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Molecular Endocrinology, doi:10.1210/me.2008-0006
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Molecular Endocrinology 22 (8): 1935-1949
Copyright © 2008 by The Endocrine Society

A Large Form of Secretogranin III Functions as a Sorting Receptor for Chromogranin A Aggregates in PC12 Cells

Lu Han, Masayuki Suda, Keisuke Tsuzuki, Rong Wang, Yoshihide Ohe, Hirokazu Hirai, Tsuyoshi Watanabe, Toshiyuki Takeuchi and Masahiro Hosaka

Department of Molecular Medicine (L.H., M.S., R.W., T.T., M.H.) and Biosignal Research Center (Y.O.), Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan; Department of Neurophysiology, Gunma University Graduate School of Medicine (K.T., H.H.), Maebashi 371-8511, Japan; and Department of Anatomy II (T.W.), Asahikawa Medical College, Asahikawa 078-8510, Japan

Address all correspondence and requests for reprints to: Masahiro Hosaka, Secretion Biology Lab, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan. E-mail: mhosaka{at}showa.gunma-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GRANIN-FAMILY PROTEINS (GRANINS), including chromogranin A (CgA), CgB, secretogranin II (SgII), SgIII, and 7B2, are localized to the secretory granules (SGs) of neuroendocrine cells (1). Granins have been shown to play at least three functions in SG formation: 1) sorting of peptide hormones to the SGs, 2) condensation of peptide hormones in the SGs, and 3) SG biogenesis (2, 3, 4, 5). First, as a sorting receptor, the membrane-associated form of carboxypeptidase E (CPE) was first demonstrated to bind and sort proopiomelanocortin (POMC) to the SGs (6). CPE was frequently found to localize with SgIII along the periphery of the SGs (7). We further showed that SgIII binds CPE, receives POMC from CPE, and transfers it to CgA (7). Thus, granins such as SgIII and CgA are involved in the sorting of POMC. Because CPE and SgIII are able to directly bind and sort peptide hormones to SGs, their sorting functions lead to the receptor-mediated sorting hypothesis (6, 7). Second, granins have been shown to condense peptide hormones with their aggregation-prone property in a weakly acidic, high-Ca2+ intragranular milieu (2, 3, 8). Because the aggregation-prone property of granins leads to the SG formation, the selective granin aggregation hypothesis has been advanced for the SG formation theory (2, 3, 8). Third, CgA was demonstrated to play an essential role in SG biogenesis in rat pheochromocytoma-derived PC12 cells (9). A decrease of CgA expression by antisense RNAs was shown to cause profound loss of dense-core SGs and impairment of the regulated secretion of exogenously expressed POMC.

This CgA-induced SG formation hypothesis has given rise to widespread controversy with the above two hypotheses (10, 11, 12). Two other granins, CgB and SgII, are also reported to form SGs in non-neuroendocrine cells, such as NIH3T3, and also COS fibroblasts (13, 14). However, Meldolesi’s group could not confirm the role of CgA in SG biogenesis using several PC12 cell sublines and nonsecretory cell lines (15). On the other hand, the role of CgA in SG biogenesis has been verified in vivo by two research groups. Mahapatra et al. (16) demonstrated that obliteration of CgA in a CgA-deleted mouse model led to decreased size and number of SGs as well as hypertension in this model. Kim et al. (17) reported that down-regulation of CgA expression by expressing antisense RNA against CgA in transgenic mice resulted in the decreased number of SGs with appearance of swelling features in the adrenal medulla. And Hendy et al. (18) reported that the CgA-deleted mice showed no obvious phenotype in endocrine functions, and their endocrine vesicle size and number were normal in the adrenal chromaffin cells. Because the CgA-deleted mice show increased expression of other granins, it should be addressed whether other granins compensate for granule formation instead of CgA.

Recently, we demonstrated that the N-terminal region of SgIII binds to cholesterol-rich lipid membranes, resulting in the formation of an SgIII-based CgA and peptide hormone complex in the SGs (19). CPE has also been shown to bind to lipid rafts within SGs (20). Thus, we postulated that to the cholesterol-rich SG-budding domain at the trans-Golgi network (TGN), SG-residential proteins with high cholesterol affinity, such as SgIII, CPE, and prohormone convertases, may be targeted for sorting peptide hormones into the SGs (19, 20, 21, 22, 23). Because SgIII receives POMC molecules from CPE and transfers them to CgA at the cholesterol-rich SG-budding domain (7), SgIII is considered to play a key function in the sorting of peptide hormones to the SGs.

