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• W2003558962 abstract "The NH2-terminal region of pro-opiomelanocortin (POMC) is highly conserved across species, having two disulfide bridges that cause the formation of an amphipathic hairpin loop structure between the 2nd and 3rd cysteine residues (Cys8to Cys20). The role that the NH2-terminal region of pro-opiomelanocortin plays in acting as a molecular sorting signal for the regulated secretory pathway was investigated by using site-directed mutagenesis either to disrupt one or more of the disulfide bridges or to delete the amphipathic loop entirely. When POMC was expressed in Neuro-2a cells, ACTH immunoreactive material was localized in punctate secretory granules in the cell body and along the neurites, with heavy labeling at the tips. ACTH was secreted from these POMC-transfected cells in a regulated manner. Disruption of both disulfide bridges or the second disulfide bridge or removal of the amphipathic hairpin loop resulted in constitutive secretion of the mutant POMC from the cells and a lack of punctate secretory granule immunostaining within the cells. We have modeled the NH2-terminal POMC Cys8to Cys20domain and have identified it as an amphipathic loop containing four highly conserved hydrophobic and acidic amino acid residues (Asp10-Leu11-Glu14-Leu18). Thus the sorting signal for POMC to the regulated secretory pathway appears to be encoded by a specific conformational motif comprised of a 13-amino acid amphipathic loop structure stabilized by a disulfide bridge, located at the NH2terminus of the molecule. The NH2-terminal region of pro-opiomelanocortin (POMC) is highly conserved across species, having two disulfide bridges that cause the formation of an amphipathic hairpin loop structure between the 2nd and 3rd cysteine residues (Cys8to Cys20). The role that the NH2-terminal region of pro-opiomelanocortin plays in acting as a molecular sorting signal for the regulated secretory pathway was investigated by using site-directed mutagenesis either to disrupt one or more of the disulfide bridges or to delete the amphipathic loop entirely. When POMC was expressed in Neuro-2a cells, ACTH immunoreactive material was localized in punctate secretory granules in the cell body and along the neurites, with heavy labeling at the tips. ACTH was secreted from these POMC-transfected cells in a regulated manner. Disruption of both disulfide bridges or the second disulfide bridge or removal of the amphipathic hairpin loop resulted in constitutive secretion of the mutant POMC from the cells and a lack of punctate secretory granule immunostaining within the cells. We have modeled the NH2-terminal POMC Cys8to Cys20domain and have identified it as an amphipathic loop containing four highly conserved hydrophobic and acidic amino acid residues (Asp10-Leu11-Glu14-Leu18). Thus the sorting signal for POMC to the regulated secretory pathway appears to be encoded by a specific conformational motif comprised of a 13-amino acid amphipathic loop structure stabilized by a disulfide bridge, located at the NH2terminus of the molecule. Endocrine cells and peptidergic neurons contain a regulated secretory pathway, in addition to the “default” constitutive secretory pathway present in all cells (1Brownstein M.J. Russell J.T. Gainer H. Science. 1980; 207: 373-378Crossref PubMed Scopus (645) Google Scholar). Prohormones and proneuropeptides are intracellularly routed through the endoplasmic reticulum and Golgi apparatus to the trans-Golgi network (2Kelly R.B. Science. 1985; 230: 25-32Crossref PubMed Scopus (761) Google Scholar, 3Orci L. Ravazzola M. Amherdt M. Perrelet A. Powell S.K. Quinn D.L. Moore H.-P.H. Cell. 1987; 51: 1039-1051Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 4Sossin W.S. Fisher J.M. Scheller R.H. J. Cell Biol. 1990; 110: 1-12Crossref PubMed Scopus (91) Google Scholar, 5Kelly R.B. Cell. 1993; 72: 43-53Abstract Full Text PDF PubMed Scopus (213) Google Scholar). In the trans-Golgi network they are actively sorted away from other proteins and packaged into the granules of the regulated secretory pathway where they are proteolytically processed to biologically active peptides that are released from the cell upon stimulation (6Schnabel E. Mains R.E. Farquhar M.G. Mol. Endocrinol. 