Download The unique proline-rich domain of parotid proline

Journal of Cell Science 109, 1637-1645 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS1183
1637
The unique proline-rich domain of parotid proline-rich proteins functions in
secretory sorting
Lisa E. Stahl, Rhonda L. Wright, J. David Castle and Anna M. Castle*
Department of Cell Biology and the Molecular Biology Institute, University of Virginia Health Sciences Center, Charlottesville, VA
22908, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
When expressed in pituitary AtT-20 cells, parotid prolinerich proteins enter the regulated pathway. Because the
short N-terminal domain of a basic proline-rich protein is
necessary for efficient export from the ER, it has not been
possible to evaluate the role of this polypeptide segment as
a sorting signal for regulated secretion. We now show that
addition of the six-amino acid propeptide of proparathyroid hormone to the proline-rich protein, and especially to
a deletion mutant lacking the N-terminal domain, dramatically accelerates intracellular transport of these polypeptides. Under these conditions the chimeric deletion mutant
is stored as effectively as the full-length protein in dense
core granules. The propeptide does not function as a
sorting signal in AtT-20 cells as it does not reroute a constitutively secreted reporter protein to the regulated
pathway. During transit, the propeptide is cleaved from the
chimeric polypeptides such that the original structures of
the full-length and the deletion mutant proline-rich
proteins are reestablished. We have also found that the percentage stimulated secretion of the proline-rich proteins
increases incrementally (almost twofold) as their level of
expression is elevated. The increase reflects an enrichment
of these polypeptides in the granule pool and its incremental nature suggests that sorting of proline-rich proteins
involves an aggregation-based process. Because we can now
rule out contributions to sorting by both N- and C-terminal
segments of the proline-rich protein, we deduce that the
unique proline-rich domain is responsible for storage. Thus
at least some of the determinants of sorting for regulated
secretion are protein-specific rather than universal.
INTRODUCTION
cells and types of secretory products (Colomer et al., 1994).
Thus the sorting machinery may be more diverse than originally anticipated.
Several efforts have been made to identify structural determinants within secretory proteins that may function in sorting.
Among endocrine and exocrine proteins, structural regions
have been identified that are either necessary or sufficient for
targeting to granules (Stoller and Shields, 1989; Chu et al.,
1990; Cool et al., 1995; Arrandale and Dannies, 1994; Castle
et al., 1992). Many, but not all, of these regions are located at
or near the N termini of the polypeptides. No bona fide
consensus structural signal has been identified, although
common structural motifs involved in sorting have been postulated (Kizer and Tropsha, 1991; Gorr and Darling, 1995).
However, there is limited insight regarding how these domains
function in sorting.
We have been examining the mechanisms of sorting of
salivary proline-rich proteins (PRPs). PRPs are elongate
proteins lacking disulfide bonds and they have an unusually
simple structure consisting of three domains: a short Nterminal segment, a central proline-rich domain of repeating
cassettes, and a short C-terminal segment. They are efficiently
targeted to secretion granules in parotid acinar cells (Arvan and
In endocrine and exocrine secretory cells, storage granules are
formed from a subset of the proteins that are transported
through the secretory pathway. Molecular sorting is required
to determine which proteins are retained in these granules for
stimulus-dependent exocytosis. Several studies have implicated the function of specific cellular machinery and interactions among secretory proteins, especially selective aggregation (Chanat and Huttner, 1991; Kuliawat and Arvan, 1994;
Colomer et al., 1994), in these sorting events (reviewed by
Arvan and Castle, 1992; Bauerfeind and Huttner, 1993). Transfection of cultured cells with cDNAs encoding secretory
proteins from different cell types has shown that several
exogenous proteins are sorted as efficiently as the endogenous
secretory products (e.g. Moore and Kelly, 1985; Burgess et al.,
1985; Moore et al., 1983; Dickerson et al., 1987), suggesting
that targeting for regulated secretion might involve a common
mechanism. However, other proteins are not sorted as efficiently as endogenous products (e.g. Fennewald et al., 1988;
Castle et al., 1992; Schmidt and Moore; 1994; Reaves et al.,
1990; Stoller and Shields, 1989), and recent work has
suggested that sorting processes may differ among types of
Key words: Proline-rich protein, Protein sorting, Regulated
secretion, AtT-20 cell
1638 L. E. Stahl and others
Castle, 1986; Blair et al., 1991), and when expressed from
cloned cDNAs in a pituitary cell line, AtT-20, they are stored
in modest amounts in the dense core granules (Castle et al.,
1992; Castle and Castle, 1993). Deletion of the N-terminal
thirteen amino acid segment (transition region) from a basic
PRP eliminated storage of the protein but also resulted in its
inefficient transport from the ER. Because the ability to detect
regulated secretion of biosynthetically labeled proteins is compromised by slow ER export, we were not able to determine
whether the transition region was truly needed for storage in
granules or whether the deletion mutant (PRP∆T) would be
stored if its ER export was efficient. Consequently, we have
sought ways to restore more rapid ER export of PRP∆T so as
to allow examination of its storage in dense core granules.
We now report that rapid export of PRP∆T can be achieved
by attaching the hexapeptide prosequence of parathyroid
hormone (PROPTH) to its N terminus. We selected PROPTH for
this approach for several reasons. First, PROPTH is normally
removed rapidly and efficiently from PTH during intracellular
transport (Habener et al., 1979; Hellerman et al., 1984) and we
hoped that similar processing might occur from the chimera,
thereby generating PRP∆T. Second, PROPTH is thought to
function in translocation into the ER but does not affect postER transport through the secretory pathway in GH4 cells
(Wiren et al., 1988). Third, PROPTH is short and its strongly
basic and hydrophilic character differs from the amphipathic
nature of hydrophobic helical motifs that have been postulated
to function in post-Golgi sorting for regulated secretion (Kizer
and Tropsha, 1991; Gorr and Darling, 1995). Finally, the PTH
signal and propeptide have been used in chimeras previously
and have enabled efficient transport of recombinant fibronectin
polypeptides (Schwarzbauer et al., 1987). We now show that
upon restoring efficient ER export to PRP∆T, PROPTH is
indeed cleaved and PRP∆T is stored in secretory granules.
Moreover, the fraction of PRP∆T present in a stimulusreleasable (granule) pool approximately doubles with
increased expression. Thus we have clarified that the native Nterminal domain of PRP is not necessary for granule storage
provided that ER export is efficient. Rather the storage of PRP
is probably mediated by self-interactions of its unique prolinerich domain.
MATERIALS AND METHODS
Antibodies
Anti-ACTH (adrenocorticotropic hormone) antiserum and the polyclonal antibody against PRPs were characterized previously (Blair et
al., 1991; Castle and Castle, 1993). The polyclonal antibody against
the vesicular stomatitis protein G (Indiana strain) was a gift from Dr
Robert Wagner (University of Virginia).