SgIII was first identified as a 1B1075 clone whose corresponding mRNA exhibited differential expression between brain and nonneural tissues (24, 25). Mice missing the 1B1075 gene are fertile and exhibit no overt behavioral, locomotor, or morphological abnormalities, except that they have short ears. Although SgIII has not been as extensively studied as other granins, it was demonstrated that the levels of both SgIII and POMC mRNA increase more than 30-fold in the Xenopus, when it is moved to a black background from a white background, resulting in an increase in melanophore-stimulating hormone for color adaptation (26). The coordinated induction of SgIII and POMC messages suggests their involvement in a finely integrated secretory function. Indeed, we demonstrated that SgIII binds POMC in AtT-20 cells (7). However, although SgIII is highly expressed in pancreatic β-cells, their derived cell line MIN6, pituitary glands, and their derived cell line AtT-20, it has not been identified in adrenal chromaffin cells and their derived PC12 cells (8), where CgA is sorted correctly to the SGs.

PC12 cells have long been used for prohormone sorting and processing studies and for SG biogenesis studies (2, 3, 27). Thus, we think it important to explore whether PC12 cells have a distinct prohormone-sorting mechanism without SgIII or a common mechanism with an SgIII analog. In the present study, we identified a large and noncleaved SgIII in PC12 cells, which brings CgA to the SGs in a manner similar to that seen in other endocrine cells. We demonstrated remarkable ability of SgIII to bind the aggregated form of CgA molecules, which coaggregate at least adrenomedullin, a major peptide hormone in chromaffin cells, in their core. Furthermore, we found SgIII directly binds to adrenomedullin. Through this study, we also attempted to compromise the two conflicting hypotheses, the receptor-mediated sorting hypothesis and the selective granin aggregation hypothesis, for the SG formation, because Dikeakos and Reudelhuber (28) commented on the peptide hormone sorting study that despite the fundamental importance of the peptide hormone sorting process, the nature of the sorting signals for entry of proteins into dense-core secretory granules remains a source of vigorous debate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CgA Lacking the SgIII-Binding Domain Cannot Reach SGs at the Tips of the Processes
Because SgIII has not been identified in adrenal glands and their derived PC12 cells, the sorting mechanism of CgA remains to be elucidated in these cell types (8). To investigate this further, we studied the SG-targeting capacity of FLAG-tagged CgA constructs containing the SgIII-binding domain in PC12 cells, listed on the left panel of Fig. 1Go. As an SG-residential protein, we chose another granin-family protein, SgII. The SgIII-binding domain-containing full-length CgA 1–444, CgA 1–112, and CgA {Delta} (17–38) were localized densely to the tips of the processes and the perinuclear Golgi area (Fig. 1AGo, B, and C). All of them were virtually colocalized with SgII, suggesting that as long as CgA constructs contain the SgIII-binding domain, they are properly sorted to the SGs in PC12 cells, as seen in other endocrine cell-types (29). Notably the N-terminal disulfide loop has been demonstrated to be essential for the correct sorting of CgB in PC12 cells (30). We found that the mutant CgA {Delta}(17–38) lacking this disulfide loop was properly sorted to the SGs at the tips of the processes (Fig. 1CGo). However, when the SgIII-binding domain of CgA was deleted, the mutant CgA {Delta}(41–109) was not sorted to the SGs (Fig. 1DGo). Thus, the SgIII-binding domain is essential for the sorting of CgA to the SGs in PC12 cells.


Figure 1
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  		Fig. 1. Localization of CgA Constructs in PC12 Cells

PC12 cells transiently expressing CgA 1–444-FLAG (A), CgA 1–112-FLAG (B), CgA {Delta}(17–38)-FLAG (C), and CgA {Delta}(41–109)-FLAG (D) were first immunoreacted with mouse anti-FLAG antibody followed by FITC-labeled antimouse IgG (green, left), and rabbit anti-SgII antiserum followed by Texas Red-labeled anti-rabbit IgG (red, middle). Merged images of FLAG (green) and SgII (red) staining are presented on the right. E, To mark the Golgi area, PC12 cells expressing CgA {Delta}(41–109)-FLAG were immunostained with mouse anti-FLAG and sheep polyclonal anti-rat TGN38 antibody. Note that CgA constructs (A–C) with the SgIII-binding domain (CgA 41–109) are punctately stained with SgII-positive granules at the tips of the cell processes (arrowheads). In contrast, CgA {Delta}(41–109) (D and E) lacking the SgIII-binding domain, is restricted to the Golgi area (arrow). Bar, 20 µm. F, PC12 cell extracts (20 µg) expressing CgA-FLAG constructs were run on an SDS-PAGE for immunoblotting with the antibodies to FLAG to evaluate their expression.

