Impaired Prohormone Convertases in
Cpefat/Cpefat Mice*
Yemiliya
Berman
,
Nino
Mzhavia,
Ann
Polonskaia, and
Lakshmi A.
Devi§
From the Department of Pharmacology and § Kaplan
Comprehensive Cancer Center, New York University School of Medicine,
New York, New York 10016
Received for publication, September 18, 2000, and in revised form, October 12, 2000
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ABSTRACT |
A spontaneous point mutation in the coding region
of the carboxypeptidase E (CPE) gene results in a loss of CPE
activity that correlates with the development of late onset obesity
(Nagert, J. K., Fricker, L. D., Varlamov, O., Nishina,
P. M., Rouille, Y., Steiner, D. F., Carroll, R. J.,
Paigen, B. J., and Leiter, E. H. (1995) Nat.
Genet. 10, 135-142). Examination of the level of neuropeptides
in these mice showed a decrease in mature bioactive peptides as a
result of a decrease in both carboxypeptidase and prohormone convertase
activities. A defect in CPE is not expected to affect endoproteolytic
processing. In this report we have addressed the mechanism of this
unexpected finding by directly examining the expression of the major
precursor processing endoproteases, prohormone convertases PC1 and PC2
in Cpefat mice. We found that the levels of PC1
and PC2 are differentially altered in a number of brain regions and in
the pituitary. Since these enzymes have been implicated in the
generation of neuroendocrine peptides (dynorphin A-17,
-endorphin,
and
- melanocyte-stimulating hormone) involved in the control of
feeding behavior and body weight, we compared the levels of these
peptides in Cpefat and wild type animals.
We found a marked increase in the level of dynorphin A-17, a decrease
in the level of
-melanocyte-stimulating hormone, and an alteration
in the level of C-terminally processed
-endorphin. These results
suggest that the impairment in the level of these and other peptides
involved in body weight regulation is mainly due to an alteration in
carboxypeptidase and prohormone convertase activities and that this may
lead to the development of obesity in these animals.
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INTRODUCTION |
Peptide hormones and neuropeptides are synthesized as propeptides
that undergo a series of modifications before they are released as
active peptides. A number of enzymes involved in the processing of
neuropeptides have been identified and characterized (Ref. 2 and
references therein). These include subtilisin/kexin-like pro-proteins
such as furin, prohormone convertase 1 (PC1,1 also known as PC3),
PC2, PACE4, PC4, PC5/PC6, and PC7/PC8. Carboxypeptidases are required
to remove cleavage site residues, usually Lys and Arg, from the C
terminus of peptide hormone and neuropeptides after the action of
prohormone convertases. Carboxypeptidase E (CPE, also known as CPH), a
metallocarboxypeptidase with neuroendocrine distribution, is the major
enzyme responsible for the C-terminal trimming of the majority of
peptide hormones and neuropeptides (3). Support for this comes from a
mouse model lacking active CPE (designated
Cpefat) that shows accumulation of C-terminally
extended peptides. Although a full-length messenger RNA transcript is
produced in these mice, the translated mutant protein is degraded in
the endoplasmic reticulum due to a point mutation (serine 202 to
proline) in a highly conserved region of the enzyme (1). Thus, these
mice show deficiency in C-terminal trimming of basic residues (1,
4-8). In addition, these mice exhibit impaired endoproteolytic
processing as characterized by precursor accumulation (4) and/or
altered gene expression (6, 7). These changes point to an alteration in
the endoproteolytic processing of neuropeptides and hormone precursors
in Cpefat mice.
It is well established that feeding behavior is regulated by a number
of peptides (Ref. 9 and references therein). Among them, dynorphin A-17
(Dyn A-17) (9) and
-endorphin 1-31 (Ref. 10 and references therein)
have been shown to stimulate feeding, whereas acetylated forms of
-melanocyte-stimulating hormone (
-MSH) exert a tonic inhibition
on feeding behavior (Ref. 11 and references therein). On the other
hand, postranslational processing of
-endorphin through N-terminal
acetylation and C-terminal proteolysis eliminates effects on food
intake (10). Dynorphins,
-endorphin, and
-MSH are peptides
derived from prodynorphin (ProDyn) and proopiomelanocortin (POMC) by
the action of prohormone convertases (PC1 and PC2) followed by the
removal of C-terminal basic residues by CPE (3).
A number of studies (both in vitro as well as in
vivo) implicate PC1 and PC2 in ProDyn processing (12-14). Studies
using mice lacking PC2 activity (PC2 K/O) show a complete lack of Dyn
A-8 and substantially reduced levels of Dyn A-17 and Dyn B-13 (13, 14).
Thus PC2 appears to be involved in the generation of Dyn A-17, Dyn A-8,
and Dyn B-13 from ProDyn under physiological conditions (13, 14). As in
the case of ProDyn, PC1 and PC2 have been implicated in POMC processing
(Ref. 15 and references therein). The cleavage profile of POMC by PC1
produces a pattern very similar to the one normally found in the
anterior pituitary, high levels of adrenocorticotropic hormone (ACTH)
and
-lipotropin (
-LPH) and low levels of
-endorphin (15, 16).
PC2 on its own does not release ACTH from POMC but cleaves POMC into
N-terminally extended corticotropin containing the joining peptide,
-MSH, and
-endorphin (15, 16). A combination of PC1 and PC2
produces a pattern similar to that seen in the neurointermediate lobe, high levels of
-MSH, corticotropin-like intermediate lobe peptide (CLIP),
-endorphin, and low levels of ACTH and
-LPH (15). Thus,
PC1 and PC2 are distinct proprotein convertases acting alone or
together to produce a set of tissue-specific maturation products in the
brain and pituitary. The mechanism by which the defect in CPE reduces
the processing of these peptide hormones and neurotransmitters is not
well understood.
In the present study we tried to determine if the absence of CPE leads
to alterations in the level of PCs (protein activity and mRNA),
which in turn could affect the endoproteolytic processing of ProDyn and
POMC in the brain, and pituitary of Cpefat mice.
We found that the maturation of PC1 is decreased and the levels of PC2
are increased in Cpefat mice as compared with
wild type (WT) mice. We also found that the proteolytic processing of
ProDyn and POMC are decreased in Cpefat mice,
resulting in reduced levels of Dyn A-8, Dyn B-13, and
-MSH among
other bioactive peptides.
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EXPERIMENTAL PROCEDURES |
Animals--
Mice were bred at The Jackson Laboratory as
described previously (1). The identity of Cpefat
(
/
) animals was confirmed by genotyping using MIT primers (D8MT69 F
and R; D8MIT131 F and R) from Research Genetics according to the
protocol supplied by The Jackson Laboratory. Nonobese littermates (+/
) or (+/+) referred to as WT were used as controls. The age of the
animals ranged from 17 to 20 weeks.
