A Conserved alpha -Helix at the Amino Terminus of Prosomatostatin Serves as a Sorting Signal for the Regulated Secretory Pathway*

Rania MouchantafDagger §, Ujendra KumarDagger , Traian Sulea, and Yogesh C. PatelDagger ||

From Dagger  Fraser Laboratories, Departments of Medicine and Neurology and Neurosurgery, Pharmacology, and Therapeutics, McGill University, Royal Victoria Hospital and Montreal Neurological Institute, Montreal, Quebec H3A 1A1, Canada, and the  National Research Council Biotechnology Research Institute, Montreal, Quebec H4P 2R2, Canada

Received for publication, March 20, 2001, and in revised form, April 13, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mammalian prosomatostatin (PSST) contains the bioactive peptides SST-14 and SST-28 at the COOH-terminal end of the molecule and a putative sorting signal in the propeptide segment for targeting the precursor to the regulated secretory pathway. The NH2-terminal segment of PSST consists of an amphipathic alpha -helix, which has been totally conserved throughout vertebrate evolution. We have analyzed the PSST-(3-15) region for sorting function by alanine scanning and deletional mutagenesis. Mutants created were stably expressed in AtT-20 cells. Regulated secretion was studied by analyzing basal and stimulated release of SST-14 LI and by immunocytochemistry for staining of SST-14 LI in punctate granules. Deletion of the PSST-(3-15) segment blocked regulated secretion and rerouted PSST for constitutive secretion as unprocessed precursor. Alanine scanning mutagenesis identified the region Pro5-Gln12 as being important in precursor targeting, with Leu7 and Leu11 being critical. Molecular modeling demonstrated that these two residues are located in close proximity on a hydrophobic surface of the alpha -helix. Disruption of the alpha -helix did not impair the ability of PSST to be processed at the COOH terminus to SST-14 and SST-28. Processing, however, was shifted to the early compartments of the secretory pathway rather than storage granules and was relatively inefficient.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Secretory cells such as neuroendocrine, exocrine, and mast cells contain two distinct pathways for protein secretion, a constitutive secretory pathway (CSP)1 that transports proteins to the cell surface by bulk flow and a regulated secretory pathway (RSP) that releases secretory proteins from a granular storage pool in response to specific stimuli (1, 2). Proteins destined for secretion are initially synthesized as precursors on ribosomes, translocated into the lumen of the endoplasmic reticulum, and transported through the Golgi stacks to the trans-Golgi network (TGN). Here the protein is sorted via clathrin-coated vesicles into the RSP consisting of dense core secretory granules or the CSP through small nonclathrin-coated vesicles, which exit from the TGN and rapidly migrate to the plasma membrane (2-6). A major unanswered question is the mechanism for sorting prohormone and proneuropeptide precursors in the TGN into either the CSP or RSP (4-6). Sorting is an active process that requires some form of recognition of the secretory protein (2, 4-6). It is one step in a multistep cascade during which the prohormone is concentrated over 100-fold, packaged with other granular proteins, extruded into budding secretory vesicles, and proteolytically processed into smaller mature products. These events may be interdependent, and their temporal and spatial relationship remains poorly understood (5, 6). Three models have been proposed to explain how proteins are sorted to the RSP. The first proposes that regulated secretory proteins possess an intrinsic ability to form aggregates leading to packaging of condensed products into secretory granules, thereby sorting them away from soluble proteins that are carried off by bulk flow in small vesicles. Support for this model comes from the tendency of a number of secretory granule proteins such as prolactin, growth hormone, the chromogranins, carboxypeptidase E (CPE), and prohormone convertase 2 (PC2) to aggregate at the mildly acidic pH in the TGN (7-12). However, other proteins, such as fibronectin, that aggregate easily are not targeted into the RSP, and modifications on proteins such as chromogranin B and insulin-like growth factor-1 result in missorting without affecting aggregation (13-15). Furthermore, GH does not aggregate in the acidic environment of the TGN in COS-7 cells but does so in AtT-20 cells, and blockade of acidification with chloroquin and bafilomycin A1 is without effect on the ability of these hormones to aggregate in secretion granules in GH4C1 cells, suggesting that aggregation alone is not sufficient for sorting into secretory granules (7). The second model assumes that regulated secretory proteins contain sorting signals in the form of specific-sequence motifs or conformational epitopes that allow them to be sorted from constitutive secretory proteins by a receptor-mediated mechanism at the level of the TGN (1, 2). The third model combines features of the first two mechanisms and assumes that there is initial interaction of the regulated secretory protein with a receptor, which then triggers the formation of an aggregate that is packaged into secretion granules. Several lines of evidence suggest that the propeptide is recognized by the sorting apparatus and that the structural domains that serve as recognition signals are dominant, since fusion of a constitutively secreted protein to a hormone (e.g. GH) targets the hybrid protein to the RSP and deletion of sorting signal domains results in mistargeting to the CSP (16-22). A sorting sequence domain has been described in the prosegment of POMC, enkephalin, SST, chromogranins, and PC1 (18-23). The most compelling arguments for a specific sequence sorting motif have come from studies of POMC and prosomatostatin (PSST) (18, 19, 21, 24-28). In the case of POMC, structure-function and molecular modeling studies have identified a sorting signal motif in the NH2-terminal segment made up of a disulfide bond constrained amphipathic hairpin loop that binds to a sorting receptor identified as membrane-associated CPE (Fig. 1) (21, 25). Molecular modeling has revealed a similar putative sorting motif in two other precursors, proenkephalin and proinsulin (25). Mutation of the binding site on CPE or in vitro antisense depletion of CPE or genetic obliteration of CPE in the CPEfat mouse leads to missorting of POMC, proenkephalin, and proinsulin (25, 26, 29). Not all secretory proteins, however, are recognized for sorting by CPE. For instance, chromogranin A, which possesses a RSP sorting domain similar to that in POMC, does not use CPE as a sorting receptor, suggesting the existence of other sorting receptors (26). PSST is another well characterized precursor that has been suggested to harbor a sorting signal (18, 19, 28). Mammalian PSST is processed post-translationally at COOH-terminal dibasic and monobasic sites to yield SST-14 and SST-28, respectively (30, 31). In addition, cleavage at an unknown site at the NH2-terminal region has been implicated in generating the decapeptide PSST-(1-10) without any known biological activity (32). The PSST-(1-10) sequence is conserved throughout vertebrate evolution (33), and deletion of this region results in missorting of the mutant precursor (28) (Fig. 1). A comparison of the amino acid sequence and secondary structure of the PSST NH2-terminal segment with that of 14 other prohormones that have been shown experimentally to be sorted to secretory vesicles in AtT-20 cells has identified a common motif consisting of a degenerate amphipathic alpha -helix (34). This consensus sorting sequence in the case of PSST lies within residues 3-15 and differs from the disulfide bond containing hairpin loop structure in POMC, proenkephalin, and proinsulin (Fig. 1). In the present study, we have analyzed the PSST-(3-15) segment as a sorting signal by alanine scanning and deletional mutagenesis. We show that Leu7 and Leu11, which form part of a contiguous hydrophobic patch on the surface of the alpha -helix, are critical for sorting function and that COOH-terminal processing of PSST to SST-14 and SST-28 can occur constitutively but is relatively inefficient in the absence of correct precursor targeting to the RSP.


