PC5-A-mediated Processing of Pro-neurotensin in Early Compartments of the Regulated Secretory Pathway of PC5-transfected PC12 Cells*

Among the members of the proprotein convertase (PC) family, PC1 and PC2 have well established roles as prohormone convertases. Another good candidate for this role is PC5-A that has been shown to be present in the regulated secretory pathway of certain neuroendocrine tissues, but evidence that it can process prohormones is lacking. To determine whether PC5-A could function as a prohormone convertase and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-neurotensin/neuromedin N (pro-NT/NN) into active peptides. Our data showed that in transfected PC12 cells, PC5-A was converted from its 126-kDa precursor form into a 117-kDa mature form and, to a lesser extent, into a C-terminally truncated 65-kDa form of the 117-kDa product. Metabolic and immunochemical studies showed that PC5-A was sorted to early compartments of the regulated secretory pathway where it colocalized with immunoreactive NT. Furthermore, pro-NT/NN was processed in these compartments according to a pattern that differed from that previously described in PC1- and PC2-transfected PC12 cells. This pattern resembled that previously reported for pro-NT/NN processing in the adrenal medulla, a tissue known to express high levels of PC5-A. Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway and suggest a role for this enzyme in the physiological processing of pro-NT/NN.

Pro-neuropeptide/hormone cleavage at basic residues (most often dibasic sequences) is a general phenomenon by which active peptides are processed from their precursor in neuroendocrine tissues. The identity of the cellular proteases that effect these cleavages has long remained elusive. Recently, a family of proprotein convertases (PCs) 1 with the ability to cleave protein precursors at basic residues was identified by homology with the yeast Kex2 protease (reviewed in . Among the members of this family, particular attention has been paid to PC1 (also named PC3) and PC2 (4 -7) as potential prohormone convertases because of their reported association with the regulated secretory pathway of neuroendocrine cells. Evidence was provided that PC1 and PC2 were both involved in the processing of a number of prohormone precursors including proinsulin (8 -10), proglucagon (11)(12)(13), and pro-opiomelanocortin (14 -16). Furthermore, the two enzymes were found to differ in their cleavage site specificity, thus leading to differential processing of these precursors.
More recently, another member of the PC family, PC5 (also designated PC6), has also been proposed to play the role of a prohormone convertase (17)(18)(19)(20). Two PC5 isoforms generated from a single gene by alternative splicing have been identified (17)(18)(19). PC5-A is a soluble form that exhibits a widespread distribution in endocrine and non-endocrine tissues, being particularly abundant in the intestine and adrenals. PC5-B is membrane-bound, and its distribution is restricted to the lungs, intestine, and adrenals (3,(17)(18)(19). A recent study showed that PC5-A and PC5-B were sorted to different subcellular compartments when stably transfected in AtT-20 cells (20). Thus, PC5-A was stored into regulated secretory vesicles, whereas PC5-B was retained in the Golgi. In addition, PC5-A was found to colocalize with pro-glucagon and PC2 in the secretory granules of the pancreatic ␣-cell (20). Taken together, these data strongly suggest that PC5-A might function as a prohormone convertase in the regulated secretory pathway. However, in contrast to PC1 and PC2, virtually nothing is known about the ability of PC5 to process neuropeptide/hormone precursors in neuroendocrine cells. Recent studies have suggested that PC5 could be involved in the bioactivation of Mü llerian inhibiting substance in the Sertoli cells of the developing testis (21) and in the proteolytic processing of the membrane-associated receptor protein tyrosine phosphatase in vascular endothelial cells (22). The ability of PC5 to cleave these two proteins, which cannot be considered as classical prohormones, was demonstrated by cotransfecting enzyme and substrate into non-endocrine cells.
A useful neuroendocrine cell model to investigate the role of PC5 as a prohormone convertase is the rat pheochromocytoma PC12 cell line. PC12 cells have been shown to be devoid of both PC1 and PC2 and to express very low levels of PC5 (3). In addition, PC12 cells synthesize a number of neuropeptide precursors including the neurotensin/neuromedin N (NT/NN) precursor which they poorly process (23)(24)(25). Pro-NT/NN, the structure of which is depicted in Fig. 1, is predominantly expressed in the brain and in the distal small intestine (26 -30). It is also found in other tissues such as the adrenals (31)(32)(33)(34). Pro-NT/NN processing in the brain, gut, and adrenals yields different combinations of biologically active peptides (26,35) as summarized in Fig. 1. Undifferentiated PC12 cells do not express pro-NT/NN but can be induced to express very high levels of the precursor by a combination of nerve growth factor (NGF), forskolin, dexamethasone, and lithium (36,37). We have recently shown that stable transfection of PC12 cells with either PC1 or PC2 led to differential processing of pro-NT/NN, PC1 mimicking the pro-NT/NN processing pattern observed in the gut and PC2 reproducing that observed in the brain (38,39). In order to determine whether PC5-A may function as a prohormone convertase in the regulated secretory pathway and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-NT/NN into active peptides.
