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Differential subcellular distribution of PC1, PC2 and furin in bovine adrenal medulla and secretion of PC1 and PC2 from this tissue
Neuroscience Letters, 143 (1992) 143 145 ¢~) 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
143
NSL 08877
Differential subcellular distribution of PC1, PC2 and furin in bovine adrenal medulla and secretion of PC1 and PC2 from this tissue R. Kirchmair a, C. Egger ", R Gee b, R. Hogue-Angeletti b, R. Fischer-Colbrie ~', A. Laslop ~ a n d H. Winkler ~ '~Department o/Pharmacology, University ~['Innsbruck, Innsbruck (Austria) and bDevelopmental Biology and Cancer, Alhert Einstein ('ollege Of Medicbw, Bronx, N Y ~USA ; (Received 4 May 1992: Revised version received 29 May 1992; Accepted 29 May 1992)
Key words." Endopeptidase: Secretion: Chromaffin granule The subcellular distribution of PC l, PC2 and furin was determined in bovine adrenal medulla by immunoblotting of fractions obtained by density gradient centrifugation. PC1 and PC2 were found to be confined Io chromaffin granules whereas furin (C-terminal-peptide) was absent from these organelles. Stimulation of bovine adrenal medulla by carbamoylcholine chloride induced the secretion of PC1 and PC2. The secreted enzymes had the same molecular size as PC1 and PC2 present in chromaffin granules.
Processing of propeptides requires cleavage at pairs of basic residues [10]. Three enzymes have been proposed to represent the required endoproteases. These include furin [1, 14, 22], PC1 [16, 17, 19] and PC2 [16, 18]. These two latter enzymes are found only in neuronal and endocrine cells [16, 18], whereas furin has a more widespread distribution [8]. Cleavage of prohormones by PC1 and PC2 requires [2] that these enzymes are colocalized in secretion granules. In fact both enzymes were found in purified chromaffin granules [5, 9], however a detailed subcellular distribution has not yet been obtained. For furin on the other hand it was claimed that this enzyme is confined to the Golgi region of constitutively secreting cells as shown by immunohistochemistry [4, 12]. In the present paper we have compared the subcellular distribution of these enzymes in an endocrine tissue, i.e. the bovine adrenal medulla, which secretes by the regulated pathway. In addition we have determined whether PCI and PC2 are secreted, when the gland is stimulated by carbamoylcholine chloride. For subcellular tYactionation [20] a large granule fraction was isolated from bovine adrenal medulla and subjected to sucrose density gradient centrifugation. The gradient fractions were diluted with 0.3 M sucrose (a third of volume added) and centrifuged to sediment all
Correspondence: H. Winkler, Department of Pharmacology, University of Innsbruck, Peter-Mayr-Strage la, A-6020 Innsbruck, Austria. Fax: 43-512-507-2419.
cell particles. The sediments were used for immunoblotting [7]. Antisera were raised in rabbits [9] against synthetic peptides coupled to keyhole limpet haemocyanin via a cysteine residue. For PC1 an 18-mer corresponding to the C-terminal amino acids 709 726 [17], for PC2 a 19mer corresponding to amino acids 592 610 [17], for furin a 22-mer corresponding to the rat sequence amino acids 772-793 [11] and for glycoprotein III a 16-mer corresponding to the bovine sequence amino acids 1 16 [13] were used. For secretion experiments perfusates were collected from retrogradely perfused bovine glands [7, 15] during control periods and during stimulation with carbamoylcholine chloride. After dialysis and freeze drying the samples were analysed by immunoblotting. Fig. 1. demonstrates the subcellular distribution of the three enzymes in bovine adrenal medulla. The position of chromaffin granules in the gradient is indicated by the peak of glycoprotein III immunoreactivity in the dense fractions [6]. The distribution of PCI and PC2 exactly matches that of the granule marker. Small amounts of this marker and of the enzymes are also present in the top fractions of the gradient where membranes of damaged chromaffin granules equilibrate. Previous studies [5, 9] have shown that purified chromaffin granules contain these enzymes, the present results establish that these two enzymes are specifically confined to chromaffin granules and apparently not found in other cell compartments. Since the large dense core vesicles of neurones, at least
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9
PC1 iC
10
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PC2 C
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85
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PC1
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45-
30-
70
Pc2 90"
FUR4N Fig. 1. Subcellular distribution of PC 1, PC2, furin and glycoprotein III. A large granule fraction of bovine adrenal medulla was subjected to density gradient centrifugation. The gradient fractions were analyzed by one-dimensional immunoblotting. The fractions from I to 10 correspond to the fractions from the bottom to the top of the gradient.