Construction of vectors
PROPTH-PRP∆T and PROPTH-PRP
The signal and pro sequences of preproPTH were synthesized by the
polymerase chain reaction (PCR) with a HindIII restriction site
included at the 5′ end and an EcoRI site at the 3′ end. The prepro
fragment was cloned into HindIII and EcoRI sites of pBluescript
(Stratagene, Inc., CA). Fragments encoding PRP and PRP∆T lacking
the signal sequence were synthesized by PCR with an EcoRI site at
the 5′ end and cloned into pBluescript containing the prepro sequence
of PTH. The extra bases resulting from inclusion of the EcoRI site
were deleted by oligonucleotide-directed mutagenesis according to
the instructions from Bio-Rad (Muta-Gene kit, Bio-Rad Laboratories,
Richmond, CA). Mutated clones were screened by limited DNA
sequencing, and the selected constructs were sequenced in entirety to
confirm their structure. For expression in eukaryotic cells, the cDNA
fusion constructs were excised from pBluescript and cloned into the
HindIII/BamHI site of the pRhR1100 expression vector (provided by
Dr Tim Reudelhuber, Montreal Cancer Research Institute, Quebec)
which is driven by the Rous sarcoma virus long terminal repeat (Chu
et al., 1990).
HISPTH-PRP∆T
HISPTH-PRP was obtained by site-directed mutagenesis of a restriction fragment containing the signal and propeptide sequences of
preproPTH and 93 base pairs of PRP∆T. The sequence of the entire
mutagenized fragment was confirmed by sequencing. The mutagenized fragment was then attached to the remainder of PRP∆T using
standard cloning techniques.
tG and PROPTH-tG
The cDNA for tG was a gift from Dr J. Rose and was cloned into the
pLEN expression vector (Neufeld et al., 1987). The signal and propeptide sequences of preproPTH with added 5′ SalI and 3′ HindIII sites
were synthesized by PCR, inserted into pBluescript, and the primary
structure was confirmed by DNA sequencing. tG cDNA lacking
nucleotides encoding a signal sequence was also synthesized by PCR,
and it was inserted into the plasmid containing the signal and propeptide sequence of preproPTH. The amino acid sequence across the
junction of PTH propeptide and tG was KRSKLKTS where the underlined amino acids result from HindIII. The final construct was cloned
into the pLEN expression vector.
Cell culture and transfection
Mouse pituitary AtT-20 D16v cells were cultured as described previously (Castle et al., 1992). DNA transfections were carried out by
calcium phosphate precipitation. Selection of stable transfectants and
screening for expression of polypeptides by western blotting were also
performed as described (Castle et al., 1992).
Metabolic labeling and immunoprecipitation
Cells expressing PRP, PROPTH-PRP, PROPTH-PRP∆T or HISPTHPRP∆T were plated in 24-well plates with 1×105 cells/well. After 4872 hours in culture, cells were labeled for 15 hours with 0.4 mCi/ml
[3H]proline (Amersham Corp.) and chased as specified in individual
experiments. The cells and medium containing secreted proteins were
harvested and radiolabeled PRP-related polypeptides or ACTH were
immunoprecipitated and quantitated by SDS-PAGE and scintillation
counting as previously described (Castle et al., 1992).
Level of expression was measured following a 15 hour labeling
period with [3H]proline. Radiolabeled PRP-related polypeptides in the
medium and cell lysates were quantitated and the level of expression
was calculated as the total amount of radiolabeled PRP-related
polypeptide synthesized during the labeling (sum of radiolabeled
polypeptide from the medium and cell lysate). The data were
corrected for the number of prolines in each molecule and total
trichloroacetic acid-precipitable counts in each sample. The level of
expression of ACTH-related peptides was estimated from the same
experiment as the sum of radiolabeled proopiomelanocortin, ACTH
biosynthetic intermediate and ACTH. The data were corrected for the
number of prolines in each molecule and total trichloroacetic acidprecipitable counts in each sample.
To measure the percentage stimulated secretion, duplicate wells of
cells were labeled for 15 hours, chased first for 6 hours in the absence
of secretagogue and then chased for an additional 3 hours in the
absence (basal secretion) or the presence (stimulated secretion) of 5
mM 8-Br-cAMP. Radiolabeled PRP- or ACTH-related polypeptides
from both chase media and cell lysates were quantitated and the per-
Sorting of proline-rich proteins 1639
centage stimulated secretion was calculated as the difference between
stimulated and basal secretion of radiolabeled polypeptides, expressed
as the percentage of total (chases + cell extract).
Cells expressing tG and PROPTH-tG were plated in 35 mm dishes
at a density of 3×105 cells per dish. After 48-72 hours, the cells were
labeled for 1 hour with 0.25 mCi/ml Expre35S35S-label (ICN Radiochemicals, Irvine, CA) in minimal essential medium (Gibco BRL,
Gaithersburg, MD) supplemented with 15% dialyzed Nuserum, 20
mM Hepes, 4 mM glutamine or for 15 hours with 0.15 mCi/ml
Expre35S35S-label in the same medium supplemented with 2.5 µg/ml
of methionine and 4 µg/ml of cystine. Chases were carried out in the
same medium containing excess cold methionine and cystine plus
Trasylol as proteinase inhibitor (50 kallikrein units/ml).
Determination of ER exit rates
To estimate ER exit rates, cells were labeled with [3H]proline for 15
hours and chased for different lengths of time. At each timepoint,
medium and cells were harvested, immunoprecipitated with anti-PRP
antibody and digested with Endoglycosidase H (Endo H) and
analyzed as described previously (Castle et al., 1992). Endo Hsensitive and Endo H-resistant bands were quantitated by densitometric scanning. Half-times of exit from the ER were obtained from
log-linear plots of the fraction of each polypeptide that was Endo Hsensitive versus time.
Quantitation of stimulated secretion using western
blotting
AtT-20 cells expressing PROPTH-PRP and PROPTH-PRP∆T were
plated at equal cell densities in duplicate dishes. After 2-3 days, cell
monolayers were washed thoroughly and incubated in serum-free
medium with or without 5 mM 8-Br-cAMP for 3 hours. Cells were
lysed and assayed for protein (BCA protein assay; Pierce, Rockford,
IL) and DNA content (West et al., 1985). Aliquots of the medium
were analyzed by SDS-PAGE followed by western blotting with the
anti-PRP antibody. Bound antibody was detected with 125I-conjugated
secondary antibody and the amount quantitated by phosphoimager
analysis using ImageQuant software (Molecular Dynamics). The
values were normalized to DNA content, and the net stimulated
secretion was calculated as the amount of PRP secreted in the
presence minus the amount secreted in the absence of 8-Br-cAMP.
Automated microsequencing of PROPTH-PRP∆T
AtT-20 cells expressing PROPTH-PRP∆T were biosynthetically
labeled for 20 hours with 1 mCi/ml [3H]proline and labeled PROPTHPRP∆T polypeptides were immunoprecipitated. The immunoprecipitates were heated for 15 minutes at 55°C in SDS sample buffer
(Laemmli, 1970) containing 5 mM dithiothreitol and then treated with
10 mM iodoacetamide for 2 hours at 20°C in the dark. Samples were
resolved on 12.5% tube gels which were sliced into 1 mm slices and
eluted with 5 mM ammonium acetate. Bovine serum albumin (0.5
nM) was added as a carrier and the eluted material was exchanged
into 0.01% SDS and less than 0.38 mM Tris using a Centricon microconcentrator (Amicon, Beverly MA). This preparation was loaded
onto a sequencer (Applied Biosystems 470-A,Foster City, CA) and
sequenced by Edman degradation at the University of Virginia Biomolecular Facility.