Our data show that in PC5-A-transfected PC12 cells the convertase is sorted to early compartments of the regulated secretory pathway where it colocalizes with immunoreactive NT and processes pro-NT/NN with a pattern that differs from that previously described in PC1-and PC2-transfected PC12 cells (39). This pattern resembles that observed for pro-NT/NN processing in the adrenal medulla (26,35), a tissue known to express high levels of PC5-A (3,17). Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway of neuroendocrine cells and suggest a role for this enzyme in the physiological processing of pro-NT/NN.

EXPERIMENTAL PROCEDURES
PC12 Cell Culture and Transfection-PC12 cells were grown as described previously (24,39). For transfection experiments, the cells were cultured in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 10% horse serum. The mPC5-A cDNA was subcloned in the eukaryotic expression vector pcDNA 3 (Invitrogen, Leek, The Netherlands). PC12 cells were transfected by electroporation as previously reported for PC1 and PC2 transfections (39). Two PC12/PC5 clones (E5.9 and E5.C) were selected for the studies reported here by Western blotting with anti-PC5-A antiserum. Wild type and transfected PC12 cells at 60 -80% confluency were stimulated with optimal concentrations of NGF (200 ng/ml), dexamethasone (1 M), forskolin (1 M), and LiCl (20 mM) for 48 h. The cells were extracted and analyzed for PC5-A expression and pro-NT/NN processing as described below.
Western Blot Analysis-Cells were washed with PBS and homogenized in phosphate buffer (50 mM Na 2 HPO 4 , pH 7.4) containing 1 mM EDTA, 0.3 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, 0.5% Nonidet P40, and 0.9% NaCl. The extracts were centrifuged, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent, following the procedure recommended by the manufacturer. Western blotting was performed as described previously (39) using an antiserum directed against the sequence DY-DLSHAQSTYFNDPK representing residues 116 -132 consisting of the PC5 N-terminal sequence following the potential activation site RTKR (20). This antiserum has been shown to recognize pro-PC5-A (126 kDa), mature PC5-A (116 kDa), and a C-terminally truncated form of mature PC5-A (65-70 kDa) (17,20). In some experiments, the PC5 antiserum was preadsorbed by incubation with its synthetic antigen (5 M) for 1 h at 37°C.
RNA Isolation and Analysis-Cells were washed with PBS. Total RNA from the cell lines was extracted and submitted to Northern blot analysis as described previously (39). mPC5 and rpro-NT/NN (gift from Paul R. Dobner, Massachusetts University, Worcester) were radiolabeled by random priming with 5Ј-[␣-32 P]dCTP (Ͼ3000 Ci/mmol; ICN). The labeled probes were added to the prehybridization mix at a concentration of 1-3 ϫ 10 6 cpm/ml and hybridized overnight at 42°C. In some experiments, the blots were washed out and rehybridized with a control labeled probe encoding GAPDH. The films were scanned, and mRNA bands were quantified by densitometry relative to GAPDH mRNA.
Biosynthetic Studies-Biosynthetic analyses of the metabolic fate of PC5 were performed as described previously (40). Briefly, PC12 cells that had reached 80% confluency were stimulated for 48 h, washed with PBS, and then switched for 1 h to a methionine-free medium (RPMI 1640) (Life Technologies, Inc.), supplemented with 0.5% calf serum. Subsequently, cells were labeled with [ 35 S]methionine (100 Ci/ml) (Mandel Scientific Co., Ontario, Canada). At the end of the incubation period, the media were removed, and cells were disrupted in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA and 20 g/ml phenylmethylsulfonyl fluoride) by incubation on ice for 20 min. The media and cell lysates were pre-cleared in two steps using normal rabbit serum and protein A-agarose and immunoprecipitated with the PC5 antiserum described above. All immunoprecipitations were performed as described previously (40). The immunoprecipitation products were resolved by electrophoresis on 8% polyacrylamide gels followed by treatment with Entensify (NEN Life Science Products) and autoradiography.
For stimulation of granule content release, PC5-A-transfected PC12 cells and AtT-20 cells (20) were pulse-labeled for 10 min with [ 35 S]methionine (100 Ci/ml) (Mandel Scientific Co., Ontario, Canada), then chased for 30 min in methionine-free medium. This medium was replaced for 50 min with fresh methionine-free medium without inducers and without or with 5 mM 8-Br-cAMP. The media were immunoprecipitated and resolved on SDS-polyacrylamide gel electrophoresis as described above.