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Fig. 1.   Comparison of the NH2-terminal PSST sequences of anglerfish I, catfish I, mouse, rat, and human precursors. The boldface leucine residues separated by 3 amino acids are highly conserved hydrophobic residues predicted to play a crucial role in the formation of an alpha -helix. Shown for comparison are sequences of human procortistatin (PCST) (which features a leucine-containing alpha -helix not at the NH2 terminus but further downstream in the molecule), anglerfish PSSTII NH2-terminal sequence (which does not feature an alpha -helix), and human PC1 (which displays an alpha -helix comparable with that in mammalian PSST. Also compared is the disulfide bond constrained amphipathic hairpin loop motif in POMC, proenkephalin, and insulin postulated to act as a sorting signal for binding to carboxypeptidase E. Chromogranin A, which also contains this motif, however, does not interact with CPE.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Synthetic peptides were obtained as follows. SST-14 and SST-28 were from Bachem (Marina del Rey, CA); Tyr0 SST-14 was from Peninsula Laboratories (Belmonte, CA); acetonitrile and trifluoroacetic acid were purchased from Fisher; heptafluorobutyric acid was obtained from Pierce; pepstatin-A, 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenylmethylsulfonyl fluoride were from Sigma. Forskolin (FSK) was purchased from Calbiochem; Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Life Technologies, Inc. Ser-X-tend was obtained from Irvine Scientific (Santa Anna, CA). All other reagents were of analytical grade and were obtained from various suppliers.

Construction of Wild Type and Mutant PSST cDNAs-- cDNA for wild type rat prepro-SST was constructed in the expression vector pTEJ8. Using rat prepro-SST as template, a series of mutants were created by the PCR overlap extension technique (35) (Fig. 2): (i) alanine scanning mutagenesis substituting Ala for each of the 13 residues from Ser3 to Leu15 in PSST; (ii) NH2-terminal deletion mutant deleting residues 3-15 of PSST (Delta NPSST); and (iii) insertional mutants substituting Lys13 with KR or RTKR. To construct the mutants, two fragments were created separately, which included a 5' fragment containing the desired mutation using primer A and a reverse primer and a 3' fragment using primer B and a forward primer where the forward primer and reverse primers are mirror images of each other. The fragments were then ligated in a third PCR to generate the full-length mutant PPSST cDNA. For example, to create Ser3 right-arrow Ala, primers A and 2 were used to synthesize the 5' fragment of PPSST, and primers B and 1 were used to generate the 3' fragment. Primer A (5'-ATT CATA AGC TTG CCG CCA CCA TGC TGT CCT GCC GT-3' (forward)) was designed to contain HindIII endonuclease restriction site, Kozak consensus sequence, and initiation codon. Primer B (5'-TAG TAG ATG AAT TCC TAA CAG GAT GTG GAA TGT-3' (reverse)) contained 3'-flanking sequence and stop codon followed by an EcoRI restriction site.


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Fig. 2.   Schematic illustration of the alanine substitution PSST NH2-terminal mutants (A), PSST-(3-15) deletion mutant, Delta N PSST (B), and mutants substituting Lys13 for KR and Lys13 for RTKR (C).