RESULTS
As discussed in the Introduction, we have added PROPTH to
PRP∆T in an attempt to overcome its very slow rate of ER
export. Initially, two chimeric cDNAs were constructed,
PROPTH-PRP∆T and PROPTH-PRP (Fig. 1). PROPTH-PRP∆T,
encodes the PTH signal and propeptide (Hendy et al., 1981)
attached to amino acid residue 17 of PRP∆T. Amino acids 21-
33 of PRP are deleted in the PRP∆T portion of the construct
(Castle et al., 1992). PROPTH-PRP, encodes the same PTH
segment attached to the N terminus (residue 17) of full-length
PRP. Full-length PRP containing its native signal sequence and
transition domain was included in some of the studies (Castle
et al., 1992).
PROPTH-PRP∆T and PROPTH-PRP cDNAs were transfected
into AtT-20 cells and stable cell lines were created. We
selected cell lines with different levels of expression of
PROPTH-PRP∆T, PROPTH-PRP and PRP. The level of
expression of all PRP-related polypeptides varied over a 100fold range (Tables 1, 2 and 3) but in all cases was less than
20% of the level of endogenous ACTH-related peptides.
Notably, the level of expression of ACTH-related peptides was
nearly the same in all the clones examined (Table 1, legend).
Acceleration of ER export by PROPTH
Rates of export from the ER were estimated by following the
kinetics of the loss of sensitivity to Endo H digestion (Castle
et al., 1992). As shown by the values of t1/2ER in Table 1, the
presence of PROPTH causes a dramatic acceleration in transport
of PRP∆T. PROPTH-PRP∆T exited the ER with a t1/2ER of 1.31.4 hours in contrast to >7 hours for PRP∆T (Table 1).
Similarly, PROPTH-PRP exhibited a t1/2ER of 1.1 hours. The
independence of the export rate on level of expression for both
PROPTH-PRP∆T and PROPTH-PRP also contrasts with the
results obtained for PRP, for which t1/2ER was ~6 hours for a
Fig. 1. Schematic structure of PRP and tG constructs. Full-length
basic PRP consists of the signal sequence (PREPRP; wavy line), the
transition domain (TPRP; filled in rectangle), the proline-rich domain
(PPRP; elliptical shapes) and the C-terminal domain, (CPRP; open
rectangle) (Castle et al., 1992). PROPTH-PRP is a chimera of the
signal (PREPTH; wavy line) and propeptide of PTH (PROPTH;
rectangle) with full-length PRP. PROPTH-PRP∆T consists of the
PTH signal and propeptide fused to the N terminus of PRP∆T, which
lacks amino acids 21-33 (Castle et al., 1992). The sequence of
PROPTH is KSVKKR. Note that amino acids 17-20 of the transition
domain (small filled-in rectangle) are present within this construct.
HISPTH-PRP∆T has the identical structure as PROPTH-PRP∆T except
that the sequence of HISPTH propeptide is HSVLHR. PROPTH-tG is a
chimera of the signal and propeptide of PTH with the truncated G
protein of vesicular stomatitis virus, tG (Moore and Kelly, 1985).
1640 L. E. Stahl and others
Table 1. Kinetics of exit PRP-related polypeptides from
the ER
Construct
PROPTH-PRP∆T
PROPTH-PRP∆T
PRP∆T
PRP∆T
PROPTH-PRP
PROPTH-PRP
PRP
PRP
PRP
PRP
Clone
LOEPRP
20
43
26
1
39
4
A2
1A1
B9
B35
3.3
66.0
2.9
12.0
1.0
60.0
2.9
21.5
59.4
97.5
Table 2. 8-Br-cAMP-dependent secretion of PRP-related
polypeptides
t1/2ER (hours)
1.3
1.4
7.0
9.7
1.1
1.1
6.0*
2.5*
1.8
1.8
LOEPRP represents level of expression of PRP-related polypeptides
corrected to total trichloroacetic acid-precipitable counts. It was determined
as described in Materials and Methods. The values shown were normalized to
the lowest expressing clone, PROPTH-PRP 39. The level of expression of
ACTH-related polypeptides determined from the same experiments had an
average value of 506 ± 20 (s.e.m.).
t1/2ER is the halftime of exit of PRP-related polypeptides from the ER. It
was determined as described in Materials and Methods.
*Values of t1/2ER were taken from Table 3 of Castle et al. (1992).
low expressing clone and increased to a plateau of 1.8 hours
for progressively higher expressing clones (Table 1 above;
Castle et al., 1992). Thus the presence of the propeptide
bypasses the concentration-dependent step during transport
from the ER, which is normally mediated by the transition
domain of PRP.
Secretion and storage of PROPTH-PRP∆T and
PROPTH-PRP
As the PROPTH chimeras were transported from the ER with
rapid and essentially identical kinetics, it was now possible to
evaluate whether the presence of the transition region of PRP
was truly necessary for targeting to storage granules. We used
stimulation of secretion of radiolabeled cells by a secretagogue
to assess whether the chimeric polypeptides were routed to the
regulated pathway. Secretion of both chimeric polypeptides
was stimulated with 8-Br-cAMP, indicative of their presence
in secretory granules. The percentage of total radiolabeled
PROPTH-PRP∆T and PROPTH-PRP undergoing stimulated
secretion is shown in Table 2 and will be referred to as the percentage stimulated secretion throughout this work. The results
show that the percentage stimulated secretion of PROPTHPRP∆T and PROPTH-PRP is the same (10-20%) for comparable levels of expression. Therefore, in this context, the transition region appears unnecessary.
For both chimeric polypeptides, we also observed that the
percentage stimulated secretion exhibited a single-step
increase with increased level of expression. For several
different clones expressing low levels of PROPTH-PRP∆T or
PROPTH-PRP (representative examples are clones 20 and 39,
respectively, in Table 2) the percentage stimulated secretion
was ~10%. At higher levels of expression (e.g. clones 10 and
8 in Table 2), the percentage stimulated secretion increased to
~20% but did not rise significantly beyond this level with yet
higher levels of expression (e.g. clones 43 and 4 in Table 2).
To confirm that the differences in stimulus-dependent
secretion of the chimeric polypeptides do not reflect variations
in intrinsic storage capacity or secretory response, we
measured 8-Br-cAMP-stimulated secretion of radiolabeled
Construct
PROPTH-PRP∆T
PROPTH-PRP∆T
PROPTH-PRP∆T
PROPTH-PRP
PROPTH-PRP
PROPTH-PRP
PRP
PRP
PRP
PRP
Clone
LOEPRP
20
10
43
39
8
4
A2
1A1
B9
B35
3.3
13.2
66.0
1.0
10.0
60.0
2.9
21.5
59.4
97.5
%
Stim. sec.