 
PC12 Cells Express a Large-Sized Immunoreactive SgIII at Relatively Low Levels
Because the CgA construct lacking the SgIII-binding domain was not sorted to the SGs in the tips of the processes (Fig. 1DGo), we assumed the presence of an SgIII-like molecule in PC12 cells. Initially, we examined the Northern and Western blots for SgIII expression. By the Northern blot analysis using 10 µg total RNA, an SgIII message was detected weakly in PC12 and more strongly in the pituitary and endocrine cell lines (AtT-20 and MIN6) but not in the adrenal gland, liver, and nonendocrine cell line CHO (Fig. 2AGo). By the Western blot with 20 µg cell extracts, SgIII bands were detected in the brain, pituitary cell, and pancreatic β-cell lines (AtT-20, INS-1, and MIN6) and not in the adrenal glands, PC12 cells, and nonendocrine controls such as the liver and CHO cells (Fig. 2BGo, upper panel). However, by the blot with 100 µg cell extracts, immunoreactive SgIII was detected in the PC12 cells and weakly in the adrenal glands (Fig. 2BGo, lower panel). Because the immunoreactive SgIII displayed a larger size (75–80 kDa) compared with the three bands of approximately 70, 55, and 35 kDa in the other endocrine cell lines, we looked for the presence of SgIII mRNA in PC12 cells by RT-PCR and found the identical SgIII mRNA by DNA sequencing (data not shown).


Figure 2
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  		Fig. 2. Tissue and Cell-Type Distribution of SgIII Analyzed by Northern Blot and Western Blot

A, Northern blot. Total RNA (10 µg) from the indicated tissues and cell lines were run on an agarose gel, whose blot was hybridized with a 32P-labeled cDNA probe for SgIII. B, Western blot. Cell extracts (20 µg was used in the upper panel; an indicated dosage was used in the lower panel) from the indicated tissues, and cell lines were subjected to SDS-PAGE for immunoblotting with the antibody to SgIII.

 
Because SgIII is expressed at relatively low levels in the adrenal glands, we compared SgIII-expressing cells with CgA-positive cells in the glands. CgA was strongly immunostained in the adrenal medulla, whereas SgIII was weak in the same region (data not shown). Similarly, CgA was strongly immunostained from the Golgi area to the long processes in PC12 cells, whereas SgIII immunostaining was weak, although there was evidence of SgIII from staining along the long processes and the Golgi area (data not shown).

Large-Sized SgIII Is a Noncleaved Form
We presumed that the large form of SgIII may result from excessive glycosylation in the PC12 cells. To address the difference in the glycosylation of SgIII, we tested three different treatments: 1) tunicamycin, 2) glycosidase F, and 3) trifluoromethanesulfonic acid (TFMS). First, both the PC12 and INS-1 cells were cultured in the presence of tunicamycin for an indicated time period. The size of SgIII decreased with time, and the size differed between the PC12 and INS-1 cells (supplemental Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Second, by deglycosylation with glycopeptidase F, the N-linked oligosaccharides were removed from SgIII, but the PC12-derived SgIII and INS-1-derived SgIII differed in size (supplemental Fig. 1B). Third, each cell extract was subjected to treatment with TFMS to cleave their glycosidically linked carbohydrate chains. This TFMS treatment again did not make the two SgIII species identical in size (supplemental Fig. 1C). These results indicate that the N-glycosylation of SgIII does not account for its larger form in PC12 cells.

We then presumed that SgIII may be cleaved differently in PC12 cells and identified the N-terminal amino acid sequence of SgIII. To purify SgIII molecules from PC12 cells, the cell extract was chromatographed on diethylaminoethyl (DEAE)-Sepharose two times and then subjected to Macro-Prep High S support chromatography. SgIII-rich fractions were identified and combined and were then run through an amino acid sequence analyzer. The N-terminal sequence was XPKPEGSQDKSLHNRELXA (Fig. 3Go), which corresponds to rat SgIII 23–41, the region immediately after the signal peptide cleavage. This finding indicated that the large-sized SgIII in PC12 cells is a noncleaved form. The noncleaved form of SgIII suggests both PC12 cells and adrenal glands lack endoproteases specific for SgIII, which are usually found in other endocrine cells (31).


Figure 3
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  		Fig. 3. Purification of a Large Form of SgIII

Left panel, The SgIII-rich fraction of PC12 cells from Macro- Prep High S support chromatography was subjected to SDS-PAGE, followed by Coomasie brilliant blue (CBB) staining. C.E., Chromatographically enriched fraction. Right panel, Cells (100 µg for PC12, 20 µg for INS-1) were subjected to SDS-PAGE for immunoblotting with the antibody to SgIII to show the position of a large form of SgIII from PC12 (left lane). The N-terminal sequence was determined as described in the text.

 
To further confirm the distinct processing of SgIII in PC12 and INS-1 cells, we compared the cleavage patterns by infecting an adenoviral SgIII-FLAG to each cell line. A FLAG-tagged, size-enlarged SgIII band was detected in addition to that of the endogenous SgIII by the anti-SgIII antibody (supplemental Fig. 2, upper panel, lanes 1 and 2), whereas large-sized SgIII alone was detected by anti-FLAG antibody (upper panel, lanes 3 and 4). When FLAG-tagged SgIII was expressed in INS-1 cells, size-enlarged SgIII bands were detected in addition to the two endogenous SgIII bands (lower panel, lanes 1 and 2), whereas anti-FLAG antibody revealed the size-enlarged SgIII bands alone (lower panel, lanes 3 and 4). The processing pattern of FLAG-tagged SgIII bands was identical to that of the endogenous SgIII, and FLAG-tagged SgIII remained noncleaved in the PC12 cells.