For metabolic studies of pro-NT/NN biosynthesis, PC12 cells that had reached 80% confluency were stimulated for 48 h, washed with PBS, and incubated for 1 h in a methionine-free medium (ICN Biomedicals, France). The cells where then labeled with [ 35 S]methionine/cysteine (100 Ci/ml) (ICN Biomedicals) for 5 min. At the end of the pulse period, the cells were chased for different times in complete medium, washed with PBS, and homogenized in lysis buffer (10 mM Tris, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, and 100 g/ml phenylmethylsulfonyl fluoride). All incubations were carried out at 37°C with optimal concentrations of NGF, dexamethasone, forskolin, and LiCl as described above. Cell lysates were centrifuged, and the supernatant was pre-cleared using normal rabbit serum 1:100 and 10% pansorbin (Calbiochem). After pre-clearing, the supernatants were immunoprecipitated with a pro-NT/NN antiserum (gift from Paul Dobner, University of Massachusetts, Worcester, MA) at a 1:500 dilution and protein A-Sepharose (Sigma). The pro-NT/NN antiserum was obtained by immunizing rabbits with a fusion protein containing amino acids 8 -169 of prepro-NT/NN fused to the bacterial Trp E protein (23). The immunoprecipitation products were resolved by electrophoresis on 15% polyacrylamide gels and revealed by autoradiography.
Immunohistochemical Studies-PC5 and NT antigens were visualized either separately (single label) or jointly (double label) in transfected and non-transfected (wild type) PC12 cells using indirect immunofluorescence. In a separate set of experiments, PC5 immunostaining Various combinations of peptides generated by dibasic cleavages are present in tissues that express pro-NT/ NN. Thus, NT and NN are the major products found in the brain; NT and large NN are predominant in the gut; and NT, large NN, and large NT are the main products found in the adrenals (26,35). was combined to the immunodetection of either MG-160, a membrane sialoglycoprotein associated with the medial cisternae of the Golgi apparatus (41) or Syntaxin-6, a newly discovered member of the syntaxin family predominantly localized to the trans-Golgi network (42).
For both series of experiments, cells were grown on polylysine-coated coverslips. At approximately 50% confluence, they were stimulated or not for 48 h as described above. After stimulation, cells were rinsed twice in 0.1 M phosphate buffer and fixed with 4% paraformaldehyde in phosphate buffer for 15 min at room temperature. They were then washed with 0.1 M Tris-buffered saline (TBS), pH 7.4, and blocked for 20 min in TBS containing 0.5% bovine serum albumin and 3% normal goat serum.
For single labeling experiments, cells were then incubated overnight at 4°C with either PC5 or NT primary antibody diluted in TBS containing 0.05% Triton X-100, 0.5% bovine serum albumin, and 1% normal goat serum. The PC5 antiserum (raised in rabbit) was the same as the one used for Western blot analyses and immunoprecipitation, and was utilized at a dilution of 1:4000. The anti-NT antibody (raised in rat) was purchased from Eugene Tech International (Ridgefield Park, NJ) and used at a dilution of 1:1000. After thorough rinsing in TBS, cells immunolabeled with the PC5 antiserum were revealed with a FITCconjugated goat anti-rabbit IgG (Jackson ImmunoResearch) and cells immunolabeled with the NT antiserum with a Texas Red-conjugated goat anti-rat IgG (Jackson ImmunoResearch). Both secondary antibodies were diluted 1:100 with TBS and applied for 45 min at room temperature. Specificity controls included omission of either primary antibody or use of primary antisera preabsorbed for 1 h at 37°C with 5 M antigenic peptide.
For double-labeled experiments, cells were co-incubated overnight at 4°C with PC5 and either NT, MG-160, or Syntaxin-6 antibodies diluted in TBS containing 0.02% Triton X-100 and 1% normal goat serum. PC5 and NT antibodies were the same as above and were used at dilutions of 1:2000 and 1:000, respectively. The MG-160 antibody (mouse monoclonal antibody 10A8) was a generous gift from Dr. N. K. Gonatas (University of Pennsylvania, Philadelphia) and was used at a dilution of 1:200. The Syntaxin-6 antibody (monoclonal mouse IgG1 isotype) was purchased from Transduction Laboratories (Lexington, KY) and was used at a dilution of 3 g/ml. At the end of the incubation, cells were rinsed in TBS and incubated for 2 h at room temperature with a mixture of FITC-conjugated goat anti-rabbit IgGs and Texas Red-conjugated goat anti-rat IgGs (for PC5/NT double-labeling experiments) or of Texas Red-conjugated goat anti-rabbit IgGs and FITC-conjugated goat anti-mouse IgGs (for PC5/MG-160 and PC5/Syntaxin-6 experiments). All secondary antibodies were diluted at 1:100 and purchased from Jackson ImmunoResearch. All cells were then rinsed in TBS, mounted in Aquamount, and examined by confocal microscopy.
Labeled cells were visualized with either a Leica CLSM or a Zeiss LSM 410 inverted confocal microscope each equipped with an argon/ krypton laser. FITC signals and Texas Red signals were imaged by exciting samples with 488 and 568 nm, respectively. All images were acquired as single optical sections through the middle of the cells and averaged over 32 scans/frame. Imaged were stored on optical discs and archived and labeled using Adobe Photoshop.