10.3±1.2
18.3±1.3
21.0±2.1
9.4±1.1
24.6±1.6
18.2±1.1
6.1±0.5*
5.2±0.8*
11.0±2 †
9.0
‡
LOEPRP represents level of expression of PRP-related polypeptides
normalized to total trichloroacetic acid precipitable cpm. It was determined as
described in Materials and Methods.
% Stim. sec. represents the difference between stimulated (+ 8Br-cAMP)
and basal (−8-Br-cAMP) secretion of radiolabeled PRP-related polypeptides
expressed as percentage of total (chases + cell extract). Detailed description
of the experiment is presented in Materials and Methods. Data are presented
as the mean of four experiments ± s.e.m.
*Data taken from Castle et al. (1992).
†Mean of two experiments ± deviation from the mean.
‡Result of a single experiment.
ACTH-related peptides in untransfected AtT-20 cells and
selected clones expressing PROPTH-PRP∆T. As shown in
Table 3, the percentage stimulated secretion of ACTH-related
peptides was essentially the same in all cell populations.
The studies of stimulated secretion suggested that low and
high expressing clones contain different fractions of PROPTHPRP∆T and PROPTH-PRP in the stimulus-releasable (granule)
pool. These differences should be reflected in the fraction of
PROPTH-PRP∆T and PROPTH-PRP in the post-Golgi (Endo Hresistant) pool. We compared the percentage of cell-associated
Endo H-resistant PROPTH-PRP∆T and PROPTH-PRP at
different times of chase following a long-term labeling for high
and low expressing clones (Fig. 2). For each clone this value
decreases rapidly during the first hour probably reflecting
release of PROPTH-PRP∆T and PROPTH-PRP via the constitutive pathway and reaches a plateau with longer times of chase.
At all timepoints, the percentage of cell-associated Endo Hresistant PROPTH-PRP∆T and PROPTH-PRP is higher (by
~10% on average) in a high expressing clone than in a low
expressing clone.
Our results using radiolabeled polypeptides imply that the
level of expression has a significant influence on the distribution of PROPTH-PRP and PROPTH-PRP∆T among the major
Table 3. 8-Br-cAMP-dependent secretion of ACTH
Construct
Clone
LOE
PROPTH-PRP∆T
% Stim.
sec. ACTH
PROPTH-PRP∆T
PROPTH-PRP∆T
20
43
AtT-20
3.3
66.0
NA
49
47
49
LOE PROPTH-PRP∆T refers to the level of expression of PROPTH-PRP∆T
in each clone.
% Stim. sec. of ACTH was calculated as described for PRP-related
polypeptides (Materials and Methods).
NA, not applicable.
Sorting of proline-rich proteins 1641
Fig. 2. Cell-associated Endo H-resistant PROPTHPRP∆T and PROPTHPRP in low- and high-expressing
clones. Low expressing clones, PROPTH-PRP∆T 20 (d)
and PROPTH-PRP 39 (.), and high expressing clones,
PROPTH-PRP∆T 43 (j) and PROPTH-PRP 4 (m) were
labeled with [3H]proline for 15 hours to approach
steady state and chased for varying lengths of time up to
four hours. At each timepoint, radiolabeled secreted and
cell-associated polypeptides were immunoprecipitated
and subjected to Endo H digestion followed by SDSPAGE and fluorography. Radiolabeled PROPTH-PRP∆T
and PROPTH-PRP were quantitated by densitometry,
and Endo H-resistant, cell-associated PROPTH-PRP∆T
and PROPTH-PRP were expressed as the percentage of
total (secreted + cell-associated) radiolabeled protein.
intracellular pools, the granules and the ER. Because steadystate labeling enriches for the slowest turnover pools of
protein, to a first approximation, the granule and ER pools may
be represented by Endo H-resistant and Endo H-sensitive
PRPs, respectively. Using the clones expressing PROPTHPRP∆T as examples, at steady state the fraction of Endo Hresistant PROPTH-PRP∆T is 0.62 in clone 43 (high level of
expression) and only 0.40 in clone 20 (low level of expression)
(Fig. 2). Based on these fractions and the difference in level of
expression, it is possible to predict that the granule pool in
clone 43 should be 31-fold higher (0.62/0.4×20) than in clone
20 while the ER pool (calculated on basis of Endo H-sensitive
PROPTH-PRP∆T should be only 13-fold higher (0.38/0.6×20)
in clone 43 than in clone 20. We have confirmed the predicted
increase in the granule pool experimentally by comparing the
stimulated discharge of PROPTH-PRP∆T by clones 43 and 20
using quantitative western blotting (see Table 4). The results
indicate a 33-fold higher stimulated secretion of PROPTHPRP∆T by clone 43. Applying the same analysis to PROPTHPRP clones 39 and 4, the predicted fold difference in stimulated secretion of 88 (0.53/0.36×60) is in good agreement with
the experimental value of 83 (see Table 4).
Secretion and storage of full-length PRP
Our previous study showed that the percentage stimulated
secretion of full-length PRP did not increase when levels of
expression of PRP varied over the range corresponding to 0.64% of ACTH (Table 2, this work; Castle et al., 1992). In view
of the increased percentage stimulated secretion of the PROPTH
chimeras observed at higher levels of expression, we have now
measured 8-Br-cAMP-stimulated secretion of full-length PRP
in clones expressing it at yet higher levels. Interestingly, an
approximately twofold increase in the percentage stimulated
secretion was observed when the level of expression of PRP
was increased (Table 2). Yet, as in the case of PROPTH-PRP∆T
and PROPTH-PRP, no further significant rise in the percentage
stimulated secretion occurred at still higher levels of
expression (Table 2). Thus the secretion of full-length PRP
shows the same trend as that of the chimeric polypeptides. But
overall, the percentage stimulated secretion is lower at all
levels of expression.
The role of the PTH propeptide
The presence of the chimeric polypeptides in regulated
secretion has lead us to examine in more detail the potential
role(s) of PROPTH in sorting of PRPs. We considered two possibilities. First, we examined whether storage might relate to
cleavage of the propeptide, reflecting an interaction with the
processing enzyme (Journet et al., 1993). Second, we examined
whether the propeptide might serve as a sorting signal.