Noncleaved Large SgIII Is Functional for CgA Binding and SG Targeting
To examine whether the noncleaved large SgIII is functional for binding to CgA, we made two glutathione S-transferase (GST)-tagged CgA constructs: full-length CgA 1–444 and CgA {Delta}(41–109) lacking the SgIII-binding domain. GST-CgA 1–444 pulled down three SgIII bands of approximately 70, 55, and 35 kDa in size from AtT-20, MIN6, and INS-1 cell extracts under an intragranular milieu of pH 5.5 and 10 mM Ca2+, whereas GST-CgA {Delta}(41–109) pulled down no SgIII bands (Fig. 4AGo). In contrast, CgA 1–444 pulled down noncleaved large SgIII of 75–80 kDa from PC12 cell extracts, whereas CgA {Delta}(41–109) did not (Fig. 4AGo, bottom panel).


Figure 4
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  		Fig. 4. Binding of CgA to SgIII in PC12 Cells

A, The constructs GST-CgA 1–444 and GST-CgA {Delta}(41–109), and GST alone, were incubated with cell extracts from AtT-20, MIN6, INS-1, or PC12 in an intragranular milieu (pH 5.5 and 10 mM Ca2+). After incubation, the GST constructs and GST alone were separated from the cell extract by glutathione beads and subjected to SDS-PAGE for immunoblot with the antibody to SgIII. B, PC12 (P) and INS-1 (I) cell extracts were immunoprecipitated by rabbit polyclonal antibody to SgIII (upper panel) or mouse monoclonal antibody to CgA (bottom panel). In both immunoprecipitation experiments, control immunoprecipitation was performed with either rabbit polyclonal or mouse monoclonal antibody to SgII, an SG-residential protein. The starting fraction (lanes 5 and 6; 20 µg extracted protein except lane 5 in bottom panel) and immunoprecipitates (lanes 1–4) were analyzed by an SDS-PAGE and immunoblot using the antibodies to CgA (upper panel) and SgIII (bottom panel). The cell extract for lane 5 in the bottom panel is 100 µg PC12 cell extract.

 
When PC12 and INS-1 cell extracts were precipitated by the anti-SgIII antibody, CgA was recovered in the precipitates (Fig. 4BGo, upper panel). Conversely, SgIII was recovered in the precipitates by the anti-CgA antibody (Fig. 4BGo, bottom panel). To prove specificity of the CgA and SgIII immunoblot, we used the SgII-specific polyclonal antibody (Fig. 4BGo, upper panel) and monoclonal antibody (Fig. 4BGo, bottom panel) for immunoprecipitation. The SgII-specific antiserum didn’t pull down SgIII and CgA (Fig. 4BGo). The precipitates of SgIII and CgA from PC12 cells are less than that from INS-1 cells. These findings suggest that low levels of SgIII also form a complex with CgA, but not with SgII, in PC12 as well as INS-1 cells.

Because SgIII requires the SG membrane-binding domain SgIII 40–186 for its targeting to the SGs in AtT-20 and MIN6 cells (19), we expressed three FLAG-tagged constructs: full-length SgIII 1–471, SgIII {Delta}(187–373) lacking the CgA-binding domain, and SgIII {Delta}(40–186) lacking the SG membrane-binding domain, to examine the targeting of SgIII in PC12 cells. As an SG-residential protein, we used SgII. SgIII 1–471 and SgIII {Delta}(187–373) were delivered to the tips of the processes in addition to the perinuclear Golgi area (Fig. 5Go). However, when the mutant SgIII lacking the SG membrane-binding domain was expressed, it was localized restrictedly to the Golgi area and failed to target to the SGs (Fig. 5Go). These findings suggest that the noncleaved, large SgIII is functional in terms of CgA binding and SG membrane binding in PC12 cells.


Figure 5
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  		Fig. 5. Localization of SgIII Constructs in PC12 Cells

PC12 cells transiently expressing FLAG-SgIII constructs SgIII 1–471 (A), SgIII {Delta}(187–373) (B), and SgIII {Delta}(40–186) (C and D) were first immunoreacted with the mouse anti-FLAG antibody followed by FITC-labeled antimouse IgG (green, left), rabbit anti-SgII (A–C), and sheep polyclonal anti-rat TGN38 antibody (D) followed by Texas Red-labeled antirabbit or antisheep IgG (red, middle). Merged images of FLAG (green), SgII (A–C; red), and TGN38 (D; red) staining are presented on the right. E, PC12 cells (20 µg) expressing SgIII constructs were run on an SDS-PAGE for immunoblotting with the antibodies to FLAG to show their expression. Note that SgIII constructs with the SG membrane-binding domain (SgIII 40–186) are colocalized with punctately stained SgII-positive SGs at the tips of the cell processes (arrowhead), which appear yellow on the right. In contrast, SgIII {Delta}(40–186) lacking the SG membrane-binding domain is restricted to the Golgi area (C and D; arrow). Bar, 20 µm.