Tissue Preparation--
WT and Cpefat
mice were decapitated between 10:00 a.m. and 12:00 noon. Whole brains
and pituitaries were collected and dissected into regions or
immediately frozen on dry ice. For regional distribution studies brains
were dissected into seven regions as described by Glowinski and
Iversen (17). Frozen tissues were stored at
70 °C until use.
PC2 Enzyme Assay--
For PC2 activity determination, frozen
tissues were extracted with 50 mM Tris-Cl, pH 7.5, containing 0.5% Triton X-100, 10% glycerol, 1 µM E-64,
1 µM pepstatin, 10 µM leupeptin, 300 µM phenylmethylsulfonyl fluoride, and 5.0 µg/ml
aprotinin (buffer A). After sonication, extracts were centrifuged at
14,000 × g for 20 min at 4 °C. Supernatants were
collected, separated into aliquots, and stored at
70 °C until
further use. PC2 activity was measured using 200 µM
Pyr-Arg-Thr-Lys-Arg-aminomethylcoumarin (American Peptide Co.) as a
substrate in 100 mM sodium acetate buffer, pH 5.0, containing 1 mM CaCl2 and 0.1% Tx-100 in the
presence of a protease inhibitor mixture (0.28 mM
N-tosyl-L-phenylalanine chloromethyl ketone,
0.14 mM
N-
-p-tosyl-L-lysine chloromethyl ketone, 1 µM E-64, 1 µM pepstatin A, and 10 µM captopril). The inhibitor mixture was included to
protect the substrate from nonspecific enzymatic degradation by other
proteolytic enzymes. All incubations were at 37 °C for 30 min to
4 h. Parallel samples were incubated with 1 µM human
C-terminal (CT) peptide 1-31 (a gift from Dr. I. Lindberg, Louisiana
State University Medical Center). The release of
7-amino-4-methylcoumarin was measured using a PerkinElmer Life Sciences
spectrofluorimeter (
excitation = 360 nm;
emission = 480 nm), and the amount of product formed was calculated using free
7-amino-4-methylcoumarin as a standard. The activity inhibited by the
C-terminal peptide was taken as PC2 activity.
Inactivation of PC2 by C-terminally Extended Dyn-and
Leu-Enk-derived Peptides--
Recombinant PC2 (10-50 ng) (a gift from
Dr. I. Lindberg, Louisiana State University Medical Center) was
incubated with 100 µM Dyn A-17 and 5-8 concentrations of
C-terminally extended peptides (shown in Table II) for 30 min at
37 °C in 0.1 M sodium acetate buffer, pH 5.0, containing
0.1% Triton X-100 and 1 mM CaCl2. The reactions were terminated by boiling for 10 min. The pH of the reaction
mixture was adjusted to 7.5 with 1 M Tris-Cl, pH 8.0, followed by incubation with 6 ng of CPB/100 µl of reaction mixture for another 30 min at 37 °C, and the assay was terminated by
boiling. The Dyn A-8 released from Dyn A-17 was quantified by
radioimmunoassay (RIA) as described previously (18, 19).
Western Blot Analysis of CPE, CPD, ProDyn, POMC, PC1, and
PC2--
Before Western blot analysis buffer conditions were optimized
to exclude the possibility of enzyme and precursor degradation during
extraction. Two different buffers were tested and compared with the
buffer used for PC2 activity determination (buffer A). These buffers
contained different combinations of protease inhibitors and/or
denaturing agents. The composition of the tested buffers were 50 mM Tris-HCl, pH 7.4 containing 10% glycerol, 2% SDS, 300 µM phenylmethylsulfonyl fluoride, 10 µM
leupeptin, 1 µM pepstatin A, 1 µM
E-64, 5 µg/ml aprotinin (buffer B), and 50 mM Tris-HCl, pH 7.4, containing 1 mM leupeptin, 10 mM EDTA,
1% Triton X-100, 10% glycerol (buffer C). Tissue homogenates of
various brain regions or of a whole brain or pituitary from WT and
Cpefat mice extracted with buffer A were
subjected to SDS gel electrophoresis on 10% (CPE, CPD, PC1, PC2) or
16% gels (ProDyn, POMC) and analyzed by Western blotting. CPE and CPD
antisera (a gift from Dr. L. Fricker, Albert Einstein College of
Medicine) were directed against the C-terminal region of the enzymes
(20, 21). The 13 S antiserum, directed against mid-portion of Dyn B-13,
was used to detect ProDyn (4). The anti-POMC antiserum (Phoenix
Pharmaceuticals, Inc.), directed against the N-terminal amino acid
sequence 27-52, was used at a dilution 1:1000. The anti-PC1 and
anti-PC2 antisera, directed against the N- and C-terminal regions of
the enzymes, respectively, were used at a dilution of 1:1000 (14). The
blots were normalized by tubulin using anti-tubulin antiserum (Sigma) at a dilution of 1:2000. Western blots were visualized using an ECL kit
(Pierce). Densitization of blots was done using NIH Image software.
RNA Analysis--
Total cellular RNA was extracted from mouse
brain and pituitary using a TRIzol reagent kit (Life Technologies,
Inc.). Total RNA (30 µg) was electrophoresed through 1.5% agarose
gel containing 2.2 M formaldehyde using 0.04 M
MOPS, pH 7.0, buffer containing 10 mM sodium acetate and 1 mM EDTA. RNA was transferred to nitrocellulose (Gene screen
membrane, PerkinElmer Life Sciences) overnight, and the filter
was air-dried and baked at 80 °C for 2 h. After
prehybridization the blots were hybridized with appropriately labeled
probes as described previously (22). Antisense cRNA probes were
synthesized using SP6 (POMC) or T7 (PC1, PC2, ProDyn) RNA polymerase in
the presence of [
-32P]CTP using a
Riboprobe® in vitro transcription system
(Promega). The fragments used to generate each of the riboprobes were
as follows: POMC, a 923-base pair EcoRI/HindIII
fragment from a full-length mouse POMC cDNA clone (23), and ProDyn,
a 300-base pair HindIII/BamHI fragment of the
main exon of the rat ProDyn gene (24, 25). Rat (r) PC1 and rPC2
cDNAs were obtained using polymerase chain reaction as described
previously (26). The rPC1 probe (492 base pairs) was 97% identical to
mouse PC1, corresponding to nucleotides 715-1206. The rPC2 probe (450 base pair) was 95% identical to mouse PC2, corresponding to
nucleotides 878-1326. The blots were washed three times for 15 min at
room temperature with 2× SSC (1× SSC = 0.15 M NaCl
and 0.015 M sodium citrate), 0.05% SDS followed by
2-3 washes for 15 min at 65-70 °C with 0.2× SSC, 0.1% SDS and
exposed to a phospho screen for 3 h or overnight at room
temperature. Autoradiograms were quantified by scanning densitometry
and normalized to 18 S RNA visualized by ethidium bromide staining.
Peptide Extraction--
For peptide analysis whole brains were
homogenized in 0.1 M CH3COOH at 100 °C (10 volumes for the brain) and incubated at this temperature for 15 min.
