Identification of a Novel Prohormone Sorting Signal-Binding Site on Carboxypeptidase E, a Regulated Secretory Pathway-Sorting Receptor
Chun-Fa Zhang,
Christopher R. Snell and
Y. Peng Loh
Section on Cellular Neurobiology (C.-F.Z., Y.P.L.) Laboratory
of Developmental Neurobiology National Institute of Child Health
and Human Development National Institutes of Health Bethesda,
Maryland 20892
Novartis Institute for Medical Research
(C.R.S.) London, WCIE 6BN, United Kingdom
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ABSTRACT
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Sorting of the prohormone POMC to the regulated
secretory pathway necessitates the binding of a sorting signal
to a sorting receptor, identified as membrane carboxypeptidase E (CPE).
The sorting signal, located at the N terminus of POMC consists of two
acidic (Asp10,Glu14)
and two hydrophobic (Leu11,
Leu18) residues exposed on the surface of an
amphipathic loop. In this study, molecular modeling of CPE predicted
that the acidic residues in the POMC-sorting signal bind specifically
to two basic residues, Arg255 and
Lys260, present in a loop unique to CPE,
compared with other carboxypeptidases. To test the model, these two
residues on CPE were mutated to Ser or Ala, followed by baculovirus
expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes
containing CPE mutants with either Arg255 or
Lys260, or both residues substituted, showed no
binding of
[125I]N-POMC1-26
(which contains the POMC-sorting signal motif), proinsulin, or
proenkephalin. In contrast, substitution of an
Arg147 to Ala147 at a
substrate-binding site, Arg 259 to
Ala259 and Ser202 to
Pro202, in CPE did not affect the level of
[125I]N-POMC1-26
binding when compared with-wild type CPE. Furthermore, mutation of the
POMC-sorting signal motif (Asp10,
Leu11, Glu14,
Leu18) eliminated binding to wild-type CPE.
These results indicate that the sorting signal of POMC, proinsulin, and
proenkephalin specifically interacts with
Arg255 and Lys260 at a
novel binding site, independent of the active site on CPE.
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INTRODUCTION
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Peptide hormones and neuropeptides, unlike other secretory
proteins that are secreted constitutively, are specifically packaged
into granules of the regulated secretory pathway (RSP), uniquely
present in neuroendocrine cells. They are released in a
calcium-dependent manner upon stimulation. These molecules are
synthesized as larger precursors (prohormones/proneuropeptides) at the
endoplasmic reticulum (ER). They are then transported and sorted at the
trans-Golgi network into secretory granules (1, 2) where
they are processed to bioactive peptides. For many prohormones, sorting
to the RSP secretory granules is a prerequisite for processing.
However, much remains to be learned about the sorting mechanism. Our
previous studies have provided evidence that the sorting of prohormones
to the RSP at the trans-Golgi network occurs by a
receptor-mediated mechanism (3, 4). A sorting signal motif that is
sufficient and necessary for targeting the prohormone POMC to the RSP
has been identified (5, 6, 7). This motif consists of two acidic residues
(Asp10, Glu14) and two aliphatic hydrophobic
residues (Leu11, Leu18) exposed on the surface
of an amphipathic loop (residues 820) (Fig. 1
). Molecular modeling of proenkephalin,
using POMC as a template and the x-ray crystal structure of insulin,
revealed a similar sorting signal motif in both these molecules (Fig. 1
). As in POMC, two acidic residues and two aliphatic hydrophobic
residues were exposed on the surface of a loop structure in the N
terminus of proenkephalin and in the A and B chains of insulin. In
addition, GH, which was found to be constitutively secreted from
Cpefat/Cpefat mouse anterior pituitary (9),
also has a similar motif, as revealed by the x-ray crystal
structure (PDB ID: 1HGU; Brookhaven National Laboratories, Brookhaven,
NY). However, in chromogranin A, which does not depend on
carboxypeptidase E (CPE) for sorting to the regulated secretory pathway
(8), such a motif was not evident. Analysis to date suggests that the
POMC-sorting motif is present in other (pro)hormones that use CPE as a
sorting receptor.

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Figure 1. Low-Energy Conformers for POMC1-24,
Proenkephalin1-41, and the x-Ray Structure of Porcine
Insulin (11 )
The sorting signal motif already described (6 ) for N-POMC corresponds
to residues D10, L11, E14, and
L18. The corresponding residues in the modeled
proenkephalin structure (residues D18, I19,
E29, L32) are shown, as are the corresponding
residues in the x-ray structure of porcine insulin (LA16,
EA17, EB13, and LB17). The
-carbon atoms of the proposed sorting motif overlay with an RMS fit
of 1 Å.
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Using N-POMC1-26, which contains the POMC-sorting
signal motif, as a ligand in binding studies, a sorting receptor was
identified as membrane CPE (3, 7). Two other lines of evidence further
indicate that CPE is a sorting receptor in vivo. First,
depletion of CPE in Neuro 2a cells by antisense RNA resulted in the
missorting of POMC (3), proenkephalin, and proinsulin (8) to the
constitutive pathway. Second, Cpefat/Cpefat
mice with severely depleted levels of a mutant CPE in the pituitary (9, 10) showed constitutive secretion of large amounts of POMC (9). In
addition, these mutant mice did not show the normal meal-stimulated
rise in circulating CCK, consistent with a role of CPE as a sorting
receptor for packaging hormones into secretory granules of the RSP
(11).