the adrenergic ones, are very similar to chromaffin granules in their protein composition [23] these results indicate that the neuronal vesicles also contain PCI and PC2. In fact their mRNAs have already been found in nervous tissue [16]. The antisera against a synthetic peptide present in rat furin immunostained in rat (results not shown) and bovine (Fig. 1) adrenal medulla a band of Mr 90000. As shown previously [12] in cells transfected with cDNA for rat furin a 94 kDa protein is immunoprecipitated by furin antibodies. Our results now demonstrate that there is apparently a good cross-immunoreactivity between the rat and bovine enzyme. The subcellular distribution of furin differed markedly from those of PC 1 and PC2 (see Fig. 1). The enzyme was found in the top fraction of the gradient where microsomal elements including mem-
Fig. 2. Secretion of PC1 and PC2 from adrenal medulla. Perfusates were collected from bovine adrenal medulla during control and stimulation periods with carbamoylcholine chloride and subjected to onedimensional immunoblotting. CG. chromaffin granule membranes: C, control perfusate; S, perfusate during stimulation• Amounts of protein used were: PC 1: CG 20 pg, C 125 pg, S 250 pg; PC2: CG 10 pig, C 75 pg, S 150pg.
branes of the Golgi region are found [21]. This is in agreement with previous immunohistochemical data on transfected COS-1 and BSC-40 cells [4, 12]. However in these cells a regulated pathway of secretion is absent. The present study now establishes that in cells with secretory granules furin (or to be more specific, at least its C-terminal region) is not sorted into these organelles. This is consistent with the view [8] that this enzyme is primarily not involved in the processing of secretory propeptides. Fig. 2 demonstrates that PC1 and PC2 appear in perfusates of bovine adrenal medulla stimulated with carbamoylcholine chloride. It has been previously shown [9] that in bovine chromaffin granules a significant amount of PC2 is soluble whereas by far the larger part of PC 1 is membrane-bound. In agreement, we found in the perfusates a strong immunostaining for PC2, but only a rather weak one for PC1. Thus the present study establishes that these two enzymes represent further compo-
145
nents of the secretory cocktail released from this organ and in analogy probably also from neurons containing large dense core vesicles resembling chromaffin granules. It should be emphasized that the secreted forms of PC1 and PC2 had the same molecular sizes as the soluble and membrane-bound form of these two enzymes [9]. Secretion of PC1 and PC2 from mammalian cells normally producing these enzymes has not been demonstrated previously, however GH4 cells transfected with PC1 and PC2 cDNA also secreted them [3]. In this case, only secreted PC1 had the same size as the intracellular one, whereas PC2 secreted into the medium was smaller than the one stored cellularly. In chromaffin granules PC1 and PC2 can now be included into the ever growing group of peptides which are both membrane-bound, but also secreted together with the catecholamines and chromogranins. Other members of this group are dopamine fl-hydroxylase, glycoprotein III, and the other peptide converting enzymes carboxypeptidase H and the alpha-amidating enzyme [23]. This work was supported by the Fonds zur F6rderung ,ter wissenschaftlichen Forschung (Austria), by the Dr. Legerlotz-Stiftung and by funds from NIH (Grant NS 22697, R.H.-A.) 1 Barr, P.J., Mason, O.B., Landsberg, K.E., Wong, RA., Kiefer, M.C. and Brake, A.J., cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues, DNA and Cell Biol., 10 (1991) 319 328. 2 Benjannet, S., Rondaeu, N., Day, R., Chrdtiem M. and Seidah, N.G., PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues, Proc. Natl. Acad. Sci. USA, 88 (1991) 3564-3568. 3 Benjannet, S., Reudelhuber, T., Mercure, C., Rondeau. N., Chrdtien, M. and Seidah, N.G., Pro-protein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity and intracellular environment, J. Biol. Chem., in press. 4 Bresnahan, RA., Leduc, R., Thomas, L., Thorner, J., Gibson, H.L., Brake, A.J., Barr, RJ. and Thomas, G., Human fur gene encodes a yeast KEX2-1ike endoprotease that cleaves pro-fl-NGF in viw~, J. Cell Biol., 111 (1990)2851 2859. 5 Christie, D.L., Batchelor, D.C. and Palmer, D.J., Identification of kex2-related proteases in chromaffin granules by partial amino acid sequence analysis, J. Biol. Chem., 266 (1991) 15679 15683. 6 Fischer-Colbrie, R., Zangerle, R., Frischenschlager, 1., Weber. A. and Winkler, H., Isolation and immunological characterization of a gtycoprotein from adrenal chromaffin granules, J. Neurochem.. 42 (1984) 1008 1016. 7 Fischer-Colbrie, R. and Frischenschlager, I., Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranins B, and enkephalin-containing peptides, J. Neurochem., 44 (1985) 1854-1861. 8 Hatsuzawa, K., Hosaka, M., Nakagawa, T., Nagase, M., Shoda, A., Murakami, K. and Nakayama, K., Structure and expression of mouse furin, a yeast Kex2-related protease, J. Biol. Chem., 265 (1990) 22075 22078.