After centrifugation (13,000 × g for 30 min at
4 °C) the supernatants were concentrated on Speed Vac (Savant) and
stored at
20 °C. Anterior lobe (AL) and neurointermediate lobe of
the pituitary (NIL) were homogenized with buffer used for PC2
extraction (buffer A, glycerol was excluded from the buffer composition, 100 µl/structure). Upon removal of a small aliquot for
PC2 activity determination (to ensure the accuracy of dissection into
AL and NIL), an equal volume of 2 M CH3COOH was
added to the remainder. After centrifugation, the supernatants were
concentrated on Speed Vac and stored at
20 °C. Before RIA and/or
gel exclusion chromatography, samples were resuspended in methanol:0.1
N HCl (v:v).
Size Exclusion Chromatography and RIAs--
Gel exclusion
chromatography was performed on Superdex ® peptide
HR 10/30 column (Amersham Pharmacia Biotech). Samples in a total volume
of 50-100 µl (usually 2-3 brains combined together) were applied to
the column and separated with 1% formic acid containing 0.02%
protease-free bovine serum albumin (Sigma). Individual extracts of AL
and NIL of the pituitary were applied to the column in a total volume
of 25 µl and separated with 30% acetonitrile in 0.1% trifluoroacetic acid. The flow rate was 0.5 ml/min, and 0.5 ml fractions were collected. Fractions were dried, resuspended, and subjected to RIA to quantitate ProDyn/Dyn (14, 18, 19)- or POMC-derived
peptides. For POMC-derived peptides (
-endorphin,
-MSH, and ACTH)
RIAs were carried out as described for ProDyn/Dyn-derived peptides.
-Endorphin antiserum (National Institute of Drug of Abuse) was used
at a titer of 1:90,000; this antiserum has an IC50 of 30 fmol/tube and completely cross-reacts with
-LPH. ACTH antiserum
(Peninsula Laboratories, Inc.) was used at 1:75,000 final dilution;
this antiserum has an IC50 of 100 fmol/tube and completely
cross-reacts with human ACTH 18-39 but does not cross-react with
-LPH or
-endorphin.
-MSH antiserum (Peninsula Laboratories, Inc.) was used at a dilution of 1:30,000; this antiserum has an IC50 of 20 fmol/tube and exhibits 67% cross-reactivity
with (Nle4, D-Phe7)-
-MSH but does not cross-react
with ACTH or
-endorphin.
Characterization of POMC Peptides by Reverse Phase (RP)
HPLC--
For RP-HPLC, 200-250 µl of material containing the
highest immunoreactivity after fractionation of the NIL of the
pituitary on Superdex peptide column were injected into a peptide C18
column (Vydac) and eluted with a gradient of 80% acetonitrile in 0.1% trifluoroacetic acid (solvent B) against 0.1% trifluoroacetic acid
(solvent A). For the fractionation of
-endorphin and ACTH, the
gradient was 30-35% B for the initial 5 min, 35-45% B for the next
45 min, and then to 50% B over the next 5 min (27). For the
fractionation of
-MSH, the gradient was 15-45% B for the first 30 min and then to 65% B over the next 10 min. The flow rate was 1 ml/min, and 0.5-ml fractions were collected, concentrated in Speed Vac
concentrator and dissolved in methanol/HCl for RIA. Synthetic
-endorphin, ACTH, CLIP, and
-MSH peptides (from Peninsula Laboratories) were used as standards to calibrate the column.
 |
RESULTS |
CPE activity is deficient in Cpefat mice
since a single mutation in the coding region results in an unstable
enzyme that is quickly degraded before maturation and transport to the
Golgi apparatus (20). To confirm the absence of mature CPE in
Cpefat mice, we carried out Western blot
analysis with an antiserum that detects both pro-CPE as well as mature
CPE. We found a 53-kDa band corresponding to mature CPE in the brain of
WT mice (Fig. 1). This size is consistent
with the form of mature CPE previously reported in various bovine and
rat tissues (28). In contrast, we found an immunoreactive band of ~56
kDa that corresponds to the size of pro-CPE (i.e.
the precursor form of CPE with 14 additional N-terminal
amino acids) in Cpefat mice. Mature CPE (53 kDa)
is not seen in these animals, which is consistent with previous
findings (4). The absence of mature CPE in
Cpefat mice could result in a compensatory
up-regulation of other related carboxypeptidases such as CPD. By
Western blot analysis we found a protein band of 180 kDa, representing
the major form of CPD in all regions of both WT and
Cpefat animals (Fig. 1). This size is consistent
with the predominant form of CPD previously reported in bovine
pituitary and other tissues (21, 29). Careful comparison of the
relative levels of CPD between WT and Cpefat
mice show no significant differences, suggesting that the lack of
active CPE does not result in an increase in CPD levels in Cpefat mice.

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Fig. 1.
Western blot analysis of CPE (total brain)
and CPD in the brain regions of WT (+/+) and
Cpefat( / ) mice. Approximately 15 µg of protein from total brain or each region was subjected to gel
electrophoresis and Western blotting using the polyclonal antiserum
directed against CPE and CPD or tubulin antiserum and analyzed as
described under "Experimental Procedures." The positions and
molecular weights of prestained protein standards (Sigma) are
indicated.
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Previous studies found an accumulation of C-terminally extended
peptides in Cpefat mice (1, 4-8). In addition,
these mice exhibit an accumulation of partially processed intermediates
as well as the precursor, ProDyn (4). However, these studies did not
examine the extent of ProDyn accumulation in various brain regions. We
examined this as well as the relative level of another peptide hormone
precursor, POMC, in Cpefat mouse brain regions.
We found a predominant band of 30 kDa, representing ProDyn in all brain
structures (Fig. 2). The level of
immunoreactive ProDyn is 1.5-4.0-fold higher in
Cpefat mouse brain regions as well as in
pituitary as compared with WT mice. Western blot analysis of POMC in
pituitaries shows a predominant band of 23 kDa and minor bands of 32 and 28 kDa (Fig. 2). The 30-32-kDa form of POMC has been previously
reported in various cell culture systems (30-32). The other bands
probably represent unglycosylated forms or processing intermediates of POMC. We found an increase in the level of all these forms in Cpefat animals. These results indicate that
there is an accumulation of both ProDyn and POMC precursors in
Cpefat mice.

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Fig. 2.
ProDyn and POMC immunoreactivity in WT (+/+)
and Cpefat ( / ) mouse brain regions and
pituitary. Tissue samples from each brain region (30 µg) and
pituitary (10 µg for POMC analysis) were subjected to immunoblot
analysis with ProDyn (13 S) or POMC antisera as described under
"Experimental Procedures." The blots were reprobed with a
monoclonal anti-tubulin antiserum (Sigma) for normalization and
analyzed as described. The position and the size of molecular weight
markers are indicated on the left margin.
