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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02608

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Interaction between secretogranin III and carboxypeptidase E facilitates prohormone sorting within secretory granules

Masahiro Hosaka^1,*, Tsuyoshi Watanabe^2,*, Yuko Sakai², Takeshi Kato³ and Toshiyuki Takeuchi^1,

¹ Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
² Department of Anatomy II, Asahikawa Medical College, Asahikawa 078-8510, Japan
³ Laboratory of Molecular Recognition, Graduate School of Integrated Science, Yokohama City University, Yokohama 236-0027, Japan

View larger version (73K): [in a new window]   Fig. 1. Intragranular localization of SgIII and CPE in rat pituitary mammotropes (A), rat pancreatic ß-cells (B), and mouse AtT-20 cells (C). CPE is visualized with 15 nm gold particles (A-C), SgIII is visualized with 10 nm immunogold particles (A-C). Insulin (B) and POMC-derived ACTH-containing peptides (C) are labeled with 5 nm immunogold particles. Note that SgIII (arrowheads) and CPE (arrows) are preferentially localized in the periphery of the SGs, and the SgIII and CPE are occasionally co-localized at the same sites in the periphery of the SGs (white arrows). Bar, 100 nm.

View larger version (32K): [in a new window]   Fig. 2. Co-immunoprecipitation of CPE and SgIII. (A,B) AtT-20 and MIN6 cell lysates were subjected to immunoprecipitation using rabbit antibody to CPE (A) or SgIII (B). In the CPE immunoprecipitation experiment (A), control immunoprecipitation was performed with monoclonal antibody to insulin receptor, a non-SG-residential protein, because the antibody to CPE was monoclonal. In the SgIII immunoprecipitation experiment (B), preimmune serum was used for control immunoprecipitation, because the antibody to SgIII was polyclonal. The starting fraction (lane 3) and immunoprecipitates (lanes 1, 2) were analyzed by SDS-PAGE and immunoblot using antibodies to SgIII, CPE, CgA, ß-granin and SgII. SgII blot was performed for the SgIII immunoprecipitation experiment to show whether aggregation-prone granins such as SgII are co-precipitated or not (B, bottom panels). As shown in Fig. 2B, CPE and CgA were pulled-down but SgII was not in both AtT-20 and MIN6 cells. Asterisks indicate the pro-form of SgIII. Both A and B (n=7)-10. (C) In vitro pull-down by GST-SgIII, GST-CgA and GST-CPE. GST-fused proteins, SgIII 23-471, CgA 1-444 and CPE 23-477 (20 µg each), were immobilized on glutathione beads and incubated with AtT-20 cell extract (1.0 ml) for the pull-down experiments. A fifth of each pull-down product was analyzed by SDS-PAGE and immunoblotting using antibodies to SgIII, CgA and CPE. Asterisks indicate the pro-form of SgIII (n=5). (D) Enzyme assay of the GST-CPE fusion protein. CPE activity was assayed using dansyl-Gly-Lys as a substrate by HPLC-fluorophotometry. Rat cortex supernatant was used as a positive control and GST alone was used as a negative control for CPE enzyme activity. The enzyme activity was characterized for CPE by 1 mM CoCl2 and 1 mM GEMSA (n=3). (E) Binding between SgIII and CPE at pH 7.4 or 5.5 with or without 10 mM Ca2+. GST-SgIII (lanes 1-4) and GST (lanes 5-8) (20 µg each) were incubated with AtT20 cell extract (1.0 ml) at either pH 7.4 or 5.5, and either 10 mM Ca2+ (+) or Ca2+ (–). Next, the cell lysates alone (lanes 9 and 10) and cell lysates with GST-SgIII were analyzed by SDS-PAGE, and were immunoblotted with the antibody to CPE. One-twentieth of the cell extract was run on lane 3 (A,B), lane 5 (C) and lanes 9 and 10 (E). n=3.

View larger version (40K): [in a new window]   Fig. 3. Analysis of binding domains between SgIII and CPE. To investigate the specific binding domains of SgIII (A) and CPE (B), four GST-SgIII fusion proteins, full-length SgIII 23-471, SgIII 23-186, SgIII 187-373 and SgIII 374-471, were tested for binding to CPE; and five GST-CPE fusion proteins, pro-form CPE 23-477, mature form CPE 43-477, CPE 131-365, CPE 23-281 and CPE 332-477, were tested for binding to SgIII. GST-fusion proteins immobilized on glutathione beads were incubated with AtT-20 cell extract at pH 5.5 with 10 mM Ca2+. The precipitates were detected by antibody to CPE (upper panel) or antibody to SgIII (lower panel) antibody. Asterisks indicate the pro-form of SgIII. One-twentieth of the cell extract was run on lane 6 (A) and lane 7 (B) n=4 (A) and n=3 (B).