The following forward and reverse primers bind to the same region of rat prepro-SST cDNA and were designed to contain the desired mutation. Ser3, 5'-ACCGGGGCGCCCGCGGACCCCAGA-3' (forward) and 5'-TCTGGGGTCCGCGGGCGCCCCGGT-3' (reverse) (nt 66-90); Asp4, 5'-ACCGGGGCGGCCCTCGGCCCCCAGACTCCGTCA-3' (forward) and 5'-TGACGGAGTCTGGGGGGCCGAGGGCGCCCCGGT-3' (reverse) (nt 66-98); Pro5, 5'-GCGCCCTCGGACGCCAGACTCCGTCA-3' (forward) and 5'-TGACGGAGTCTGGCGTCCGAGGGCGC-3' (reverse) (nt 72-98); Arg6, 5'-TCGGACCCCGCACTCCGTCAGTTTCT-3' (forward) and 5'-AGAAACCTGACGAGTGCGGGTCCGA-3' (reverse) (nt 78-104); Leu7, 5'-CGGACCCCAGAGCTCGTCAGTTTCTG-3' (forward) and 5'-CAGAAACTGACGAGCTCTGGGGTCCG-3' (reverse) (nt 90-116); Arg8, 5'-ACCCCAGACTCGCTCAGTTTCTGCA-3' (forward) and 5'-TGCAGAAACTGAGCGAGTCTGGGGT-3' (reverse) (nt 83-108); Gln9, 5'-ACCCCAGACTCCGTGCGTTTCTGCAGAA-3' (forward) and 5'-TTCTGCAGAAACGCACGGAGTCTGGGGT-3' (reverse) (nt 83-111); Phe5, 5'-AGACTCCGTCAGGCTCTGCAGAAGT-3' (forward) and 5'-ACTTCTGCAGAGCCTGACGGAGTCT -3' (reverse) (nt 88-113); Leu11, 5'-CTCCGTCAGTTTGCGCAGAAGTCTCTG-3' (forward) and 5'-CAGAGACTTCTGCGCAAACTGACGGAG-3' (reverse) (nt 91-118); Gln12, 5'-TCAGTTTCTGGCGAAGTCTCTGGCGGCT-3' (forward) and 5'-AGCCGCCAGAGACTTCGCCAGAAACTGA-3' (reverse) (nt 96-124); Lys13, 5'-CAGTTTCTGCAGGCCTCTCTGGCGGCT-3' (forward) and 5'-AGC CGC CAG AGA GGC CTG CAG AAA CTG-3' (reverse) (nt 96-123); Ser14, 5'-AGTTTCTGCAGAAGGCTCTGGCGGCTGCCA-3' (forward) and 5'-TGGCAGCCGCCAGAGCCTTCTGCAGAAACT-3' (reverse) (nt 98-127); Leu15, 5'-CTGCAGAAGTCTGCAGCGGCTGCCACC-3' (forward) and 5'-GGTGGCAGC CGC TGCAGACTTCTGCAG-3' (reverse) (nt 103-138); Lys13 right-arrow KR, 5'-CAGTTTCTGCAGAAGAGGTCTCTGGCGGCT-3' (forward) and 5'-AGCCGCCAGAGACCTCTTCTGCAGAAACTG-3' (reverse) (nt 96-120); Lys13 right-arrow RTKR, 5'-CAGTTTCTGCAGAGGACAAAGAGGTCTCTGGCGGCT -3' (forward) and 5'-AGCCGCCAGAGACCTCTTTGTCCTCTGCAGAAACTG-3' (reverse) (nt 96-120); and Delta NPSST, 5'-GGTGTCACCGGGGCGCCCGCGGCTGCCACCGGGAA-3' (forward) and 5'-TTCCCGGTGGCAGCCGCGGGCGCCCCGGTGACACC-3' (reverse) (nt 60-134, segment with deletion of nt 79-117).

PCR was carried out with 50 ng of PPSST cDNA in 100 µl containing 20 mM Tris-HCl, 200 µM dNTPs, 1.5 mM MgCl2, 6% Me2SO, and 2 units of Pfu (Stratagene) using the following conditions: denaturation at 94 °C for 80 s, anealing at 59 °C for 50 s, and extension at 72 °C for 60 s for 25 cycles followed by extension at 72 °C for 10 min. PCR products were separated by agarose gel electrophoresis, and the amplified bands were electroeluted and purified. Fragments B-1 and A-2 were then fused in a ligation reaction using flanking primer pair A and B. After PCR ligation, the products were digested to completion with EcoRI and HindIII, and the purified fragments were subcloned into HindIII-EcoRI multiple cloning sites of pTEJ8. All recombinant plasmid constructions were verified by sequencing of double-stranded DNA (University Core DNA Service, University of Calgary, Alberta, Canada), and at least two independent clones of each mutant were independently transfected.

Cell Culture and Transfection-- AtT-20 mouse anterior pituitary cells were cultured in Dulbecco's modified Eagle's medium with 5% fetal bovine serum supplemented with Ser-X-tend in an atmosphere of 5% CO2 and 95% air in a humidified incubator at 37 °C. Cells were plated in 100 × 20-mm Petri dishes and transfected at 50% confluency with 3-5 µg of the appropriate plasmid construct by LipofectAMINE (Life Technologies) and stable G418 (0.861 mg/ml)-resistant nonclonally selected cells were propagated for study.

