The RGD Motif and the C-terminal Segment of Proprotein Convertase 1 Are Critical for Its Cellular Trafficking but Not for Its Intracellular Binding to Integrin alpha 5beta 1*

Carole RovèreDagger §, José Luis, Jean-Claude Lissitzkyparallel , Ajoy BasakDagger **, Jacques Marvaldi, Michel ChrétienDagger **, and Nabil G. SeidahDagger Dagger Dagger

From the Dagger  Laboratories of Biochemical and Molecular Neuroendocrinology, the Protein Engineering Network of Centres of Excellence, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec H2W 1R7 Canada, the  Laboratoire de Biochimie Cellulaire CNRS-Unité Propre de Recherche de l'Enseignement Supérieur Associe'e 6032, Faculté de Pharmacie, 27 Blvd. Jean Moulin, 13385 Marseille, cedex 05, France, and parallel  INSERM U387 Hôpital Ste Marguerite, 270 Blvd. Ste Marguerite, 13009 Marseille, cedex 29, France

    ABSTRACT
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INTRODUCTION
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Cellular trafficking of subtilisin/kexin-like precursor convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin alpha 5beta 1 associates primarily with the zymogens proPC1, proPC1-Delta C (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as alpha 5beta 1.

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INTRODUCTION
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Limited proteolysis of prohormones and proproteins is a general mechanism generating bioactive peptides and proteins in a regulated fashion. Often, this processing step involves specific cleavage(s) post single basic or pairs of basic residues. Recently, seven mammalian serine proteinases related to yeast kexin and bacterial subtilisins were identified and shown to be responsible for such conversions. These proprotein convertases (PCs)1 were called furin (also called PACE), PC1 (PC3), PC2, PACE4, PC4, PC5 (PC6), and PC7 (LPC, SPC7, or PC8) (for reviews and updates, see Refs. 1-5). Whereas furin, PACE4, PC5, and PC7 exhibit a widespread tissue distribution, PC4 is predominantly found in testicular germ cells. The expression of PC1 and PC2 is mostly restricted to endocrine and neural cells. All of these PCs exhibit an N-terminal signal peptide, followed by a prosegment, a catalytic domain, a P domain, and an enzyme-specific C-terminal segment (4, 5). The most conserved catalytic domain of eukaryotic precursor convertases exhibits the closest similarity to bacterial subtilisins and contains the catalytic triad Asp, His, and Ser, as well as the oxyanion hole Asn (4, 5). In contrast to the relatively short bacterial subtilases, the catalytic domain of eukaryotic convertases is extended at the C terminus by a 140-150 amino acid P domain, the function of which is less well understood.

The P domain has been shown to be critical for the zymogen cleavage of prokexin (6), pro-PACE4 (7), proPC2 (8), and proPC1 (9); for the substrate cleavage activity of furin (10, 11); and for the sorting of PC2 (12) and PC1 (13). Based on molecular modeling, it has recently been suggested that the P domain is an independently folded structure and that in PC1 it consists of 8-stranded beta -barrels with a well organized inner hydrophobic core (14). Furthermore, because it is different from subtilisins, the dibasic cleavage specificity of PCs may require the presence of a large number of acidic (Asp and Glu) residues within the substrate binding region. It was suggested that the P domain, unique to PCs, contributes to the stabilization of their catalytic subunit (14). Studies of various chimeras implied that the P domain is necessary to both fold and maintain the catalytic pocket in an active form and to regulate the calcium and pH dependence of PCs (15).

Within the P domain, a conserved RRGDL sequence is observed in six (except for PC7) of the seven mammalian PCs, and its RG dipeptide is always present in all PC orthologues (13). For PC7, which exhibits an RRGSL sequence, the C-terminal border is more extended (16). It has recently been suggested that the RGD sequence in PCs lies in between two beta -strands (beta 3 and beta 4), which contain 6 of the predicted 22 residues forming the inner hydrophobic core of the P domain (14). Because an RGD sequence is found in a number of proteins that bind cell adhesion integrins, such as fibronectin and vitronectin, it was originally suggested that this motif might be important for the interaction of the PCs with integrins (17). Binding of fibronectin to its integrin receptor was shown to be dependent on the RGD sequence and to be abrogated when replaced by RGE (18). The conservation of the RGD motif in mammalian PCs suggested a structural and/or functional role, which may be common to all of them.

In a previous report, we showed that the RGD sequence of PC1 is critical for the efficient folding and zymogen processing of proPC1 (89 kDa) into PC1 (83 kDa) within the endoplasmic reticulum (ER) and for its ability to be processed into the C-terminally truncated 66-kDa PC1-Delta C (13). Furthermore, previous studies demonstrated that the 66-kDa form of PC1 is formed inside granules (19-22). Accordingly, data derived from vaccinia virus expression of the RGE mutant of PC1 suggested that the RGD motif is important for the sorting of PC1 into the regulated secretory pathway (13).