9 Kirchmair, R., Gee, P., Hogue-Angeletti, R., Laslop, A., FischerColbrie, R. and Winkler, H., Immunological characterization of the endoproteases PC1 and PC2 in adrenal chromaffin granules and in the pituitary gland, FEBS Lett., 297 (1992) 302 305. 10 Lindberg, 1., The new eukaryotic precursor processing proteinases, Mol. Endocrinol.,5(1991) 1361 1365. 11 Misumi, Y., Sohda, M. and Ikehara, Y., Sequence of the eDNA encoding rat furin, a possible propeptide-processing endoprotease, Nucleic Acids Res., 18 (1990) 6719. 12 Misumi, Y., Oda, K., Fujiwara, T., Takami, N., Tashiro, K. and lkehara, Y.. Functional expression of furin demonstrating its intracellular localization and endoprotease activity tk)r processing of proalbumin and complement pro-C3. J. Biol. Chem., 266 (1991) 16954 16959. 13 Palmer, D.J. and Christie, D.L., The primary structure of glycoprotein Ill from bovine adrenal medullary chromaffin granules, J. Biol. Chem., 265 (1990) 6617 6623. 14 Roebroek, A.J.M., Schalken, J.A., Leunissen, J.A.M., Onnekink, C., Bloemers, H.P.J. and Van den Ven, W.J.M., Evolutionary conserved close linkage of the c-fed/fps prolo-oncogene and genetic sequences encoding a receptor-like protein, EMBO J., 5 (1986) "~9 7 -,0-. '~'~ -1 15 Schneider, F.H., Smith, A.D. and Winkler, H., Secretion from the adrenal medulla: biochemical evidence for exocytosis, Br. J. Pharmac. Chemother.,31 (1967) 94 104. 16 Seidah. N.G., Gaspar, L., Mion, P., Marcinkiewicz, M., Mbikay, M. and Chretien, M., cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases, DNA Cell Biol., 9 (1990) 415 424. 17 Seidah. N.G., Marcinkiewicz, M., Benjannet, S., Gaspar, L., Beaubien, G., Mallei, M.G., Lazure, C., Mbikay, M. and Chretien, M., Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2, Mol. Endocrinol., 5 (1991) 111 122. 18 Smeekens, S.P. and Steiner, D.F., Identification of a human insulinoma eDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing pro/ease Kex2, J. Biol. ('hem., 265 (1990) 2997 3000. 19 Smeekens, S.P., Avruch, A.S., LaMendola, J., Chan, S.J. and Steiner. D.F., Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans, Proc. Natl. Acad. Sci. USA, 88 (1991) 340 344. 20 Smith, A.D. and Winkler, H., The localization of lysosomal enzymes in chromaffin tissue, J. Physiol., 183 (19661 179 188. 21 Trifaro, J.M., Duerr, A.C. and Pinto, J.E.B., Membranes of the adrenal medulla: a comparison between the membranes of the golgi apparatus and chromaffin granules, Mol. Pharmacol., 12 (1976) 536 545. 22 Van den Ven, W.J.M., Voorberg, J., Fontijn, R., Pannekoek, H., Van dcn Ouweland, A.M.W., Van Duijnhoven, H.L.P., Roebroek, A.J.M. and Siezen, R.J., Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes, Mol. Biol. Rep., 14 (1990) _65 _7,. 23 Winkler, H. and Fischer-Colbrie, R., Common membrane proteins ofchromaffin granules, endocrine and synaptic vesicles: properties, tissue distribution, membrane topography and regulation of synthesis, Neurochem. Int., 17 (1990) 245 262.