Secretion Studies-- Stably transfected AtT-20 cells were cultured in 35-mm diameter six-well plastic Petri dishes and grown to 80-90% confluency, after which they were prepared for studies of basal and stimulated secretion of immunoreactive SST-14 (SST-14 LI). Following removal of the feeding medium, groups of five wells were incubated with Dulbecco's modified Eagle's medium plus 1% bovine serum albumin containing phenylmethylsulfonyl fluoride and pepstatin-A (20 µg/ml each). To study regulated secretion, cells were incubated with 20 µM FSK or 10-7 M TPA for 4 h. Media were then harvested and centrifuged at 1000 × g for 5 min to remove detached cells, and the supernatant was acidified to pH 4.8 with 1 M acetic acid and stored at -20 °C pending radioimmunoassay (RIA) analysis of SST-14 LI. Attached cells were extracted by scraping into 1 M acetic acid containing phenylmethylsulfonyl fluoride and pepstatin-A (20 µg/ml each) on ice. The cell suspension was further extracted by sonication followed by centrifugation at 5000 × g for 30 min. The supernatant was stored at -20 °C for RIA and HPLC analysis.

HPLC-- Pooled acidified secretion media and cell extracts were diluted 1:7 with 0.1% trifluoroacetic acid and concentrated using Waters Sep-Pak C18 cartridges. The adsorbed peptides were analyzed by HPLC on a C18 µBondapak reverse phase column using a Waters HPLC system as previously described (30, 31). The column was eluted at room temperature (21 °C) at 1 ml/min with 12-55% acetonitrile and a 0.2% heptafluorobutyric acid gradient over 150 min. The column effluent was monitored for UV absorbance at 214 and 280 nM. Fractions were spiked with 10 µl of 10% bovine serum albumin, and stored at -20 °C until further use. 30-100-µl aliquots from each fraction were rotary-evaporated with a Speedvac and assayed for SST-14 LI by RIA.

RIA of SST-14 LI-- RIA for SST-14 LI was performed using a rabbit anti-SST antibody (R149), [125I]Tyr0 SST-14 radioligand, synthetic SST-14 standards, and a bovine serum albumin-coated charcoal separation method (30, 31). Antibody R149 is directed against the central segment of SST-14 and detects SST-14 as well as the molecular forms extended at the amino terminus of the peptide such as SST-28 and PSST.

Immunofluorescence Microscopy-- The cellular localization of SST-14 LI in AtT-20 cells expressing wild type and mutant PSST forms was characterized by fluorescence immunocytochemistry (36). Stably transfected AtT-20 cells were plated at 1.25 × 105 cells/well in 24-well plates coated with 50 mg/ml polyornithine. On day 3 at ~60-70% confluency, cells were washed twice in PBS and fixed in 2% paraformaldehyde (in 0.1% PBS) for 20 min on ice. Cells were then permeabilized with 0.2% Triton X-100 in (0.1% PBS) for 5 min at room temperature, washed three times in PBS and incubated with R149 anti-SST-14 antibody (diluted 1:1000) for 8-12 h at 4 °C. The cells were washed with PBS and incubated for 90 min at 20 °C with Cy3-conjugated goat anti-rabbit secondary antibody (1:200). For staining the Golgi apparatus, cells were washed twice in PBS and incubated for 5 h with wheat germ agglutinin conjugated to fluoroscein (1:1000). Finally, cells were washed twice with PBS, mounted with immunofluor and viewed under a Zeiss LSM 410 confocal microscope. Images were obtained as single optical sections taken through the middle of cells and averaged over 32 scans/frame.

Secondary Structure Prediction and Model Building-- The secondary structure of rPSST (residues Ala1-Cys92) was predicted with the NPS@ consensus secondary structure prediction algorithm (37) using 11 secondary structure prediction methods: SOPM, SOPMA, HNN, DPM, DSC, GOR-I, GOR-III, GOR-IV, PHD, PREDATOR, and SIMPA96. A structural model of the predicted alpha -helical region Pro5-Thr19 was constructed from standard geometries using the BIOPOLYMER module in SYBYL 6.6 molecular modeling software (Tripos Inc., St. Louis, MO). NH2 and COOH termini were blocked with acetyl and methylamino groups, respectively. Structural refinement was carried out by energy minimization using an AMBER 4.1 all-atom force field (38) and a distance-dependent (4R) dielectric constant.

Statistical Analysis-- Results are expressed as mean ± S.E. Statistical analysis was carried out by one-way analysis of variance followed by Dunnet's significance test. Significance was indicated by a p value of < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Basal and Stimulated Release of WT PPSST-- AtT-20 cells expressing WT PSST released total SST-14 LI at a low basal rate of 0.54 ± 0.08 ng/ml/4 h representing 8.3% of total cell content (Fig. 3A). FSK stimulated SST-14 LI secretion 1.9-fold, whereas TPA demonstrated a 3.3-fold stimulation of SST-14 LI release. By immunocytochemistry, SST-14 LI displayed punctate localization in vesicular structures in both the main cell body throughout the cytoplasm as well as in cell processes (Fig. 4, A-C). These results provide both morphological and functional evidence that PSST is properly sorted to the RSP in AtT-20 cells displaying low basal secretion and positive response to secretagogue stimulation, thereby making these cells an appropriate model for studying PSST sorting to the RSP.


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Fig. 3.   Comparison of basal, FSK, and TPA stimulated secretion of SST-14 LI from AtT-20 cells stably expressing WT PSST (A), Delta N PSST (B), Lys13 to Lys-Arg mutant (C), and Lys13 to Arg-Thr-Lys-Arg mutant (D). Transfected cells were incubated for 4 h with control medium or medium containing FSK 20 µM or TPA 100 nM. At the end of the incubation, cell extracts (CE) and media (M) were separately assayed for SST-14 LI. Values shown are mean ± S.E. of five measurements from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.