In the present study, molecular modeling of CPE was carried out to
predict the RSP sorting signal-binding domain. Two basic residues on
CPE to which the two acidic residues of the RSP-sorting signal motif on
the prohormones could bind were identified. Mutagenesis of those two
basic residues, expression of the mutant CPEs in Sf9 cells using the
baculovirus system, and prohormone binding studies with Sf9 cell
membranes containing mutant CPEs were carried out to test the model.
The study identified residues Arg255 and Lys260
on CPE as the putative prohormone sorting signal-binding site,
providing further evidence in support of specific prohormone-CPE
interaction at the molecular level to mediate sorting of prohormones to
the regulated secretory pathway.
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RESULTS
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Identification of Putative Sorting Signal Motifs on Proenkephalin
and Proinsulin
Molecular modeling of N-POMC1-26 has previously
revealed a RSP-sorting signal motif on POMC consisting of residues
Asp10, Leu11, Glu14,
Leu18, exposed on the surface of an amphipathic loop
between Cys8 and Cys20 (Fig. 1
) (6). Deletion
and site-directed mutagenesis to disrupt the amphipathic loop of
N-POMC1-26 (5, 6) led to the missorting of POMC to the
constitutive secretory pathway. Here we show by immunocytochemistry
that transfection of POMC mutated at the four exposed
residues, Asp10, Leu11, Glu14,
Leu18, into Neuro 2a cells resulted in no punctate
immunostaining in the neurites (Fig. 2C
),
consistent with lack of packaging of mutant POMC into dense core
secretory granules. These cells revealed primarily perinuclear
immunostaining of mutant POMC that overlapped with wheat germ
agglutinin staining of the Golgi (Fig. 2D
). This is in contrast to
cells expressing wild-type POMC that showed punctate immunostaining
along and at the tips of neurites (Fig. 2A
), typical of hormone
packaging into regulated dense-core secretory granules. Untransfected
cells did not show any positive immunostaining (Fig. 2B
). These results
support our prediction that residues Asp10,
Leu11, Glu14, Leu18 on
N-POMC constitute the motif that is necessary for sorting POMC into the
RSP.

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Figure 2. Immunolocalization of WT and Mutant POMC
Transfected into Neuro 2a cells
A and B, Rhodamine staining for WT POMC in transfected (A) and
untransfected Neuro 2a cells (B). Note the punctateness in the staining
along the neurites (arrows). C, Rhodamine staining for
mutant POMC in transfected Neuro 2a cells. D, FITC-wheat germ
agglutinin staining of the Golgi (arrows) for the cells
presented in panel C. Bar, 10 µm.
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Molecular modeling of proenkephalin1-41 also revealed a
sorting motif similar to POMC, consisting of two acidic and two
hydrophobic residues: D18, I19,
E29, and L32 (Fig. 1
). Likewise, a similar
motif was found on the surface of insulin as revealed by the x-ray
structure (12) (Fig. 1
). This motif on insulin is maintained in
proinsulin as revealed by nuclear magnetic resonance studies (13).
Thus, putative RSP-sorting signal motifs similar to POMC are present in
other prohormones that could interact with CPE to facilitate sorting to
the regulated secretory pathway.
Molecular Modeling of CPE
Molecular modeling of CPE was carried out to identify a possible
binding site for the POMC-sorting signal. Membrane CPE contains a pre-
(signal peptide) sequence followed by a proregion at the N terminus of
the enzyme (Fig. 3A
). The sorting
signal-binding site and the C-terminal hydrophobic tail for membrane
anchoring are shown in Fig. 3A
. Since the CPE inhibitor, guanidinoethyl
mercaptosuccinic acid, was previously shown not to inhibit
N-POMC1-26 binding to bovine pituitary intermediate lobe
secretory granule CPE (3), the modeling focused on domains that were
unique to CPE, outside of the catalytic site. The family of
carboxypeptidases A, B, T, M, N, and E were aligned using Pileup
(Genetics Computing Group, Madison, WI) taking as reference
points the conserved zinc-binding and catalytic residues. The location
of the binding motif for the RSP-sorting signal was predicted to be
unique to CPE, exposed on the protein surface, and have suitable side
chains to bind to the acidic side chains (Asp10 and
Glu14) in the RSP-sorting signal (N-POMC8-20).
High-resolution x-ray crystallographic structures are available for
carboxypeptidase A, B and T, of which the latter is the most homologous
to CPE. Sequence alignment of CPE and CPT readily identified
CPE254-273 as an inserted loop with the required
structural properties. The structural analysis predicted this sequence
to be hydrophilic, surface located, and to have two ß-turns at
positions 255 and 261. The sequence CPE254-273 was modeled
and manually docked to N-POMC as described in Materials and
Methods to give the bound conformation shown in Fig. 3B
(the two
structures have been drawn apart slightly to better view the side chain
complementarity between the signal motif and receptor-binding
domain).

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Figure 3. Diagrammatic Representation and Model of CPE
A, Diagrammatic representation of the CPE molecule showing the pre- and
proregion, the sorting signal-binding site and C-terminal hydrophobic
membrane-binding tail (MBD). B, Model of N-POMC and
CPE254-273 showing the complementarity of
Arg255 (R255), Lys260
(K260) in CPE, and Asp10 (D10) and
Glu14 (E14) in N-POMC.
