive release of this mutant POMC ().
These data suggest that there is information in the 13 amino acids of
the hairpin loop region of POMC that is essential to sort POMC
correctly to the regulated secretory pathway.
-terminal POMC outside the
hairpin loop were necessary for sorting to the secretory granules, 78
amino acids spanning the entire region from Lys
to
Arg
(Fig. 4) were deleted. ACTH
was
found in punctate secretory granules in these cells
(Fig. 2 F), similar to those of the wild type POMC, and
ACTH products (ACTH
and ACTH
)
were secreted in a regulated manner (). These results
suggest that there is no information in the
-melanocyte-stimulating hormone or joining peptide region of POMC
that is necessary for sorting to the dense core granules.
Molecular Modeling of N-POMC
The data presented
here identify the regulated secretory pathway sorting signal for POMC
as a highly conserved region consisting of a 13-amino acid hairpin loop
conformation (CQDLTTESNLLAC
) stabilized by one
disulfide bridge. A three-dimensional model of this
NH
-terminal region of POMC shows that the Cys
to Cys
sequence of 13 amino acids assumes a
heart-shaped loop with two lobes, one centered on
Asp
-Leu
and the other on
Thr
-Glu
(Fig. 6). The structure is clearly
amphipathic with a cluster of hydrophobic side chains ( i.e. Cys
-Leu
-Leu
-Ala
-Cys
)
at the base of the heart and a hydrophilic region at both upper lobes
of the heart-shaped loop (Fig. 6). The disulfide bridge between
Cys
and Cys
locks this domain in a rigid
conformation. If the POMC sequences known for different species are
compared (Fig. 1 A), the residues that are conserved in
this region in all species would correspond to
Asp
-Leu
-Glu
-Leu
(Fig. 1 A). It is therefore likely that some or all
of the side chains of these residues within the amphipathic loop play
an important role in the sorting mechanism.
Figure 6:
Three-dimensional computer diagram of the
NH-terminal region of POMC. The figure shows the
Cys
/Cys
disulfide bridge ( yellow) and
the amphipathic loop portion of the lowest energy structure for
POMC
.
to Pro
) containing the amphipathic
loop and both disulfide bridges were deleted, the mutant POMC was
sorted to the constitutive secretory pathway in Neuro-2a cells
(24) . We now show that eliminating just the 13-amino acid
amphipathic loop (Cys
to Cys
) was sufficient
to cause the mutant POMC to be secreted constitutively. This mutant
POMC was not processed in the Neuro-2a cells, further suggesting
routing through the constitutive secretory pathway. These results
provide evidence that firmly establishes for the first time that the
13-amino acid amphipathic loop region of N-POMC is necessary for
sorting POMC to the regulated secretory pathway, and there appears to
be no other essential sorting signals in the rest of the POMC molecule.
/Cys
). These results are further supported
by the observation that, in both salmon II and trout B POMC
(25, 26, 27) where the first disulfide bridge
(Cys
/Cys
) has been disrupted by a natural
mutation of Cys
to a Phe, there was no adverse effect on
regulated secretion and thus no adverse effect on targeting
(28, 29) .
1-26 mutations. Roy et al. (22) found
that both of these mutant proteins were secreted in a regulated manner,
were localized to regulated secretory granules in the cells, and were
processed in the cells. In our study ( Fig. 2and )
and that of Cool and Loh
(24) , the same mutant POMC proteins
were secreted constitutively, were not localized to regulated secretory
granules, and were primarily not processed. Differing methods of
analysis and the low number of experiments by Roy et al. (22) may account for the differences between the results.
1-26 mutant. Furthermore, data for the analysis of
secretion of the C8S mutant were not shown, but the authors concluded
that this mutant was secreted in a regulated manner
(22) . On
the contrary, our secretion data for this and other mutants,
representing the mean from four experiments, provide conclusive proof
that this C8S mutant protein as well as the NH
-terminally
deleted (Cys
to Cys
loop deletion) mutant are
secreted via the constitutive secretory pathway.
-endorphin in the cell extract only as evidence for targeting POMC
to the regulated secretory pathway is ambiguous, because another group
has shown that the Neuro-2a cell line can process POMC directly to
-endorphin, which is at the COOH terminus of this prohormone,
efficiently bypassing the
-lipotrophic hormone intermediate
(30) . The subcellular site for this aberrant cleavage is as yet
undetermined in the Neuro-2a cell line. Additionally, four antisera
were used, each for a different assay in the studies of Roy et al. (22) , each of which reacted with different regions of the
POMC molecule. Thus there was a lack of continuity in following one
POMC product ( e.g.
-endorphin) from immunocytochemical
localization through processing and secretion. In contrast, antisera to
only the ACTH portion of the POMC molecule were used for our entire
study.
