Post-translational processing of beta-secretase (beta-amyloid-converting enzyme) and its ectodomain shedding. The pro- and transmembrane/cytosolic domains affect its cellular activity and amyloid-beta production.

Processing of the beta-amyloid precursor protein (betaAPP) by beta- and gamma-secretases generates the amyloidogenic peptide Abeta, a major factor in the etiology of Alzheimer's disease. Following the recent identification of the beta-secretase beta-amyloid-converting enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant betaAPP(sw) beta-secretase product C99. BACE is palmitoylated at three Cys residues within its transmembrane/cytosolic tail and is sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE is shed into the medium and that the extent of ectodomain shedding is palmitoylation-dependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a decrease in the alpha-secretase product APPsalpha. Although there is little increase in the generation of Abeta by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one lacking the prosegment leads to enhanced Abeta levels. These findings suggest that the shedding of BACE may play a role in the amyloidogenic processing of betaAPP.

Regional and cellular localization of the neuroendocrine prohormone convertases PC1 and PC2 in the rat central nervous system.

PC1 and PC2 are two major enzymes involved in the processing of protein precursors directed to the regulated secretory pathway. Whereas transcripts encoding both enzymes are widely distributed in the central nervous system, information regarding the localization of proteins themselves is still lacking. In an attempt to gain insight into the neurobiologic roles of PC1 and PC2, both enzymes were immunolocalized in the rat brain by using C-terminally directed antibodies, which respectively recognize the 87-kDa PC1 and the 75 and 68-kDa PC2 forms. Adjacent sections immunoreacted with PC1 or PC2 antibodies exhibited selective patterns of immunostaining in regions well characterized with respect to their biosynthesis of multiple neuropeptides such as the cerebral cortex, hippocampus, and hypothalamus. PC1 signal intensity was generally weaker than that of PC2, although both enzymes displayed extensive overlapping patterns of expression. As assessed by double-labeling experiments at the cellular level, PC1 and PC2 immunoreactive signals were localized within the trans-Golgi network and nerve terminals, in keeping with the biosynthetic pathways of neuropeptides. Immunoreactive fibers were detected in many areas throughout the brain but were particularly densely distributed in the hypothalamus and the brainstem. Both enzymes were also localized within dendrites of numerous neurons, supporting the hypothesis that dendritic neuropeptide maturation and release may occur in a large number of brain regions. Taken together, our results provide new evidence that both convertases are efficiently targeted to the neuronal regulated secretory pathway and are well poised to process protein precursors in biologically active end-products within the mammalian brain.

Endoproteolytic processing of integrin pro-alpha subunits involves the redundant function of furin and proprotein convertase (PC) 5A, but not paired basic amino acid converting enzyme (PACE) 4, PC5B or PC7.

Several integrin alpha subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-alpha5, alpha6 and alphav by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-alpha subunit substrates in vitro; the defective pro-alpha chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-alpha subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin alpha-subunit precursors. In conclusion, these results indicate that integrin alpha-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.

Biosynthesis and enzymatic characterization of human SKI-1/S1P and the processing of its inhibitory prosegment.

Biochemical and enzymatic characterization of the novel human subtilase hSKI-1 was carried out in various cell lines. Within the endoplasmic reticulum of LoVo cells, proSKI-1 is converted to SKI-1 by processing of its prosegment into 26-, 24-, 14-, 10-, and 8-kDa products, some of which remain tightly associated with the enzyme. N-terminal sequencing and mass spectrometric analysis were used to map the cleavage sites of the most abundant fragments, which were confirmed by synthetic peptide processing. To characterize its in vitro enzymatic properties, we generated a secreted form of SKI-1. Our data demonstrate that SKI-1 is a Ca(2+)-dependent proteinase exhibiting optimal cleavage at pH 6.5. We present evidence that SKI-1 processes peptides mimicking the cleavage sites of the SKI-1 prosegment, pro-brain-derived neurotrophic factor, and the sterol regulatory element-binding protein SREBP-2. Among the candidate peptides encompassing sections of the SKI-1 prosegment, the RSLK(137)- and RRLL(186)-containing peptides were best cleaved by this enzyme. Mutagenesis of the latter peptide allowed us to develop an efficiently processed SKI-1 substrate and to assess the importance of several P and P' residues. Finally, we demonstrate that, in vitro, recombinant prosegments of SKI-1 inhibit its activity with apparent inhibitor constants of 100-200 nM.

The prosegments of furin and PC7 as potent inhibitors of proprotein convertases. In vitro and ex vivo assessment of their efficacy and selectivity.

All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.

Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization.

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.

The subtilisin/kexin family of precursor convertases. Emphasis on PC1, PC2/7B2, POMC and the novel enzyme SKI-1.

Proopiomelanocortin (POMC) is a precursor to various, bioactive peptides including ACTH, beta LPH, alpha MSH, and beta endorphin (beta END). Processing of POMC at dibasic residues is tissue-specific and is performed by either PC1 alone (resulting in ACTH and beta LPH, anterior pituitary corticotrophes) or by a combination of PC1 and PC2 (yielding alpha MSH and beta END, pituitary neurointermediate lobe and hypothalamus). The PC2-specific binding protein 7B2 is intimately involved in the zymogen activation of proPC2 into PC2. Structure-function studies of these enzymes demonstrated the presence of N- and C-terminal domains, as well as specific amino acids within the catalytic segment that influence the degree of activity of each enzyme and the interaction of PC2 with 7B2. The tissue distribution, plasticity of expression, and the multiple precursors that are differentially cleaved by PC1 and/or PC2, predict a wide array of combinatorial activities of these convertases within the endocrine and neuroendocrine system. The phenotypic consequences of the absence of genetic expression of either PC1 or PC2 are now explored using knockout mice and in human patients suffering from obesity and diabetes.

The LIM homeobox protein mLIM3/Lhx3 induces expression of the prolactin gene by a Pit-1/GHF-1-independent pathway in corticotroph AtT20 cells.

mLIM3, a member of the LIM homeobox family, was recently demonstrated to be critical for proliferation and differentiation of the pituitary cell lineage. Using a pool of degenerate oligonucleotides we determined the DNA sequence ANNAGGAAA(T/C)GA(CIG)AA as the set preferentially recognized by mLIM3. A nearly identical sequence is found in the prolactin (PRL) promoter, within a 15-mer stretch from nucleotides (nts) -218 to -204 which is highly conserved between human, rat, and bovine. In order to test the hypothesis of a transcriptional effect of mLIM3 on the prolactin promoter, stable transfectants of mLIM3 cDNA in AtT20 tumor cells revealed that PRL mRNA expression was induced in 3 separate stable clones. Gel retardation experiments performed using nuclear extracts isolated from one of the AtT20/mLIM3 stable transfectants revealed affinity towards the 15-mer element of the PRL promoter. From these results, we propose that the PRL promoter element (nts -218 to -204) could be functional in vivo. Finally, we demonstrate that in AtT20 cells prolactin mRNA expression is not induced by the Pit-1/GHF-1 pathway and that growth hormone mRNA is not detected concomitantly with prolactin. We conclude that mLIM3 may play a key role in inducing PRL gene expression in lactotrophs by binding to a conserved motif close to a Pit-1/GHF-1 site within the proximal PRL promoter.

Residues unique to the pro-hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity.

The prohormone convertase PC2 is one of the major subtilisin/kexin-like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin-like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2-specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2-specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site-directed mutagenesis of Tyr194 and of the oxyanion hole Asp309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into beta-endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D-PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co-immunoprecipitations and Western blots, binding assays indicate that Tyr194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp309 is critical for the binding of proPC2 with pro7B2.

The integrity of the RRGDL sequence of the proprotein convertase PC1 is critical for its zymogen and C-terminal processing and for its cellular trafficking.

In order to define the functional importance of the conserved RRGDL motif in the P-domain of the mammalian proprotein convertases(PCs) we generated and cellularly expressed three mutant PC1 vaccinia-virus (VV) recombinants: ARGDL-PC1, RAGDL-PC1 and RRGEL-PC1. Functionally, these mutants caused a decreased level of processing of pro-opiomelanocortin (POMC) into beta-lipotropic pituitary hormone (beta-LPH), especially in the constitutively secreting BSC40 cells. Pulse-chase analyses demonstrated that, in part, this effect was due to both an increased degradation of the mutant PC1s within the endoplasmic reticulum and to a diminished level of zymogen processing in the same compartment. In addition, within cells containing secretory granules such as PC12 and GH4C1 cells, such mutations prevented the C-terminal auto-processing of PC1 into the fully mature 66 kDa form stored in the secretory granules of regulated cells. Since the 66 kDa PC1 is the most active form of the enzyme, it is proposed that the RRGDL sequence is critical for the generation of maximal intracellular PC1 activity. In regulated cells, co-expression of POMC with PC1 or its mutants together with the general PC inhibitor alpha1-antitrypsin Portland (alpha1-PDX), which acts primarily within the constitutive secretory pathway, demonstrated that the latter completely inhibited the formation of beta-LPH by PC1 mutants, whereas it only partially inhibited the ability of wild-type PC1 to process POMC. This suggests that RRGDL mutations prevent PC1 from entering secretory granules and hence the formation of the 66 kDa PC1, and result in the mis-sorting of PC1 mutants towards the constitutive secretory pathway. This conclusion was further supported by immunocytochemical data demonstrating that RRGDL mutants exhibit an intracellular localization pattern different from that of the granule-associated wild-type PC1,but similar to that of the Golgi-localized convertase PC5-B.