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Binding of N-POMC1-26 to Recombinant
CPE
To test the above model of the prohormone sorting signal-binding
site, in vitro binding assays using recombinant CPE were
established. Full-length membrane CPE was expressed in Sf9 insect cells
using the baculovirus expression system. Figure 4A
2 shows a confocal
micrograph of Sf9 cells infected with recombinant virus carrying
full-length CPE and immunostained with an N-terminal CPE antibody
previously described (3). Note that most of the staining is highly
concentrated around the cells, indicating that the CPE is primarily
localized to the plasma membrane. No immunostaining was found in cells
infected with CPE using a preimmune antiserum (Fig. 4A
1).
Fig 4B
shows a Western blot of CPE from Sf9 cell membranes. Cells
infected with the AcNPV virus carrying the vector alone without CPE
(lane 1), and uninfected cells (lane 2) showed no CPE expression,
whereas cells expressing full-length CPE showed a correctly sized band
of
55 kDa that was stained with CPE antibodies directed against the
proregion (lane 3). Similar results were obtained with antibodies
directed against the N terminus of CPE (Fig. 8A
, lanes 13). Very
little CPE staining was observed in Western blots of the medium,
indicating minimal secretion of membrane CPE (data not shown). This
further supports the immunocytochemical data that the recombinant CPE
expressed in Sf9 cells is primarily anchored to the plasma membrane and
most, if not all, of it is in the proform.

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Figure 4. Immunolocalization and Western Blot of CPE
A1, FITC immunofluorescence of CPE in infected Sf9 cells
stained with preimmune antiserum. A2, Confocal micrograph
showing FITC immunofluorescence staining of CPE on the surface of
CPE-infected Sf9 cells. Bar, 10 µm. B, Western blot of
CPE in cell membranes from uninfected cells (lane 1), Sf9 cells
infected with AcNPV alone (lane 2), and Sf9 cells infected with mouse
CPE (lane 3). The CPE proregion antisera nos. 1 and 2 mixed 1:1 were
used at 1:2000 dilution.
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Figure 8. [125I]N-POMC1-26 Binding
to CPE
A, Western blot of CPE from membranes (20 µg protein) of Sf9 cells
expressing WT and the various CPE mutations (M1M9 shown in Fig. 7 )
using the CPH52 antiserum. Uninfected cell membranes (lane 1) and
cells infected with AcNPV virus alone (lane 2) showed no CPE
expression. WT CPE (lane 3) and mutant CPEs, M1M9, in infected cells
are shown (lanes 412, respectively). B, Counts per min
[125I]N-POMC1-26 bound to WT and mutant CPEs
on Sf9 cell membranes. The numbers under the bars
correspond to the Western blot numbering described in panel A. Values
shown are the mean ± SEM assayed in triplicate from
one membrane preparation for each mutation. The nonspecific membrane
binding (lanes 1 and 2) was not subtracted from the bars shown (lanes
312). Results shown are representative of a total of three different
infections and membrane preparations for each mutant. C, The counts per
min [125I]N-POMC1-26 bound shown in panel B
was normalized for the amount of CPE present in each sample, determined
in arbitrary units by scanning the Western blots shown in panel A. The
normalized counts per min for WT CPE control was made equal to 100%,
and normalized values of the negative controls (lanes 1 and 2) or
mutants (lanes 412) were calculated as percent relative to wild type.
The numbers under the bars correspond to the Western
blot numbering described in panel A.
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An intact cell-binding assay using N-POMC1-26, which
contains the RSP-sorting signal, was carried out to determine whether
the membrane CPE in Sf9 cells was exposed to the cell surface. Sf9
cells were fixed and then incubated with N-POMC1-26
followed by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)
carbodimide-HCl (EDC). The cells were then assayed by immunostaining
with N-POMC1-26 antibodies. Figure 5
shows the immunocytochemical
localization of CPE on the plasma membrane of infected Sf9 cells
expressing CPE (Fig. 5B
), but not in uninfected cells (Fig. 5A
).
Uninfected cells incubated with N-POMC1-26 showed no
N-POMC1-26 immunostaining on the cell surface (Fig. 5C
1),
in contrast to cells that were infected with CPE (Fig. 5D
).
Furthermore, Sf9 cells infected with CPE showed no immunostaining when
incubated with a different POMC peptide,
ß-endorphin1-31, which does not contain the RSP-sorting
signal motif (Fig. 5C
2). These results indicate that
N-POMC1-26 bound specifically to CPE, which was anchored
in the Sf9 cell membranes facing the outside of the cells after
exocytosis. This is further substantiated by the observation that the
use of Triton X-100, which renders the cells permeable to proteins, was
not necessary for the binding of N-POMC1-26 to CPE to
occur (data not shown).