 
CgA Targeting to SGs Is Blocked by the Overexpression of the SG Membrane-Binding Domain-Lacking SgIII
Although noncleaved SgIII can function as a binding partner for CgA, we wondered whether SgIII could target a large quantity of CgA to the SGs in PC12 cells (Fig. 2Go). We attempted to show that SG membrane-binding domain-lacking SgIII {Delta}(40–186) blocks the targeting of endogenous CgA to SGs but found that there was already an excess amount of endogenous CgA molecules present in the PC12 cells. The same as CgA, endogenous SgIII was not affected by CgA mutant expression (supplemental Fig. 3). Thus, we introduced FLAG-tagged SgIII {Delta}(40–186) and hemagglutinin (HA)-tagged CgA simultaneously to the PC12 cells at either a 2:1 or 10:1 ratio. Their expression levels reflected the ratio of the two DNA constructs used for transfection by immunoblotting (Fig. 6AGo). Actually, between 2:1 and 10:1 transfection, the densitometric ratio of CgA-HA blots was 1.1:1.0, whereas that of SgIII {Delta}(40–186) was 1:4.4 (data not shown). At the 2:1 ratio of FLAG-tagged SgIII {Delta}(40–186) and HA-tagged CgA, the CgA-HAs were localized densely at the tips of the cell processes in addition to the perinuclear Golgi area, whereas the SgIII {Delta}(40–186)-FLAG was restricted to the Golgi area (Fig. 6BGo). In contrast, at the 10:1 transfection ratio, the CgA-HAs were localized restrictedly to the perinuclear Golgi area marked by TGN38, as were the SgIII {Delta}(40–186)-FLAGs (Fig. 6BGo). Endogenous CgA was not affected in traffic by SgIII {Delta}(40–186) overexpression, suggesting the presence of an exceeding amount of CgA molecules relative to exogenous SgIII {Delta}(40–186) expression (Fig. 6BGo, bottom panel).


Figure 6
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  		Fig. 6. Effect of SgIII Lacking the SG Membrane-Binding Domain on the Sorting of CgA to SGs

A, FLAG-tagged SgIII {Delta}(40–186) and HA-tagged CgA 1–444 were cotransfected to PC12 cells at either a 2:1 or 10:1 molar ratio (see Materials and Methods). After cotransfection, PC12 cells (20 µg) were run on an SDS-PAGE for immunoblotting with the antibodies to HA or FLAG to show their expression. B, PC12 cells transfected with FLAG-tagged SgIII {Delta}(40–186) and HA-tagged CgA 1–444 at either a 2:1 or 10:1 molar ratio were immunoreacted with the mouse anti-FLAG antibody followed by FITC-labeled antimouse IgG (green, left), rat anti-HA, sheep polyclonal antirat TGN38, and rabbit anti-CgA antibody followed by Texas Red-labeled antirat, antisheep, or antirabbit IgG (red, middle). Merged images of FLAG, HA, and TGN38 staining are presented on the right. With the 2:1 ratio, HA-CgA was sorted to the SGs at the tips of the processes, but with the 10:1 ratio, HA-CgA sorting to the SGs was blocked. Bar, 20 µm. C, HA-tagged CgA 1–444 was transfected to PC12 cells with FLAG-tagged SgIII {Delta}(40–186) by the 2:1 or 10:1 ratio shown in A. The transfected cells were pulse-labeled with [35S]methionine/cysteine for 2 h. The cells were further chased for 1 h in DMEM with or without 10 mM carbachol stimulation. The radiolabeled cell extracts (Cell) and culture media (Medium) were immunoprecipitated with the anti-HA antibody. The precipitates were subjected to SDS-PAGE for fluorography (left panel). The radioactivity ratio between the cell extract and culture medium was quantified by BAS 1800II (Fujifilm) (right panel). All experiments were independently repeated at least three times.

 
We further confirmed these morphological data by pulse-labeling CgA-HA with [35S]methionine/cysteine and chasing the CgA-HA with or without 10 mM carbachol stimulation. We transfected PC12 cells with the FLAG-tagged SgIII {Delta}(40–186) and HA-tagged CgA at a 2:1 ratio and a 10:1 ratio. The 35S radioactivity in the CgA-HA was evaluated by densitometric recording of the fluorography image in the PC12 cell extract and culture medium (Fig. 6CGo, top). At the 2:1 transfection ratio, over 75% of the [35S]CgA-HAs were detected in the cell extract, and the rest of the radioactivity was found in the culture medium (Fig. 6CGo, bottom). Upon carbachol stimulation, the radioactivity retained in the cell extract decreased to approximately 40%, whereas the radioactivity in the culture medium increased to over 60%, indicating that the CgA was secreted to the culture medium by carbachol stimulation. By contrast, in the 10:1 ratio experiment, approximately 65% of the [35S]CgA-HA was secreted into the culture medium, and the rest (35%) remained in the cell extract, even in the nonstimulating state, as seen in a carbachol-stimulating condition (Fig. 6CGo, bottom). Thus, we suggest that CgA-HA is secreted through a constitutive secretory pathway when the regulated pathway is blocked by excessively expressed SgIII {Delta}(40–186).