In the present study, we investigated the functional importance of the RGD sequence in the cellular sorting of PC1 in AtT20 cells that normally express this enzyme endogenously. This was achieved using stable transfectants of PC1-RGD and PC1-RGE containing a Myc epitope tag. Biosynthetic and immunocytochemical data provided strong evidence for the critical importance of the RGD motif in the sorting of PC1 to secretory granules. Cell adhesion assays showed that in vitro, a synthetic 14 amino acid (aa) peptide spanning the RGD sequence of PC1 can bind to integrins. In contrast, co-expression studies in AtT20 cells provided evidence for an RGD-independent ER-localized binding of integrin alpha 5beta 1 to proPC1, proPC1-Delta C (missing the 137-aa C-terminal segment Delta C but still containing the RGD sequence (23)) and proPC2.

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mPC1-Myc Constructions-- The sequences of native mouse (m)PC1 and its D520E (RGD/RGE) mutant described in Ref. 13 were modified to include a 10-aa Myc antigen (underlined) inserted at the N terminus of mature mPC1, resulting in the sequence RSKR110EQKLISEEDL111SVQD of full-length mPC1 (24). For this purpose, two complementary oligonucleotides containing the cohesive ends of the Psp1406-I restriction endonuclease (italics) were synthesized, annealed, and phosphorylated. Their sequences were as follows: sense, 5'-CGTTCAGTTGAACAAAAGCTCATCTCAGAGGAAGATCTT-3', and antisense, 5'-CGAAGATCTTCCTCTGAGATGAGCTTTTGTTCAACTGAA-3'. The double-stranded oligonucleotide was then ligated into the Psp1406-I site of the cDNA of mPC1. The mutated mPC1-RGD-Myc and mPC1-RGE-Myc were then subcloned either into the 5' HindIII/SalI 3' sites of PMJ602 vector, and vaccinia virus recombinants were isolated, as described Ref. 20, or into the 5' HindIII/EcoRV 3' sites of pcDNA3 (Invitrogen).

Vaccinia Virus-- The isolation of recombinant vaccinia viruses (vvs) expressing mPC1 and mPC2, as well as their RGD mutants, was previously reported (13, 20, 25). The recombinant vv:mPOMC was a generous gift of Dr. Gary Thomas (Portland, OR). We have similarly isolated vaccinia virus recombinants of the human (h) integrins alpha 5 and beta 1, kindly provided by Dr. E. Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA). GH4C1 or AtT20 cells were infected with the specified plaque-forming units (pfu)/cell vv:mPC1-RGD, vv:mPC2-RGD, vv:mPC1-RGE, vv:mPC2-RGE, vv:mPC1-RGD-Myc, vv:mPC1-RGE-Myc, vv:mPOMC, vv:halpha 5, and vv:hbeta 1.

Stable and Transient Transfectants-- Full-length mPC1-RGD-Myc and mPC1-RGE-Myc cDNAs cloned into the pcDNA3 vector (Invitrogen) or an empty pcDNA3 vector were transfected into AtT20 cells using a DOSPER reagent as suggested by the manufacturer (Life Technologies, Inc.). Six G418-resistant clones expressing each enzyme were isolated. One of the clones expressing the highest amount of mPC1-RGD-Myc was chosen for further analyses. All mPC1-RGE-Myc G418-resistant clones expressed low levels of the enzyme, as estimated by Western blots using an enhanced chemiluminescence kit (Boehringer Mannheim) and the Myc-specific antibody at a dilution of 1:2000. Based on immunoprecipitation of the radiolabeled protein, the highest expressor (clone I) was selected for further investigation by biosynthetic analyses and by immunocytochemistry.

Biosynthetic Analyses-- Seventeen hours postinfection with vv recombinants, GH4C1 cells (3 × 106) were radiolabeled with 200 µCi of [35S]Met for 3 h. The media and cell extracts were immunoprecipitated with either an N-terminal mPC1-antibody (20); an antiserum to beta -endorphin (26), or a Myc-specific monoclonal antibody (Myc-1-9E10.2) obtained from ATCC (clone CRL-1729). The immunoprecipitates were resolved by SDS-PAGE on either 8% or 15% polyacrylamide gels, and the dried gels were autoradiographed (20, 26). All biosynthesis experiments were performed at least twice. For stable transfectants, AtT20 cells were pulsed for 15 min with [35S]Met followed by a chase of 1 or 3 h in the presence or absence of 5 mM of the secretagogue 8-Br-cAMP (27).