1989; 3: 1223-1235Crossref PubMed Scopus (148) Google Scholar, 7Milgram S.L. Mains R.E. J. Cell Sci. 1994; 101: 737-745Google Scholar). The nature of the sorting signal involved in directing prohormones and proneuropeptides to these regulated secretory granules has not been determined, because no consensus amino acid sequences have been found. The adrenocorticotropin/endorphin prohormone pro-opiomelanocortin (POMC) 1The abbreviations used are:POMCpro-opiomelanocortinN-POMCNH2-terminal POMCACTHadrenocorticotropic hormoneACTHiACTH immunoreactive materialHPLChigh pressure liquid chromatography. is representative of a class of prohormones that is directed to the regulated secretory pathway in neuroendocrine cells (8Burgess T.L. Craik C.S. Matsuuchi L. Kelly R.B. J. Cell Biol. 1987; 105: 659-668Crossref PubMed Scopus (44) Google Scholar, 9Arvan P. Kuliawat R. Prabakaran D. Zavacki A.M. Elahi D. Wang S. Pilkey D. J. Biol. Chem. 1991; 266: 14171-14174Abstract Full Text PDF PubMed Google Scholar, 10Pimplikar S.W. Huttner W.B. J. Biol. Chem. 1992; 267: 4110-4118Abstract Full Text PDF PubMed Google Scholar, 11Loh Y.P. Thorn N.A. Treiman M. Petersen O.H. Molecular Mechanisms in Secretion. Munksgaard, Copenhagen1988: 525-539Google Scholar). Other members of this class of prohormones include pro-enkephalin, pro-vasopressin, pro-dynorphin, and pro-oxytocin, all of which have in common in their primary amino acid sequence one or more pairs of cysteine residues that may form a disulfide bridge and thus a hairpin loop structure in their NH2-terminal regions (Fig. 1 A). For example, in POMC the first 26 amino acids contain 4 cysteine residues that are highly conserved (Fig. 1 A) (12Douglass J. Civelli O. Herbert E. Annu. Rev. Biochem. 1984; 53: 665-715Crossref PubMed Scopus (326) Google Scholar) and have been experimentally shown to form a hairpin loop stabilized by two disulfide bridges (Cys2/Cys24 and Cys8/Cys20) (13Bennett H.P.J. Seidah N.G. Benjannet S. Solomon S. Chretien M. Int. J. Pept. Protein Res. 1986; 27: 306-313Crossref PubMed Scopus (23) Google Scholar) (Fig. 1 B). pro-opiomelanocortin NH2-terminal POMC adrenocorticotropic hormone ACTH immunoreactive material high pressure liquid chromatography. Recent work in our laboratory (14Tam W.H.H. Andreasson K.A. Loh Y.P. Eur. J. Cell Biol. 1993; 62: 294-306PubMed Google Scholar) has demonstrated that the NH2-terminal hairpin loop consisting of 26 amino acids of POMC was sufficient for targeting a non-native protein, chloramphenicol acetyltransferase, to the regulated secretory pathway in AtT20 cells. However, the first 10 amino acids of NH2-terminal POMC, which lacked the formation of the hairpin loop, were insufficient for sorting chloramphenicol acetyltransferase to the regulated secretory pathway. These data suggested that residues 1-26 contained information necessary for sorting POMC to the regulated secretory pathway. In this study, site-directed mutagenesis was used to identify the specific molecular motif within N-POMC encoding this sorting signal. A unique 13-amino acid amphipathic loop structure, stabilized by one disulfide bridge, at the NH2terminus of POMC was identified as the signal essential for the sorting of this prohormone into the regulated secretory pathway of the mouse neuroendocrine cell line, Neuro-2a. Bovine POMC cDNA (kindly donated by Dr. S. Nakanishi) was subcloned into the cloning vector, dsM13p18 (15Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). Using an Amersham mutagenesis system (version 2, RPN 1523), substitution mutations were made by annealing the ssM13-POMC to a 39-nucleotide probe complementary to positions 72-111 in POMC. The probe contained glycine-to-cysteine mutations in position 82, 101, or both, and after annealing was treated with DNA polymerase (Klenow fragment) and T4-ligase to convert the hybrid to dsM13-POMC. The non-mutant strand was nicked with NciI, digested with exonuclease III, and polymerized by DNA polymerase I and T4 ligase. The constructs were transfected into JM101 bacteria and plated. The M13-POMC phages were isolated, and the inserts were sequenced. Wild type and mutated POMC cDNA were cut from the M13 vector by PstI and blunt ended by T4 polymerase, and NheI linkers were ligated. The mixture was digested with NheI and HincII, and the gel was purified. The POMC cDNA was ligated to a NheI- and SmaI-digested pMSG expression vector