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To examine if the apparent increase in the precursor levels is due to
enhanced mRNA levels, we carried out Northern blot analyses. The
levels of ProDyn (seen as an ~2.4-kilobase band) or POMC (seen as an
~1.2-kilobase band) mRNAs in WT are comparable with those in
Cpefat mice, indicating that the changes in
precursor levels are not accompanied by modifications in ProDyn or POMC
gene expression (Fig. 3). It should be
pointed out that Cpefat mice with an average
weight of 33.7 ± 1.5 g exhibit no change in POMC mRNA
levels compared with WT mice (27.6 ± 0.6 g;
p < 0.01). In contrast, Cpefat
mice with an average weight of 47.6 ± 1.5 g exhibit a
2-3-fold increase in POMC mRNA level in the pituitary as compared
with WT mice (data not shown). These data suggest that POMC mRNA
changes became evident as mice grow and develop severe obesity.

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Fig. 3.
Northern blot analysis of ProDyn, PC1, and
PC2 mRNAs from brain and POMC mRNA from pituitary of WT and
Cpefat mice. Northern blot analysis
was carried out as described under "Experimental Procedures." Blots
were normalized with 18 S RNA using levels estimated by ethidium
bromide staining (lower panels). The positions and sizes of
molecular mass markers (Promega) are indicated on the left
margin.
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The lack of a significant difference in ProDyn and POMC mRNA
contents would suggest that the increase in precursor amounts could be
due to changes in the level of the processing enzymes. We attempted to
determine if the mRNA levels of the two PCs implicated in ProDyn
and POMC processing (namely PC1 and PC2) are altered in
Cpefat mice. We found that PC1 and PC2 mRNA
are detected as major bands of 3.0 and 2.8 kilobases, respectively,
both in Cpefat and WT mice (Fig. 3) and that
there are no significant differences in PC1 or PC2 mRNA levels in
these mice. Taken together, the results (showing no changes in gene
expression) would suggest that the precursor accumulation could be
mainly due to alterations in the level of active enzymes in
Cpefat mice.
We compared the levels of PC1 and PC2 in Cpefat
and WT mice by Western blot analysis. The PCs are synthesized as
inactive zymogens, and their maturation involves a series of
endoproteolytic steps such as the removal of the pro-domain and
truncation of the C terminus presumably by autocatalysis (Refs. 2,
33-35, and 36 and references therein). To exclude the possibility of
artifactual processing of PCs during extraction, three buffers with
different combinations of protease inhibitors and/or denaturing agents
(see "Experimental Procedures" for details) were tested, and the
buffer that preserved the enzymatic activity but blocked nonspecific hydrolysis during extraction was used for further analysis. We found a
decrease in the level of 68-kDa PC1 and an increase in the level of
87-kDa in different brain regions and pituitary of Cpefat mice (Fig.
4A). The 87-kDa form has been
shown to be present in a partially active state before the secretory
granules, where it is processed to a maximally active state (36).
Quantitation of the enzyme levels shows that the 68-kDa form is 1.5-5
times lower in Cpefat mice compared with WT mice
in all regions examined (Fig. 4A, lower panel).
The largest decrease is detected in the pituitary, cerebellum,
hippocampus, striatum, and cortex followed by midbrain, pons, and
medula oblongata and hypothalamus. It should be noted that we found a
concomitant increase in the 87-kDa form of the enzyme in all the
regions examined (Fig. 4A, upper panel).
Nonetheless, the total level of PC1 (sum of the levels of 87 and 68 kDa) in all brain regions of Cpefat mice is
lower compared with WT animals. These data suggest that the apparent
decrease in the 68-kDa form of PC1 could result not only from a
decrease in enzyme maturation but also from other changes at the
translational and/or post-translational level.

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Fig. 4.
PC1 and PC2 immunoreactivities in WT (+/+)
and Cpefat ( / ) mouse brain regions and
pituitary. Upper panels, Western blot analyses. Tissue
samples (15 µg) from each brain region and pituitary were subjected
to immunoblot analysis with polyclonal N-terminal-directed PC1
antiserum (A) or C-terminal-directed PC2 antiserum
(B) and monoclonal anti-tubulin antiserum as described under
"Experimental Procedures." Lower panels, relative
abundance of the 68-kDa form of PC1 and 68-71-kDa forms of PC2.
Results represent the mean ± S.E. (n = 4). Mean
values of the 68-kDa band of PC1 or 68-71-kDa bands of PC2 from WT are
taken as 100%, and corresponding PC1 and PC2 bands from
Cpefat mice are expressed as % WT. The position
and the size of molecular weight markers are indicated on the left
margin.
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PC2 is seen as a major protein of 68-71-kDa forms in all brain regions
examined (Fig. 4B). When compared with WT mice, the level of
immunoreactive PC2 is increased by ~1.3-2.0-fold in every brain
region as well as in the pituitary of Cpefat
mice (Fig. 6). We further examined this unexpected finding by testing
if the increase in ir-PC2 results in an increase in PC2 activity.
For this we used a simple assay that takes advantages of the 31-amino
acid C-terminal peptide derived from human 7B2 to selectively inhibit
PC2 (37), thus allowing specific determination of the enzyme activity
in tissue homogenates. We found that PC2 activity is also increased
about 1.3-2-fold in all brain regions and pituitary of
Cpefat mice as compared with WT mice (Table
I). Moreover, this is in good correlation
with the increase in PC2 immunoreactivity seen in
Cpefat animals.
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Table I
PC2 activity in brain regions and pituitary of WT and Cpefat
mice
Data represent the mean ± S.E. of triplicate determinations from
three animals. The enzyme extraction and assay were carried out as
described under "Experimental Procedures." Enzyme activity
represents the activity inhibited by 1 µM human CT
peptide 1-31.
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To better understand this paradoxical finding as to how decreased
processing could result from an increased level of PC2, we attempted to
determine if peptides that accumulate in Cpefat
mice, i.e. peptides with C-terminally basic residue
extensions, would inhibit PC2. We found that Dyn B-14, Dyn A-7, and Dyn
A-6 inhibited recombinant PC2 (Table II).
Dyn A-7 with double Arg extensions is the most potent, and peptides
with single Arg extensions are more potent than those with single Lys
extensions. It is possible that a substantial accumulation of these
peptides and/or other peptides with C-terminal basic residues within
the secretory granules could lead to a significant inhibition of PC2 in
Cpefat mice. Indeed, previously it has been
reported that processing of ProDyn and Dyn A-17 is less efficient in
the absence of CPE (13). Another possibility is that a portion of the
neuroendocrine protein 7B2, with the CT peptide 1-18 containing
Lys-Lys at the C terminus, remains associated with the protein and,
thus, inhibits the enzyme, whereas in WT mice this inhibition is
removed by CPE. It has been previously shown that CT peptide 1-18 is a
powerful inhibitor of PC2, whereas CT peptide 1-16 (lacking the
C-terminal Lys-Lys) displays little inhibitory effect even at extremely
high concentrations (38).
Next, we examined the effect of altered PC1 and PC2 levels on the
extent of processing of two peptide precursors, namely ProDyn and POMC.