Immunocytochemical Analysis of mPC1-RGD-Myc- and mPC1-RGE-Myc-expressing Cells-- Because the monoclonal antibody to Myc was not found to be suitable for immunofluorescence cytochemistry, mPC1-Myc was localized in transfected AtT20 cells by using the peroxidase/diamino benzamidine technique (28). The primary Myc-1-9E10.2 antibody was used at a dilution of 1:100, and the secondary antibody consisted of horseradish anti-mouse IgG coupled to peroxidase used at a 1:75 dilution (Amersham Pharmacia Biotech).

Cell Adhesion Assays-- The synthetic peptides GRGDSP and GRGESP were purchased from Bachem. The synthetic 14-mer mPC1 peptides, TIEYSRRGD520LHVTL, representing aa 512-525 (24), and its D520E mutant TIEYSRRGE520LHVTL, were prepared by solid phase synthesis using fast Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a 431A ABI-solid phase peptide synthesizer. Their quality and purity were verified by reverse phase high pressure liquid chromatography and by fast atom bombardment mass spectrometry, giving the expected molecular masses of 1658 and 1672 Da, respectively. Adhesion substrates were prepared by coating flat-bottom 96-well microtiter plates for 1 h at 37 °C with 50 µl of vitronectin (prepared as described in Ref. 29) at 1 µg/ml. Coated wells were then blocked with 1% bovine serum albumin in phosphate-buffered saline for 1 h. CHO cells were obtained in a single cell suspension by treatment of subconfluent cell monolayers with 0.53 mM EDTA in phosphate-buffered saline, washed twice with Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin (adhesion buffer) and preincubated for 1 h at 4 °C with synthetic peptides. Cells (50,000 cells in 50 µl) were added to each well and allowed to adhere to the substrata for 1 h at 37 °C in a cell culture incubator. Unattached cells were removed by gently washing three times with adhesion buffer and residual attached cells were fixed by 1% glutaraldehyde. After staining by 0.1% crystal violet, cells were lysed with 1% SDS, and the absorbance was measured at 600 nm by a microplate reader (model 960 from Metertech).

Co-immunoprecipitation of Integrin alpha 5beta 1 with PC1 and PC2-- AtT-20 cells were solubilized with 50 mM Tris-HCl, pH 8, 200 mM NaCl, 1 mM EDTA, and 1% Triton X-100 containing 0.5% bovine serum albumin and a mixture of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 500 units/ml aprotinin, 1 µg/ml leupeptin, 1 µM pepstatin, 1 mM iodoacetamide, and 1 mM o-phenanthroline). Samples were incubated for 5 h at 4 °C with 10 µg/ml of purified rabbit polyclonal antibodies against the C-terminal portion of the alpha 5-integrin subunit. Control experiments included a preincubation of the anti-alpha 5 antibody with excess of the C-terminal peptide immunogen. Immunoprecipitated material was submitted to SDS-PAGE on a 7% polyacrylamide gel and blotted onto nitrocellulose. For Western blot analyses, polyclonal antibodies against the N terminus of PC1 and the C terminus of PC2 were used. The labeled proteins were detected using the above enhanced chemiluminescence system.

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MATERIALS AND METHODS
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Insertion of a Myc Tag at the N-terminal of Mature mPC1 Does Not Affect Its Autoactivation or Its Ability to Process POMC-- In order to differentiate the expression of exogenous PC1 from its endogenous homologue in cells, we introduced a 10-aa Myc epitope tag between the prosegment and the start of the catalytic subunit. This strategic site was chosen because it was previously shown that insertion of a FLAG-epitope tag at a similar site in furin did not affect its enzymatic properties (30). As a test for the effect introducing the Myc tag, we overexpressed vaccinia virus recombinants of PC1-RGD and PC1-RGE in GH4C1 cells (devoid of endogenous PC1 (31)) and compared their biosynthesis to those of their Myc-tagged derivatives PC1-RGD-Myc and PC1-RGE-Myc. As shown in Fig. 1, the insertion of a Myc epitope does not affect the autocatalytic conversion (32) of proPC1 (89 kDa) into PC1 (83 kDa) for either the wild type enzyme or its RGE mutant. Because the extent of processing of PC1-RGD and PC1-RGD-Myc into the 66-kDa PC1-Delta C is quite similar (Fig. 1A), this demonstrates that the Myc tag does not significantly affect the late processing (19-22) of the 83-kDa form of the wild type PC1 into its 66-kDa derivative. In addition, our data show that the Myc monoclonal antibody allows the immunoprecipitation of radiolabeled Myc-tagged PC1s (Fig. 1B). Finally, it is clear that no 66-kDa form of PC1-RGE-Myc is secreted into the medium (Fig. 1, A and B) and that both PC1-RGE and PC1-RGE-Myc exhibit lower levels in the medium as compared with their RGD parents (Fig. 1A) (13). Furthermore, overexposure of the autoradiogram did not reveal the presence of any trace of 66-kDa PC1-RGE in either media (not shown). Intracellularly, because both the N-terminal PC1 antibody (Fig. 1A, CTL) and the monoclonal Myc antibody (Fig. 1B, RGD and RGE) detect either a nonspecific protein or the endogenous (nonsecretable) c-Myc protein migrating closely to the 66-kDa position (33) (Fig. 1B), it is difficult to assess the level of intracellular 66-kDa PC1.