View larger version (117K): [in a new window]   Fig. 4. Intragranular localization of SgIII, CPE and peptide hormones PRL and POMC. (A) WT mouse (WT) mammotropes, (B) Cpefat mouse (fat/fat) mammotropes, (C) WT corticotropes and (D) fat/fat mouse corticotropes. SgIII and CPE were visualized with 15 and 10 nm gold particles, respectively. PRL (A,B) and POMC-derived ACTH-containing peptides (C,D) were labeled with 5 nm gold particles. CPE observed in the WT mammotropes and corticotropes (A,C, respectively) was not found in the fat/fat mammotropes and corticotropes (B,D, respectively), although these ultrathin sections were immunostained with a less-diluted antibody to CPE (1:10 at dilution). By contrast, SgIII was detected as in Fig. 1. Note that anti-ACTH-antibody-reacting POMC-derived peptides are distributed from the peripheral to the intragranular region of the SGs (C,D). Bar, 100 nm.

View larger version (36K): [in a new window]   Fig. 5. In vitro pull-down of POMC-derived peptides by GST-CPE, GST-SgIII and GST-CgA. The GST-fused proteins, SgIII 23-471, CgA 1-448 and CPE 23-477 (20 µg each), were immobilized on glutathione beads and incubated with AtT-20 cell extract (1.0 ml) for pulling down POMC-derived peptides. For GST pull-down control, although GST alone did not bring down POMC-derived peptides, GST-7B2 27-210 was used as a negative control to show that particular SG-residential proteins are able to bring down POMC-derived peptides. POMC-derived peptides in the pulled-down precipitates were analyzed by SDS-PAGE and immunoblotting using the antibody to ACTH. One-twentieth of the cell extract was run on lane 5 (n=5).

View larger version (29K): [in a new window]   Fig. 6. Compensatory expression of SgIII and CgA for CPE in the Cpefat mouse. (A) Pituitary homogenates from wild-type mice (WT, lane 1) and the Cpefat mouse (fat/fat, lane 2) were analyzed by immunoblotting and probed with antibodies to CPE, SgIII, CgA and -tubulin. The blots were visualized using the ECL kit. (B) The relative levels of CPE, SgIII, CgA and -tubulin were determined by quantitative radio-immunoblotting with pituitary gland homogenates from five WT mice and five Cpefat mice. The blots were reacted with polyclonal or monoclonal antibody to CPE, SgIII, CgA and -tubulin (see upper panel) and were probed with 125I-labeled secondary antibody. The signals were measured using a BAS2000 and were normalized by the signal obtained from an -tubulin blot as an internal control. The levels of CPE, SgIII, CgA and -tubulin in the Cpefat pituitary were expressed by assuming those in the WT pituitary as 100%.

View larger version (17K): [in a new window]   Fig. 7. Secretion of IR-ACTH from the WT and Cpefat pituitaries. (A) IR-ACTH content in the WT and Cpefat (fat/fat) pituitaries. The IR-ACTH content is expressed as ng/pituitary. Values are mean±s.e.m. (n=4). (B) IR-ACTH secretion from the WT and Cpefat pituitaries. A pituitary placed in a column was perifused with the buffer described in the Materials and Methods. The buffer was run at a flow rate of 1 ml/minute, and perifusate was collected every 4 minutes, thus each fraction number indicates 4 ml of perifusate aliquot. IR-ACTH is measured in each fractionated perifusate. Relative IR-ACTH (% of total pituitary content) released from the WT () and Cpefat (fat/fat) () pituitary (n=4). IR-ACTH secretion was stimulated with 10–9 M CRH and then 60 mM KCl at an indicated position (fr#2-5, CRH; fr#11-14, KCl).

View larger version (44K): [in a new window]   Fig. 8. Transfer of POMC from CPE to SgIII and CgA. (A) Effect of CgA, SgIII and MBP on POMC-binding capacity of CPE or SgIII. GST-CPE 23-477 or GST-SgIII 23-471 was incubated with the AtT-20 cell extract to fully bind POMC molecules. POMC-bound GST-CPE or GST-SgIII was incubated with increasing doses of recombinant CgA, SgIII or MBP, as indicated by bold figures on the top panel. GST-CPE or GST-SgIII was then pulled down with glutathione beads, and subjected to SDS-PAGE for POMC immnoblotting. In lane 9, one-twentieth of AtT-20 cell extract was run as a control. (B) In vitro pull-down of SgIII or CgA by anti-ACTH antibody. After POMC-bound GST-CPE was incubated with recombinant SgIII or CgA, POMC was pulled down by the anti-ACTH antibody. The precipitates were subjected to SDS-PAGE and immunoblotting for SgIII and CgA. Upper panel shows SgIII bound to POMC, and lower panel shows CgA bound to POMC. As a control IP experiment, preimmune serum was used instead of the anti-ACTH antibody (left panels shown as Control IP).

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