Analysis of Pro-NT/NN Processing Products-The specificities of the NT (29G), NN (NN-Ah), and K6L (K6L-Af) antisera used here as well as the radioimmunoassay procedures employing these antisera have been previously described in detail (43). Briefly, the NT antiserum reacts with the free C terminus of NT; the NN antiserum recognizes the free N terminus of NN; and the K6L antiserum detects the free N terminus of the sequence that follows the Lys 85 -Arg 86 site in rat pro-NT/NN. These antisera cross-react weakly (Ͻ1%) with antigenic sequences that are internal to proneurotensin or proneurotensin fragments. Another anti-NN antiserum (NMN, kindly provided by Robert Carraway, Worcester) was used. This antiserum detects the free C terminus of NN and cross-reacts poorly (Ͻ3%) with NT or with C-terminally extended NN sequences (28).
Portions of acid extracts from PC12 cells were citraconylated, submitted to Arg-directed tryptic digestion (24,40), and assayed for Nterminal immunoreactive NN (iNN). The value of CTiNN thus obtained provides an index of the total amount of pro-NT/NN (either processed or unprocessed) that was synthesized and stored in the cells at the time of extraction. Cell extracts were fractionated by reverse-phase HPLC as described elsewhere (24,40). The fractions were assayed for their content in iNT, C-terminal iNN, N-terminal iNN, and iK6L. Because of the above-described antisera specificity, these assays measure the amounts of precursor products that are processed at the Lys 163 -Arg 164 , Lys 148 -Arg 149 , Lys 140 -Arg 141 , and Lys 84 -Arg 85 sequences, respectively. The products detected after HPLC were NT, NN, large NT, and large NN that eluted with retention times of 40, 43, 69, and 71 min, respectively. The results were normalized for the amount of protein in each extract. The percentages of cleavage at the Lys 163 -Arg 164 , Lys 148 -Arg 149 , Lys 140 -Arg 141 , and Lys 84 -Arg 85 sequences were calculated by dividing post-HPLC iNT, C-terminal iNN, N-terminal iNN, and iK6L, respectively, by CTiNN and by multiplying these ratio values by 100.

PC5-A Expression, Biosynthesis, and Release in PC5-A-transfected PC12
Cells-Northern blot analysis of PC12 cells stimulated by a mixture of NGF, forskolin, dexamethasone, and lithium revealed that wild type PC12 cells expressed only low amounts of the 3.8-kilobase pair PC5-A mRNA in contrast to the stimulated transfected E5.9 and E5.C clones that expressed high amounts of the transcript ( Fig. 2A). In Western blot analysis performed with an N-terminally directed PC5-A antiserum, two proteins of 126 and 117 kDa were specifically labeled (Fig. 2B). They correspond to pro-PC5-A and mature PC5-A (pro-PC5-A minus its prosegment), respectively, as shown recently in PC5-A-transfected AtT-20 cells (20). Neither of these bands were visible when the PC5 serum was preabsorbed with 5 M synthetic antigen (Fig. 2C). PC5-A enzyme levels were higher in clone E5.9 than in clone E5.C, in contrast to the amounts of PC5-A mRNA that were higher in E5.C than in E5.9 cells (compare Figs. 2, A and B). Clone E5.9 produced the 117-kDa form of PC5-A in much larger amounts than the 126-kDa protein in contrast to clone E5.C and wild type PC12 cells that exhibited higher levels of the 126-kDa precursor form than the 117-kDa protein (Fig. 2B). The reason for the apparent discrepancy between PC5 mRNA and protein levels in clone E5.C is not known. Similar unexplained variations have been previously observed in PC2-transfected PC12 cells between PC2 mRNA and protein levels (39).
Pulse-chase analysis of the biosynthetic fate of PC5-A in PC12 cells (clone E5.9) revealed, after a 5-min pulse with and proteins (50 g) were analyzed by Northern and Western blotting as described under "Experimental Procedures." A, Northern blot analysis of PC5-A, pro-NT/NN, and GAPDH mRNAs. Densitometric analysis of the blots indicated that, after normalizing for GAPDH mRNA levels, PC5 mRNA levels were increased 30-and 18-fold in clones E5.C and E5.9, respectively, as compared with wild type PC12 cells, whereas pro-NT/NN mRNA levels (summing the 1.1-and 1.5-kilobase pair bands) were about 2-fold lower in clones E5.C and E5.9 than in wild type PC12 cells. B and C, Western blot analysis of PC5-A with the PC5 antiserum that had been preabsorbed with its synthetic antigen (C) or not preabsorbed (B). Note the complete extinction of the 126-and 117-kDa bands in C as compared with B. Densitometric analysis of the gels showed that 126-kDa PC5 levels were increased 3-and 4-fold and that 117-kDa PC5 levels were increased 5-and 20-fold in clones E5.C and E5.9, respectively, as compared with wild type PC12 cells.