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Fig. 1.   Vaccinia virus expression of mPC1-RGD-Myc and its RGE mutant in GH4C1 cells. GH4C1 (3 × 106) cells were infected with either vv:wild type (control (CTL)), vv:mPC1 (RGD), vv:mPC1-Myc (RGD/myc), vv:mPC1-RGE, or vv:mPC1-RGE-Myc (RGE/myc), each at 2 pfu/cell for native mPC1 (RGD) and 3 pfu/cell for the RGE mutants. Seventeen hours postinfection, the cells were pulse-labeled with [35S]Met for 3 h. The cell extracts and media were immunoprecipitated with either a PC1 N-terminal antibody recognizing the segment 84-100 of mPC1 (20,24) or a Myc mouse monoclonal antibody. The immunoprecipitates were separated on an 8% SDS-polyacrylamide gel and fluorographed as described (13). The resulting autoradiogram also depicts the migration position of proPC1 (89 kDa), PC1 (83 kDa), and the C-terminally truncated PC1-Delta C (66 kDa).

As a functional test for the Myc-tagged PC1 activity, we compared the ex vivo ability of PC1 and PC1-Myc enzymes to process POMC into beta -lipotrophic hormone (26). As shown in Fig. 2, the levels of secreted beta -lipotrophic hormone are similar in the presence or absence of the Myc epitope tag. This demonstrates that the introduction of the Myc tag at the selected position did not affect the intracellular enzymatic activity or cleavage selectivity of PC1. Finally, and in accordance with the lower overall levels of PC1-RGE and PC1-RGE-Myc as compared with their RGD parents (Fig. 1), we note that the former produces less beta -lipotrophic hormone than the latter, as was previously observed for the RGE mutant of PC1 (13).


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Fig. 2.   Comparative POMC processing by mPC1 and mPC1-Myc. GH4C1 (3 × 106) cells were infected with 2 pfu/cell vv:mPOMC together with either 2 pfu/cell vv:wild type (control (CTL)), vv:mPC1 (RGD), vv:mPC1-Myc (RGD/myc), 3 pfu/cell vv:mPC1-RGE, or vv:mPC1-RGE-Myc (RGE/myc). Seventeen hours postinfection, the cells were pulse-labeled with [35S]Met for 3 h. Cell lysates and media were immunoprecipitated with an antibody specific to beta -endorphin (26). The immunoprecipitates were separated on a 15% SDS-PAGE gel, and the dried gel was autoradiographed. The migration positions of unprocessed POMC and of beta -LPH are emphasized.

Stable Transfectants of PC1-RGD-Myc and PC1-RGE-Myc in AtT20 Cells-- In order to study the importance of the integrity of RGD motif on the sorting of PC1 to secretory granules, we chose the widely used AtT20 cells (which endogenously synthesize large amounts of wild type PC1) as the ideal cell type to perform this investigation. We thus generated stable transfectants of both PC1-RGD-Myc and PC1-RGE-Myc and selected the highest expressing clones for each, based on levels estimated by Western blots using the Myc antibody (Fig. 3). Although at least three clones exhibiting high expression levels of PC1-RGD-Myc were isolated (we only show one of them), all positive PC1-RGE-Myc clones revealed relatively much lower intracellular levels of Myc-tagged PC1. Again, no 66-kDa form of PC1 was observed for the latter clones (Fig. 3). We thus selected one of them (clone I) for biosynthetic analyses (Fig. 4) and immunocytochemical localization (Fig. 5).


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Fig. 3.   Western blot of stable transfectants of mPC1-RGD-Myc and mPC1-RGE-Myc in AtT20 cells. Cell extracts of stable transfectants of mPC1-RGD-Myc (one clone) and mPC1-RGE-Myc (clones I-IV) in AtT20 cells (50 µg of total protein/lane, as estimated by a Bio-Rad assay) were resolved by SDS-PAGE on an 8% acrylamide gel. As a control, we also obtained a stable transfectant of the pcDNA3 vector alone. The separated proteins were probed with a Myc monoclonal antibody at a dilution of 1:2000. The proteins were visualized by enhanced chemiluminescence. The migration positions of PC1 and PC1-Delta C and the positions of the molecular mass markers are shown.