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Fig. 4.   Immunofluorescence localization of SST-14 LI in AtT-20 cells expressing WT PPSST (A-C) and PPSST mutants Lys13 to KR (D-F), Leu7 to Ala (G-I), and Lys13 to Ala (J-L). The left panels show SST-14 LI identified by Cy3 red fluorescence. The middle panels show wheat germ agglutinin (WGA) localized as green fluorescence in the Golgi, and the right panels show the cellular colocalization. Note the presence of SST-14 LI throughout the cytoplasm in A-C and J-L. SST-14 LI is absent in cytoplasmic granules in D-F and G-I and colocalized with WGA in the Golgi region. Similar localization was seen in the case of the Delta PSST-(3-15) and Leu13 to Ala mutants (not shown). Scale bar, 25 µM.

Delta NPSST, KR, and RTKR Substitution Mutants-- To assess the sorting function of the NH2-terminal domain of PSST, we created a deletion mutant in which the Ser3 to Leu15 residues were removed. In addition, two other mutants were created, replacing the putative monobasic Lys13 processing site with RTKR (a classic furin motif) or the dibasic motif KR to enhance NH2-terminal PSST cleavage endogenously by the prohormone convertase furin or PC1/PC2, respectively. AtT-20 cells stably expressing the Delta NPSST mutant released SST-14 LI at a high basal rate (7.1 ± 0.33 ng/ml/4 h) representing ~50% of total cell content (Fig. 3B). Release was unresponsive to FSK or TPA stimulation during a 4-h incubation (7.12 ± 0.38 and 7.83 ± 0.35 ng/ml SST-14 LI, respectively) (Fig. 3B). Similar results were obtained with the RTKR and KR substitution mutants, which showed even higher basal release of SST-14 LI of 72 and 81% of cell content, respectively, with no response to FSK and TPA stimulation (Fig. 3, C and D). These results were correlated with immunocytochemistry. Contrary to WT PSST expression in AtT-20 cells, SST-14 LI in cells expressing the KR substitution mutant was localized to a perinuclear area that was immunopositive for wheat germ agglutinin (WGA) and corresponded to the TGN (Fig. 4, D-F). Similar results were obtained with Delta NPSST and RTKR mutants. Constitutive secretion, absence of secretagogue responsiveness, and lack of SST-14 LI staining in punctate granules suggest that the PSST-(1-15) domain harbors important information that is essential for sorting PSST correctly to the RSP.

Alanine Substitution Mutants-- Having found that the amino-terminal 3-15 domain of PSST contains a potent sorting signal, we proceeded to map specific amino acid residues involved by alanine scanning mutagenesis. Mutants were stably expressed in AtT-20 cells and characterized for basal and regulated secretion, and the results were correlated with immunocytochemistry. Basal release of SST-14 LI from the Ser3, Asp4, Lys13, Ser14, and Leu15 mutants was <10% of total cellular content, comparable with that of WT PSST (Fig. 5). Pro5, Arg6, Arg8, Gln9, Phe10, and Gln12 mutants, however, exhibited somewhat higher levels of basal SST-14 LI release compared with wild type (~15% of cell content/4 h). Substitution of the Leu7 and Leu11 residues with Ala resulted in a dramatic increase in basal secretion to 72 and 70% of total cell content, respectively, comparable with the amounts found with the Lys13 to KR and Lys13 to RTKR substitutions. These results were correlated with the ability of the AtT-20 cell transfectants to respond to stimulation with FSK (20 µM) or TPA (100 µM) for 4 h (Table I). The Ser3, Asp4, Lys13, Ser14, and Leu15 mutants all displayed increased release of SST-14 LI in response to both FSK and TPA. Like WT PSST, the Lys13, Ser14, and Leu15 mutants showed a 2-fold increase in secretion in response to FSK, whereas the Ser3 and Asp4 mutants exhibited somewhat reduced 1.6-1.7-fold stimulation. Both WT and the five responsive mutants displayed differentially greater sensitivity to TPA compared with FSK stimulation. Thus, TPA induced a 3-fold increase in SST-14 LI release from WT PSST and a 2-4-fold increase in the case of the Ser3, Asp4, Lys13, Ser14, and Leu15 mutants. In contrast, FSK produced an approximate doubling of SST-14 LI release from WT and the five responsive mutants. Ala substitution of the 8 amino acid residues from Pro5 to Gln12 rendered all of these mutants totally unresponsive to both FSK and TPA stimulation (Table I). The ability of the mutants to respond to secretagogues was correlated with the granular morphology of the cells. Fig. 4 depicts the subcellular distribution of immunofluorescent SST-14 LI in representative point mutants. As an example of a mutant displaying high basal secretion and loss of regulated secretion, the Leu7 point mutant showed immunofluorescent SST-14 localized in a perinuclear area, which overlapped the distribution of wheat germ agglutinin staining (Fig. 4, G-I). Unlike WT PSST cells, SST-14 LI was not identified in the cell body of the two mutants. As an example of a point mutant that continued to display regulated secretion, the Lys13 mutant displayed a punctate pattern of staining throughout the cytoplasm similar to WT PSST, implying proper PSST targeting to secretory granules (Fig. 4, J-L).


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Fig. 5.   Percentage of basal SST-14 LI released into the medium of AtT-20 cells transfected with Ala substitution mutants of the PSST-(3-15) region. Mean values in each case are shown as numbers above the bars (mean ± S.E.; n = 5). Values shown are representative of three independent experiments.