[ 35 S]methionine, two intracellular immunoreactive PC5 products migrating with apparent molecular masses of 126 and 117 kDa (Fig. 3A). Both bands progressively diminished in intensity with time (Fig. 3A). In the media, the 117-kDa product was observed after a 30-min chase and gradually accumulated with time (Fig. 3B). Densitometric analysis of the data indicated that 30, 48, and 78% of the total 117-kDa PC5-A produced were secreted into the medium at chase times of 30, 60, and 120 min, respectively. Whereas the total amount of PC5-A (126-ϩ 117-kDa forms, cells and medium) varied less than 10% during the first 120 min of chase, the amount of 117-kDa PC5-A relative to total PC5-A increased from 53% at time 0 of chase to 59, 66, and 82% after 30, 60, and 120 min of chase, respectively (Fig. 3, A  and B). These results may suggest that 126-kDa PC5-A was converted to the 117-kDa form, as reported in PC5-A-transfected AtT-20 cells (20), in which the two PC5-A products of 126 and 117 kDa were unequivocally identified by microsequencing as pro-PC5-A and zymogen-cleaved PC5-A, respectively (20).
Very low amounts of a 65-kDa form of PC5-A were detected at both 120 and 240 min (Fig. 3B). This form presumably corresponds to C-terminally truncated, mature PC5-A, the biosynthesis of which has been observed in PC5-A-transfected AtT-20 cells (20).
In order to investigate whether PC5-A was sorted to the regulated secretory pathway in PC12 cells, PC5-A-transfected PC12 cells (clone E5.9) were compared with PC5-A-transfected AtT-20 cells for their ability to release PC5-A proteins when stimulated with 5 mM 8-Br-cAMP (Fig. 3C). The release of both the 117-and 65-kDa forms of PC5-A was increased by the secretagogue in both cell lines (Fig. 3C). However, the ratio of the density of the 117-kDa over the 65-kDa band in stimulated conditions, as evaluated by densitometric analysis of the autoradiograms, was approximately 7 times higher in PC12 cells than in AtT-20 cells. Densitometric analysis also revealed that the increase in protein release was approximately 50% higher for the 65-kDa PC5-A product than for the 117-kDa protein, in both PC12 and AtT-20 cells. This result was confirmed in four independent experiments and demonstrates that the 117-and 65-kDa forms of PC5-A can enter the secretory granules.

Pro-NT/NN Synthesis and Processing in PC5-A-transfected PC12 Cells-Stimulated wild type and PC5-A-transfected
PC12 cells expressed high levels of the 1.1-and 1.5-kilobase pair pro-NT/NN mRNAs ( Fig. 2A). These two mRNA species have been shown to differ in their polyadenylation site (37). Pro-NT/NN mRNA levels were paralleled by CTiNN concentrations that measure the total amount of intracellular pro-NT/NN (processed ϩ unprocessed) stored during the 48-h induction period (Table I). Further processing analysis showed that wild type PC12 cells generated low amounts of pro-NT/ NN-derived products (Table I). In contrast, pro-NT/NN processing was markedly increased in the PC5-A transfectants. They produced similar amounts of NT and large NN. They also yielded substantial quantities of large NT, even in higher amount than NT and large NN for clone E5.C. Both clones, however, exhibited only low levels of NN. Immunoreactive K6L was undetectable (not shown), thus indicating that the Lys 85 -Arg 86 dibasic in pro-NT/NN was not cleaved by PC5 in our transfectants. From these data, the percentages of cleavage at the three C-terminal Lys-Arg doublets in pro-NT/NN were calculated for each PC12/PC5-A clone (Table I). PC5-A cleaved pro-NT/NN with an order of preference for the dibasic sites that was Lys 163 -Arg 164 Ͼ Lys 148 -Arg 149 Ͼ Ͼ Lys 140 -Arg 141 .
To determine the time frame of pro-NT/NN processing, pulse-chase experiments were performed with wild type PC12 cells and clone E5.9 (Fig. 4). After a 5-min pulse with [ 35 S]methionine/cysteine, a labeled protein with an apparent molecular mass of 17.3 kDa corresponding to that of pro-NT/NN was immunoprecipitated. The amount of pro-NT/NN in wild type PC12 cells remained virtually constant over the first 60-min period of chase and decreased over the next 60-min period (Fig.  4B), presumably due to constitutive secretion of the precursor. In contrast, pro-NT/NN started to decrease after 15 min and sharply declined between 30 and 60 min of chase in clone E5.9 (Fig. 4B). This rapid decrease of pro-NT/NN likely resulted from the fact that the precursor undergoes processing in PC5transfected PC12 cells. Processing products (such as large NT or large NN) could not be detected in the pulse-chase experiments presented in Fig. 4. This is probably due to the fact pro-NT/NN was weakly labeled after a 5-min pulse and that according to Table I newly processed large NT and large NN would represent only a fraction of pro-NT/NN. When E5.9 cells were pulse-labeled for 20 min, a labeled protein with a molecular mass of 13.2 corresponding to that of large NN, the major large pro-NT/NN processing product in E5.9 cells (Table I) visible (not shown). Taken together, the data indicate that pro-NT/NN processing in PC5-transfected PC12 cells is a rapid event starting between 15 and 30 min following pro-NT/NN synthesis.