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Fig. 4.   Biosynthetic analysis of stable transfectants of mPC1-Myc in AtT20 cells. AtT20 cells (3 × 106) stably transfected with either mPC1-Myc-RGD or mPC1-Myc-RGE in pcDNA3 were pulse-labeled for 15 min with [35S]Met and chased for either 1 or 3 h in the presence (+) or absence (-) of 5 mM 8-Br-cAMP. The mPC1-Myc proteins in the cells and media were analyzed under the same conditions as in Fig. 1 using the Myc monoclonal antibody. The resulting autoradiogram also depicts the migration positions of proPC1 (89 kDa), PC1 (83 kDa), and the C-terminally truncated PC1-Delta C (66 kDa).


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Fig. 5.   Comparative immunocytochemistry of mPC1-RGD-Myc and mPC1-RGE-Myc in AtT20 cells. Distribution pattern of mPC1-RGD-Myc (A) and mPC1-RGE-Myc (B) revealed with a Myc monoclonal antibody using the diamino benzamidine-immunoperoxidase technique (28). Immunostaining to mPC1-RGD-Myc was found paranuclearly and in some tips of the cellular extensions (A, dark triangles). This pattern resembles that of ACTH in AtT20 cells (34). In contrast, for the mPC1-RGE-Myc, immunostaining was predominantly found paranuclearly (B), suggesting a Golgi-like localization similar to that reported for the TGN marker TGN38 (34). Furthermore, within cellular extensions, very little Myc immunoreactivity was found (B, arrows). Magnification, × 500; scale bar, 25 µm.

The stable transfectants were pulse-labeled with [35S]Met for 15 min, followed by a chase of either 1 or 3 h in the presence or absence of 8-Br-cAMP (Fig. 4). Under these conditions, after 1 h of chase, the ratio of the levels of PC1-RGD-Myc (83 kDa) in cAMP treated versus control cells was 1.8, as compared with 3.1 for the 66-kDa derivative. In contrast, the ratio of the levels of secreted 83-kDa PC1-RGE-Myc in the presence versus absence of cAMP was 0.9. At the 3 h-chase time, the corresponding values for the secreted 83- and 66-kDa RGD forms were 1.1 and 2.7, respectively. Concomitantly, the corresponding intracellular ratios of the 83- and 66-kDa forms of PC1-RGD-Myc were 0.9 and 0.5, respectively. Thus, following the 3-h chase, practically all the 83-kDa form of PC1 is converted into PC1-Delta C (which lacks the C-terminal 137 aa but still contains the RGD sequence), and cAMP can only stimulate the secretion of the latter form. These results are in agreement with the fact that the 66-kDa form is primarily found in granules, whereas the 83-kDa form is mostly in the trans-Golgi Network (TGN) (19-22). At the 3-h chase period, the level of secreted 83-kDa PC1-RGE-Myc is also slightly lower in the presence of cAMP. These data strongly suggest that the majority of PC1-RGE-Myc exit the cells via the unregulated, constitutive secretory pathway. Finally, it seems that the major intracellular form of PC1-RGE-Myc exhibits a lower molecular mass than the secreted form and that its levels did not appreciably diminish after 3 h chase, suggesting a low turnover of this likely immature ER-associated form.

As shown in Fig. 5A, immunostaining for PC1-RGD-Myc was observed both paranuclearly and at the cellular tips (dark triangles). This pattern is very similar to that previously observed in AtT20 cells in our laboratory with the endocrine hormone ACTH, which is localized both at the TGN (paranuclear) and in secretory granules (cellular extension tips) (34). In contrast, in the case of PC1-RGE-Myc most of the Myc immunoreaction was found paranuclearly (Fig. 5B) and the staining at the cellular extension tips was very weak (Fig. 5B, arrows). This pattern resembles that of constitutively secreted proteins such as alpha 1-PDX in AtT20 cells and of resident Golgi proteins, such as TGN38 (34). These data agree with those in Fig. 4, whereby PC1-RGD-Myc can enter the regulated secretory pathway, whereas PC1-RGE-Myc exits the cell predominantly via the constitutive pathway.

Interaction of PC1 with Cell Adhesion Integrins-- The conserved RRGDL sequence in convertases (4, 13) suggested that PCs might interact with cell surface integrins that recognize RGD-containing sequences. In that context, phage display revealed that integrins alpha 5beta 1 and alpha vbeta 3 recognize an RRGDL sequence (35). Thus, in order to test the possibility that PC1 can interact with integrins, we synthesized a 14-aa peptide spanning the RRGDL sequence of structure: TIEYSRRGD520LHVTL (24), as well as its D520E (RGE) mutant. For cell adhesion assays, we chose CHO cells, which bind vitronectin in an RGD-dependent fashion (36). We thus compared the ability of the synthetic mPC1-RGD and RGE peptides, as well as the model hexapeptides GRGDSP and GRGESP to compete with vitronectin in the binding of CHO cells. Thus, the data in Fig. 6 show that the peptide mPC1-RGD is about 2.5-fold more potent in competing with vitronectin for CHO cells as compared with the synthetic GRGDSP peptide. As expected, both RGE-containing peptides do not prevent vitronectin from binding CHO cells. These data suggested that in vitro mPC1 could interact with integrins, such as those found on the cell surface of CHO cells.