                              
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Table I
Comparison of basal and FsK- and TPA-stimulated secretion of SST-14 LI from AtT-20 cells stably transfected with WT and Ala substitution mutant
Values are mean ± S.E. n = 5. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Effect of NH2-terminal PSST Mutations on COOH-terminal Processing to SST-14 and SST-28-- To characterize the products of PSST processing, cell extracts and media from AtT-20 cell transfectants were fractionated by HPLC followed by RIA of the eluting fractions (Fig. 6). The elution positions of the peaks obtained were compared with those of synthetic SST-14 and SST-28 or of purified PSST chromatographed under identical conditions. Table II compares the percentages of SST-14, SST-28, and unprocessed PSST derived from HPLC chromatograms. Extracts of cells of WT transfectants displayed three peaks coeluting with synthetic SST-14 (retention time 67 min), SST-28 (retention time 73 min), and PSST (retention time 111 min), representing 65, 28, and 7% of total immunoreactivity, respectively (Fig. 6B). SST-14 LI released basally consisted entirely of two peaks corresponding to SST-14 (70%) and SST-28 (30%) (Fig. 6B). A similar ratio of SST-14 to SST-28 was obtained in FSK and TPA stimulated release medium (data not shown). The Leu15 to Ala mutant displayed comparable HPLC profiles to WT PSST in both cell extracts and media. Thus, PSST was efficiently processed intracellularly to SST-14 and SST-28 (67 and 24% of SST-14 LI, respectively). SST-14 and SST-28 were also the principal immunoreactive species released into the medium; the peak corresponding to PSST released from these cells comprised 9% of the total released immunoreactivity. In contrast, mutants characterized by diversion of PSST from the RSP to the CSP (Delta NPSST, Lys13 to KR, Lys13 to RTKR, Leu7 to Ala, Leu11 to Ala) displayed a different HPLC profile of SST-14, SST-28, and unprocessed PSST (Fig. 6, C-F, Table II). In the case of the Leu7 to Ala mutant, despite the missorting of PSST to the RSP, the precursor was efficiently cleaved intracellularly to SST-14 and SST-28 (59 and 31%, respectively); a third peak corresponding to full-length PSST accounted for 10% of the total intracellular immunoreactivity (Fig. 6E, left panel). In contrast to cell extracts, however, the HPLC profile of SST-14 LI released basally in the medium was very different, with only small amounts of processed SST-14 and SST-28 (14 and 18%, respectively); the major product released into the medium of these cells was full-length PSST, accounting for 68% of total SST-14 LI (Fig. 6E, right panel). As expected, the pattern of release after FSK or TPA stimulation was identical to that of basal release, since neither secretagogue provoked regulated release from these cells (data not shown). Similar results were observed in the case of the Leu11 to Ala mutant and the Delta NPSST, Lys13 to KR, and Lys13 to RTKR mutants, all of which displayed efficient intracellular PSST processing to SST-14 and SST-28 (~62 and 25-31%, respectively), with a small 7-13% peak corresponding to unprocessed PSST. However, the major form released into the medium both basally and in response to secretagogue stimulation was unprocessed PSST, accounting for 51-73% of total released immunoreactivity. These results indicate that PSST that fails to be targeted to the RSP can still be processed to SST-14 and SST-28 in TGN compartments. However, PSST targeting is critical for efficient processing of the releasable pool of SST-14 and SST-28.


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Fig. 6.   HPLC profile of SST-14 LI in cell extracts (left panels) and secretion media (right panels) from AtT-20 cells transfected with WT (B), Delta N PSST (C), Lys13 to KR mutant (D), Leu7 to Ala mutant (E), Leu11 to Ala mutant (F), and Leu15 to Ala mutant (G). A illustrates the elution position of synthetic SST-14 (retention time 67 min) and SST-28 (retention time 73 min) detected by absorbance at 214 nM. The elution position of PSST (111 min) is indicated. Profiles are representative of four experiments.

                              
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Table II
Comparison of the percentage of SST-14, SST-28 and unprocessed PSST derived from HPLC chromatograms of cell extracts and media from AtT-20 cells expressing WT or mutant PSST

Molecular Modeling of rPSST-- We constructed a structural model of the Pro5 to Thr19 sequence of rPSST based on the secondary structure prediction data (Fig. 7A). This model reveals an amphipathic alpha -helix with a hydrophobic face formed by the side chains of Leu7, Phe10, Leu11, and Leu15 residues and a polar face comprising the side chains of Arg6, Arg8, Gln9, Gln12, and Lys13 residues (Fig. 7B). The side chains of Leu7 and Leu11 residues that are essential for high activity are located in close proximity to each other on the hydrophobic surface of the alpha -helical structure. It is noteworthy that for all of the point mutants created for this study that contain a single amino acid residue mutated to alanine, the alpha -helical structure is highly probable due to the strong propensity of alanine to adopt the alpha -helical conformation (39). The inactivity of the Leu7 to Ala and Leu11 to Ala mutants can be attributed to the removal of critical side chains from the hydrophobic surface, which probably forms a binding interface for the propeptide, rather than to global conformational changes introduced by the mutations.


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Fig. 7.   A, consensus secondary structure prediction for rPSST Ala1 to Cys92 sequence. h, alpha -helix; b, beta  strand. Leu7 and Leu11 are in boldface type. B, structural model of rPSST Pro5-Thr19 sequence. Two orthogonal views are shown. Residues forming the hydrophobic face of the alpha -helix are labeled. The side chains of Leu7 and Leu11 are shown in ball and stick representations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have shown that the PSST-(3-15) segment, which comprises an amphipathic alpha -helix, acts as a sorting signal for directing PSST to the RSP and that residues Leu7 and Leu11 separated by one turn on the alpha -helix are critical determinants of precursor sorting. Disruption of the NH2-terminal alpha -helix does not impair the ability of PSST to be processed at the COOH terminus to SST-14 and SST-28. Processing, however, is shifted to early compartments of the secretory pathway instead of storage granules and is relatively inefficient.