Alpha1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway.

We studied the extent of cellular inhibitory activity of alpha1-antitrypsin Portland (alpha1-PDX), a potent inhibitor of proprotein convertases of the subtilisin/kexin type. We compared the inhibitory effects of alpha1-PDX on the intracellular processing of two model precursors (pro-7B2 and POMC) mediated by six of the seven known mammalian convertases, namely furin, PC1, PC2, PACE4, PC5-A, PC5-B, and PC7. The substrates selected were pro7B2, a precursor cleaved within the trans-Golgi network (TGN), and pro-opiomelanocortin, which is processed in the TGN and secretory granules. Biosynthetic analyses were performed using either vaccinia virus expression in BSC40, GH4C1, and AtT20 cells, or stable transfectants of alpha1-PDX in AtT20 cells. Results revealed that alpha1-PDX inhibits processing of these precursors primarily within the constitutive secretory pathway and that alpha1-PDX is cleaved into a shorter form by some convertases. Evidence is presented demonstrating that in contrast to the full-length alpha1-PDX (64 kDa), the cleaved (56 kDa) secreted product does not significantly inhibit furin activity in vitro. Cellular expression of alpha1-PDX results in modified contents of mature secretory granules with increased levels of partially processed products. Biosynthetic and immunocytochemical analyses of AtT20/alpha1-PDX cells demonstrated that alpha1-PDX is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as the cleaved product.

In vitro characterization of the novel proprotein convertase PC7.

Biochemical and enzymatic characterization of the novel proprotein convertase rat PC7 (rPC7) was carried out using vaccinia virus recombinants overexpressed in mammalian BSC40 cells. Pro-PC7 is synthesized as a glycosylated zymogen (101 kDa) and processed into mature rPC7 (89 kDa) in the endoplasmic reticulum. No endogenously produced soluble forms of this membrane-anchored protein were detected. A deletion mutant (65 kDa), truncated well beyond the expected C-terminal boundary of the P-domain, produced soluble rPC7 in the culture medium. Enzymatic activity assays of rPC7 using fluorogenic peptidyl substrates indicated that the pH optimum, Ca2+ dependence, and cleavage specificity of this enzyme are largely similar to those of furin. However, with some substrates, cleavage specificity more closely resembled that of yeast kexin, suggesting differential processing of proprotein substrates by this novel convertase. We examined the rPC7- and human furin-mediated cleavage of synthetic peptides containing the processing sites of three proteins known to colocalize in situ with rPC7. Whereas both enzymes correctly processed the pro-parathyroid hormone tridecapeptide and the pro-PC4 heptadecapeptide, neither enzyme cleaved a pro-epidermal growth factor hexadecapeptide. Thus, this study establishes that rPC7 is an enzymatically functional subtilisin/kexin-like serine proteinase with a cleavage specificity resembling that of hfurin. In addition, we have demonstrated that rPC7 can correctly process peptide precursors that contain the processing sites of at least two potential physiological substrates.

Comparative functional role of PC7 and furin in the processing of the HIV envelope glycoprotein gp160.

The intracellular proteolytic processing of HIV envelope glycoprotein gp160 into gp120/gp41 is an essential step for virus infectivity. Several convertases, belonging to the pro-protein convertase family, have been proposed as candidate gp160 processing enzymes. Here we demonstrate using RT-PCR that resting human T4 lymphocytes weakly express PC7, furin, and PC5 mRNA whereas lymphocytes activated under conditions favoring HIV replication express 5-10-fold higher levels of furin and PC7. In this report, we examined the capability of the newly cloned convertase PC7 to cleave gp160 into gp120/gp41 and compared it to furin. This was carried out in a cell-based assay whereby both gp160 and the cognate convertase were co-expressed in the constitutively secreting BSC40 cells and in the regulated AtT20 cells, as well as using two in vitro assays which examined the cleavage of gp160 or of a synthetic peptide spanning the cleavage site. The data demonstrate that PC7 can cleave specifically and in a cell-type specific manner gp160 into gp120gp41, suggesting that both furin and PC7 are so far the major PC-like candidate gp160 convertase in T4 lymphocytes.