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Fig. 6.   Attachment of CHO cells to vitronectin in the presence of increasing amounts of competing peptides. CHO cells (50,000 cells/50 µl) were added to each well and allowed to adhere to a substrata of vitronectin (1 h at 37 °C). Attached cells were fixed with 1% glutaraldehyde, stained with 0.1% crystal violet, and then lysed with 1% SDS. The amount of adherent cells was estimated from the absorbance at 600 nm. The mean of triplicate measurements and the S.E. (less than 10%) are shown. RGD, ; RGE, diamond ; RRGDL, open circle ; RRGEL, triangle .

In order to test the possible intracellular binding of full-length mPC1 with integrins and the dependence of this plausible interaction on the RGD sequence, we selected alpha 5beta 1, because it binds preferentially RRGDL sequences as compared with alpha vbeta 3 (35). AtT20 cells were triply infected with vv:alpha 5, vv:beta 1, and the vaccinia recombinants of either mPC1-RGD, mPC1-RGE, mPC1-Delta C, mPC2-RGD, or mPC2-RGE, in the presence or absence of brefeldin A (BFA). The fungal metabolite BFA is known to disassemble the Golgi complex and produce fusion of the cis-, medial-, and trans-Golgi (but not the TGN) with the ER (37). We first assessed the levels of intracellular expression of each protein by Western blots and did not observe significant differences between each infection (Fig. 7). Notice that either in the presence or absence of BFA more than 50% of proPC1 is converted into PC1 and that in the case of the RGE mutant it remains mostly as proPC1-RGE. In contrast, and in accordance with the known slower proPC2 to PC2 conversion late along the secretory pathway (20), we observed a preponderance of proPC2 in the cells with a minor fraction being converted into PC2 (66 kDa) only in the absence of BFA. The production of the 71-kDa proPC2, in which the prosegment is cleaved at an internal site, has been previously reported (20, 21).


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Fig. 7.   Expression levels of vaccinia virus-expressed mPC1 and mPC2 and their RGD/RGE mutants in AtT20 cells co-expressing human integrin alpha 5beta 1. AtT20 (1 × 107) cells were infected for 2 h at 27 °C with 1 pfu/cell each: vv:alpha 5 and vv:beta 1 together with either vv:wild type (WT), vv:mPC1 (RGD (top)), vv:mPC1-RGE (RGE (top)), vv:mPC2 (RGD (bottom)), or vv:mPC2-RGE (RGE (bottom)). The cells were then incubated for 17 h in the presence or absence of 5 µg/ml of BFA. Cell lysates were then separated by SDS-PAGE on a 7% polyacrylamide gel and then analyzed by Western blot using an N-terminal PC1 and a C-terminal PC2 antiserum (20). Notice the endogenous 89-kDa PC1 and 66-kDa PC1-Delta C in the cells infected with the wild type (WT) virus control. The arrows at the right on the upper and lower panels depict the positions of the 72-kDa proPC1-Delta C and the 71-kDa proPC2 intermediate (20, 22).

Fig. 8 depicts the results of immunoprecipitation of cell extracts using an alpha 5-specific antibody followed by Western blotting analysis of the immunoprecipitates by SDS-PAGE using either an N-terminal PC1 antiserum or a C-terminal PC2-antibody. Control experiments in which the anti-alpha 5 antibody was preincubated with an excess (10 µg) of the C-terminal peptide immunogen confirmed the specificity of the immunoprecipitations (not shown). Unexpectedly, proPC1-Delta C (72 kDa) and both RGD and RGE forms of both proPC1 (89 kDa) and proPC2 (75 kDa) co-immunoprecipitate with alpha 5beta 1 in the presence of BFA, suggesting that at the level of the ER, these zymogens can interact with alpha 5beta 1 in an RGD-independent manner. Furthermore, proPC1 or proPC1-Delta C, neither of which can exit the ER (20, 22), bind alpha 5beta 1 primarily in the presence of BFA. The low level of these zymogens observed in the absence of BFA may be related to their short residence time in the ER. Only a relatively minor amount of PC1-Delta C (66 kDa) and less so of PC1 (83 kDa) (seen upon overexposure of the gel) bind alpha 5beta 1, even though their levels are not negligible (Fig. 7). Thus, these data suggest that the C-terminal 137-aa Delta C segment of PC1 is not critical for this interaction which occurs in the ER. The data also show that proPC2 binds alpha 5beta 1 in both the presence and absence of BFA, in accordance with its long residence time in the ER (20). It is difficult at the moment to eliminate the possibility that at the level of the TGN proPC2 remains bound to alpha 5beta 1, because some of the carbohydrate chains of PC2 always remain sensitive to endoglycosidase H (20). The above data demonstrated that the co-immunoprecipitation of alpha 5 and the PCs is not simply due to their overexpression, as we do not see any immunoprecipitates for PC1-RGD and PC1-Delta C in absence of BFA. An additional control included the co-expression of rPC7 (16) with alpha 5beta 1, which showed no co-immunoprecipitation even in the presence of BFA (not shown). Finally, when immunoprecipitations were performed with PC-specific antisera, Western blots showed that alpha 5 is detected (not shown). In conclusion, although the specific binding of alpha 5beta 1 to the neural and endocrine convertases occurs in the ER, neither the RGD sequence of proPC1 and proPC2 nor the Delta C polypeptide of proPC1 is critical for the interaction of these zymogens with alpha 5beta 1.