Several previous studies have shown that the prosegment of PSST harbors a sorting signal (18, 19, 28). For instance, an SST fusion protein consisting of the signal peptide and proregion of anglerfish PSSTI fused to alpha -globin is sorted to the RSP in transfected GH3 cells, whereas the alpha -globin gene joined to the beta -lactamase signal peptide is degraded in the secretory compartment (18). Anglerfish PSSTI transfected in Rin5F cells is directed to the RSP, whereas anglerfish PSSTII is mainly targeted to the CST (19). A fusion protein comprising the first 54 residues of rPSST and the last 48 amino acids of anglerfish PSSTII is correctly targeted to the RSP (19). Deletion of the rPSST-(1-10) segment results in selective blockade of the mutant precursor from sorting into a TPA-responsive (but not cAMP-responsive) secretory compartment (28). These findings suggest that NH2-terminal sequences of rPSST (and probably anglerfish PSSTI but not anglerfish PSSTII) contain intracellular targeting information (19). Molecular modeling of rPSST reveals an alpha -helix at residues 5-19 with the side chains of residues Leu7, Phe10, Leu11, and Leu15 forming a contiguous hydrophobic patch on the helix surface (Fig. 7). This domain is highly conserved in all known vertebrate PSST molecules as well as in the SST-related precursor procortistatin (PCST) (where it is located not at the NH2 terminus but further downstream at residues 19-35) but is not present in anglerfish PSSTII consistent with the targeting data obtained for this precursor experimentally (Fig. 1). The secondary structure predictions of a dozen other prohormones that are known to be targeted to the RSP also reveal a common amphipathic alpha -helix similar to the one in PSST that qualifies as a putative sorting signal (34). We have analyzed the PSST NH2-terminal alpha -helix as a sorting signal by detailed mutagenesis. Deletion of the PSST-(3-15) segment blocked regulated secretion of SST-14 LI in response to both TPA and FSK and rerouted PSST for constitutive secretion as unprocessed precursor. Similar results were obtained with two other mutants in which the Lys13 residue was substituted with RTKR (a classic furin motif) or the dibasic motif KR to enhance NH2-terminal PSST cleavage endogenously by furin or PC1/PC2, respectively. Analysis of NH2-terminal processing by NH2-terminus-specific RIA confirmed a 2- and 3-fold increase in cleavage of a PSST-(1-10)-like product from these mutant precursors (data not shown). An additional possibility for the missorting is that insertion of extra basic residues at Lys13 disrupted the alpha -helix. Thus, removal of the PSST NH2-terminal alpha -helix by mutagenesis or endogenously by endoproteolysis resulted in precursor missorting. The complete abrogation of TPA- and FSK-stimulatory responses by the three deletion mutants differs from the results of Sevarino et al., who found that deletion of the rPSST-(1-10) segment induced only partial loss of regulated secretory responses to TPA but not FSK (28). Since the NH2-terminal domain of PSST is crucial for precursor targeting to secretory granules, it is surprising to find that the precursor is normally processed at the NH2 terminus to generate PSST-(1-10). The site of processing has been postulated to be Lys13, although this region does not qualify as a substrate for monobasic cleavage by a prohormone convertase (PC)-like enzyme (32, 40). Recent studies suggest that NH2-terminal processing of PSST is effected by the novel protease subtilisin-kexin isoenzyme SKI-1, which cleaves at Leu11 (40, 41). The biological significance of PSST NH2-terminal processing is unclear, although it is known that PSST-(1-10) cleavage is relatively inefficient compared with that of SST-14 and SST-28 and is highly tissue-specific with moderate production of the peptide in stomach and brain and virtually none in islet cells or intestinal mucosa. Even in antral D-cells, the site of maximum PSST-(1-10) synthesis, only a small subpopulation of secretory granules (30% in rats, 10% in humans) contain the peptide (41). Our finding that deletion of the PSST-(3-15) domain results in missorting of the precursor suggests that endogenous NH2-terminal PSST processing must be a late event, distal to the TGN sorting process, and its function may be to target the precursor to a subpopulation of secretory granules. Alanine scanning mutagenesis identified the region Pro5-Gln12 as being important in precursor targeting, with Leu7 and Leu11 being critical. These results complement the modeling data and suggest that these two residues located in close proximity on the hydrophobic surface of the alpha -helix may provide a binding interface for interaction with a putative sorting receptor. Recently, an amphipathic alpha -helix in the COOH-terminal segment of PC1 with critical leucine residues at Leu745 and Leu749 has also been reported to mediate targeting of the convertase to the RSP (22). Unlike PSST, the COOH-terminal region of PC1 contains two segments of ~40 residues, each of which can independently target the convertase to the RSP and which both harbor alpha -helices. These results provide direct evidence that an alpha -helix in PSST and PC1 mediates the targeting of the two proproteins to the RSP and suggest that an alpha -helical structure common to a number of prohormones may serve as a general sorting signal. The alpha -helix sorting signal differs from the disulfide bond constrained amphipathic hairpin loop structure shown to be a sorting signal for POMC (21, 24, 26). The critical elements of this motif comprise residues DLEL at the apex of the loop and Cys8/Cys20 residues that form a disulfide bridge (21). Molecular modeling has revealed a similar putative disulfide bond constrained sorting motif in proenkephalin and proinsulin (26).