Functional analysis of human PACE4-A and PACE4-C isoforms: identification of a new PACE4-CS isoform.

There are seven known subtilisin/kexin-like proprotein convertases responsible for the processing of numerous precursors at either pairs or specific single basic residues. Three members, PACE4, PC4 and PC5, exhibit alternative splicing of their RNAs resulting in the generation of multiple isoforms differing in their C- or N-terminal segments. In this study we examined the biosynthesis, functional activity and cellular localization of two of these isoforms, namely the full length PACE4-A and the C-terminally truncated PACE4-C which lacks 11 amino acids at the end of its chaperone-like P-domain. We report the existence of a new isoform, termed PACE4-CS, which is a C-terminally shortened version of PACE4-C. Cellular expression results demonstrated that PACE4-A codes for a functional secretable enzyme capable of cleaving pro7B2 into 7B2. In contrast, PACE4-CS is not secreted since it remains in the endoplasmic reticulum as an inactive zymogen form, thereby emphasizing the importance of the integrity of the P-domain. Microsequencing of the intracellular PACE4-CS protein in two cell lines revealed that it is proPACE4-CS with an N-terminal trimming reminiscent of the action of a dipeptidylpeptidase recognizing the motifs X-Ala and X-Pro.

Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases.

In order to define the enzymes responsible for the maturation of the precursor of nerve growth factor (proNGF), its biosynthesis and intracellular processing by the pro-protein convertases furin, PC1, PC2, PACE4, PC5 and the PC5 isoform PC5/6-B were analysed using the vaccinia virus expression system in cells containing a regulated and/or a constitutive secretory pathway. Results demonstrate that in both cell types furin, and to a lesser extent PACE4 and PC5/6-B, are the best candidate proNGF convertases. Furthermore, two processed NGF forms of 16.5 and 13.5 kDa were evident in constitutively secreting cell lines such as LoVo and BSC40 cells, whereas only the 13.5 kDa form was observed in AtT20 cells, which contain secretory granules. Both forms display the same N-terminal sequence as mature NGF, and were also produced following site-directed mutagenesis of the C-terminal Arg-Arg sequence of NGF into Ala-Ala, suggesting that the difference between them is not at the C-terminus. Co-expression of proNGF with furin and either chromogranin B or secretogranin II (but not chromogranin A) in BSC40 cells eliminated the 16.5 kDa form. Data also show that N-glycosylation of the pro-segment of proNGF and trimming of the oligosaccharide chains are necessary for the exit of this precursor from the endoplasmic reticulum and its eventual processing and secretion. Sulphate labelling experiments demonstrated that proNGF is processed into mature NGF following the arrival of the precursor in the trans-Golgi network. This comparative study shows that the three candidate mammalian subtilisin/kexin-like convertases identified process proNGF into NGF and that the nature of the final processed products is dependent on the intracellular environment.

Comparative cellular processing of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases.

We present here the pulse and pulse-chase analysis of the biosynthesis of the envelope glycoprotein gp160 and its intracellular processing by the subtilisin/kexin-like convertases furin, PACE4, PC1, PC5 and its isoform PC5/6-B. We demonstrate that furin and to a much lesser extent PACE4, PC5/6-B and PC1 are candidate enzymes capable of processing gp160 intracellularly. Furthermore we show that furin can also process gp160/gp120 into gp77/gp53 products by cleavage at the sequence RIQR/GPGR just preceding the conserved GPGR structure found at the tip of the hypervariable V3 loop. The results show that processing into gp120 could occur at or before the trans-Golgi network (TGN) where sulphation of the oligosaccharide moieties of gp160 was detected. In contrast, the formation of gp77/gp53 by furin is a late event occurring after exit from the TGN. Our data also revealed that the alpha glucosidase I inhibitor N-butyldeoxynojirimycin, although affecting the oligosaccharide composition of gp160, does not impair the processing of either gp160 or gp120 by either furin or PACE4. Finally, the co-expression of the [Arg355, Arg358]-alpha-1-antitrypsin Portland variant was shown to potently inhibit the processing of both gp160 and gp120 by these convertases.

Cellular processing of the neurotrophin precursors of NT3 and BDNF by the mammalian proprotein convertases.