The processing profile of ProDyn was characterized in the brain and the
profile of POMC in the AL and NIL of the pituitary. Immunoreactive
peptides were measured with specific RIAs (14, 18, 19) in fractions
after size exclusion chromatography. We found that the levels of ir-Dyn
A-8 and ir-Dyn B-13 (products of monobasic processing) are ~6- and
2.0-fold lower, respectively, in Cpefat as
compared with WT mouse brains (Fig. 5,
left top and bottom panels). At the same time the
levels of ir-Dyn A-17 (the precursor of Dyn A-8) were increased by
~2-fold in Cpefat as compared with WT mouse
brain (Fig. 5, left middle panel). To determine if the
reduction in the levels of Dyn A-8 and Dyn B-13 were due to C-terminal
extension of basic residues (Lys and/or Arg), the fractions were
treated with CPB (a treatment that removes C-terminal basic residue
extensions) after separation on a gel filtration column. We found that
this treatment does not cause a substantial alteration in the amount of
ir-Dyn A-8 and ir-Dyn B-13 in fractions from WT brain but causes an
increase in the levels of these peptides in
Cpefat brain (Fig. 5, right top and
bottom panels). A 5-fold increase in the level of ir-Dyn A-8
and ~2.5-fold increase in the levels of ir-Dyn B-13 is seen that
essentially restores the levels of ir-Dyn peptides in
Cpefat mouse brain to the levels in WT brain. In
contrast, CPB treatment does not cause a substantial increase in the
level of ir-Dyn A-17 in Cpefat mouse brain (Fig.
5, right middle panel). We found that a decrease in the
amount of fully processed peptides is accompanied by an increase in the
higher molecular weight intermediates in Cpefat
mouse brain (Fig. 5). These results are consistent with the reduced endoproteolytic processing in Cpefat animals due
to alterations in the levels of PC1 and PC2 activity.

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Fig. 5.
Gel filtration profile of WT (solid
line) and Cpefat (dotted
line) mouse brain extracts. Upper panel,
schematic representation of ProDyn precursor showing possible paired
and single basic cleavage sites. Various products of post-translational
processing are shown. The black portions represent the
opioid core of Leu-Enk. R, arginine; K, lysine;
-NE, -neo-endorphin; SP, signal peptide.
Lower panels, analysis of ir-Dyn A-8, ir-Dyn B-13, and
ir-Dyn A-17 in fractions after gel filtration chromatography on
Superdex® peptide 10/30 column. The fractions were
analyzed for ir-Dyn A-8, ir-Dyn B-13, and ir-Dyn A-17 before
( CPB) and after (+CPB) carboxypeptidase B
treatment as described under "Experimental Procedures." Molecular
weight calibration standards are: cytochrome c, 12.4 kDa;
aprotinin, 6.5 kDa; dynorphin 32, 3.98 kDa; Dyn A-17, 2.15 kDa; and Dyn
A-8, 0.98 kDa.
|
|
To better understand how differentially altered levels of PC1
(decreased) and PC2 (increased) affect neuropeptide and hormone processing, we examined the processing profile of POMC, a well studied
peptide hormone with a distinct processing profile in the pituitary. AL
corticotrophs express very high levels of PC1 mRNA but very little
PC2 mRNA, whereas NIL melanotrophs express high levels of PC2
mRNA and low levels of PC1 mRNA (26). We examined the forms of
POMC-derived peptides by size exclusion chromatography and RP-HPLC in
WT and Cpefat mice. The POMC-processing pattern
is different in the AL from that seen in the NIL (Fig.
6). In the AL of WT animals, two
ir-
-endorphin peaks are seen, and their sizes of (~ 10 and 3.5 kDa) are consistent with
-LPH and
-endorphin (Fig.
6A). The levels of both these peptides are reduced in
Cpefat animals (Fig. 6A). In contrast, a
single peak of
-endorphin is seen in NIL of WT animals, and its
level does not change in Cpefat mice (Fig.
6D). The ir-ACTH peptides appear as two peaks, and their
sizes are consistent with ACTH (1-39) and CLIP (ACTH 18-39). The relative levels of these peptides vary in the two lobes of WT mice
pituitary (Fig. 6, B and E). Both peptides are
substantially reduced in both lobes in Cpefat
mice (Fig. 6, B and E). ir-
-MSH appears as a
single peak in both Al and NIL of the pituitary of WT mice, and its
levels are significantly reduced in both lobes of
Cpefat mice (Fig. 6, C and
F). It seems likely that the differences in PCs leads to the
differences in peptide levels observed between the pituitary lobes of
WT and Cpefat mice. Since size exclusion
chromatography does not differentiate among different molecular forms
of
-endorphin,
-MSH, and ACTH peak fractions from gel filtration
chromatography of NIL were analyzed by RP-HPLC. By this analysis we
found four peaks of ir-
-endorphin corresponding to the desacetyl
-endorphin 1-31,
-N-acetyl
-endorphin 1-31,
-N-acetyl
-endorphin 1-27, and
-N-acetyl
-endorphin 1-26 in WT mice (Fig.
7). In contrast, a similar analysis of
Cpefat mice reveals an increase in
-N-acetyl
-endorphin 1-31, with a substantial
decrease of
-N-acetyl
-endorphin 1-27 and a complete loss of
-N-acetyl
-endorphin 1-26. These data point
to a reduction in the C-terminal processing of
-endorphin in
Cpefat mice. The major component of the ACTH
immunoreactivity in the WT and Cpefat NIL has
the retention time of CLIP (Fig. 7). We found that the level of ir-CLIP
is reduced enormously in Cpefat mice as compared
with WT mice (Fig. 7). Interestingly, the main form of
-MSH
immunoreactivity in the WT mice elutes as diacetyl-
-MSH; a low
levels of desacetyl
-MSH and monoacetyl-
-MSH are also seen (Fig.
7). We found that the relative proportions of nonacetylated and
acetylated forms of
-MSH in WT mouse pituitary are similar to those
reported previously (39). Although the same forms are detected in
Cpefat mice, the levels of all these forms were
highly reduced (Fig. 7). These data demonstrate that POMC processing in
both lobes of the pituitary of Cpefat mice is
greatly reduced at the level of endo- and exoproteolysis, and this
significantly affects the level of differentially processed peptides.
Taken together these results support the notion that changes in
the level of PC1 and PC2 enzymes affect the processing of neuropeptides
and peptide hormones, requiring the action of both convertases for
complete cleavage of the precursors.

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Fig. 6.
Gel filtration profile of WT (solid
line) and Cpefat (dotted
line) mouse anterior and neurointermediate lobe of the
pituitary extracts. Upper panel, schematic
representation of POMC precursor, showing possible paired basic
cleavage sites. The various domains of POMC precursor molecule are
noted as ACTH, -LPH, -, -, and -MSH-stimulating hormone,
CLIP, -endorphin ( -End), and joining peptide
(JP). The signal sequence (SP) and dibasic amino
acid cleavage recognition sites are indicated in the figure.