SgIII Can Bind and Sort Aggregated Forms of CgA to SGs
Although SgIII is essential for the sorting of CgA to the SGs, we wondered whether a small quantity of SgIII could bind a large quantity of CgA in PC12 cells (Fig. 2Go). To explore whether SgIII is able to bind aggregated forms of CgA, we made three recombinant constructs, SgIII 23–471, CgA 1–444, and CgA 41–109, and examined their aggregation properties (Fig. 7AGo). With increasing concentrations, CgA 1–444 began to aggregate in an intragranular milieu of weakly acidic and high-calcium conditions (middle panel), whereas the aggregation of SgIII 23–471 was unaffected by changes in concentration (top panel). Because CgA reportedly makes an aggregation with the N-terminal disulfide loop between Cys17 and Cys38 of CgA (32), we examined the aggregation property of the disulfide loop-lacking CgA 41–109, which contains the SgIII-binding domain. As expected, CgA 41–109 did not aggregate as a function of the concentration changes (Fig. 7AGo, bottom panel).


Figure 7
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  		Fig. 7. Binding of SgIII to an Aggregated Form of CgA

A, Aggregation properties of CgA and SgIII. The SgIII 23–471, CgA 1–444, or CgA 41–109 was incubated at an indicated concentration in an intragranular milieu at pH 5.5 and 10 mM Ca2+, and then centrifuged. Pellets (P, precipitates of aggregated proteins) and supernatants (S) were directly subjected to SDS-PAGE. Note that CgA 1–444 forms an aggregate with its increasing dosage, whereas SgIII 23–471 and CgA 41–109 lacking the N-terminal disulfide loop do not. B, In vitro binding of CgA 1–444 or CgA 41–109 to GST-SgIII, GST-SgIII {Delta}(187–373), or GST-7B2. GST-SgIII, GST-SgIII {Delta}(187–373), or GST-7B2 (0.1 µg each) was incubated with either CgA 1–444 or CgA 41–109 at an indicated dosage. After incubation, each GST-fusion protein was precipitated by the anti-GST antibody. The precipitates were subjected to SDS-PAGE and immunoblotted for SgIII, CgA, or 7B2. The binding of CgA 1–444 to GST-SgIII increased with the CgA 1–444 dosage (top panel), whereas the binding of CgA 41–109 to GST-SgIII was constant despite an increase of the CgA 41–109 dosage (second panel).

 
We next incubated either CgA 1–444 or CgA 41–109 at increasing concentrations with GST-SgIII, GST-SgIII {Delta}(187–373) lacking the CgA-binding domain, or GST-7B2 at a constant concentration and then precipitated each fusion protein using the anti-GST antibody. The anti-GST antibody precipitated CgA 1–444 with increasing CgA concentrations, and the amounts of CgA precipitates were plateaued over 4.0 µg against 0.1 µg GST-SgIII (Fig. 7BGo, first panel). In contrast, the CgA 41–109 yielded a constant precipitate with the GST-SgIII (second panel), indicating that the SgIII-binding domain of CgA can bind SgIII as a monomer but does not have the capacity to form aggregation (Fig, 7AGo, bottom panel). As negative controls, GST-SgIII {Delta}(187–373) and GST-7B2 precipitated no CgA 1–444 (third and fourth panels). Then the binding ratios between SgIII and CgA were calculated on a molar basis. With increasing amounts of CgA (0.1, 0.5, 1.0, 2.0, 4.0, and 8.0 µg), the molar binding ratios between GST-SgIII and CgA 1–444 were 1:0.9, 1:4.3, 1:6.2, 1:9.8, 1:11.3, and 1:11.9 (Fig. 7BGo, first panel). On the other hand, the molar ratios between GST-SgIII and CgA 41–109 were always 1:0.9 with different amounts of CgA 41–109 (second panel). These data suggest that a single SgIII molecule can bind both single and multiple CgA molecules in aggregation. Taken together with the immunocytochemical findings in Fig. 6BGo, we concluded that a small quantity of SgIII is sufficient to target a large quantity of CgA to the SGs in PC12 cells.