In order to define the enzymes responsible for the maturation of the precursors of brain-derived neurotrophic factor (proBDNF) and neurotrophin-3 (proNT3), we have analysed their biosynthesis and intracellular processing by the proprotein convertases furin, PC1, PC2, PACE4, PC5 and its isoform PC5/6-B. In these studies, we utilized a vaccinia virus expression system in either BSC40 or the furin activity-deficient LoVo cells. Results demonstrated that in both cells furin and, to a lesser extent, PACE4 and PC5/6-B effectively process proBDNF and proNT3. Furthermore, we have determined that human proNT3 is sulfated, suggesting that processing of proNT3 occurs following the arrival of the precursor to the Trans Golgi Network.

7B2 is a specific intracellular binding protein of the prohormone convertase PC2.

Biosynthetic pulse-chase analyses have previously demonstrated that the prohormone convertase PC2 is first synthesized as a precursor pro-PC2 and that zymogen activation to PC2 occurs following the slow exit of pro-PC2 from the endoplasmic reticulum (ER) and its concentration within the trans-Golgi network (TGN). The endocrine and neural protein 7B2 is first synthesized as a nonglycosylated precursor (pro-7B2), which is cleaved within the TGN by a furin-like ubiquitous convertase at the RRKRR155S site to generate 7B2. In this report, we demonstrate that within the ER, pro-7B2 binds pro-PC2 but not any of the other convertases furin, PC1, PACE4, or PC5. This specific binding is Ca2+ dependent and does not require an N-glycosylated pro-PC2. Mutagenesis of the RRKRRS sequence demonstrated that the intact hexapeptide is critical for this binding, because the latter was abolished by mutations of the RR152 and greatly diminished by mutations of either the R151 or S156 residues of pro-7B2. Once the complex is formed in the ER, it is then transported to the TGN where furin or a furin-like convertase cleaves both precursors, even when present as a complex. We also provide evidence that following zymogen cleavage, 7B2 remains bound to PC2, suggesting the presence of at least one other Ca(2+)-dependent binding site within the 7B2 sequence. Coexpression of 7B2 and PC2, although resulting in an elevation of the level of pro-PC2, did not eliminate the processing of pro-PC2 to PC2.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function studies on the biosynthesis and bioactivity of the precursor convertase PC2 and the formation of the PC2/7B2 complex.

Site directed mutagenesis of the prohormone convertase PC2 was used to define the effect of certain residues on the zymogen activation of proPC2 and on its binding to the neuroendocrine protein 7B2. These included the oxyanion hole Asp309 (D309N), the N-terminal Glu25 (E25Q and E25K) of proPC2 and the Asp519 (D519E) of the RGD motif within the P-domain of PC2. Heterologous vaccinia virus expression of the wild type and mutant PC2's in endocrine pituitary cells such as AtT20 and GH3 cells demonstrated that the most dramatic effect was observed with the D309N mutant which no longer bound pro7B2 and which exhibited a significant reduction in its capacity to produce beta-endorphin from pro-opiomelanocortin (POMC).

Maintained PC1 and PC2 expression in the AtT-20 variant cell line 6T3 lacking regulated secretion and POMC: restored POMC expression and regulated secretion after cAMP treatment.

Two variant cell lines were recently established from parent AtT-20 cells. Whereas HYA.15.10.T.2 have a reduced level of secretory granules, HYA.15.6.T.3 are completely devoid of both the regulated pathway of secretion and of dense-core secretory granules. AtT-20 cells normally express the processing enzymes PC1, PC2, furin, carboxypeptidase E, and peptidylglycine alpha-amidating monooxygenase, as well as proopiomelanocortin, chromogranin B, and 7B2. We measured the expression of these mRNAs in both variant cell lines. Although some differences in mRNA level were noted, HYA.15.10.T.2 and HYA.15.6.T.3 cell lines maintained their expression of the processing enzymes and of 7B2. Furthermore, PC1 and PC2 were shown to be functionally active in the HYA.15.6.T.3 cells. In contrast, proopiomelanocortin and chromogranin B mRNA levels were no longer detectable in HYA.15.6.T.3 cells. Interestingly, stimulation of the HYA.15.6.T.3 cells with cAMP restored proopiomelanocortin mRNA, beta-endorphin immunoreactivity, and dense-core granules. Furthermore, at the ultrastructural level, beta-lipotropin immunoreactivity was detected in granules of cAMP-induced HYA.15.6.T.3 cells. Finally, depolarization of cAMP-induced HYA.15.6.T.3 cells with 56 mM potassium chloride resulted in a marked increase in the release of beta-endorphin immunoreactivity. These observations demonstrate that cAMP restores the regulated pathway of secretion in HYA.15.6.T.3 cells, which under untreated conditions do not demonstrate regulated release. These variant cell lines are unique models to understand better the relationship of the regulated pathway and the expression of the processing enzymes.