143
NSL 08877
Differential subcellular distribution of PC1, PC2 and furin in bovine adrenal medulla and secretion of PC1 and PC2 from this tissue R. Kirchmair a, C. Egger ", R Gee b, R. Hogue-Angeletti b, R. Fischer-Colbrie ~', A. Laslop ~ a n d H. Winkler ~ '~Department o/Pharmacology, University ~['Innsbruck, Innsbruck (Austria) and bDevelopmental Biology and Cancer, Alhert Einstein ('ollege Of Medicbw, Bronx, N Y ~USA ; (Received 4 May 1992: Revised version received 29 May 1992; Accepted 29 May 1992)
Key words." Endopeptidase: Secretion: Chromaffin granule The subcellular distribution of PC l, PC2 and furin was determined in bovine adrenal medulla by immunoblotting of fractions obtained by density gradient centrifugation. PC1 and PC2 were found to be confined Io chromaffin granules whereas furin (C-terminal-peptide) was absent from these organelles. Stimulation of bovine adrenal medulla by carbamoylcholine chloride induced the secretion of PC1 and PC2. The secreted enzymes had the same molecular size as PC1 and PC2 present in chromaffin granules.
Processing of propeptides requires cleavage at pairs of basic residues [10]. Three enzymes have been proposed to represent the required endoproteases. These include furin [1, 14, 22], PC1 [16, 17, 19] and PC2 [16, 18]. These two latter enzymes are found only in neuronal and endocrine cells [16, 18], whereas furin has a more widespread distribution [8]. Cleavage of prohormones by PC1 and PC2 requires [2] that these enzymes are colocalized in secretion granules. In fact both enzymes were found in purified chromaffin granules [5, 9], however a detailed subcellular distribution has not yet been obtained. For furin on the other hand it was claimed that this enzyme is confined to the Golgi region of constitutively secreting cells as shown by immunohistochemistry [4, 12]. In the present paper we have compared the subcellular distribution of these enzymes in an endocrine tissue, i.e. the bovine adrenal medulla, which secretes by the regulated pathway. In addition we have determined whether PCI and PC2 are secreted, when the gland is stimulated by carbamoylcholine chloride. For subcellular tYactionation [20] a large granule fraction was isolated from bovine adrenal medulla and subjected to sucrose density gradient centrifugation. The gradient fractions were diluted with 0.3 M sucrose (a third of volume added) and centrifuged to sediment all
Correspondence: H. Winkler, Department of Pharmacology, University of Innsbruck, Peter-Mayr-Strage la, A-6020 Innsbruck, Austria. Fax: 43-512-507-2419.
cell particles. The sediments were used for immunoblotting [7]. Antisera were raised in rabbits [9] against synthetic peptides coupled to keyhole limpet haemocyanin via a cysteine residue. For PC1 an 18-mer corresponding to the C-terminal amino acids 709 726 [17], for PC2 a 19mer corresponding to amino acids 592 610 [17], for furin a 22-mer corresponding to the rat sequence amino acids 772-793 [11] and for glycoprotein III a 16-mer corresponding to the bovine sequence amino acids 1 16 [13] were used. For secretion experiments perfusates were collected from retrogradely perfused bovine glands [7, 15] during control periods and during stimulation with carbamoylcholine chloride. After dialysis and freeze drying the samples were analysed by immunoblotting. Fig. 1. demonstrates the subcellular distribution of the three enzymes in bovine adrenal medulla. The position of chromaffin granules in the gradient is indicated by the peak of glycoprotein III immunoreactivity in the dense fractions [6]. The distribution of PCI and PC2 exactly matches that of the granule marker. Small amounts of this marker and of the enzymes are also present in the top fractions of the gradient where membranes of damaged chromaffin granules equilibrate. Previous studies [5, 9] have shown that purified chromaffin granules contain these enzymes, the present results establish that these two enzymes are specifically confined to chromaffin granules and apparently not found in other cell compartments. Since the large dense core vesicles of neurones, at least
144
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PC1 iC
10
I
PC2 C
94GPflf 67-
85
o o
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£
o
m
45-
30-
70
Pc2 90"
FUR4N Fig. 1. Subcellular distribution of PC 1, PC2, furin and glycoprotein III. A large granule fraction of bovine adrenal medulla was subjected to density gradient centrifugation. The gradient fractions were analyzed by one-dimensional immunoblotting. The fractions from I to 10 correspond to the fractions from the bottom to the top of the gradient.