SgIII Binds Adrenomedullin
Although large SgIII is functional for CgA sorting to the SGs in PC12 cells, it is not known which peptide hormones are sorted by SgIII. Thus, we examined the expression levels of candidate hormones and growth factors including neuropeptide Y (NPY), enkephalin, adrenomedullin, nerve growth factor (NGF), and neurotrophin 3 (NT-3) by Northern blot. Among these, we noted a high-level expression of adrenomedullin compared with a moderate expression of NGF and NT-3 and virtually no expression of NPY and enkephalin even with the use of 10 µg total RNA (data not shown). In terms of processing enzyme expression in PC12 cells, both PC1/3 and PC2 were not detected in adrenals and PC12 cells, as reported previously (31). On the other hands, cathepsin L was detected ubiquitously in the brain, adrenal glands, pituitary, AtT-20, and PC12 cells, although cathepsin L is reportedly responsible for the processing of preproenkephalin in the chromaffin granules (33). Because we noted adrenomedullin highly expressed in PC12 cells, we examined cellular localization of adrenomedullin by immunostaining. Adrenomedullin was localized punctately with CgA (Fig. 8AGo).


Figure 8
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  		Fig. 8. Coaggregation of Adrenomedullin with CgA

A, Immunostaining of adrenomedullin and CgA in PC12 cells. PC12 cells were immunoreacted with the goat anti-adrenomedullin antibody followed by Alexa488 antigoat IgG (green, left) or rabbit anti-CgA antibody followed by Texas Red-labeled antirabbit (red, middle). Merged image is presented on the right. Bar, 20 µm. B, Pull-down of adrenomedullin by GST-fused SgIII and CgA. GST-fused proteins, SgIII, CgA, 7B2, and GST alone (GST), were immobilized on glutathione beads, and glutathione beads (G-beads) were incubated with PC12 cell extract for pulling down adrenomedullin. The precipitates were detached from glutathione beads and adrenomedullin measured by RIA. For control, 7B2, GST alone, and glutathione beads were used. n = 4. C, Coaggregation of adrenomedullin with CgA. Adrenomedullin 21–182 was incubated with or without 1 µg/ml CgA 1–444 at an indicated concentration in an intragranular milieu at pH 5.5 and 10 mM Ca2+ and then centrifuged. Adrenomedullin in a pellet fraction (aggregated form) and that in a supernatant fraction were measured by RIA. A coaggregation form of adrenomedullin was expressed by the ratio of adrenomedullin in the pellet fraction to that in the supernatant fraction plus pellet fraction.

 
Because adrenomedullin is localized to SGs, we wondered whether SgIII targets adrenomedullin to SGs or not. To investigate this possibility, we examined the binding of SgIII and CgA to adrenomedullin by an in vitro pull-down assay with three GST-fused constructs immobilized onto glutathione beads: GST-SgIII 23–471, GST-CgA 1–448, and GST-7B2 27–210. Unexpectedly, both GST-SgIII and GST-CgA pulled down adrenomedullin (Fig. 8BGo), suggesting that SgIII navigates adrenomedullin together with CgA to SGs.

Because CgA is known to condense peptide hormones with its aggregates in a weakly acidic, high-Ca2+ milieu of SGs (2, 3, 8), we next examined whether CgA coaggregates adrenomedullin using an adrenomedullin 21–182 fragment and a full-length CgA fragment (Fig. 8CGo). Although adrenomedullin 21–182 alone barely precipitates as an aggregate with its increasing concentration, adrenomedullin was coaggregated in an increasing manner with a constant quantity of CgA. Thus, a large quantity of CgA appears to involve at least adrenomedullin in its aggregates to sort it to the SGs by the lead of SgIII.

Small Interfering RNA (siRNA)-Directed SgIII Depletion Impairs Intracellular Retention of CgA and Adrenomedullin
To confirm the role of SgIII in sorting CgA to SGs, we knocked down SgIII by siRNA. Because transfection of siRNA constructs was inefficient in PC12 cells, we used siRNA-SgIII with pEGFP-N2 vector encoding green fluorescent protein (GFP) to identify siRNA-SgIII-transfected cells by GFP. We sucked up cytosol from a GFP-marked single cell by a patch pipette and assessed SgIII knockdown by RT-PCR (Fig. 9AGo). SgIII mRNA was barely detected by RT-PCR. In contrast, other mRNAs for CgA, adrenomedullin, and SgII stayed unchanged either by siRNA-SgIII or by nonspecific siRNA. Furthermore, transfection of pEGFP-N2 only did not reduce SgIII expression at all compared with nontransfection control (data not shown). We then examined the effect of siRNA-SgIII on CgA and adrenomedullin traffic (Fig. 9BGo). As expected, CgA was lost from the cytoplasm except the perinuclear Golgi area (cell shape is indicated by broken circle). On the other hand, CgA was stained over the cytoplasm including the perinuclear Golgi area in nontransfected cells, which are indicated as GFP negative. Adrenomedullin also decreased from the cytoplasm by siRNA-SgIII. In contrast, adrenomedullin stained over the whole cytoplasm in nontransfected cells. Because we used SgII as a noninteracting protein with SgIII or CgA (Figs. 1Go and 5Go), SgII staining was unchanged (Fig. 9CGo, bottom). Thus, we suggest that SgIII is essential for the retention of CgA and adrenomedullin inside the cells. Summarizing with Fig. 6Go, CgA and adrenomedullin require SgIII for their targeting to SGs.