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Figure 5. Immunocytochemical Localization of CPE on
Uninfected (A) and Infected (B) Sf9 Cells
Note the FITC immunofluorescence on the plasma membrane of the
CPE-infected cells. N-POMC1-26 was incubated with
uninfected (C1) and CPE-infected cells (D); and
ß-endorphin1-31 was incubated with CPE-infected cells
(C2). The ligands on the cells were then cross-linked, followed by
immunodetection using antibodies against these peptides. Note the
N-POMC1-26 immunofluorescence on the surface of infected
cells, indicating binding of N-POMC1-26 to CPE on the
plasma membrane, but not uninfected cells. Note also the lack of
staining of CPE-infected cells incubated with
ß-endorphin1-31. Bar, 10 µm.
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A cell membrane-binding assay was established to demonstrate specific
binding of prohormones to recombinant CPE. Figure 6A
shows the effect of increasing
concentrations of N-POMC1-26 on
[125I]N-POMC1-26 binding to recombinant
membrane CPE. CPE bound [125I]N-POMC1-26
specifically in a N-POMC1-26-displaceable manner. However,
ßh-endorphin1-31 did not displace the
binding. Scatchard analysis showed a dissociation constant
(Kd) of 9.2 µM (Fig. 6B
). To further
demonstrate binding specificity, mutant N-POMC1-26 in
which the essential residues, Asp10, Leu11,
Glu14, Leu18, in the sorting signal motif (Fig. 1
) were mutated to Ala10, Ser11,
Ala14, Ser18, was tested. Table 1
shows that there was no binding of
[125I] mutant N-POMC1-26 to CPE compared
with N-POMC1-26.

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Figure 6. Displacement of
[125I]N-POMC1-26
A, Dose-dependent displacement of
[125I]N-POMC1-26 binding to CPE on infected
Sf9 cell membranes by N-POMC1-26 (squares)
or ß-endorphin1-31 (diamonds). Each data
point represents n = 3 ± SEM. B, Scatchard plot
of displacement of [125I]N-POMC1-26 binding
to CPE on Sf9 cell membranes.
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Binding of N-POMC1-26 to CPE Mutants
A series of CPE mutants was made to test the model (Fig. 3B
)
predicting that residues Arg255 and Lys260 were
essential for the binding of the prohormone-sorting signal. The nine
mutants that were made are shown in Fig. 7
. Figure 8A
shows Western blot analysis of the
relative levels of CPE in cell membranes from Sf9 cells expressing the
wild-type CPE and the various mutants (M1M9, lanes 412). The level
of background binding of ligand to membranes from uninfected cells and
cells infected with AcNPV virus alone is shown in lanes 1 and 2,
respectively. Figure 8B
shows the counts per min of
[125I]N-POMC1-26 binding to the Sf9 cell
membranes. While there was significant binding of
[125I]N-POMC to wild-type (WT) CPE (lane 3), there was
only background binding to CPE mutants with Arg255 (lanes 4
and 5) or Lys260 (lanes 6 and 7) substituted by Ala or Ser.
Similarly, mutant CPEs with both Arg255 and
Lys260 substituted by Ala or Ser (lanes 8 and 9) also
showed only background [125I]N-POMC1-26
binding. The results indicate that both these basic residues are
essential for N-POMC1-26 binding to CPE. To further
demonstrate the highly specific nature of N-POMC1-26
binding to these two basic residues, another basic residue in the
domain, Arg259, which is adjacent to Lys260,
was mutated to Ala to determine whether the ligand could discriminate
between the basic residues. This mutant CPE (lane 10) showed
[125I]N-POMC1-26 binding similar to WT CPE,
indicating that Arg259 was not involved in the interaction
of N-POMC1-26 with CPE. Mutation of one of the CPE
enzymatic substrate-binding sites Gln146
-Arg147 -Gln148 to Ala146
-Ala147 -Ala148 had no significant effect on
[125I]N-POMC1-26 binding (lane 11). Also,
since the Cpefat/Cpefat mouse carries a
mutation in Ser202 Pro202 (9), this
mutation was made to determine whether
[125I]N-POMC1-26 can bind to this mutant
form of CPE. Membranes from Sf9 cells expressing this mutant contained
lesser amounts of mutant CPE relative to WT CPE (Fig. 8A
, compare lanes
3 and 12), presumably due to degradation in the ER or in
proteosomes after exit from the ER (14). As a result, there was reduced
binding of [125I]N-POMC1-26 to
CPE202 (Fig. 8B
, lane 12). However, when the binding was
corrected for the amount of mutant CPE202 present relative
to WT control, the relative binding was similar to control (Fig. 8C
, lane 12 vs. lane 3). The amount of all the other CPE mutants
present on the Sf9 cell membranes was similar to WT CPE. Analysis of
the relative binding of [125I]N-POMC1-26
correcting for the mutant CPE present (Fig. 8C
), compared with the WT
CPE, further confirmed that mutation of Arg255 and
Lys260 (lanes 49) eliminated binding of
[125I]N-POMC1-26 to CPE, but mutations to
Arg259 (lane 10) or the substrate binding site (lane 11)
had no effect.

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Figure 7. Partial Amino Acid Sequence of WT CPE Showing a
Substrate-Binding Domain, the Cpefat/Cpefat
Mouse Mutation (Ser202), and the Prohormone Sorting
Signal-Binding Domain
The nine CPE mutations (M1-M9) and the residues substituted are shown.
The amino acid letter codes are as follows: N, Asn; R, Arg; P, Pro; A,
Ala; C, Cys; K, Lys; S, Ser.