Lower panels, analysis of ir- -endorphin, ir-ACTH, and
ir- -MSH. Acid extracts of the anterior and neurointermediate lobes
of the pituitary were subjected to gel filtration chromatography, and
the fractions were analyzed for ir- -endorphin, ACTH, and -MSH as
described under "Experimental Procedures." Molecular mass
calibration standards are cytochrome c, 12.4 kDa; ACTH, 4.58 kDa; -endorphin, 3.48 kDa; and -MSH, 1.66 kDa.
|
|

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Fig. 7.
Representative HPLC analysis of molecular
forms of -endorphin, ACTH, and
-MSH in WT (solid line) and
Cpefat (dotted line) NIL
of the pituitary. The dashed line indicates the
acetonitrile gradient. The fractions with highest immunoreactivity
after gel-filtration chromatography were pooled and analyzed by RP-HPLC
as described under "Experimental Procedures." -Endorphin, ACTH,
and -MSH immunoreactive peaks that correspond to the elution time of
the respective standards are indicated. -EP,
-endorphin; ac-EP, -N-acetyl
-endorphin; -MSH, desacetyl -MSH;
ac-MSH- monoacetyl -MSH; diac-MSH,
diacetyl -MSH.
|
|
 |
DISCUSSION |
The primary finding of the present study is that the two major
neuropeptide-processing endoproteases of the secretory pathway, namely
PC1 and PC2, are altered in mice lacking active CPE. The level of the
more active form of PC1 is decreased with a concomitant increase in the
lesser active form of the enzyme in Cpefat mice.
Furthermore, the level and activity of PC2 are increased in these mice.
These enzymes are regulated at multiple levels. PC1 and PC2 are
synthesized as zymogens and require proteolytic release of the
pro-region for activity. PC1 undergoes an autocatalytic intramolecular
processing of its N-terminal pro-fragment in the endoplasmic reticulum,
resulting in an 87-kDa active enzyme (33, 40). This 87-kDa form is
targeted to the regulated secretory pathway, where it is further
shortened by removal of 135 amino acids from its C-terminal tail,
leading to a 66-kDa form (Ref. 36 and references therein). The
C-terminal tail of PC1 has been reported to act as an inhibitor of the
enzyme (36). It is possible that in Cpefat mice,
accumulation of the C-terminal tail with an Arg-Arg at the C-terminal
end inhibits PC1 activity, thus preventing further activation of the
enzyme and leading to an increase in lesser active forms of the protein
(Fig. 4A). It is possible that in WT mice CPE removes the
C-terminal extension of the C-terminal tail of PC1, releasing the
inhibition. Another possibility is that in
Cpefat mice PC1 is inhibited by endogenous
inhibitors such as the newly discovered proSAAS (41). In
vitro studies show that purified proSAAS inhibits PC1 activity,
and overexpression of proSAAS in AtT-20 cells leads to a reduction in
the extent of POMC processing (41).
PC2 is also synthesized as a zymogen of 75 kDa that undergoes
proteolysis to yield 68-71-kDa forms (33-35). The binding protein, 7B2, regulates the maturation of PC2. 7B2 contains two domains, a
21-kDa N-terminal domain required for PC2 maturation and a CT region
that inhibits PC2 at nanomolar concentrations. There is evidence that
the 7B2 CT peptide is cleaved at the internal paired basic site, most
likely by PC2 itself (38). It is likely that the resultant peptide (CT
peptide 1-18) remains associated with PC2, inhibiting the enzyme. We
found that C-terminal basic residue-containing peptides inhibit
PC2 activity. The chronic inhibition of PC2 in Cpefat mice may result in a compensatory
increase in the level of the enzyme (Fig. 4B and Table I).
Thus efficient removal of the pro-segment, C-terminal tail and
endogenous inhibitors might represent multiple regulatory steps in the
activation of PC1 and PC2 and hence in the processing of peptide hormones.
A considerable body of evidence implicates PC1 and PC2 in the
endoproteolytic processing of POMC (Ref. 39 and references therein).
PC1 is capable of cleaving POMC to ACTH and
-LPH in heterologous
expression systems. Overexpression of PC1 in AtT20 cells speed up
initial steps of POMC processing, which leads to a more extensive
cleavage of the precursor to smaller products by PC2 (42). Antisense
RNA to PC1 has also been shown to block the processing of POMC in AtT20
cells. The reduction in the level of ir-ACTH and
-LPH in AL of
Cpefat mice (Fig. 6, A and
B) is in a good agreement with the decreased level of the
active form of PC1. ACTH and
-LPH are derived from POMC by PC1 in
the corticotropic cells of the anterior pituitary and are further
processed by PC2 in NIL with the formation of
-MSH, CLIP
(ACTH18-39),
-LPH, and
-endorphin. A decrease in the level of
ir-ACTH in AL of Cpefat mice leads to a
reduction in the amount of
-MSH and CLIP formed by PC2 in NIL (Figs.
6, E and F) despite increased levels of the enzyme. The level of ir-
-endorphin in NIL (Fig. 6D) is
virtually unchanged, indicating that the latter may be formed in
vivo by the action of PC2 directly from the POMC precursor. This
is consistent with results from heterologous expression systems, where
it was shown that PC2 is able to process POMC, leading to the formation of
-endorphin (16). These data are consistent with the hypothesis that the production of peptides requiring the action of both PC1 and
PC2 is reduced in Cpefat mice pituitary. An
impaired pattern of processing is also seen for ProDyn. PC2 is the
major enzyme involved in the generation of small mature Dyn peptides
(13, 14). We found that the PC2 activity is increased about two times
in Cpefat mice as compared with WT; however, the
same degree of increase in the processing of ProDyn is not seen. It
should be pointed out that the PC2 activity was determined under
conditions that are substantially different than the physiological
environment where the concentration of C-terminally extended peptides
required to inhibit PC2 activity could be relatively high. Although
these concentrations can occur in an immature or mature secretory
granule, which would result in partial inhibition of PC2 activity,
tissue homogenization might result in a significant dilution of
these peptides and, thus, release the enzyme from the inhibitory
intracellular effect. Thus, the 2-fold increase in PC2 activity
determined in vitro might not represent the actual activity
in the context of the cellular environment.
We found an accumulation of the acetylated form of
-endorphin 1-31,
a substantial decrease in the acetylated form of
-endorphin 1-27,
and an absence of acetylated
-endorphin 1-26 in
Cpefat mice pituitary (Fig. 7). This finding is
in a good agreement with a previous report showing that CPE catalyzes
the conversion of
-endorphin 1-27 into
-endorphin 1-26 (43). It
is possible that CPE also catalyzes the C-terminal proteolysis of
acetylated
-endorphin 1-31, which terminates in Gln; in the absence
of CPE, we found an accumulation of the latter in
Cpefat mice (Fig. 7). In the absence of CPE one
would expect an increase in the ir amount of
-endorphin 1-31 in the
hypothalamus of Cpefat mice. ir-
-endorphin
1-31 is a potent stimulator of feeding (Ref. 10 and references
therein). In contrast,
-endorphin processing through C-terminal
proteolysis or N-terminal acetylation eliminates the effect on food
intake. Altogether, these data indicate that an accumulation of Dyn
A-17 and
-endorphin 1-31 with a concomitant decrease in the amount
of ir-acetylated
-MSH (potent inhibitor of feeding) may contribute
to the development of obesity in Cpefat mice.