-globin,
-globin fusion protein was targeted
to the regulated secretory pathway in GH3 cells
(33) . The
presence of a signal that could sort
-globin to the regulated
pathway in pro-somatostatin appears to reside in the first 82 amino
acids. However, by deleting different regions of the pro-somatostatin
molecule, it was determined that multiple sorting signals might exist
in the somatostatin molecule, each of which could independently cause
sorting to the regulated secretory pathway
(34) . POMC may also
contain domains that are not sufficient by themselves to cause sorting
but that may act synergistically with the N-POMC amphipathic loop to
enhance sorting to the regulated secretory pathway. A previous study
suggests that the carboxyl terminus of POMC may contain information
that could influence sorting
(21) . However, our previous work
with the N-POMC-chloramphenicol acetyltransferase chimeric constructs
(14) and the
2-26 POMC mutant
(24) suggests
that the COOH-terminal region is not an absolute necessity for sorting
to the regulated secretory pathway.
-terminal regions of POMC from different species
indicates that in addition to the conserved cysteine residues at
positions 2, 8, 20, and 24, which form a pair of disulfide bridges
(Cys
/Cys
and
Cys
/Cys
), there are also four other highly
conserved amino acids,
Asp
-Leu
-Glu
-Leu
. It
is likely that these residues in the amphipathic loop motif play a role
in the sorting mechanism, perhaps through ionic/hydrophobic
interactions with a putative sorting receptor. It is also interesting
to note that, when only the amphipathic loop region (Cys
to
Cys
) was deleted, the two remaining cysteine residues at
positions 2 and 24 could potentially form a disulfide bridge, creating
a smaller loop with acidic and hydrophobic residues. However, this
mutant was not sorted to the regulated secretory pathway, suggesting
that this loop, if formed, was not enough to cause sorting. Immediately
following the last cysteine residue in the hairpin loop of POMC
(Cys
) is another set of highly conserved residues,
Asp
-Leu
-Glu
-Val
.
The similarity of the acidic and hydrophobic components of these
residues to the
Asp
-Leu
-Glu
-Leu
motif in the amphipathic loop suggests that they could
potentially play a role in sorting to the regulated secretory pathway.
However, helical wheel analyses of the other amino acids in this region
( e.g. Pro
, Pro
, and
Pro
) suggest that they would probably not allow the
formation of an amphipathic loop. The lack of targeting to the
regulated secretory pathway of the mutants with the amphipathic loop
deleted (Cys
to Cys
and Cys
to
Cys
) shows that these residues
(Asp
-Leu
-Glu
-Val
)
cannot compensate for the 13-amino acid amphipathic loop motif. This
conclusion was further supported by our finding that deletion of the
Lys
to Arg
region (Fig. 4, 78
Amino Acid Deletion) had no effect on the sorting of this mutant
POMC.
and Glu
are poised in a
position within the amphipathic loop structure of N-POMC that is ideal
for ionic interactions with a sorting receptor protein. Whereas a
putative ``receptor'' has long been proposed
(37) , no
protein has yet been found to play the role of ``universal''
receptor for the numerous pro-proteins found in the regulated secretory
pathway. However, there is evidence from previous studies that
N-POMC
can bind in a pH-dependent manner (optimum
pH,
5.5-6.0) to the luminal side of bovine intermediate lobe
secretory vesicle membranes
(14) and that this binding is
protease-sensitive.
(
)
Thus, the binding of the
N-POMC
amphipathic loop to a receptor is one
potential mechanism by which POMC is actively sorted to the regulated
secretory pathway.
-terminal region of POMC is a
13-amino acid amphipathic loop conformational signal, dependent upon
the integrity of the disulfide bridge and thus upon the stabilization
of the amphipathic loop for its activity. The presence of an active
sorting signal in POMC for the regulated secretory pathway suggests the
possibility that a putative receptor, located in the trans-Golgi
network, recognizes the prohormone molecule and thus is responsible for
sorting it to the newly forming regulated secretory granule. The model
we present for this amphipathic loop sorting signal can now be used as
a template by other researchers for molecular modeling to identify
similar regions in other prohormones.
Table: Stimulated secretion of ACTH from Neuro-2a
cells
from Neuro-2a
cells is in response to K
/Ca
. To
investigate the release of ACTH
, Neuro-2a cells,
transiently transfected with cDNA using Lipofectin, were exposed to a
buffer containing high concentrations of
K
/Ca
, and the ACTH
released into the buffer was assayed by radioimmunoassay. The
values represent the mean ± S.E. for four experiments. Values
are the percent of the total released.
-terminal POMC; ACTH,
adrenocorticotropic hormone; ACTH
, ACTH immunoreactive
material; HPLC, high pressure liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.