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Interestingly, no binding of
[125I]N-POMC1-26 to soluble CPE was detected
despite using 3 times the amount of CPE as was used for
membrane-binding assays (data not shown), indicating that CPE must be
anchored to membrane to bind N-POMC1-26.
Binding of Proinsulin and Proenkephalin to CPE and Mutants
Evidence that proinsulin and proenkephalin bind to the same
receptor, CPE, as N-POMC1-26 is shown in Table 2A
.
[125I]N-POMC1-26 binding to recombinant
membrane CPE was completely displaced by 10 µM proinsulin
and proenkephalin. Figure 9C
shows that
the displacement of [125 I]N-POMC1-26 by
proinsulin is competitive. Specific binding of proinsulin to CPE was
further demonstrated by the immunostaining of proinsulin on the surface
of intact Sf9 cells infected with CPE (Fig. 9B
), but not on uninfected
cells (Fig. 9A
).

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Figure 9. Displacement of
[125I]N-POMC1-26 Binding to CPE
A and B, FITC immunofluorescence of proinsulin on the plasma membrane
of cells infected with CPE (B) and uninfected cells (A). Proinsulin was
incubated with uninfected and CPE-infected cells. The ligand on the
cells was then cross-linked followed by immunodetection using an
antibody that cross-reacts with proinsulin. Note the proinsulin
immunofluorescence on the surface of infected cells, indicating binding
of proinsulin to CPE on the plasma membrane, but not on uninfected
cells. Bar, 10 µm. C, Dose-dependent displacement of
[125I]N-POMC1-26 binding to CPE on infected
Sf9 cell membranes by proinsulin. Each data point represents n =
3 ± SEM.
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The binding of iodinated proenkephalin and proinsulin to WT CPE, mutant
CPE202, and the mutant CPEs with substitutions in
Arg255 and Lys260 for Ser is shown in Table 2B
.
[125I]proenkephalin and [125I]proinsulin
bound to WT and mutant CPE202 in a similar manner. However,
binding of proenkephalin and proinsulin to single (M2) (data not shown)
or double Arg255 and Lys260 (M6) substituted
CPE mutants (Table 2B
) were poor, or none at all, indicating that these
two residues are also necessary for the binding of the putative
RSP-sorting signal of these two prohormones (Fig. 1
), similar to
N-POMC1-26.
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DISCUSSION
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Molecular modeling studies predicted that a domain on CPE between
Arg255 and Lys260 could be involved in binding
the RSP-sorting signal motif of POMC. To test this model, a binding
assay using recombinant full-length CPE or mutant CPEs expressed in Sf9
cells was used. [125I]N-POMC1-26 bound
specifically to recombinant pro-CPE localized on the plasma membranes
of Sf9 cells, and the binding was displaced by unlabeled
N-POMC1-26 in a dose-dependent manner. The Kd
of 9.2 µM was similar to the previously reported
Kd (6 µM) for bovine pituitary intermediate
lobe se-cretory granule membrane CPE (7). Thus, the
N-POMC1-26 binding was unaffected by the presence of the
15-amino acid proregion present in the recombinant CPE. The specificity
of the binding was fur-ther shown by the lack of binding of mutated
N-POMC1-26, which has the four exposed residues
(Asp10, Leu11, Glu14,
Leu18) in the motif (see Fig. 1
) substituted for
Ala10, Ser11, Ala14,
Ser18. This result further illustrates that these residues
are essential for binding to the sorting receptor CPE. Molecular
modeling studies suggest that there is ionic interaction between the
acidic residues within the POMC RSP- sorting signal (Asp10,
Glu14) with the two basic residues Arg255 and
Lys260 in the CPE-binding domain. This is also consistent
with the pH dependence of the binding of the RSP-sorting signal with
CPE in vitro (3) and sorting to the RSP in vivo
(15). Experimental evidence for this interaction was provided by the
demonstration that when Arg255 and Lys260 on
mouse CPE were substituted individually or together for Ala or Ser,
binding of N-POMC1-26 to these CPE mutants expressed on
Sf9 cell plasma membranes was eliminated. The precision of the ligand
binding to these two basic residues was exemplified by the
demonstration that substitution of Arg259, adjacent to
Lys260, had no effect on the binding of
N-POMC1-26. Furthermore, when residues
Gln146-Arg147-Gln148 on CPE, of
which Arg147 is involved in the binding of the COOH group
of the basic residues of the substrate (16), were mutated, there was no
significant effect on the binding of N-POMC1-26.
The ionic interaction involving Arg255 and
Lys260 on CPE appears not to be limited to the POMC
RSP-sorting signal, since mutation of these two basic residues on CPE
also eliminated binding of proinsulin and proenkephalin. Both these
prohormones have predicted RSP-sorting signal motifs with two acidic
and two aliphatic hydrophobic residues in a configuration similar to
Asp10, Leu11, Glu14,
Leu18 in POMC (Fig. 1
). Therefore, the Arg255
to Lys260 domain on the sorting receptor CPE, which is
highly conserved among all species where the sequence is known, appears
to be a common binding site for several putative prohormone RSP-sorting
signals.