We found that POMC mRNA levels were not increased in the brain and
pituitary of Cpefat mice as compared with WT.
However, we did see an increase in POMC mRNA in
Cpefat as they develop severe obesity (data not
shown). This finding is consistent with a previous report showing no
significant difference in POMC mRNA levels between 5-week-old fatty
and lean rats, even though significantly higher POMC mRNA levels
were observed in 12-week-old fatty rats compared with lean littermates
(44). Thus the difference in POMC mRNA content between lean and
obese animals becomes apparent as they grow and develop severe obesity. Moreover, it has been demonstrated that leptin, usually rising with
obesity, stimulates expression of anorexigenic peptides such as POMC
(45, 46). However, low concentrations of leptin (usually observed with
overfeeding or moderate obesity) did not affect the expression of POMC
mRNA level (Ref. 46 and references therein).
The distinguishing characteristics of mice homozygous for the
Cpefat mutation are early and severe
hyperproinsulinemia, transient hyperglycemia, but no
hypercorticosteronemia (1). Moderate obesity develops progressively,
starting between 8 and 12 weeks of age. The molecular mechanism by
which inactivation of CPE leads to the development of obesity in these
animals is unclear. Our data are consistent with a deficit in CPE
activity affecting maturation of major endoproteolytic enzymes (PC1 and
PC2) as well as neuropeptides derived from ProDyn and POMC. An abnormal
neuropeptide and hormone precursor processing is a general phenomenon
in Cpefat mice. Defects in the production of
neuropeptides such as
-MSH,
-endorphin, dynorphins,
melanin-concentrating hormone, neurotensin (5), gastrin (6, 7), and
cholecystokinin (8) suggest multiple roots for the development of
obesity. Critical to understanding the etiology of obesity is detailing
the processes that occur after initiation of the obesity-stimulating
event. It remains to be elucidated if the obesity in
Cpefat mice is induced by the disruption of the
melanocortin- and leptin-signaling pathway (47, 48) or a new
uncharacterized pathway. Five novel peptides derived from a common
precursor proSAAS have been identified in the brain of mice lacking
active CPE (41). It is possible that some of these or hitherto
undiscovered peptides may promote obesity through a novel and/or
already established pathway.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Edward Leiter (The Jackson
Laboratory, Bar Harbor, ME) for providing us with
Cpefat mice, Dr. Lloyd Fricker (Albert Einstein
College of Medicine) for CPE and CPD antisera, Drs. Nabil Seidah
(Clinical Research Institute of Montreal) and Richard Mains (University
of Connecticut Health Center) for PC1, PC2, and POMC plasmids, and Dr.
Iris Lindberg (Louisiana State University Medical Center) for purified
recombinant PC2, human CT peptide 1-31, and peptides for the
generation of PC1 and PC2 antisera.
 |
FOOTNOTES |
*
This work is supported in part by National Institutes of
Health Grants DA00342 (to Y. B.) and NS26880 and DA00458 (to
L. A. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology,
New York University School of Medicine, New York, NY 10016. Tel.:
212-263-7119; Fax: 212-263-7133; E-mail:
bermay01@med.nyu.edu.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M008499200
 |
ABBREVIATIONS |
The abbreviations used are:
PC, prohormone
convertase;
CPE, CPB, and CPD carboxypeptidase E, B, and D,
respectively;
Dyn A-17, dynorphin A-17;
-MSH,
-melanocyte-stimulating hormone;
ProDyn, prodynorphin;
POMC, proopiomelanocortin;
ACTH, adrenocorticotropic hormone;
-LPH,
-lipotropin;
CLIP, corticotropin-like intermediate lobe peptide;
WT, wild type;
CT, C-terminal;
RIA, radioimmunoassay;
MOPS, 4-morpholinepropanesulfonic acid;
r-, rat;
ir-, immunoreactive;
AL, anterior lobe;
NIL, neurointermediate lobe;
RP, reverse phase;
HPLC, high performance liquid chromatography.
 |
REFERENCES |
1.
|
Nagert, J. K.,
Fricker, L. D.,
Varlamov, O.,
Nishina, P. M.,
Rouille, Y.,
Steiner, D. F.,
Carroll, R. J.,
Paigen, B. J.,
and Leiter, E. H.
(1995)
Nat. Genet.
10,
135-142[Medline]
[Order article via Infotrieve]
|
2.
|
Steiner, D. F.
(1998)
Curr. Opin. Chem. Biol.
2,
31-39[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Fricker, L. D.
(1991)
in
Peptide Biosynthesis and Processing
(Fricker, L. D., ed)
, pp. 199-230, CRC Press, Inc., Boca Raton, FL
|
4.
|
Fricker, L. D.,
Berman, Y. L.,
Leiter, E. H.,
and Devi, L. A.
(1996)
J. Biol. Chem.
271,
30619-30624[Abstract/Free Full Text]
|
5.
|
Rovere, C.,
Viale, A.,
Nahon, J.-L.,
and Kitabgi, P.
(1996)
Endocrinology
137,
2954-2958[Abstract]
|
6.
|
Udupi, V.,
Gomez, P.,
Song, L.,
Varlamov, L.,
Reed, J. T.,
Leiter, H.,
Fricker, L. D.,
and Greeley, G. H.
(1997)
Endocrinology
138,
1959-1963[Abstract/Free Full Text]
|
7.
|
Lacourse, K. A.,
Friis-Hansen, L.,
Rehfeld, J. F.,
and Samuelson, L. C.
(1997)
FEBS Lett.
416,
45-50[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Cain, B. M.,
Wang, W.,
and Beinfeld, M. C.
(1997)
Endocrinology
138,
4034-4037[Abstract/Free Full Text]
|
9.
|
Inui, A.
(2000)
Pharmacol. Rev.
52,
35-61[Abstract/Free Full Text]
|
10.
|
Bray, G. A.
(1989)
Am. J. Clin. Nutr.
50,
891-902[Abstract]
|
11.
|
Mountjoy, K. G.,
and Wong, J.
(1997)
Mol. Cell. Endocrinol.
128,
171-177[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Dupuy, A.,
Lindberg, I.,
Zhou, Y.,
Akil, H.,
Lazure, C.,
Chretien, M.,
Seidah, N. G.,
and Day, R.
(1993)
FEBS Lett.
337,
60-65[CrossRef]
|
13.
|
Day, R.,
Lazure, C.,
Boudreault, A.,
Limperis, P.,
Dong, W.,
and Lindberg, I.