Immunohistochemical Localization of PC5-A, NT, MG-160, and Syntaxin-6 in PC5-A-transfected PC12 Cells-In stimulated transfected PC12 cells (clone E5.9), PC5-A immunoreactivity was highly punctate and distributed throughout the cytoplasm of the cells (Fig. 5A). Characteristically, the immunolabeling was more intense within the perinuclear core of the cells than at their periphery or in their processes (Fig. 5,  A, E, and F). Stimulated wild type PC12 cells showed only weak PC5-A immunolabeling, consistent with the low endogenous expression of PC5 in these cells (not shown). Both transfected and non-transfected cells were devoid of immunolabeling when the primary antibody was omitted or was preabsorbed with synthetic antigen (not shown). Stimulated E5.9 cells immunoreacted with an NT antibody (which recognizes both short and N-terminally extended forms of NT) also exhibited intense punctate labeling (Fig. 5B). This labeling, however, was distinct from that of PC5 in that NT immunofluorescent puncta were more heavily concentrated at the periphery of cell bodies and at the level of processes (Fig.  5B). No NT labeling was observed in unstimulated PC12 cells (not shown). In stimulated, transfected PC12 cells double-labeled for PC5-A and NT, there was only minimal overlap between the two labels in the central perinuclear core (in which PC5 immunoreactivity predominated) and in neural processes (in which NT immunoreactivity predominated) (Fig. 5C). In the mid-cytoplasmic zone, however, several PC5-immunoreactive vesicles were also immunoreactive for the NT antigen (Fig. 5D).
Stimulated Release of Pro-NT/NN Processing Products-In order to determine whether PC5-A-generated pro-NT/NN processing products could be released through the regulated secretory pathway, E5.9 cells were depolarized in high potassium, and immunoreactive NT was assayed in the extracellular medium. Fig. 6 shows that a 30-min depolarization resulted in a 4-fold increase over basal secretion in immunoreactive NT. HPLC analysis of the secreted immunoreactive NT indicated that it consisted of NT (55%) and large NT (45%) in high potassium conditions, whereas, in basal conditions, NT and large NT amounted to 47 and 53% of immunoreactive NT, respectively. Note that these proportions are somewhat different from those found intracellularly for the two components in E5.9 cells. This could reflect a higher rate of degradation in the extracellular medium of the small as compared with the large form of NT.

DISCUSSION
The present study clearly establishes that PC5-A can function as a prohormone convertase in the regulated secretory pathway of neuroendocrine cells. This was demonstrated by stably expressing PC5-A in PC12 cells and showing that the enzyme was colocalized with pro-NT/NN in early compartments of the regulated secretory pathway where it efficiently processed the precursor.
Western blot analysis and pulse-chase studies showed that the biosynthesis of PC5-A in transfected PC12 cells led to the formation of the 126-, 117-, and 65-kDa proteins previously described in a PC5-A-transfected AtT-20 cell line (20). In the latter, these three forms were shown to correspond to pro-PC5-A, to the active form of the enzyme produced by excision of the N-terminal prosegment of pro-PC5-A and to a C-terminally truncated form of the active 117-kDa enzyme, respectively (20). However, the amounts of the 65-kDa protein that were detected in the PC5-A-transfected PC12 and AtT-20 cell lines differed, the former displaying considerably lower levels of the 65-kDa product than the latter (20). This difference was also evident in release experiments which showed that, upon stimulation, TABLE I Analysis of pro-NT/NN processing in wild type and PC5-A-transfected PC12 cells PC12 cells were stimulated with NGF, dexamethasone, forskolin, and Li ϩ for 48 h. The amounts of CTiNN and pro-NT/NN-derived peptides (see Fig. 1) and the percentages of cleavage at the dibasic sites were determined as described under "Experimental Procedures." The values are the means Ϯ S.E. from two independent experiments performed in duplicate. WT, wild type.

FIG. 4. Pulse-chase analysis of pro-NT/NN biosynthesis in PC5-A-transfected PC12 cells.