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Fig. 8.   Co-immunoprecipitation of human integrin alpha 5beta 1 with mPC1 and mPC2 and their RGD/RGE mutants in AtT20 cells. AtT20 (1 × 107) cells were infected for 2 h at 27 °C with 1 pfu/cell each: vv:alpha 5 and vv:beta 1, together with either vv:wild type control (WT), vv:mPC1 (RGD (left)), vv:mPC1-RGE (RGE (left)), vv:mPC2 (RGD (right)), or vv:mPC2-RGE (RGE (right)). The cells were then incubated for 17 h in the presence or absence of 5 µg/ml of BFA. Cell lysates were then immunoprecipitated with an integrin alpha 5 polyclonal antibody. The immunoprecipitates were separated by SDS-PAGE on a 7% polyacrylamide gel, followed by Western blotting of the gel using either an N-terminal mPC1 or C-terminal mPC2 antibodies (20). The migration of purified PC1 (83 kDa), proPC2 (75 kDa), and PC2 (66 kDa), as well as the positions of proPC1 (89 kDa), proPC1-Delta C (72 kDa), and PC1-Delta C (66 kDa), are indicated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented in this work represent a first attempt to define the functional significance of the conserved RGD motif in proprotein convertases. The available data clearly point out that the integrity of this motif is crucial for the stability (13) and the sorting of the convertase PC1 (see above). Thus, whereas the wild-type PC1 enters secretory granules (Figs. 4 and 5), possibly assisted by its C-terminal sorting domain Delta C (23, 38), the PC1-RGE mutant is rapidly degraded early along the secretory pathway (13), and the little that does exit the ER is secreted via the constitutive secretory pathway (Figs. 4 and 5). In agreement, we observed that all stable transfectants of PC1-RGE-Myc exhibit much lower levels of expression than the corresponding PC1-RGD-Myc clones (Fig. 3). This result is in accord with our earlier observation that the steady-state levels of vaccinia virus expressed recombinants of PC1-RGE are much lower than those of their corresponding wild type enzyme (13). We surmise that it is likely that the RGD motif plays an important role in the folding of PC1 within the ER. The structural alterations imposed by the RGD/RGE mutation are such that PC1-RGE can no longer enter secretory granules. This suggests that the RGD sequence influences either its degree of calcium-dependent aggregation and/or its possible interaction with a putative sorting receptor in the TGN/immature secretory granules, both processes proposed as likely secretory granule sorting mechanisms (39-41). Based on the predicted structure of the P domain of PC1 (14), the RGD sequence lies between the diagonally opposed third and fourth beta -strands and is likely on the hydrophilic side (13) of the predicted 8-stranded-beta -barrel structure exhibiting a Greek key motif (14). Because it is proposed that the P domain consists of two beta -sheets formed by beta -strands 1, 2, 4, and 7 and beta -strands 3, 8, 5, and 6, respectively (14), we envisage that the RGD sequence would sit in between the two beta -sheets.