The results of this study, showing highly specific binding between
prohormone and CPE in a saturable manner, are consistent with a
possible role of membrane CPE as a RSP-sorting receptor for a number of
prohormones. In addition, we have also shown that membrane CPE, which
is enzymatically poorly active (17), is tightly bound to secretory
granule membranes in a Triton X-100/1 M NaCl-insoluble
manner (7), characteristic of a membrane-bound receptor. The binding
domain in CPE for the sorting signal of POMC is independent of the
enzymatic substrate site and available only when CPE is bound to the
membrane. This further segregates the initial role of membrane CPE as a
sorting receptor in the secretory pathway vs. its subsequent
role as a soluble enzymatically active exopeptidase in the secretory
granule in removing C-terminal basic residues from cleaved peptide
hormones (17, 18).
The importance of CPE in sorting of prohormones in vivo was
demonstrated by studies in which depletion of CPE in Neuro 2a cells by
antisense CPE RNA resulted in missorting of POMC (3), proenkephalin,
and proinsulin (8) to the constitutive pathway. In addition, we showed
that Cpefat/Cpefat mice depleted of mutant CPE
exhibited constitutive secretion of large amounts of POMC from
intermediate and anterior lobes of their pituitary glands (3, 9). While recent studies with NIT 2 and NIT 3 cells, immortalized
pancreatic cell lines from Cpefat/Cpefat mice,
have shown regulated secretion of immunoreactive insulin, it was
significantly less than that from immortalized pancreatic NIT 1 cells
from control mice (19). Interestingly, there was as much mutant
CPE202 in the NIT 3 cells as WT CPE in NIT 1 cells. Given
that our present study has shown that CPE202 can exit the
ER in Sf9 cells and bind proenkephalin and proinsulin as efficiently as
WT CPE, it is possible that some CPE202 in the NIT 2 and
NIT 3 cells may have exited the ER and are able to serve as sorting
receptors. Similarly, in another study carried out by Irminger et
al. (20) using Cpefat/Cpefat mice,
regulated secretion of proinsulin was also observed in pancreatic islet
cells. The design of this release study precludes detection of any
constitutive secretion of proinsulin that may have occurred during the
stimulation period, since no parallel experiments tracking unstimulated
release of proinsulin for the same period was carried out. Therefore
the amount of stimulated secretion of proinsulin in the
Cpefat/Cpefat mice may, in fact, have been less
than that reported. In addition, like the NIT3 cells, significant
levels of immunoreactive CPE202, including the mature form,
have been detected by Naggert et al. (10) in the pancreatic
islet cells of Cpefat/Cpefat mice, although
pituitary had virtually none. Presence of the mature form in the islets
suggests that CPE202 exited the ER, since maturation has
been shown to occur in the trans-Golgi network (21). In the
context of the present study, it is therefore possible that there are
sufficient levels of immunoreactive CPE202 in the
pancreatic islets of the Cpefat/Cpefat mice to
bind and sort proinsulin to the RSP. Since CPE levels were not
determined in the islets of the mice used in the study of Irminger
et al. (20), the possibility exists that significant amounts
of CPE202 in the islets may account for their results.
Nevertheless, a different mechanism for sorting proinsulin to the RSP
in the islets, which does not exist in Neuro 2a cells, cannot be ruled
out. Further studies will clarify the role of CPE as a sorting receptor
in different cell types and for different prohormones.
In summary, molecular modeling and site-directed mutagenesis studies
have identified a putative motif between residues 255 and 260 on CPE
that can bind the prohormone RSP-sorting signal. Within this domain,
two basic residues, Arg255 and Lys260, appear
to be essential for the ionic binding of the acidic residues,
Asp10 and Glu14, within the POMC RSP-sorting
signal, as well as proenkephalin and proinsulin, which also have
predicted sorting signal motifs that contain two acidic residues.
RSP-sorting signal domains have been identified for other RSP proteins,
e.g. chromogranin B (22, 23), the processing enzymes PC5A
(24), PC2 (25), and peptidylglycine
-amidating monooxygenase
(26). The mechanism by which these sorting signal domains serve to
target these proteins into the regulated secretory pathway has not been
elucidated. Clearly, CPE does not act as a sorting receptor for
chromogranin A (7, 8) and hence other sorting receptors and mechanisms
may exist (27). However, the understanding of how the POMC sorting
signal and the proenkephalin and proinsulin putative sorting signals
can specifically interact with CPE, as elucidated from this study, is
consistent with the model of a receptor-mediated mechanism for sorting
prohormones to the RSP.
 |
MATERIALS AND METHODS
|
---|
Materials
Full-length mouse CPE cDNA was cloned from mouse Lambda ZAP II
library (Stratagene, La Jolla, CA) screening with a CPE cDNA sequence
probe. Deep Vent DNA polymerase, T4 DNA ligase, and restriction
endonucleases were from New England Biolabs (Beverly, MA). Insect Sf9
cell line, pAcGP67 expression vector, and linearized BaculoGold DNA
were from Pharmingen (San Diego, CA). N-POMC1-26 was from
Phoenix Pharmaceuticals, Inc. (Mountain View, CA), and mutated
N-POMC1-26 (Asp10, Leu11,
Glu14, Leu18, to Ala10,
Ser11, Ala14, Ser18) was custom
synthesized by Peninsula Laboratories (Belmont, CA).