and transfected into HB101 Escherichia coli bacteria. Deletion mutations were created by restriction digestion of the area to be deleted and blunt end ligation of the fragments. All the POMC cDNA mutations were verified by sequencing. Bacteria containing wild type and mutated POMC plasmids were grown in 500 ml of LB broth and purified using Qiagen Maxi Prep kits (Chatsworth, CA). Stable Neuro-2a cell lines were made by using Lipofectin (Life Technologies, Inc.) to transfect the plasmids containing wild type or mutated POMC and were selected using the hypoxanthine-guanine phosphoribosyltransferase selection marker. Ten different clones for each mutant were screened for expression of the mutant POMC, after which three final clones were selected for the experiments. The cells were grown on poly- L-lysine-coated coverslips, and expression of the POMC/mutant POMC was stimulated with 100 n M dexamethasone. After 24 h the cells were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 10% goat serum in phosphate-buffered saline, and incubated for 16 h with antibody to ACTH1–39 (DP4) (16Elkabes S. Loh Y.P. Nieburgs A. Wray S. Dev. Brain Res. 1989; 46: 85-95Crossref PubMed Scopus (59) Google Scholar) at 1:2500 dilution. The cells were washed with phosphate-buffered saline and stained using a goat anti-rabbit serum conjugated with rhodamine (Boehringer Mannheim). Wheat germ agglutinin conjugated to fluorescein (1:250 dilution) was used for staining the Golgi apparatus. The labeled cells were photographed using a Nikon Optiphot fluorescent microscope. Neuro-2a cells were grown in 60-mm plates and transfected with 20 μg of POMC or mutated POMC using Lipofectin (Life Technologies, Inc.). A comparison between stably and transiently transfected cells indicated that similar amounts of POMC or mutant POMC were produced per well of 106cells. Subsequently, transient transfections were used for the secretion experiments. After 48 h of stimulation with 100 n M dexamethasone in complete media, the transfected cells were incubated for 3 h in a basal release medium (Medium 1) containing 25 m M Hepes, 125 m M NaCl, 4.8 m M KCl, 1.2 m M KH2PO4, 5.6 m M dextrose, 1 m M CaCl2, 0.5 m M MgCl2, and 0.1% bovine serum albumin at pH 7.4. After 3 h the medium was collected and one-half of the cells received Medium 1, while the other half received a stimulated release medium (Medium 2) identical to Medium 1 except for the substitution of 51 m M K+ and 5.4 m M Ca2+. Both media contained 200 kallikrein-inactivating units/ml aprotinin and 1 m M 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (ICN Biochemicals, Aurora, OH). After 3 h of incubation in Medium 2, the medium was collected, extracted on a Sep-Pak C18, and fractionated by HPLC. The system used for HPLC was a Beckman 421 system and a Bio-Rad HiPore RP-304 column (4.6 × 250 mm). Solvent A was 0.1% trifluoroacetic acid, and solvent B was 80% acetonitrile in 0.1% trifluoroacetic acid. Samples were injected as 100-μl volumes and separated using a linear gradient from 10 to 20% solvent B in 10 min and from 20 to 70% solvent B in 60 min at a flow rate of 1 ml/min. 1-ml samples were collected and analyzed for ACTHiby radioimmunoassay using antibody DP6, which is specific for ACTH1–14but also cross-reacts with ACTH1–15, ACTH1–39, and POMC (17Estivariz F.E. Friedman T.C. Chikuma T. Loh Y.P. J. Biol. Chem. 1992; 267: 7456-7463Abstract Full Text PDF PubMed Google Scholar). The values represent the mean ± S.E. for three to four experiments. The percentage of total released ACTHiwas calculated by dividing the ACTHiin Medium 2 by the total ACTHi(i.e. ACTHiin Medium 1 + Medium 2 + the cell extract). The peptide structure was built with disulfide bridges between Cys2/Cys24and Cys8/Cys20and was subjected to 250 ps of molecular dynamics at 600 K. At each picosecond, the structure was captured and extensively minimized using a “conjugate-gradient” algorithm. All calculations were performed using the Discover and Insight programs (Biosys, Palo Alto, CA). The backbone of the loop from Cys8to Cys20is shown as an oval ribbon with the side chains shown as shaded sticks. When full-length POMC was expressed in Neuro-2a cells (18Klebe R.J. Ruddle F.H. J. Cell Biol. 1967; 43 (abstr.): 69Google Scholar, 19Olmstead J.B. Carlson K. Klebe R. Ruddle F. Rosenbaum J. Proc. Natl. Acad. Sci. U. S. A. 1970; 65: 129-136Crossref PubMed Scopus (261) Google Scholar, 20Nol G. Zollinger L. Laliberte F. Rassart E. Crine P. Boileau G. J. Neurochem. 