Proteolytic processing of the PTH propeptide
The kinetics of processing of native proPTH to PTH are very
rapid and are consistent with proteolytic cleavage occurring
within or just after exiting the Golgi complex (Habener et al.,
1979; Hellerman et al., 1984). If processing of PROPTH is
occurring in the chimeric polypeptides, then the propeptide
should be absent in secreted polypeptides. For cell-associated
polypeptides, the propeptide should be present in Endo Hsensitive forms (ER forms) and absent in Endo H-resistant
forms (Golgi/post-Golgi forms). Using cells expressing
PROPTH-PRP∆T and PRP∆T, we compared the electrophoretic
mobilities of secreted and cell-associated [3H]proline-labeled
polypeptides. Fig. 3A shows that secreted PROPTH-PRP∆T
(PRO∆T in Fig. 3A) and PRP∆T (∆T in Fig. 3A) have identical
apparent Mr values suggesting that the propeptide has been
removed from PROPTH-PRP∆T. The cell-associated Endo H
sensitive PROPTH-PRP∆T has higher apparent Mr than the
Endo H sensitive PRP∆T, indicating that the propeptide is still
attached. The cell-associated Endo H resistant PROPTHPRP∆T has an identical Mr as the secreted PRP∆T indicating
the absence of the propeptide in the post-Golgi pool of
PROPTH-PRP∆T. No band of higher Mr was ever observed in
samples of secreted or cell-associated Endo H-resistant
PROPTH-PRP∆T, suggesting that the propeptide is cleaved off
from the majority of transported PROPTH-PRP∆T, whether or
not it is stored in the granules. This is illustrated by the
identical mobility of PROPTH-PRP∆T released in the absence
and presence of 8-Br-cAMP (Fig. 3B). Together, these results
imply that the polypeptide stored in the granules of cells
expressing PROPTH-PRP∆T corresponds to PRP∆T, the
deletion mutant that was judged to be defective in storage and
transport previously (Castle et al., 1992). Analysis of secreted
and cell-associated PROPTH-PRP yielded similar results (not
shown).
To confirm that the propeptide was cleaved at the predicted
processing site (the carboxyl side of arginine-1 of the hexapeptide prosequence; Fig. 1), we performed limited N-terminal
sequencing of secreted radiolabeled PROPTH-PRP∆T. Using
[3H]proline for biosynthetic labeling, radioactivity was antici-
1642 L. E. Stahl and others
Table 4. Western blotting of stimulated secretion of
PROPTH-PRP∆T and PROPTH-PRP
PROPTH-PRP∆T
43
Net stimulated secretion (AU)
Stimulated secretion ratio
Mean simulated secretion ratio
20
130,000
3,600
36
33±6
PROPTH-PRP
4
39
100,000
1,370
73
83±9
Net stimulated secretion represents the difference in secretion of PROPTHPRP∆T or PROPTH-PRP in the presence and absence of 5 mM 8-Br-cAMP.
The data are from a sample experiment performed as described in Materials
and Methods. The values presented in arbitrary units (AU) are taken from
phosphoimager analysis and normalized for DNA content.
Stimulated secretion ratio is: (net stimulated secretion PROPTH-PRP∆T
43)/(net stimulated secretion PROPTH-PRP∆T 20) and (net stimulated
secretion PROPTH-PRP 4)/net stimulated secretion PROPTH-PRP 39). This is
the fold difference in stimulated secretion between high and low expressing
clones.
Mean stimulated secretion ratio is an average for three separate
experiments ± s.e.m.
Fig. 3. Processing of PTH propeptide from PROPTHPRP∆T chimera.
(A) Comparison of secreted (SEC) and cell-associated polypeptides
(CELL) from cells expressing PROPTH-PRP∆T (PRO∆T) or PRP∆T
(∆T) following a 15 hour labeling with [3H]proline.
Immunoprecipitated polypeptides were digested with Endo H and
immunoprecipitates were analyzed by 10% SDS-PAGE and
fluorography. Positions of the Endo H resistant (r) and Endo H
sensitive (s) forms are indicated. (B) Comparison of PROPTH-PRP∆T
polypeptides secreted in the absence or presence of a secretagogue.
Two wells of cells expressing PROPTH-PRP∆T were labeled for 15
hours with [3H]proline and chased for 6 hours in the absence of
stimulation (not shown). Subsequently the medium was replaced
with medium with (+) or without (−) 5 mM 8-Br-cAMP.
Radiolabeled PROPTH-PRP∆T polypeptides were
immunoprecipitated and analyzed by 10% SDS-PAGE and
fluorography. (C) N-terminal sequencing of radiolabeled secreted
PROPTH-PRP∆T. Cells expressing PROPTH-PRP∆T were labeled for
20 hours with [3H]proline. Secreted PROPTH-PRP∆T was prepared
for N-terminal sequencing as described in Materials and Methods.
Six cycles of Edman degradation were performed and the
radioactivity recovered in each cycle is shown.
pated in the 5th and 6th residues from the N terminus (Fig. 1;
Castle et al., 1992). As seen in Fig. 3C, these expectations were
confirmed, indicating correct cleavage of the PTH propeptide.
Mutagenesis of the processing site in PROPTHPRP∆T
Having established correct cleavage of PROPTH during intracellular transport, we sought to test the effect of preventing the
cleavage of the chimeras by mutagenesis of the processing site.
The PTH propeptide of chimeric PROPTH-PRP∆T and
PROPTH-PRP may be cleaved by PC3 or the more ubiquitous
furin (Smeekens et al., 1991; reviewed by Steiner et al., 1992;
Seidah et al., 1993). In order to alter amino acid residues
commonly recognized by these enzymes and at the same time
retain the same positive charge of the PTH propeptide, the
lysines at positions -2 and -6 were changed to histidines (Fig.
1) by in vitro mutagenesis of PROPTH-PRP∆T. The resulting
construct (HISPTH-PRP∆T) was transfected into AtT-20 cells
and stable lines with different levels of expression were
selected.
To demonstrate that the propeptide is not cleaved, we
examined the fate of HISPTH-PRP∆T after labeling with
[3H]histidine. As shown in Fig. 4, both secreted and cell-associated HISPTH-PRP∆T were labeled with [3H]histidine. Since
PRP∆T contains no histidine residues, this indicates that the
propeptide was not removed. Further, the ratio of
[3H]histidine:[3H]proline labeling for secreted HISPTH-PRP∆T
is the same as that for cell-associated HISPTH-PRP∆T (Fig. 4),
consistent with no cleavage.
Using cells expressing HISPTH-PROPTH, we tested whether
secretion of HISPTH-PROPTH was stimulated with 8-Br-cAMP.
Results presented in Table 5 show that the secretion of HISPTHPRP∆T was stimulated with 8-Br-cAMP; however, the percentage stimulated secretion was slightly lower than observed
for PROPTHPRP∆T for all levels of expression examined
(Table 2). As in the case of PROPTH-PRP∆T, PROPTH-PRP and
PRP, there was an approximately twofold jump in the percentage of stimulated secretion of HISPTH-PROPTH as the level
of expression was increased. Thus removal of the propeptide
is not essential for storage in granules.
Effect of PROPTH on the storage of truncated VSVG
To address the second possibility that PROPTH carries independent sorting information, we fused it to the truncated G
protein of vesicular stomatitis virus (tG), which lacks its Nterminal cytoplasmic and transmembrane segment (Fig. 1). By
itself, tG is secreted constitutively in AtT-20 cells (Moore and
Kelly, 1985), but it is rerouted to the regulated pathway when
fused to growth hormone (Moore and Kelly, 1986). Both tG
and the PROPTH-tG chimera (Fig. 1) were transfected into AtTTable 5. 8-Br-cAMP-dependent secretion of HISPTHPRP∆T
Construct
HISPTH-PRP∆T
HISPTH-PRP∆T
HISPTH-PRP∆T
Clone
LOE
HISPTH-PRP∆T
%
Stim. sec.