A, wild type and PC5-A-transfected (clone E5.9) PC12 cells were pulse-labeled with [ 35 S]methionine/cysteine for 5 min, then chased for 5, 15, 30, 60, and 120 min, followed by immunoprecipitation with a polyclonal rabbit anti-pro-NT/NN antiserum, and resolution of immunoprecipitated products on a 15% SDS-polyacrylamide gel. Molecular masses are given in kDa. Exposure time of the autoradiograms, 20 days. B, densitometric analysis of the 17.3-kDa protein corresponding to pro-NT/NN. The results are the mean Ϯ S.E. from three and five independent experiments with wild type PC12 cells and clone E5.9, respectively. PC12 cells released less of the 65-kDa product relative to the 117-kDa form than AtT-20 cells (Fig. 3C). As the processing step leading to the formation of the truncated 65-kDa protein has been suggested to take place in secretory granules (20), this may suggest, as further discussed below, that PC12 and AtT-20 cells differ in their ability to store and/or cleave 117-kDa PC5-A in regulated secretory vesicles.
Immunocytochemistry coupled to confocal microscopy revealed that within transfected PC12 cells (clone E5.9) PC5 immunoreactivity was most heavily concentrated within the cytoplasmic core of cell bodies where it partially colocalized with the Golgi marker MG-160 (41) and the TGN marker Syntaxin-6 (42). These results demonstrate that a major fraction of both 117-and 126-kDa forms of PC5-A are associated with the Golgi apparatus and the TGN, as previously demon-strated in AtT-20 cells (20). The distribution of PC5 was clearly more extensive, however, than that of either MG-160 or Syntaxin, and it is likely, therefore, that PC5 is also localized within vesicular elements upstream (e.g. endoplasmic reticulum) or downstream (e.g. secretory vesicles) of the Golgi apparatus. NT immunoreactivity was more sparsely and more peripherally distributed than PC5 within the cytoplasm of stimulated, transfected PC12 cells. In particular, there was no obvious accumulation of the label in the perinuclear Golgi/TGN area, as seen with PC5. There were, however, numerous NTimmunoreactive vesicle-like organelles within the mid-cytoplasmic zone of the cell that colocalized PC5 and might have corresponded to TGN and/or secretory vesicles. By contrast, there was little colocalization of the two markers in neural processes where the bulk of NT, but very little PC5-A immu- , and PC5-A/Syntaxin-6 (F and FЈ) immunolabeling in PC5-A-transfected PC12 cells (clone E5.9). A, punctuate PC5-A immunoreactivity pervades the cytoplasm of transfected cells. Note the absence of immunoreactivity in the nucleus (n) and the sparse labeling at the periphery of the cell body and in distal processes (arrowheads). B, NT immunoreactivity is also highly punctate and distributed throughout the cytoplasm of the cells. However, it is more intense than PC5 at the periphery of the cell body and within neural processes (arrowheads). Here again, the nucleus (n) is devoid of labeling. C, in double-labeled cells optically cross-sectioned through the nucleus, PC5 (green) and NT (red) immunoreactivities are largely segregated, the former predominating in the perinuclear region and the latter at the periphery of the perikaryon (arrowheads) and within processes (arrows). N, nucleus. D, in optical sections passing through zones in which PC5 (green) and NT (red) labelings overlap, numerous labeled organelles are seen to contain the two markers (yellow, arrowheads). E and EЈ, dual PC5 (E) and MG-160 (EЈ) immunolabeling. Although originally acquired in red and green, respectively, PC5 and MG-160 antigens are both imaged here using lutz pseudocolor. Note the extensive overlap between the two markers and the wider distribution of PC5 over MG-160 immunoreactivity. N, nucleus. F and FЈ, dual PC5 (F) and Syntaxin-6 (FЈ) immunolabeling. Acquisition and pseudocolor representation as in E and EЈ. Here again, there is extensive colocalization but incomplete overlap between the two markers. Note that the distribution of the TGN marker Syntaxin-6 (FЈ) is distinct from that of MG-160, a marker of medial Golgi cisternae. Scale bar, 5 m.
noreactivity, was detected. The latter finding is in marked contrast to the situation in AtT-20 cells where PC5-A and adrenocorticotropic hormone were found to colocalize in vesicular structures at the tips of cellular extensions (20).
These observations suggest that in PC5-A-transfected PC12 cells, pro-NT/NN processing occurs in early compartments of the regulated secretory pathway. They further support the possibility that sorting of PC5-A within the regulated secretory pathway may differ in AtT-20 and PC12 cells. The fact that the 117-and 65-kDa PC5-A forms were released by treatment of PC12 and At-T20 cells with 8-Br-cAMP indicates that these proteins enter regulated secretory vesicles in both cell lines. However, unlike AtT20 cells (20), PC12 cells do not appear to extensively accumulate PC5-A within mature secretory granules, given the lack of PC5 immunoreactivity detected inside PC12 processes by immunocytochemistry. Rather, the present immunohistochemical data suggest that the enzyme is predominantly stored within the Golgi apparatus, TGN, and immature secretory granules. The latter compartment might even be the main source of the PC5-A products that are released by 8-Br-cAMP, as immature secretory granules in PC12 cells have been shown to release their content upon stimulation (44). This would explain the lower levels of 65-kDa PC5-A protein found in PC12 cells as compared with AtT-20 cells, as the C-terminal cleavage of the 117-kDa protein that gives rise to the 65-kDa truncated form is thought to occur late in the regulated secretory pathway (20).