1989; 52: 1050-1057Crossref PubMed Scopus (29) Google Scholar, 21Chevrier D. Fournier H. Nault C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1993; 94: 213-221Crossref PubMed Scopus (13) Google Scholar), ACTHiwas immunocytochemically localized to punctate secretory granules in both the main cell body and along the neurites with very heavy punctate labeling at the tips of the neurites, which suggested accumulation in this region (Fig. 2 A). Wheat germ agglutinin-fluorescein isothiocyanate staining was used to show the Golgi apparatus in these cells (Fig. 2 A′). Immunoelectron microscopy confirmed that these granules were similar in size to ACTHi-containing granules described by others in the Neuro-2a cell line (22Roy P. Chevrier D. Fournier H. Racine C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 82: 237-250Crossref PubMed Scopus (19) Google Scholar, 23Chevrier D. Fournier H. Nault C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 79: 109-118Crossref PubMed Scopus (17) Google Scholar). 2D. R. Cool, M. Fenger, C. R. Snell, and Y. P. Loh, unpublished data. Analysis of the ACTHiproducts showed processing of POMC to ACTH1–39and ACTH1–14(Fig. 3). These ACTH products were secreted into the medium in a stimulated manner with high K+/Ca2+ (Table I). These results showed that POMC was sorted to the regulated secretory pathway in Neuro-2a cells, thus making this a useful cell line in which to study prohormone sorting.FIG. 3Representative immunoreactive profile of products released in a regulated manner from Neuro-2a cells transiently expressing POMC. The number of nanograms of immunoreactive peptide/fraction was determined by radioimmunoassay. The predominant peaks that co-ran with the synthetic peptide markers, ACTH1-14 and ACTH1-39, are shown. The filled circles represent unstimulated release, and the open circles represent sti mulated release in the presence of high K+/Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table I:Stimulated secretion of ACTH from Neuro-2a cellsTable I:Stimulated secretion of ACTH from Neuro-2a cellsStimulated secretion of ACTHifrom Neuro-2a cells is in response to K+/Ca2+. To investigate the release of ACTHi, Neuro-2a cells, transiently transfected with cDNA using Lipofectin, were exposed to a buffer containing high concentrations of K+/Ca2+, and the ACTHireleased into the buffer was assayed by radioimmunoassay. The values represent the mean ± S.E. for four experiments. Values are the percent of the total released. Stimulated secretion of ACTHifrom Neuro-2a cells is in response to K+/Ca2+. To investigate the release of ACTHi, Neuro-2a cells, transiently transfected with cDNA using Lipofectin, were exposed to a buffer containing high concentrations of K+/Ca2+, and the ACTHireleased into the buffer was assayed by radioimmunoassay. The values represent the mean ± S.E. for four experiments. Values are the percent of the total released. Because the Cys residues are highly conserved across species (12Douglass J. Civelli O. Herbert E. Annu. Rev. Biochem. 1984; 53: 665-715Crossref PubMed Scopus (326) Google Scholar) and are present in several prohormones (Fig. 1 A), we first determined whether the two disulfide bridges in N-POMC (Cys2/Cys24and Cys8/Cys20) were involved in the sorting signal motif. Substitution mutations were made to disrupt either one or both of the disulfide bridges (Fig. 4). Disruption of both disulfide bridges (C2S,C8S) resulted in a perinuclear ACTH immunostaining pattern, suggesting localization within the Golgi (Fig. 2 B). Wheat germ staining for the Golgi confirmed this co-localization (Fig. 2 B′). Substantial amounts of ACTHi, primarily as unprocessed POMC (Fig. 5), were released from these cells in a non-stimulated manner, characteristic of constitutive secretion (Table I). Disruption of the first disulfide bridge (Fig. 4, C2S) resulted in punctate secretory granule ACTH immunostaining at the tips and along the length of the neurites, similar to that of cells transfected with wild type POMC (Fig. 2 C). Stimulated secretion of ACTHi(ACTH1–39and ACTH1–14) from these cells was found (Table I). Disruption of the second disulfide bridge (Fig. 4, C8S) resulted in the immunostaining