the adrenergic ones, are very similar to chromaffin granules in their protein composition [23] these results indicate that the neuronal vesicles also contain PCI and PC2. In fact their mRNAs have already been found in nervous tissue [16]. The antisera against a synthetic peptide present in rat furin immunostained in rat (results not shown) and bovine (Fig. 1) adrenal medulla a band of Mr 90000. As shown previously [12] in cells transfected with cDNA for rat furin a 94 kDa protein is immunoprecipitated by furin antibodies. Our results now demonstrate that there is apparently a good cross-immunoreactivity between the rat and bovine enzyme. The subcellular distribution of furin differed markedly from those of PC 1 and PC2 (see Fig. 1). The enzyme was found in the top fraction of the gradient where microsomal elements including mem-
Fig. 2. Secretion of PC1 and PC2 from adrenal medulla. Perfusates were collected from bovine adrenal medulla during control and stimulation periods with carbamoylcholine chloride and subjected to onedimensional immunoblotting. CG. chromaffin granule membranes: C, control perfusate; S, perfusate during stimulation• Amounts of protein used were: PC 1: CG 20 pg, C 125 pg, S 250 pg; PC2: CG 10 pig, C 75 pg, S 150pg.
branes of the Golgi region are found [21]. This is in agreement with previous immunohistochemical data on transfected COS-1 and BSC-40 cells [4, 12]. However in these cells a regulated pathway of secretion is absent. The present study now establishes that in cells with secretory granules furin (or to be more specific, at least its C-terminal region) is not sorted into these organelles. This is consistent with the view [8] that this enzyme is primarily not involved in the processing of secretory propeptides. Fig. 2 demonstrates that PC1 and PC2 appear in perfusates of bovine adrenal medulla stimulated with carbamoylcholine chloride. It has been previously shown [9] that in bovine chromaffin granules a significant amount of PC2 is soluble whereas by far the larger part of PC 1 is membrane-bound. In agreement, we found in the perfusates a strong immunostaining for PC2, but only a rather weak one for PC1. Thus the present study establishes that these two enzymes represent further compo-
145
nents of the secretory cocktail released from this organ and in analogy probably also from neurons containing large dense core vesicles resembling chromaffin granules. It should be emphasized that the secreted forms of PC1 and PC2 had the same molecular sizes as the soluble and membrane-bound form of these two enzymes [9]. Secretion of PC1 and PC2 from mammalian cells normally producing these enzymes has not been demonstrated previously, however GH4 cells transfected with PC1 and PC2 cDNA also secreted them [3]. In this case, only secreted PC1 had the same size as the intracellular one, whereas PC2 secreted into the medium was smaller than the one stored cellularly. In chromaffin granules PC1 and PC2 can now be included into the ever growing group of peptides which are both membrane-bound, but also secreted together with the catecholamines and chromogranins. Other members of this group are dopamine fl-hydroxylase, glycoprotein III, and the other peptide converting enzymes carboxypeptidase H and the alpha-amidating enzyme [23]. This work was supported by the Fonds zur F6rderung ,ter wissenschaftlichen Forschung (Austria), by the Dr. Legerlotz-Stiftung and by funds from NIH (Grant NS 22697, R.H.-A.) 1 Barr, P.J., Mason, O.B., Landsberg, K.E., Wong, RA., Kiefer, M.C. and Brake, A.J., cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues, DNA and Cell Biol., 10 (1991) 319 328. 2 Benjannet, S., Rondaeu, N., Day, R., Chrdtiem M. and Seidah, N.G., PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues, Proc. Natl. Acad. Sci. USA, 88 (1991) 3564-3568. 3 Benjannet, S., Reudelhuber, T., Mercure, C., Rondeau. N., Chrdtien, M. and Seidah, N.G., Pro-protein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity and intracellular environment, J. Biol. Chem., in press. 4 Bresnahan, RA., Leduc, R., Thomas, L., Thorner, J., Gibson, H.L., Brake, A.J., Barr, RJ. and Thomas, G., Human fur gene encodes a yeast KEX2-1ike endoprotease that cleaves pro-fl-NGF in viw~, J. Cell Biol., 111 (1990)2851 2859. 5 Christie, D.L., Batchelor, D.C. and Palmer, D.J., Identification of kex2-related proteases in chromaffin granules by partial amino acid sequence analysis, J. Biol. Chem., 266 (1991) 15679 15683. 6 Fischer-Colbrie, R., Zangerle, R., Frischenschlager, 1., Weber. A. and Winkler, H., Isolation and immunological characterization of a gtycoprotein from adrenal chromaffin granules, J. Neurochem.. 42 (1984) 1008 1016. 7 Fischer-Colbrie, R. and Frischenschlager, I., Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranins B, and enkephalin-containing peptides, J. Neurochem., 44 (1985) 1854-1861. 8 Hatsuzawa, K., Hosaka, M., Nakagawa, T., Nagase, M., Shoda, A., Murakami, K. and Nakayama, K., Structure and expression of mouse furin, a yeast Kex2-related protease, J. Biol. Chem., 265 (1990) 22075 22078.