(1998)
J. Biol. Chem.
273,
829-836[Abstract/Free Full Text]
|
14.
|
Berman, Y.,
Mzhavia, N.,
Polonskaia, A.,
Furuta, M.,
Steiner, D. F.,
Pintar, J.,
and Devi, L.
(2000)
J. Neurochem.
75,
1763-1770[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Castro, M. G.,
and Morrison, E.
(1997)
Crit. Rev. Neurobiol.
11,
35-57[Medline]
[Order article via Infotrieve]
|
16.
|
Benjannet, S.,
Rondeau, N.,
Day, R.,
Chretien, M.,
and Seidah, N. G.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
3564-3568[Abstract]
|
17.
|
Glowinski, J.,
and Iversen, L. L.
(1966)
J. Neurochem.
13,
655-669[Medline]
[Order article via Infotrieve]
|
18.
|
Berman, Y.,
Devi, L.,
and Carr, K. D.
(1994)
Brain Res.
664,
49-53[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Berman, Y.,
Devi, L.,
and Carr, K. D.
(1995)
Brain Res.
685,
129-113[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Varlamov, O.,
Leiter, E. H.,
and Fricker, L. D.
(1996)
J. Biol. Chem.
271,
13981-13986[Abstract/Free Full Text]
|
21.
|
Song, L.,
and Fricker, L. D.
(1996)
J. Biol. Chem.
271,
28884-28889[Abstract/Free Full Text]
|
22.
|
Krumlauf, R.
(1996)
in
Basic DNA and RNA Protocols
(Harwood, A. J., ed)
, pp. 116-128, Humana Press Inc., Totowa, NJ
|
23.
|
Uhler, M.,
and Herbert, E.
(1983)
J. Biol. Chem.
258,
257-261[Abstract/Free Full Text]
|
24.
|
Douglas, J. O.,
McMurray, C. T.,
Garrett, J. E.,
Adelman, J. P.,
and Calavetta, L.
(1989)
Mol. Endocrinol.
3,
2070-2078[Abstract]
|
25.
|
Berman, Y.,
Devi, L.,
Spangler, R.,
Kreek, M.-J.,
and Carr, K. D.
(1997)
Mol. Brain Res.
46,
25-30[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Day, R.,
Schafer, M. K.-H.,
Watson, S. J.,
Chretien, M.,
and Seidah, N. G.
(1992)
Mol. Endocrinol.
6,
485-497[Abstract]
|
27.
|
Jaffe, S. B.,
Sobieszczyk, S.,
and Wardlaw, S. L.
(1994)
Brain Res.
648,
24-31[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Fricker, L. D.,
Das, B.,
and Angeletti, R. H.
(1990)
J. Biol. Chem.
265,
2476-2482[Abstract/Free Full Text]
|
29.
|
Song, L.,
and Fricker, L. D.
(1995)
J. Biol. Chem.
270,
25007-25013[Abstract/Free Full Text]
|
30.
|
Zhou, A,
Bloomquist, B. T.,
and Mains, R. E.
(1993)
J. Biol. Chem.
268,
1763-1769[Abstract/Free Full Text]
|
31.
|
Bruzzaniti, A.,
Marx, R.,
and Mains, R. E.
(1999)
J. Biol. Chem.
274,
24703-24713[Abstract/Free Full Text]
|
32.
|
Shen, F.-S.,
and Loh, P. Y.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5314-5319[Abstract/Free Full Text]
|
33.
|
Benjannet, S.,
Rondeau, N.,
Paquet, L.,
Boudreault, A.,
Lazure, C.,
Chretien, M.,
and Seidah, N. G.
(1993)
Biochem. J.
294,
735-743[Medline]
[Order article via Infotrieve]
|
34.
|
Shennan, K. I. J.,
Taylor, N. A.,
Jermany, J. L.,
Matthews, G.,
and Docherty, K.
(1995)
J. Biol. Chem.
270,
1402-1407[Abstract/Free Full Text]
|
35.
|
Lamango, N. S.,
Apletalina, E.,
Liu, J.,
and Lindberg, I.
(1999)
Arch. Biochem. Biophys.
362,
275-282[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Jutras, I.,
Seidah, N. G.,
Reudelhuber, T. L.,
and Brechler, V.
(1997)
J. Biol. Chem.
272,
15184-15188[Abstract/Free Full Text]
|
37.
|
Martens, G. J.,
Braks, J. A.,
Elb, D. W.,
Zhou, Y.,
and Lindberg, I.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
5784-5787[Abstract]
|
38.
|
Zhu, X.,
Rouille, Y.,
Lamango, N. S.,
Steiner, D. F.,
and Lindberg, I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4919-4924[Abstract/Free Full Text]
|
39.
|
Low, M.,
Liu, B.,
Hammer, G.,
Rubinstein, M.,
and Allen, R. G.
(1993)
J. Biol. Chem.
268,
24967-24975[Abstract/Free Full Text]
|
40.
|
Lindberg, I.
(1994)
Mol. Cell. Neurosc.
5,
263-268[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Fricker, L. D.,
McKinzie, A., A.,
Sun, J.,
Curran, E.,
Qian, Y.,
Yan, L.,
Patterson, S. D.,
Courchesne, P. L.,
Richards, B.,
Levin, N.,
Mzhavia, N.,
Devi, L.,
and Douglas, J. O..
(2000)
J. Neurosci.
20,
639-648[Abstract/Free Full Text]
|
42.
|
Zhou, A.,
and Mains, R.
(1994)
J. Biol. Chem.
269,
17440-17447[Abstract/Free Full Text]
|
43.
|
Smyth, D. G.,
Maruthainar, K.,
Darby, N. J.,
and Fricker, L. D.
(1989)
J. Neurochem.
53,
489-493[Medline]
[Order article via Infotrieve]
|
44.
|
Fukushima, M.,
Nakai, Y.,
Tsukada, T.,
Usui, T.,
Nakaishi, S.,
Naito, Y.,
Tominaga, T.,
Senoo, K.,
Fukata, J.,
Ikeda, H.,
Matsuo, T.,
and Imura, H.
(1993)
Int. J. Obes. Relat. Metab. Disord.
17,
337-341[Medline]
[Order article via Infotrieve]
|
45.
|
Flier, J. S.,
and Maratos-Flier, E.
(1998)
Cell
92,
437-440[Medline]
[Order article via Infotrieve]
|
46.
|
Ahima, R. S.,
Kelly, J.,
Elmquist, J. K.,
and Flier, J. S.
(1999)
Endocrinology
140,
4923-4931[Abstract/Free Full Text]
|
47.
|
Spiegelman, B. M.,
and Flier, J. S.
(1996)
Cell
87,
377-389[Medline]
[Order article via Infotrieve]
|
48.
|
Fisher, S. L.,
Yagaloff, K. A.,
and Burn, P.
(1999)
J. Recept. Signal Transduct. Res.
19,
203-216[Medline]
[Order article via Infotrieve]
|
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