of large (500-2000 nm) cytoplasmic vesicular-like structures containing ACTHi(Fig. 2 D). Stimulated secretion was not observed, but constitutive secretion of ACTHias unprocessed POMC (data not shown) was found (Table I). These data suggest not only that at least one disulfide bridge is necessary for sorting but also that the position of this disulfide bridge (Cys8/Cys20) was critical for sorting.FIG. 5Representative profile of ACTH immunoreactive products released constitutively from Neuro-2a cells expressing C2S,C8S mutant POMC. The number of nanograms of immunoreactive peptide/fraction was determined by radioimmunoassay, with the predominant peak identified as POMC because it co-ran with a 3H-labeled mouse POMC standard prepared as described in Loh et al. (38Loh Y.P. Parish D.C. Tuteja R. J. Biol. Chem. 1985; 260: 7194-7205Abstract Full Text PDF PubMed Google Scholar). The filled circles represent unstimulated release, and the open circles represent stimulated release in the presence of high K+/Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The hairpin loop region was further analyzed as a sorting signal by making a deletion mutant to remove the 13 amino acids forming the loop (C8QDLTTESNLLAC20) (Fig. 4, Cys8-Cys20 Loop Deletion). The Cys8to Cys20loop deletion-mutated POMC protein was localized to a perinuclear region characteristic of localization in the Golgi (Fig. 2 E). There was only constitutive release of this mutant POMC (Table I). These data suggest that there is information in the 13 amino acids of the hairpin loop region of POMC that is essential to sort POMC correctly to the regulated secretory pathway. In order to determine whether other regions of NH2-terminal POMC outside the hairpin loop were necessary for sorting to the secretory granules, 78 amino acids spanning the entire region from Lys25 to Arg101 (Fig. 4) were deleted. ACTHiwas found in punctate secretory granules in these cells (Fig. 2 F), similar to those of the wild type POMC, and ACTH products (ACTH1–39 and ACTH1–14) were secreted in a regulated manner (Table I). These results suggest that there is no information in the γ-melanocyte-stimulating hormone or joining peptide region of POMC that is necessary for sorting to the dense core granules. The data presented here identify the regulated secretory pathway sorting signal for POMC as a highly conserved region consisting of a 13-amino acid hairpin loop conformation (C8QDLTTESNLLAC20) stabilized by one disulfide bridge. A three-dimensional model of this NH2-terminal region of POMC shows that the Cys8to Cys20sequence of 13 amino acids assumes a heart-shaped loop with two lobes, one centered on Asp10-Leu11 and the other on Thr13-Glu14(Fig. 6). The structure is clearly amphipathic with a cluster of hydrophobic side chains (i.e. Cys8-Leu17-Leu18-Ala19-Cys20) at the base of the heart and a hydrophilic region at both upper lobes of the heart-shaped loop (Fig. 6). The disulfide bridge between Cys8and Cys20locks this domain in a rigid conformation. If the POMC sequences known for different species are compared (Fig. 1 A), the residues that are conserved in this region in all species would correspond to Asp10-Leu11-Glu14-LeuFig. 1 A). It is therefore likely that some or all of the side chains of these residues within the amphipathic loop play an important role in the sorting mechanism. Previous studies from our laboratory have shown that the first 26 amino acids of N-POMC contained information that was sufficient to sort a reporter protein, chloramphenicol acetyltransferase, to the regulated secretory pathway in AtT20 cells (14Tam W.H.H. Andreasson K.A. Loh Y.P. Eur. J. Cell Biol. 1993; 62: 294-306PubMed Google Scholar). In addition, we have shown that when 25 amino acids of N-POMC (Cys2 to Pro26) containing the amphipathic loop and both disulfide bridges were deleted, the mutant POMC was sorted to the constitutive secretory pathway in Neuro-2a cells (24Cool D.R. Loh Y.P. Biochimie (Paris). 