9 Kirchmair, R., Gee, P., Hogue-Angeletti, R., Laslop, A., FischerColbrie, R. and Winkler, H., Immunological characterization of the endoproteases PC1 and PC2 in adrenal chromaffin granules and in the pituitary gland, FEBS Lett., 297 (1992) 302 305. 10 Lindberg, 1., The new eukaryotic precursor processing proteinases, Mol. Endocrinol.,5(1991) 1361 1365. 11 Misumi, Y., Sohda, M. and Ikehara, Y., Sequence of the eDNA encoding rat furin, a possible propeptide-processing endoprotease, Nucleic Acids Res., 18 (1990) 6719. 12 Misumi, Y., Oda, K., Fujiwara, T., Takami, N., Tashiro, K. and lkehara, Y.. Functional expression of furin demonstrating its intracellular localization and endoprotease activity tk)r processing of proalbumin and complement pro-C3. J. Biol. Chem., 266 (1991) 16954 16959. 13 Palmer, D.J. and Christie, D.L., The primary structure of glycoprotein Ill from bovine adrenal medullary chromaffin granules, J. Biol. Chem., 265 (1990) 6617 6623. 14 Roebroek, A.J.M., Schalken, J.A., Leunissen, J.A.M., Onnekink, C., Bloemers, H.P.J. and Van den Ven, W.J.M., Evolutionary conserved close linkage of the c-fed/fps prolo-oncogene and genetic sequences encoding a receptor-like protein, EMBO J., 5 (1986) "~9 7 -,0-. '~'~ -1 15 Schneider, F.H., Smith, A.D. and Winkler, H., Secretion from the adrenal medulla: biochemical evidence for exocytosis, Br. J. Pharmac. Chemother.,31 (1967) 94 104. 16 Seidah. N.G., Gaspar, L., Mion, P., Marcinkiewicz, M., Mbikay, M. and Chretien, M., cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases, DNA Cell Biol., 9 (1990) 415 424. 17 Seidah. N.G., Marcinkiewicz, M., Benjannet, S., Gaspar, L., Beaubien, G., Mallei, M.G., Lazure, C., Mbikay, M. and Chretien, M., Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2, Mol. Endocrinol., 5 (1991) 111 122. 18 Smeekens, S.P. and Steiner, D.F., Identification of a human insulinoma eDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing pro/ease Kex2, J. Biol. ('hem., 265 (1990) 2997 3000. 19 Smeekens, S.P., Avruch, A.S., LaMendola, J., Chan, S.J. and Steiner. D.F., Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans, Proc. Natl. Acad. Sci. USA, 88 (1991) 340 344. 20 Smith, A.D. and Winkler, H., The localization of lysosomal enzymes in chromaffin tissue, J. Physiol., 183 (19661 179 188. 21 Trifaro, J.M., Duerr, A.C. and Pinto, J.E.B., Membranes of the adrenal medulla: a comparison between the membranes of the golgi apparatus and chromaffin granules, Mol. Pharmacol., 12 (1976) 536 545. 22 Van den Ven, W.J.M., Voorberg, J., Fontijn, R., Pannekoek, H., Van dcn Ouweland, A.M.W., Van Duijnhoven, H.L.P., Roebroek, A.J.M. and Siezen, R.J., Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes, Mol. Biol. Rep., 14 (1990) _65 _7,. 23 Winkler, H. and Fischer-Colbrie, R., Common membrane proteins ofchromaffin granules, endocrine and synaptic vesicles: properties, tissue distribution, membrane topography and regulation of synthesis, Neurochem. Int., 17 (1990) 245 262.