16
15
10
5.4
21.5
86.0
7±1
14±2
11±2
LOE HISPTH-PRP∆T and % Stim. sec. were determined as described in
Table 2. The mean ± s.e.m. of three experiments are presented.
Sorting of proline-rich proteins 1643
Fig. 4. Labeling of HISPTH-PRP∆T with [3H]histidine. Cells
expressing HISPTH-PRP∆T were labeled for 15 hours with 0.2
mCi/ml [3H]proline (pro) or 0.4 mCi/ml of [3H]histidine (his).
Secreted (SEC) and cell-associated (CELLS) HISPTH-PRP∆T was
recovered by immunoprecipitation and analyzed by 12.5% SDSPAGE and fluorography.
20 cells and stable cell lines secreting each construct were
created.
The glycosylation pattern of PROPTH-tG suggests that it
correctly traverses the intracellular transport pathway. As is the
case for tG (see Fig. 5A; Fig. 2, Moore and Kelly, 1985), the
predominant intracellular form of PROPTH-tG is Endo Hsensitive (Fig. 5A) indicating its presence in the ER. A minor
fraction of both cell-associated tG and PROPTH-tG is Endo Hresistant (not visible at the exposure shown) and represents the
post-Golgi pool. The small amount of intracellular Endo Hresistant tG and PROPTH-tG is consistent with the absence of
both polypeptides in secretory granules. In contrast to the intracellular polypeptides, secreted tG and PROPTH-tG are entirely
resistant to Endo H digestion (Fig. 5B).
We examined the secretion in the presence and absence of
8-Br-cAMP to determine whether PROPTH-tG is rerouted to
the regulated secretory pathway. The effect of secretagogue
was examined following a 1 hour pulse with Expre35S35S label
and a 5 hour chase. As shown in Fig. 6, no 8-Br-cAMPdependent stimulation of PROPTH-tG was observed. The same
result was obtained with tG (Fig. 6) as was expected based on
previous findings (Moore and Kelly, 1985). These results
obtained with a 1 hour labeling were confirmed with cells that
were labeled for 15 hours to approach steady state (data not
shown). Our findings suggest that PROPTH does not contain
independent sorting information for the regulated pathway.
Fig. 5. Endoglycosidase H digest of tG and PROPTHtG. Clones
expressing tG and PROPTH-tG were labeled for 2 hours with
Expre35S35S label and the secreted and cell-associated tG-related
polypeptides were immunoprecipitated. The immunoprecipitates
were subjected to Endo H digestion as indicated. (A) Cell-associated
tG and PROPTH-tG polypeptides. (B) Secreted tG and PROPTH-tG
polypeptides.
Fig. 6. Secretion of tG and PROPTH-tG. Two wells of cells
expressing tG or PROPTH-tG were labeled for 1 hour with
Expre35S35S label and chased for 5 hours without a secretagogue (1st
chase). Subsequently, cells were chased for an additional 2.5 hours in
the absence (−) or presence (+) of 5 mM 8-Br-cAMP (2nd chase).
tG-related polypeptides were immunoprecipitated and analyzed by
10% SDS-PAGE and fluorography. The slightly greater amount of
PROPTH-tG in the presence of 8-Br-cAMP during the second chase
reflects a greater number of cells in this well; the amount of PROPTHtG recovered from this well in the first chase was also greater by the
same factor. Quantitation indicates that the average stimulation of
secretion of tG and PROPTH-tG was 0% of total (average of two
experiments).
DISCUSSION
In this study we have achieved rapid intracellular transport of
a deletion mutant, PRP∆T, and of full-length PRP by attaching
to them the propeptide of PTH. Using this approach we have
gained new insight about the intracellular transport and sorting
of these unusual secretory proteins. Our findings argue against
the presence of a universal sorting signal for regulated
secretory proteins and are consistent with the notion that
protein-specific interactions are important.
Intracellular transport
The relatively slow ER export of full-length PRP suggested the
presence of an ER retention mechanism for PRPs (Castle et al.,
1992). Since polypeptides lacking either the transition domain
(PRP∆T) or the C-terminal domain (PRP∆C) also exit the ER
slowly (Castle et al., 1992), this ER retention most likely
involves the proline-rich repeats. Proline-rich domains are
known to function in protein-protein interactions (Williamson,
1994), and PRPs may bind to resident ER protein(s) via their
proline-rich repeats. For full-length PRP, ER retention
decreases with increasing concentration (level of expression)
of PRP (Castle et al., 1992). Based on this observation, we have
inferred that self-association of PRPs competes with retention
in the ER. This self-association appears to be mediated by the
transition region as its deletion causes extensive retention of
PRP∆T regardless of concentration (Castle et al., 1992).
Replacement of the transition region with the short and basic
PTH propeptide causes rapid ER export that is independent of
level of expression, suggesting that the ER retention
mechanism has been bypassed. The same results are obtained
when the PTH propeptide and the transition regions are both
present (PROPTH-PRP), suggesting that the propeptide has a
dominant effect. As pointed out by Mains et al. (1995) the PTH
propeptide exhibits a striking sequence similarity to the
propeptides of PAM and of albumin, both of which are thought
to accelerate the rate of secretion of the respective polypep-
1644 L. E. Stahl and others
tides, most likely as a consequence of enhanced ER export
(Mains et al., 1995; McCracken and Kruse, 1989). Together
with the finding that the effect of the PAM propeptide is transferrable to another protein, PC2 (Mains et al., 1995), these
results are suggestive of a specialized mechanism involving
recognition of the propeptide sequence. However, deletion of
the propeptide from PTH does not alter its own rate of transport
(Wiren et al., 1988) suggesting that not all proteins are affected
by the presence of the propeptide. Clearly, further studies will
be required to elucidate the mode of action of these propeptides.
Using both electrophoretic analysis and N-terminal microsequencing, we have demonstrated that the propeptide is
cleaved from the chimeric PROPTH-PRP∆T before secretion.
Further, processing was observed regardless of the pathway of
secretion, suggesting that cleavage occurred before or concomitantly with sorting. Therefore, the post-Golgi and secreted
forms of PROPTH-PRP∆T and PROPTH-PRP have identical
primary structures to the original PRP∆T and PRP, validating
the strategy of propeptide attachment as a bypass for slow ER
export.
Sorting and storage of PRP-related polypeptides
It is clear that by accelerating ER export, PROPTH enhances
the ability to detect granule storage of PRP and PRP∆T. We
regard the possiblity that PROPTH contains sorting information
for the regulated secretory pathway as unlikely for two reasons.