1994; 76: 265-270Crossref PubMed Scopus (52) Google Scholar). We now show that eliminating just the 13-amino acid amphipathic loop (Cys8to Cys20) was sufficient to cause the mutant POMC to be secreted constitutively. This mutant POMC was not processed in the Neuro-2a cells, further suggesting routing through the constitutive secretory pathway. These results provide evidence that firmly establishes for the first time that the 13-amino acid amphipathic loop region of N-POMC is necessary for sorting POMC to the regulated secretory pathway, and there appears to be no other essential sorting signals in the rest of the POMC molecule. The participation of the disulfide bridges in sorting was also tested by substituting a serine residue for cysteine at position 2, position 8, or both, which effectively disrupted the disulfide bridges. Disruption of the second disulfide bridge or both disulfide bridges resulted in the missorting of POMC to the constitutive secretory pathway. Mutant POMC secreted by the constitutive secretory pathway was not processed. However, disruption of the first disulfide bridge had no effect on sorting to secretory granules. Thus we conclude that sorting of POMC to the regulated secretory pathway is dependent only on the presence of the disulfide bridge closest to the amphipathic loop (Cys8/Cys20). These results are further supported by the observation that, in both salmon II and trout B POMC (25Kawauchi H. Arch. Biochem. Biophys. 1983; 227: 343-350Crossref PubMed Scopus (79) Google Scholar, 26Kitahara N. Nishizawa T. Iida K. Okazaki H. Andoh T. Soma G.I. Comp. Biochem. Physiol. B Comp. Biochem. 1988; 91: 365-370Crossref PubMed Scopus (57) Google Scholar, 27Salbert G. Chauveau I. Bonnec G. Valotaire Y. Jego P. Mol. Endocrinol. 1992; 6: 1605-1613PubMed Google Scholar) where the first disulfide bridge (Cys2/Cys24) has been disrupted by a natural mutation of Cys24to a Phe, there was no adverse effect on regulated secretion and thus no adverse effect on targeting (28Scott A.P. Baker B.I. J. Endocrinol. 1974; 63: 24P-25PGoogle Scholar, 29Baker B.I. Leatherland J.L. Scott A.P. Cell Tissue Res. 1974; 151: 481-487Crossref PubMed Scopus (13) Google Scholar). Roy et al. (22Roy P. Chevrier D. Fournier H. Racine C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 82: 237-250Crossref PubMed Scopus (19) Google Scholar) have reported similar results for two of the mutants described here, C2S,C8S and C2S. However, our results differ concerning the fate of the C8S and POMC-Δ1-26 mutations. Roy et al. (22Roy P. Chevrier D. Fournier H. Racine C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 82: 237-250Crossref PubMed Scopus (19) Google Scholar) found that both of these mutant proteins were secreted in a regulated manner, were localized to regulated secretory granules in the cells, and were processed in the cells. In our study (Fig. 2 and Table I) and that of Cool and Loh (24Cool D.R. Loh Y.P. Biochimie (Paris). 1994; 76: 265-270Crossref PubMed Scopus (52) Google Scholar), the same mutant POMC proteins were secreted constitutively, were not localized to regulated secretory granules, and were primarily not processed. Differing methods of analysis and the low number of experiments by Roy et al. (22Roy P. Chevrier D. Fournier H. Racine C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 82: 237-250Crossref PubMed Scopus (19) Google Scholar) may account for the differences between the results. First, for their secretion studies, Roy et al. (22Roy P. Chevrier D. Fournier H. Racine C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 82: 237-250Crossref PubMed Scopus (19) Google Scholar) reported the result of only one experiment for the POMC-Δ1-26 mutant. Furthermore, data for the analysis of secretion of the C8S mutant were not shown, but the authors concluded that this mutant was secreted in a regulated manner (22Roy P. Chevrier D. Fournier H. Racine C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 82: 237-250Crossref PubMed Scopus (19) Google Scholar). On the contrary, our secretion data for this and other mutants, representing the mean from four experiments, provide conclusive proof that this C8S mutant protein as well as the NH2-terminally deleted (Cys8to Cys20loop deletion) mutant are secreted via the constitutive secretory pathway. Second, the use by Roy et al. (22Roy P. Chevrier D. Fournier H. Racine C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 82: 237-250Crossref PubMed Scopus (19) Google Scholar) of the processing of POMC to β-endorphin in the cell extract only as evidence for targeting POMC to the regulated secretory pathway is ambiguous, because another group has shown that the Neuro-2a cell line can process POMC directly to β-endorphin, which is at the COOH terminus of this prohormone, efficiently bypassing the β-lipotrophic hormone intermediate (30Day N.C. Lin H. Ueda Y. Meador-Woodruff J.H. Akil H. Neuropeptides. 