ßh-Endorphin1-31 was from Peninsula
Laboratories (Belmont, CA). N-POMC1-26 and mutant
N-POMC1-26 were iodinated by Covance Inc. (Vienna, VA.)
using 125I-labeled Bolton-Hunter Reagent (Amersham,
Arlington Heights, IL). Proenkephalin and proinsulin (gifts from I.
Lindberg and K. Vad, respectively) were iodinated by Covance,
Inc., using the Chloramine T procedure. The rabbit antiserum
raised against N-POMC1-26 was a gift from Phoenix
Pharmaceuticals, Inc.; the rabbit antiserum DP3 was raised against
human ß-endorphin1-31by Covance Inc. The insulin
antiserum was purchased from Peninsula Laboratories. The antisera (nos.
1 and 2) against the proregion of CPE was a gift from L. Fricker. EDC
and N-hydroxysuccinimide were from Pierce
Immunologicals (Rockford, IL). All other chemicals were of analytical
grade.
Immunocytochemistry of Neuro 2a Cells Expressing WT and Mutant
POMCs
Mutant POMC with
D10L11E14L18
substituted for
A10,S11A14S18 was
generated by PCR using bovine POMC cDNA as a template and a mutagenic
oligonucleotide as primer. WT and mutant POMC cDNA fragments were
subcloned into the mammalian expression vector pcDNA3.1 (from
Invitrogen, Carlsbad, CA) at the restriction sites, NheI and
EcoRV. The two constructs were transformed into DH5
bacteria and plasmid DNAs were prepared. The mutant POMC cDNA was
confirmed by sequencing. Neuro 2a cells, a neuroendocrine cell line,
were transfected with WT and mutant POMC cDNA using lipofectin (Life
Technologies Inc. Gaithersburg, MD). After 48 h, the cells were
fixed, permeabilized, blocked with 10% goat serum in PBS, incubated
with antibody against ACTH (DP4, which also cross-reacts with POMC,
1:5000 dilution), and then stained with goat antirabbit serum
conjugated with rhodamine (Boehringer Mannheim, Indianapolis, IN.) (6).
Wheat germ agglutinin conjugated with fluorescein isothiocyanate (FITC,
EY Laboratories, Inc., Mateo, CA, 1:250 dilution) was used to
stain the Golgi apparatus. Images were captured using a TEC-470
charge-couple device color camera on a Nikon Optiphot epifluorescent
microscope (Nikon, Inc., Melville, NY) coupled to a Macintosh computer
using Adobe Photoshop software (Adobe Systems, Mountain View, CA).
Baculovirus Expression of WT and Mutant CPEs
Ten cDNA fragments encoding mouse Pro-CPE or mouse mutant
Pro-CPE (M1: R255 to A; M2: R255 to S; M3: K260 to A, M4: K260 to S;
M5: R255 and K260 to A; M6: R255 and K260 to S; M7: R259 to A; M8:
NRN146148 to AAA and M9: S202 to P) generated by PCR were inserted
into the pAcGP67 baculovirus expression vector. These ten constructs
were then transformed into DH5
bacteria (Life Technologies), and the
plasmid DNAs were prepared using Qiagen Maxiprep Kits (Qiagen,
Valencia, CA) and sequenced with PRISM Ready Reaction Dye Deoxy
Terminator Cycle Sequencing Kit (Perkin-Elmer, Foster City, CA). WT
and mutant Pro-CPE plasmid DNAs were cotransfected with linearized
BaculoGold AcNPV DNA (Pharmingen) into Sf9 cells, using the calcium
phosphate method. The transfected cells were maintained at 27 C in
TMN-FH medium containing 10% FBS, 50 IU/ml penicillin-streptomycin,
and 0.125 µg/ml Fungizone for 56 days. The recombinant viruses were
purified and identified by plaque assay. For Pro-CPE or mutant Pro-CPE
expression, Sf9 cells were infected with recombinant baculoviruses at a
multiplicity of infection of 5. The medium and the cells were
harvested 2.5 days post infection.
Preparation of Sf9 Cell Membranes
Infected and uninfected Sf9 cells were harvested, washed with
PBS, and homogenized in a tissue grinder (Potter-Elvehjem), with 50
strokes at 1800 rpm. The cell membranes were harvested by
centrifugation at 100,000 x g for 45 min, and the
pellet was suspended in binding buffer (50 mM MES buffer,
pH 5.5, containing 120 mM NaCl and 5 mM
KCl).
Membrane Binding Assay
The binding assay was carried out in binding buffer using 20
µg membrane protein in each assay except for Fig. 6
where 10 µg
protein were used. The total volume of the assay was 400
µl, and each assay was done in triplicate.
[125I]N-POMC1-26 was added to the binding
assay to a final concentration of 2 pM (
150,000 cpm),
except for experiments in Fig. 8
, where
450,000 cpm were used. The
binding reaction was performed at 4 C overnight. Bound
N-POMC1-26 was then precipitated by the addition of 200
µl 0.5%
-globulin and 500 µl 24% polyethelene glycol (PEG
6000) to the reaction, followed by incubation on ice for 15 min and
centrifugation at 3000 rpm for 30 min at 4 C. The supernatant was
removed and the pellet counted on a Beckman
-counter. A blank
without membranes was used for background subtraction. Specific
N-POMC1-26 binding was calculated by subtracting the
binding measured in uninfected, or AcNPV (virus without CPE)-infected
Sf9 cell membranes. For the dose-dependent displacement binding
studies, the extent of binding of
[125I]N-POMC1-26 was determined in the
presence or absence of various concentrations of unlabeled
N-POMC1-26, or proinsulin. The binding studies with
[125I] proenkephalin and [125I]proinsulin
were as described for N-POMC1-26, except that the
incubations were done at pH 6.5, the optimal pH for binding of these
prohormones, and
450,000 cpm of ligand were used.