Integrins are a major group of versatile adhesion receptors endowed with both adhesive and signaling functions. They possess shared and unique specificities both outside and inside the cell. Many of the integrins share an affinity toward the RGD recognition sequence in their extracellular matrix ligands but are still capable of distinguishing different RGD-containing proteins (18). In attempt to define the ability of the conserved RRGDL motif to bind integrins extracellularly, we tested the in vitro integrin binding of a 14-aa synthetic peptide spanning this sequence (Fig. 6). The data demonstrated that this peptide behaved similarly to a fibronectin-related hexapeptide, GRGDSP. Importantly, the RGE mutants of either the mPC1- or the fibronectin-derived peptide did not bind CHO cells, as expected from RGD-dependent integrin binding. These data suggested that secreted PC1, or a fragment thereof, could in principle bind to surface integrins such as those found on CHO cells. However, we also wanted to investigate the possible intracellular interaction of integrins with PC1 and PC2, because it is now known that the processing of the alpha -chain of certain integrins is performed within the TGN by furin-like enzymes (42). We opted to co-express alpha 5beta 1 with PC1 and another neuroendocrine convertase PC2, because phage display revealed that this integrin binds preferentially to an RRGDL-containing sequence (17, 35) found in these PCs (13). Unexpectedly, co-immunoprecipitation results demonstrated that alpha 5beta 1 binds to both proPC1 and proPC2 within the ER in an RGD-independent fashion (Fig. 8). Furthermore, our data showed that the C-terminal Delta C segment of PC1 is not implicated in this interaction. It is intriguing that primarily the zymogens proPC1 and proPC2 bind to alpha 5beta 1 in the ER, even though PC1 is formed in this organelle and remains so in the presence of BFA (Fig. 8). This might implicate the participation, at least in part, of the prosegments of PC1 and/or PC2 in this interaction. Interestingly, no RGD sequence is found in the prosegments of any PC. Recently, it was reported that motifs other than RGD can be recognized by alpha 5beta 1, e.g. an RHD sequence found in the Alzheimer's disease, causing peptide Abeta 40 and Abeta 42 (43). In that context, an RYD sequence is found in the catalytic subunit of mPC1 (aa 199-201), mPC5 (aa 205-207), and hPACE4 (aa 237-240). Thus, more work is necessary to define the critical motif(s) responsible for the binding of proPC1 and proPC2 to alpha 5beta 1 in the ER. Nevertheless, it is noteworthy that alpha 5beta 1 intracellularly binds to these zymogens even though it represents one of the most discriminating of the RGD-recognizing integrins with regard to extracellular ligand specificity (18). However, we cannot eliminate the possibility that the ER interaction of alpha 5beta 1 with proPC1 and/or proPC2 is indirect and could involve one or more intermediary proteins, such as chaperones for example. In that context, the presence of multicomponent complexes in the ER involved in the folding of proteins has recently been reported (44). Finally, similar results were also obtained in GH4C1 cells (not shown) that do not express either PC1 or PC2 (31), thereby eliminating the possibility that the endogenous enzymes interfered with our assays in AtT20 cells.

Earlier data suggested that residues within the P domain of PC1 influence the sorting of this enzyme to the regulated secretory pathway (13). Based on the above data, we can conclude that integrin alpha 5beta 1 is likely not involved in the discriminative sorting of PC1-RGD toward secretory granules and of PC1-RGE to the constitutive secretory pathway. In addition, the present data demonstrate that although the conserved RGD sequence in PC1 is required for its stability and sorting toward secretory granules, this does not seem to implicate an intracellular RGD-dependent interaction with integrin alpha 5beta 1. Rather, this integrin seems to selectively bind proPC1 and proPC2 within the ER. The understanding of the functional implication of this interaction will surely lead to a more profound definition of the cellular biology of PCs and integrins.

    ACKNOWLEDGEMENTS

We thank S. Benjannet, A. M. Mamarbachi, J. Hamelin, J. Rochemont, J. Marcinkiewicz, A. Lemieux, and F. Parat for technical help throughout this study. Many thanks are due to A. Chen for the monoclonal Myc antibody preparation. We also thank Dr. M. Marcinkiewicz for help on the immunocytochemistry of the PC1-transfectants. The secretarial assistance of Sylvie Emond is appreciated.

    FOOTNOTES

* This work was supported by Medical Research Council program Grant PG-11474 and by the Government of Canada's Network of Centers of Excellence program supported by the Medical Research Council and Natural Sciences and Engineering Research Council of Canada through the Protein Engineering Network of Centers of Excellence.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.

§ Present address: Institut de Pharmacologie du CNRS, 660 route des Lucioles, 06560 Valbonne, Sophia-Antipolis, France. Supported in part by the Fondation Fyssen (Paris, France) and by the Fonds de la Recherche en Sante du Québec (Montreal, Quebec, Canada).

** Present address: Loeb Health Research Institute, Ottawa Civic Hospital, 725 Parkdale Ave., Ottawa, Ontario K1Y 4K9, Canada.

Dagger Dagger To whom correspondence should be addressed: Clinical Research Institute of Montreal, 110 Pine Ave. West, Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5609; Fax: 514-987-5542; E-mail: seidahn{at}ircm.qc.ca.

    ABBREVIATIONS

The abbreviations used are: PC, precursor convertase; vv, vaccinia virus; POMC, pro-opiomelanocortin; ER, endoplasmic reticulum; TGN, trans-Golgi network; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; BFA, brefeldin A; mPC1, mouse PC1; pfu, plaque-forming unit(s); aa, amino acid(s).

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ABSTRACT
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