1993; 24: 253-262Crossref PubMed Scopus (8) Google Scholar). The subcellular site for this aberrant cleavage is as yet undetermined in the Neuro-2a cell line. Additionally, four antisera were used, each for a different assay in the studies of Roy et al. (22Roy P. Chevrier D. Fournier H. Racine C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1991; 82: 237-250Crossref PubMed Scopus (19) Google Scholar), each of which reacted with different regions of the POMC molecule. Thus there was a lack of continuity in following one POMC product (e.g. β-endorphin) from immunocytochemical localization through processing and secretion. In contrast, antisera to only the ACTH portion of the POMC molecule were used for our entire study. The requirement for the integrity of the disulfide bridge in sorting proteins to the regulated secretory pathway has also been demonstrated for chromogranin B (31Chanat E. Weiss U. Huttner W.B. Tooze S.A. EMBO J. 1993; 12: 2159-2168Crossref PubMed Scopus (132) Google Scholar, 32Chanat E. Weiss U. Huttner W.B. FEBS Lett. 1994; 351: 225-230Crossref PubMed Scopus (54) Google Scholar). Disruption of the disulfide bridge by the addition of the thiol-reducing agent dithiothreitol to PC12 cells caused rerouting of the endogenous chromogranin B to the constitutive pathway. The constitutively secreted chromogranin B, in the reduced state due to treatment with dithiothreitol, lacked the loop structure formed by the disulfide bridge (Fig. 1 A), implicating the involvement of this structure as a sorting signal for the regulated secretory pathway. A sorting signal may also exist for prohormones without cysteine residues, such as pro-somatostatin. In experiments in which the first 82 amino acids of the pro-region of pro-somatostatin were fused to 142 amino acids of α-globin, α-globin fusion protein was targeted to the regulated secretory pathway in GH3 cells (33Stoller T.J. Shields D. J. Cell Biol. 1989; 108: 1647-1655Crossref PubMed Scopus (102) Google Scholar). The presence of a signal that could sort α-globin to the regulated pathway in pro-somatostatin appears to reside in the first 82 amino acids. However, by deleting different regions of the pro-somatostatin molecule, it was determined that multiple sorting signals might exist in the somatostatin molecule, each of which could independently cause sorting to the regulated secretory pathway (34Sevarino K.A. Stork P. J. Biol. Chem. 1991; 28: 18507-18513Abstract Full Text PDF Google Scholar). POMC may also contain domains that are not sufficient by themselves to cause sorting but that may act synergistically with the N-POMC amphipathic loop to enhance sorting to the regulated secretory pathway. A previous study suggests that the carboxyl terminus of POMC may contain information that could influence sorting (21Chevrier D. Fournier H. Nault C. Zollinger M. Crine P. Boileau G. Mol. Cell. Endocrinol. 1993; 94: 213-221Crossref PubMed Scopus (13) Google Scholar). However, our previous work with the N-POMC-chloramphenicol acetyltransferase chimeric constructs (14Tam W.H.H. Andreasson K.A. Loh Y.P. Eur. J. Cell Biol. 1993; 62: 294-306PubMed Google Scholar) and the Δ2-26 POMC mutant (24Cool D.R. Loh Y.P. Biochimie (Paris). 1994; 76: 265-270Crossref PubMed Scopus (52) Google Scholar) suggests that the COOH-terminal region is not an absolute necessity for sorting to the regulated secretory pathway. A comparison of the NH2-terminal regions of POMC from different species indicates that in addition to the conserved cysteine residues at positions 2, 8, 20, and 24, which form a pair of disulfide bridges (Cys2/Cys24and Cys8/Cys20), there are also four other highly conserved amino acids, Asp10-Leu11-Glu14-Leu18. It is likely that these residues in the amphipathic loop motif play a role in the sorting mechanism, perhaps through ionic/hydrophobic interactions with a putative sorting receptor. It is also interesting to note that, when only the amphipathic loop region (Cys8to Cys20) was deleted, the two remaining cysteine residues at positions 2 and 24 could potentially form a disulfide bridge, creating a smaller loop with acidic and hydrophobic