Intact Cell Binding Assay
Sf9 cells were seeded in a four-well slide. At a cell density of
80% confluence, the cells were infected with recombinant baculovirus;
2.5 days post infection, the infected cells and uninfected cells were
fixed with 3% HCHO for 1 h at 22 C. After two washes with PBS,
the cells were blocked with 10% goat serum for 30 min at 22 C. They
were then incubated with the ligand, N-POMC1-26,
ßh-endorphin, or proinsulin (final concentration 100
µM) in binding buffer at 4 C overnight. The ligand was
then cross-linked by adding EDC (final concentration, 1 mM)
and N-hydroxysuccinimide (final concentration, 5
mM) to the cells at 22 C for 20 min. The cells were then
washed three times with PBS, reacted with antiserum against
N-POMC1-26 (1:500 dilution), ßh-endorphin
(1:2000 dilution), or proinsulin (1:200 dilution) at 22 C for 2 h,
followed by incubation with a second antibody coupled to FITC (1:1000)
for 1 h at 22 C. After three washes with PBS, the cells were
observed and images captured using a TEC-470 charge couple
device color camera (Optronics Engineering, Goleta, CA) on a
Nikon Optiphot epifluorescent microscope coupled to a Macintosh
8100/100 Power PC computer, using Adobe Photoshop 4.0 software.
Immunocytochemistry of CPE in Sf9 Cells
Sf9 cells were grown, infected, fixed, and blocked with goat
serum as described above. The cells were then washed three times with
PBS, reacted with CPE antiserum (CPH52, 1:500 dilution) raised
against the N terminus of CPE, at 22 C for 2 h, followed by
incubation with a second antibody coupled to FITC (1:1000) for 1 h
at 22 C. After three washes with PBS, the cells were observed and
images captured as described above or on a confocal microscope.
Western Blot Analysis of CPE
Samples were separated on 12% SDS PAGE and transferred to
nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was
probed with CPE antiserum (CPH52), or pro-CPE antisera, and CPE was
detected using the alkaline phosphatase method (picoBlue
immunoscreening kit, Stratagene).
Molecular Modeling of CPE-Binding Motif and Complex with
N-POMC
Sequence analysis of CPE and related carboxypeptidases was
performed using the GCG suite of programs (Genetics Computing Group).
The sequence (CPE254-273) was modeled with the C and N
termini constrained to 5 Å as required if the loop were to be inserted
in the CPT structure (a manuscript by C. R. Snell and Y. P. Loh
detailing the construction of CPE by homology with CPT is in
preparation). Low-energy conformers were generated using a
combination of molecular dynamics and mechanics (Discover, MSI Inc.,
San Diego, CA). Conformers were sampled every picosecond during the
molecular dynamics trajectory of 250 psec (CVFF forcefield, 600
K), and subject to extensive energy minimization using the
Newton-Raphson algorithm until the gradient was less than 0.01 kcal
Å-2. The lowest energy conformer was manually
docked with POMC in the conformation previously reported aligning the
Arg255 and Lys260 with Asp10 and
Glu14 in POMC. Molecular mechanics were used to optimize
the interaction between the two structures as described above for
CPE254-273.
Molecular Modeling of N-POMC1-26 and
Proenkphalin1-41
Molecular mechanics and molecular dynamics were performed using
Insight II and Discover (Molecular Simulations, Inc., San Diego, CA).
Each peptide sequence was assembled and disulfide bridges formed
according to the known disulfide pattern (28). Gentle molecular
dynamics at 300 K was performed for 25 psec, until the disulfide
bond lengths had optimized. These structures were then minimized using
Steepest Descents and a Newton-Raphson minimizer until convergence.
These structures were then subjected to simulated annealing using
molecular dynamics for 250 psec at 600 K, and molecular
mechanics to minimize structures, sampled every picosecond from the
dynamics trajectory, to convergence with gradient below 0.005 kcal
Å-2. This gave 250 low-energy structures for each
peptide, and from these, the lowest energy conformers were used for
further structural studies.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. K. Vad (Novo Nordisk, Bagsvaerd, Denmark),
I. Lindberg (Louisiana State University, Baton Rouge, LA), and L.
Fricker (Albert Einstein College of Medicine, Yeshiva
University) for gifts of swine proinsulin, rat proenkephalin,
and proCPE antibodies, respectively. We thank Dr. J. Bonifacino
for critical reading of this manuscript and Ms. Winnie Tam for
performing the immunocytochemistry on Neuro 2a cells transfected with
WT and mutant POMCs.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Y. Peng Loh, National Institutes of Health, Building 49, Room 5A38, Bethesda, Maryland 20892. E-mail:ypl{at}codon.nih.gov
Received for publication September 4, 1998.
Revision received January 4, 1999.
Accepted for publication January 5, 1999.
 |
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