In keeping with the predominant association of PC5 with early compartments of the regulated secretory pathway, pulsechase analysis of pro-NT/NN metabolic fate showed that pro-NT/NN cleavage was detectable as early as 15-30 min following precursor biosynthesis. Such a timing is comparable to that previously reported for secretogranin II processing in PC2transfected PC12 cells and was interpreted as indicating that secretogranin II cleavage took place within immature secretory granules (45). It is therefore tempting to speculate that the latter compartment is not only the main site of PC5 accumulation, but also of pro-NT/NN processing by PC5-A in PC12 cells.
Biochemical analysis demonstrated that pro-NT/NN maturation products were greatly increased in PC5-A-transfected as compared with wild type PC12 cells. Furthermore, the secre-tion of processed products was stimulated by depolarizing PC12 cells, indicating that they were stored in secretory vesicles. This is to our knowledge the first demonstration that PC5-A can process a neuropeptide/hormone precursor in the regulated secretory pathway. Interestingly, the pro-NT/NN processing pattern generated by PC5-A is distinct from that previously described for PC1 and PC2 in transfected PC12 cells (39). Thus, PC5-A cleaved pro-NT/NN with an order of preference for the dibasic sites that was Lys 163 -Arg 164 Ͼ Lys 148 -Arg 149 Ͼ Ͼ Lys 140 -Arg 141 . This order differs from that observed for PC1 (Lys 148 -Arg 149 Ͼ Lys 163 -Arg 164 Ͼ Ͼ Lys 140 -Arg 141 ) and for PC2 (Lys 140 -Arg 141 ϭ Lys 148 -Arg 149 ϭ Lys 163 -Arg 164 ) (39). The major consequence of these differences is the production by PC5-A of large NT in substantial amounts, a product that was barely, if at all, detectable in PC1-and PC2-transfected PC12 cells (39). PC5-A also produced NT and large NN in comparable amounts, and small amounts of NN, acting in this respect similarly to PC1 (39). PC2 differed from both PC1 and PC5-A in its ability to generate NN and NT in similar amounts and its inability to produce large NT and large NN (39).
PC1 and PC2 cleave pro-NT/NN with patterns that reproduce those observed in the gut and in the brain, respectively (39). The question arises as to whether the pro-NT/NN processing pattern observed for PC5-A in PC12 cells occurs in tissues and cells that normally express the precursor. NT has long been known to be present in chromaffin cells of the adrenal medulla (31)(32)(33)(34). Other pro-NT/NN derivatives produced in this tissue include large NT and large NN in proportions that are similar to those described here in PC5-A-transfected PC12 cells (26,35). In situ hybridization studies have shown that the adrenals exhibit one of the highest levels of PC5-A mRNA (3,17). This suggests that the enzyme might be involved in pro-NT/NN processing in this tissue. Colocalization studies of pro-NT/NN and PC5-A at the cellular and subcellular levels in the adrenal medulla should help resolve this issue. Recently, we reported that human colon cancer cell lines express pro-NT/NN (46). Some of the cell lines were found to process the precursor with a pattern similar to that described here in PC5-A-transfected PC12 cells (46). Interestingly, these cell lines expressed PC5-A but neither PC1 nor PC2 (46), thus suggesting that PC5-A might be involved in processing pro-NT/NN in human colon cancers.
PC12 cells have proven to be an extremely useful model to study the processing of pro-NT/NN by PC1, PC2, and PC5-A and to compare the processing pattern of these convertases (Ref. 39 and present study). PC12 cells were also successfully used to compare the enzymatic activity of different forms of PC1 (38). Similar to PC5-A, PC1 has been shown to undergo C-terminal processing into secretory vesicles (47,48), and the C-terminally truncated 66-kDa form of PC1 was found to be enzymatically more active than the non-truncated 87-kDa protein (38,49,50). In particular, this was demonstrated by transfecting PC12 cells either with the 87-kDa PC1 protein rendered non-cleavable by mutation of its cleavage site or with the truncated 66-kDa PC1 product and by comparing the ability of the transfected cell lines to process endogenous pro-NT/NN (38). The present study raises the question as to which of the 117and 65-kDa PC5-A proteins is responsible for the processing of pro-NT/NN. The much higher levels of the 117-kDa product as compared with the 65-kDa truncated form might suggest that the larger protein is the main pro-NT/NN cleaving enzyme in PC5-A-transfected PC12 cells. However, by analogy with PC1, it is possible that the 65-kDa PC5-A protein is the most active form of the enzyme and might contribute significantly to the processing of pro-NT/NN despite its low concentration. Future work employing a similar approach to that previously used for PC1 (38), as recalled above, might shed some light on the relative activity of both PC5-A forms.