residues. However, this mutant was not sorted to the regulated secretory pathway, suggesting that this loop, if formed, was not enough to cause sorting. Immediately following the last cysteine residue in the hairpin loop of POMC (Cys24) is another set of highly conserved residues, Asp27-Leu28-Glu31-Val34. The similarity of the acidic and hydrophobic components of these residues to the Asp10-Leu11-Glu14-Leu18motif in the amphipathic loop suggests that they could potentially play a role in sorting to the regulated secretory pathway. However, helical wheel analyses of the other amino acids in this region (e.g. Pro26, Pro32, and Pro35) suggest that they would probably not allow the formation of an amphipathic loop. The lack of targeting to the regulated secretory pathway of the mutants with the amphipathic loop deleted (Cys2 to Cys26 and Cys8to Cys20) shows that these residues (Asp27-Leu28-Glu31-Val34) cannot compensate for the 13-amino acid amphipathic loop motif. This conclusion was further supported by our finding that deletion of the Lys25 to Arg101 region (Fig. 4, 78 Amino Acid Deletion) had no effect on the sorting of this mutant POMC. Misfolding of the natural conformation of proteins has been associated with aggregation or retention and degradation in the early stages of the secretory pathway (35Marquardt T. Helenius A. J. Cell Biol. 1992; 117: 505-513Crossref PubMed Scopus (153) Google Scholar, 36Yilla M. Doyle D. Sawyer J. J. Cell Biol. 1992; 118: 245-252Crossref PubMed Scopus (21) Google Scholar). Any of the mutations (i.e. disruption of the disulfide bridges, deletion of the amphipathic loop structure, or the 78-amino acid deletion in POMC) could potentially cause such misfolding. However, we found that none of these mutations caused significant retention or degradation in the endoplasmic reticulum, because large amounts of mutant POMC were found to be secreted in a constitutive or regulated manner from these cells. How does the amphipathic heart-shaped loop of N-POMC act as a sorting signal? The modeling results indicate that the acidic side chains of Asp10and Glu14are poised in a position within the amphipathic loop structure of N-POMC that is ideal for ionic interactions with a sorting receptor protein. Whereas a putative “receptor” has long been proposed (37Chung K.N. Walter P. Aponte G.W. Moore H.P.H. Science. 1989; 243: 192-197Crossref PubMed Scopus (115) Google Scholar), no protein has yet been found to play the role of “universal” receptor for the numerous pro-proteins found in the regulated secretory pathway. However, there is evidence from previous studies that N-POMC1–76 can bind in a pH-dependent manner (optimum pH, ∼5.5-6.0) to the luminal side of bovine intermediate lobe secretory vesicle membranes (14Tam W.H.H. Andreasson K.A. Loh Y.P. Eur. J. Cell Biol. 1993; 62: 294-306PubMed Google Scholar) and that this binding is protease-sensitive. 3D. R. Cool, unpublished results. Thus, the binding of the N-POMC8–20 amphipathic loop to a receptor is one potential mechanism by which POMC is actively sorted to the regulated secretory pathway. In summary, we have provided evidence in this paper for the first time that the regulated secretory pathway sorting signal present in the NH2-terminal region of POMC is a 13-amino acid amphipathic loop conformational signal, dependent upon the integrity of the disulfide bridge and thus upon the stabilization of the amphipathic loop for its activity. The presence of an active sorting signal in POMC for the regulated secretory pathway suggests the possibility that a putative receptor, located in the trans-Golgi network, recognizes the prohormone molecule and thus is responsible for sorting it to the newly forming regulated secretory granule. The model we present for this amphipathic loop sorting signal can now be used as a template by other researchers for molecular modeling to identify similar regions in other prohormones. We thank Winnie Tam and Victor Hwang for excellent technical assistance and Drs. Ana-Maria Bamberger and Theodore C. Friedman for critical reading of the manuscript." @default.
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• W2003558962 title "Identification of the Sorting Signal Motif within Pro-opiomelanocortin for the Regulated Secretory Pathway" @default.
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