First, deletion of the propeptide from the precursor of PTH
does not affect the pathway of secretion of PTH in GH4 cells
(Wiren et al., 1988). Second, when the PTH propeptide was
fused to a reporter protein, tG, the chimeric polypeptide was
not targeted to the regulated pathway of AtT-20 cells (Fig. 6,
this work). We also considered the possibility that the propeptide might affect sorting through its interaction with the proteolytic processing machinery. An intact cleavage site has been
shown to be necessary for the storage of von Willebrand factor
in Weibel-Palade bodies, and it has been proposed that the processing enzyme may function in sorting either by facilitating
local concentration and aggregation of propeptide-containing
polypeptides or by serving as a ‘receptor’ at sites of sorting
(Journet et al., 1993). However, we have shown that disruption
of PTH propeptide processing by mutagenesis of the cleavage
site did not abrogate regulated secretion. While this would
appear to rule out a major role of the processing enzymes in
targeting of the propeptide-containing chimeras, we can’t
discount the possibility that processing enzymes might affect
the degree of storage because the percentage of 8-Br-cAMP
dependent secretion of HISPTH-PRP∆T was slightly lower than
that of PROPTHPRP∆T.
Because the major role of PROPTH is to accelerate ER export
of PRPs rather than to serve as a sorting signal, we believe that
sorting of PRPs for stimulated secretion must rely on structural
characteristics of the PRP itself. While we previously regarded
the transition domain of PRP as necessary for storage in
secretory granules (Castle et al., 1992), the present findings
argue convincingly that this is not the case. Rather the transition domain mainly facilitates efficient transport of the native
protein from the ER. Thus sorting of PRP for regulated
secretion must depend on one of the two remaining domains,
either the central proline-rich repeats or the C-terminal
segment. Our earlier study ruled out any effect of the C-
terminal segment on sorting, so by elimination, we are left with
the proline-rich repeats as being responsible. Since this domain
is unique in primary and probably higher order structure among
regulated secretory proteins, any interactions that it mediates
are likely to be specific to the PRP family.
Strikingly, regulated secretion of PRP-related polypeptides
shows a dependence on level of expression. An incremental
increase in the percentage stimulated secretion is observed
when expression is increased beyond a certain level. While
both ER and storage granule pools of PRP-related polypeptides
are elevated when the level of expression is increased, our data
clearly indicate that the storage pool is preferentially enriched
(Table 4). In contrast, the fractional storage of the endogenous
hormone ACTH was unaffected by the expression of PRPrelated polypeptides. Consequently, PRP-related polypeptides
either are stored in increased amounts in the ACTH-containing granules or are segregated into a second storage compartment with the same regulation.
Because the incremental increase in storage was observed
for the full-length PRP as well as for the chimeric polypeptides, the presence of PROPTH is not necessary for this effect.
We propose that the jump in storage is driven by the increased
steady state concentration at sites of secretory granule
formation and reflects homo- or heterotypic aggregation
involving PRP-related polypeptides. The nonlinear dependence on concentration (level of expression) is consistent with
a cooperative process which is catalyzed once a threshold concentration is reached. The jump in storage for PROPTH-PRP∆T
and PROPTH-PRP occurs at a 5- to 10-fold lower expression
level than for full-length PRP, suggesting that PROPTH may
promote somewhat different interactions than occur for fulllength PRP. In any case, the concentration-dependent storage
of PRPs is best explained as an aggregation process, and it may
reflect the same type of associations that cause PRPs to concentrate visibly within the content of parotid acinar granules
(Kousvelari et al., 1982).
In conclusion, our studies put in question the existence of a
universal sorting signal for regulated secretion as has been postulated previously (Kizer and Tropsha, 1991; Gorr and Darling,
1995). On the other hand, we wish to emphasize that the fractional storage that we have observed for PRPs and that others
have observed for several nonendocrine proteins (e.g.
Fennewald et al., 1988; Castle et al., 1992; Schmidt and Moore,
1994; Reaves et al., 1990; Stoller and Shields, 1989; Castle et
al., 1995) and free glycosaminoglycan chains (Matsuuchi and
Kelly, 1991) is typically well below the extent of storage
achieved by the endogenous hormone, ACTH. Thus we believe
that it will be important in future studies to evaluate sorting for
regulated secretion on the basis of the amount of polypeptide
stored. Cell- or protein-specific sorting signals (Cool et al.,
1995; Arrandale and Dannies, 1994; Stoller and Shields, 1989)
or highly efficient aggregation and retention mechanisms
(Kuliawat and Arvan, 1994) may be superimposed on a bulk
flow pathway leading to forming granules, and proteins that are
either absent or poorly represented in regulated secretion may
be progressively excluded from the maturing storage compartment.
We are grateful to Yun Shim for his skillful assistance in the mutagenesis and cloning of HISPTH-PRP∆T. We appreciate the helpful
advice of Drs R. Mains and B. Eipper (Johns Hopkins Medical
Sorting of proline-rich proteins 1645
School), regarding the mutagenesis of the PTH propeptide, and of
the reviewers of the submitted manuscript. We thank Dr Robert
Wagner (University of Virginia) for his gift of an antibody against
the VSV protein G, and Dr Jack Rose for his gift of the cDNA of the
truncated G protein. This work was supported by NIH Grant
DE08491.
REFERENCES
Arrandale, J. M. and Dannies, P. S. (1994). Inhibition of rat prolactin (PRL)
storage by coexpression with human PRL. Mol. Endocrinol. 8, 1083-1090.
Arvan, P. and Castle, J. D. (1986). Isolated secretion granules from parotid
glands of chronically-stimulated rats possess an alkaline internal pH and
inward-directed H+ pump activity. J. Cell Biol. 103, 1257-1267.
Arvan, P. and Castle, D. (1992). Protein sorting and secretion granule
formation in regulated secretory cells. Trends Cell Biol. 2, 327-331.
Bauerfeind, R. and Huttner, W. B. (1993). Biogenesis of constitutive
secretory vesicles, secretory granules and synaptic vesicles. Curr. Opin. Cell
Biol. 5, 628-635.
Blair, E. A., Castle, A. M. and Castle, J. D. (1991). Proteoglycan sulfation and
storage parallels storage of basic secretory proteins in exocrine cells Am. J.
Physiol. 261, C897-C905.
Burgess, T. L., Craik, C. S. and Kelly, R. B. (1985). The exocrine protein
trypsinogen is targeted into the secretory granules of an endocrine cell line:
studies by gene transfer. J. Cell Biol. 101, 639-645.
Castle, A. M., Stahl, L. E. and Castle, J. D. (1992). A 13-amino acid Nterminal domain of a basic proline-rich protein is necessary for storage in
secretory granules and facilitates exit from the endoplasmic reticulum. J.
Biol. Chem. 267, 13093-13100.
Castle, A. M. and Castle, J. D. (1993). Novel secretory proline-rich
proteoglycans from rat parotid: cloning and characterization by expression in
AtT-20 cells. J. Biol. Chem. 268, 20490-20496.
Castle, A. M., Schwarzbauer, J. E., Wright, R. L. and Castle, J. D. (1995).
Differential targeting of recombinant fibronectins based on their efficiency of
aggregation. J. Cell Sci. 108, 3827-3837.
Chanat, E. and Huttner, W. B. (1991). Milieu-induced, selective aggregation
of regulated secretory proteins in the trans-Golgi network. J. Cell Biol. 115,
1505-1519.