If there is a sorting signal, does it bind to a specific sorting receptor? Thus far, two proteins have been proposed to function as sorting signal receptors. One is the inositol 1,4,5-triphosphate receptor, which binds chromogranin A (43). This receptor, however, is only weakly expressed in secretory granules of neuroendocrine cells and therefore is unlikely to function as a general sorting receptor. The second is membrane-associated CPE expressed in high concentrations in TGN and secretory granule membranes of neuroendocrine cells (24-27). CPE binds to the POMC sorting signal motif and acts as a low affinity, high capacity sorting signal receptor (24). CPE interacts at Arg-Lys basic residues with the acidic residues in the POMC sorting signal (25). Mutation of the binding site on CPE, in vitro antisense depletion of CPE, or genetic ablation of CPE in the CPEfat mouse leads to missorting of POMC, proenkephalin, and proinsulin (25, 26). Other studies, however, have found that proinsulin is sorted to the RSP in pancreatic islets from CPE-deficient fat mice as well as in cell lines derived from pancreatic beta  cells of these mice (44, 45). Additionally, chromogranin A, which possesses a POMC-like sorting signal, does not use CPE as a sorting receptor (26). Thus, not all sorting signals are recognized by CPE, suggesting the existence of other sorting receptors. Whether there is a putative receptor that interacts with the alpha -helical sorting signal that we have identified in PSST and that is common to a number of other neuroendocrine precursors remains to be determined.

Processing of prohormones at basic residues is effected by a family of subtilisin-related mammalian Ca2+-dependent serine proteinases known as PCs with seven current members: furin, PACE4, PC1, PC2, PC4, PC5A/B, and PC7 (40). Furin, PC5B, and PC7 are membrane-bound and along with PACE4 process proteins in the CSP, whereas PC1, PC2, and PC5A process neuroendocrine precursors that are targeted to secretory granules. The cellular compartment in which cleavage occurs is controversial. Proteolytic processing of several hormone precursors (e.g. proinsulin, propresophysin, and POMC) occurs largely or exclusively in secretory granules. Immunogold labeling studies have shown that proinsulin cleavage is a post-Golgi event initiated in acidic clathrin-coated immature secretory vesicles and completed in mature uncoated granules (46). Several recent studies, however, have demonstrated that limited to extensive proteolytic cleavage of some hormone precursors can also occur proximally in the TGN (47-50). This comes as no surprise, since the converting enzymes already exist in an active form in this compartment (furin, PC1, PC5, PC7, and PACE4), and the weakly acidic (pH ~6.5) milieu would favor proprotein processing (31, 40). Conversion of SST-14 from PSST is mediated by either PC1 or PC2 (31). Although both convertases are present in secretory granules, PC1 also exists in an active form in the TGN and is therefore capable of SST-14 conversion in this compartment, whereas PC2 is optimally active in secretory granules (22, 40). Monobasic cleavage of SST-28 is effected by furin and/or PACE4 (51, 52). Blockade of PSST targeting to secretory granules by the NH2-terminal deletion and Leu7 and Leu11 PSST mutations led to an escape of large quantities of unprocessed PSST through the CSP. The remainder of the precursor, however, was retained in the TGN, where it underwent relatively efficient processing to both SST-14 and SST-28, presumably through the action of PC1 (for SST-14) and furin/PACE4 (for SST-28). These results are consistent with previous studies that have shown significant processing of PSST in the absence of secretory granules in TGN compartments (48, 50). Overall, this means that the NH2-terminal PSST conformation does not influence enzyme recognition and PSST cleavage at the COOH terminus. The main consequence of the blockade of PSST entry into secretory granules is incomplete precursor processing and retention of the cleaved mature products in Golgi vesicles. Targeting of PSST to secretory granules, therefore, subserves two purposes: to optimize processing and to package and store the mature products for regulated release.

    ACKNOWLEDGEMENTS

We acknowledge the contributions of M. Shida toward the preparation of the initial PSST mutant constructs. M. Correia provided expert secretarial assistance.

    FOOTNOTES

* This study was supported by grants from the Canadian Medical Research Council (MRC).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.

§ Supported by studentships from the Canadian Institutes of Health Research and Fonds pour la formation de chercheurs et l'aide à la recherche.

|| Distinguished Professor of the Canadian MRC. To whom correspondence should be addressed: Rm. M3-15, Royal Victoria Hospital, 687 Pine Ave. W., Montreal, H3A 1A1 Quebec, Canada. Tel.: 514-842-1231 (ext. 5042); Fax: 514-849-3681; E-mail: yogesh.patel@mcgill.ca.

Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M102514200

    ABBREVIATIONS

The abbreviations used are: CSP, constitutive secretory pathway; RSP, regulated secretory pathway; TGN, trans-Golgi network; CPE, carboxypeptidase E; PC2, prohormone convertase 2; POMC, pro-opiomelanocortin; SST, somatostatin; PSST, prosomatostatin; rPSST, rat PSST; PPSST, preprosomatostatin; TPA, 12-O-tetradecanoylphorbol-13-acetate; FSK, forskolin; PCR, polymerase chain reaction; nt, nucleotide(s); RIA, radioimmune assay; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; WT, wild type.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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