Chu, W. N., Baxter, J. D. and Reudelhuber, T. L. (1990). A targeting
sequence for dense secretory granules resides in the active renin protein
moiety of human preprorenin. Mol. Endocrinol. 4, 1905-1913.
Colomer, V., Lal, K., Hoops, T. C. and Rindler, M. J. (1994). Exocrine
granule specific packaging signals are present in the polypeptide moiety of
the pancreatic granule membrane protein GP2 and in amylase: implications
for protein targeting to secretory granules. EMBO J. 13, 3711-3719.
Cool, D. R., Fenger, M., Snell, C. R. and Loh, Y. P. (1995). Identification of
the sorting motif within pro-opiomelanocortin for the regulated secretory
pathway. J. Biol. Chem. 270, 8723-8729.
Dickerson, I. M., Dixon, J. E. and Mains, R. E. (1987). Transfected human
neuropeptide Y cDNA expression in mouse pituitary cells. J. Biol. Chem.
262, 13646-13653.
Fennewald, S. M., Hamilton, R. L and Gordon, J. I. (1988). Expression of
human preproapoAI and pre(∆pro)apoAI in murine pituitary cell line (AtT20). J. Biol. Chem. 263, 15568-15577.
Gorr, S. U. and Darling, D. S. (1995). An N-terminal hydrophobic peak is the
sorting signal of regulated secretory proteins. FEBS Lett. 361, 8-12.
Habener, J. F., Amherdt, M., Ravazzola, M. and Orci, L. (1979).
Parathyroid hormone biosynthesis: correlation of conversion of biosynthetic
precoursers with intracellular protein migration as determined by electron
microscope autoradiography. J. Cell Biol. 80, 715-731.
Hellerman, J. G., Cone, R. C., Potts, J. T., Rich, A., Mulligan, R. C. and
Kronenberg, H. M. (1984). Secretion of human parathyroid hormone from
rat pituitary cells infected with a recombinant retrovirus encoding
preproparathyroid hormone. Proc. Nat. Acad. Sci. USA 81, 5340-5344.
Hendy, G. N., Kronenberg, H. M., Potts, J. T. and Rich, A. (1981).
Nucleotide sequence of cloned cDNAs encoding human preproparathyroid
hormone. Proc. Nat. Acad. Sci. USA 78, 365-7369.
Journet, A. M., Saffaripour, S., Cramer, E. M., Tenza, D. and Wagner, D.
D. (1993). Von Willebrand factor storage requires intact prosequence
cleavage site. Eur. J. Cell Biol. 60, 31-41.
Kizer, J. S. and Tropsha, A. (1991). A motif found in propeptides and
prohormones that may target them to secretory vesicles. Biochem. Biophys.
Res. Commun. 174, 586-592.
Kousvelari, E. E., Oppenheim, F. G. and Cutler, L. S. (1982). Ultrastructural
localization of salivary acidic proline-rich proteins from Macaca fascicularis.
J. Histochem. Cytochem. 30, 274-278.
Kuliawat, R. and Arvan, P. (1994). Distinct molecular mechanisms for
protein sorting within immature secretory granules of pancreatic B-cells J.
Cell Biol. 126, 77-86.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227, 680-685.
Mains, R. E., Milgram, S. L., Keutmann, H. T. and Eipper, B. A. (1995).
The NH2-terminal proregion of peptidyl-amidating monooxygenase
facilitates the secretion of soluble proteins. Mol. Endocrinol. 9, 3-13.
Matsuuchi, L. and Kelly, R. B. (1991). Constitutive and basal secretion from
the endocrine cell line, AtT-20 J. Cell Biol. 112, 843-852.
McCracken, A. A. and Kruse, K. B. (1989). Intracellular transport of rat
serum albumin is altered by a genetically engineered deletion of the
propeptide. J. Biol. Chem. 264, 20843-20846.
Moore, H.-P. H. and Kelly, R. B. (1985). Secretory protein targeting in a
pituitary cell line: differential transport of foreign secretory proteins to
distinct secretory pathways. J. Cell Biol. 101, 1773-1781.
Moore, H.-P. H. and Kelly, R. B. (1986). Re-routing of a secretory protein by
fusion with human growth hormone sequences. Nature 321, 443-446.
Moore, H.-P. H., Walker, M. D., Lee, F. and Kelly, R. B. (1983). Expressing
a human proinsulin cDNA in a mouse ACTH secreting cell. Intracellular
storage, proteolytic processing and secretion on stimulation. Cell 35, 531538.
Neufeld, G., Mitchell, R., Ponte, P. and Gospodarowicz, D. (1987).
Expression of human basic fibroblast growth factor cDNA in baby hamster
kidney-derived cells results in autonomous cell growth J. Cell Biol. 106,
1385-1394.
Reaves, B. J., Van Itallie, C. M., Moore, H.-P. M. and Dannies, P. S. (1990).
Prolactin and insulin are targeted to the regulated pathway in GH4C1 cells,
but their storage is differentially regulated. Mol. Endocrinol. 4, 1017-1026.
Schmidt, W. K. and Moore, H.-P. H. (1994). Synthesis and targeting of
insulin-like growth factor-I to the hormone storage granules in an endocrine
cell line. J. Biol. Chem. 269, 27115-27124.
Schwarzbauer, J. E., Mulligan, R. C. and Hynes, R. O. (1987). Efficient and
stable expression of recombinant fibronectin polypeptides Proc. Nat. Acad.
Sci. USA 84, 754-758.
Seidah, N. G., Day, R. and Chretien, M. (1993). The family of pro-hormone
and pro-protein convertases. Biochem. Soc. Trans. 21, 685-690.
Smeekens, S. P., Avruch, A. S., LaMendola, J., Chan, S. J. and Steiner, D.
F. (1991). Identification of a cDNA encoding a second putative prohormone
convertase related to PC2 in AtT-20 cells and islets of Langerhans. Proc. Nat.
Acad. Sci. USA 88, 340-344.
Steiner, D. F., Smeekens, S. P., Ohagi, S. and Chan, S. J. (1992). The new
enzymology of precursor processing Endoproteases. J. Biol. Chem. 267,
23435-23438.
Stoller, T. J. and Shields, D. (1989). The propeptide of preprosomatostatin
mediates intracellular transport and secretion of alpha-globin from
mammalian cells. J. Cell Biol. 108, 1647-1656.
West, D. C., Sattar, A. and Kumar, S. (1985). A simplified in situ
solubilization procedure for the determination of DNA and cell number in
tissue cultured mammalian cells. Analyt. Biochem. 147, 289-295.
Williamson, M. P. (1994). The structure and function of proline-rich regions in
proteins. Biochem. J. 297, 249-260.
Wiren, K. M., Potts, J. T. and Kronenberg, H. M. (1988). Importance of the
propeptide sequence of human preproparathyroid hormone for signal
sequence function. J. Biol. Chem. 263, 19771-19777.
(Received 25 October 1995 - Accepted 10 March 1996)