Figure 9
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  		Fig. 9. SgIII-Directed SgIII Knockdown Impairs Intracellular Retention of CgA and Adrenomedullin

A, siRNA-directed SgIII knockdown was evaluated for SgIII, CgA, adrenomedullin, and SgII expression by RT-PCR. PC12 cells were transfected with the pEGFP-N2 and one of the SgIII-specific siRNAs (SgIII siRNA; 305si, 306si, and 307si) and nonspecific siRNA. After 48 h, cytoplasmic RNA was aspirated from GFP-positive cells by a patch pipette. The first-strand cDNA was synthesized with single-cell-derived RNA. PCR was performed with primer sets for SgIII, CgA, adrenomedullin, or SgII. Amplified DNA was run on a 2.5% agarose gel. mRNA expression was compared between SgIII siRNA (clones 1–5) and nonspecific siRNA (clones 6–10). Adr., Adrenomedullin; N.T., first-strand cDNA synthesized without template mRNA. Note that SgIII was weakly detected in two of 14 SgIII siRNA-transfected cells. In contrast, SgIII mRNA was detected in control cells (n = 16). The Pearson’s {chi}2 for independence (61 ) was statistically significant ({chi}2 = 22.86; df = 1; P = 1.75 x 10–6), indicating that SgIII mRNA was markedly suppressed by the siRNA. Other mRNAs for CgA, adrenomedullin, and SgII were detected in all tested cells, indicating that harvesting of mRNAs was not affected by the transfection of SgIII siRNA. B, SgIII immunostaining was compared between GFP-positive and -negative cells. Left, GFP; center, SgIII visualized by Texas Red; right, merge. C, Immunostaining of CgA, adrenomedullin, and SgII. Left, GFPl center, CgA (top), adrenomedullin (middle), and SgII (bottom) visualized by Texas Red; right, merge. Bar, 20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Historically, PC12 cells have been used for the study on prohormone sorting, SG formation, and SG exocytosis (4, 27, 34, 35). In studies using PC12 cells, granins were proposed as granule-forming substrates and the selective granin aggregation hypothesis was developed for the formation of SGs (1, 3, 34). Also with use of a PC12 subline where CgA expression is knocked down by antisense RNAs, Kim et al. (9) demonstrated that CgA plays an essential role in SG biogenesis. Since then, the role of granins in SG biogenesis has been controversial (10, 11, 12), and even recently, some groups suggested the key function of CgA in SG biogenesis in vivo (16, 17), and others stated that the CgA-deleted mice showed no obvious phenotype in endocrine functions and their endocrine vesicle size and number were normal in the adrenal chromaffin cells (18). Nevertheless, the study of PC12 and chromaffin cells has contributed enormously to our understanding of SG biogenesis.

Although SgIII is highly expressed in pancreatic β-cells and their derived cell lines MIN6 and INS-1 and pituitary glands and their derived cell line AtT-20, SgIII has not been identified in adrenal chromaffin cells and their derived PC12 cells (8), where CgA is correctly sorted to SGs (36). By this reason, we have encountered the criticism that there must be a distinct sorting mechanism for CgA in PC12 and adrenal chromaffin cells different from that seen in pituitary and pancreatic endocrine cells (8). In fact, in this study, we identified a noncleaved large form of SgIII in PC12 cells and rat adrenal glands. Because PC12 cells lack substantial amounts of prohormone processing enzymes (31), SgIII appears to stay in a large form, unlike that in other endocrine cells. Although this large form of SgIII is able to bind and target CgA to SGs, its expression level was lower than that in pituitary glands and other endocrine cell lines including AtT-20, MIN6, and INS-1. SgIII protein was detected only by a 5-fold overload of cell extracts on the gel.

The SgIII molecule consists of at least three functional regions: the N-terminal side of SgIII 23–186 containing a cholesterol-binding domain, the middle region of SgIII 214–373 that binds to CgA, and the C-terminal side of SgIII 374–471 that binds to CPE (7, 19, 29). SG membranes are reported to consist of cholesterol-rich lipid bilayers whose cholesterol content is 65 mol% in the bovine pituitary neural lobe (20) and 40–45 mol% in the PC12, AtT-20, and MIN6 cells (37). Consistently, we showed that SgIII binds to cholesterol-rich liposomes in a cholesterol-dependent manner up to content of 65 mol% (19). With the lack of SgIII 40–186, SgIII {Delta} (40–186) cannot target to the tips of processes in PC12 cells (Fig. 5Go). Furthermore, the overexpression of SgIII {Delta} (40–186) hampered the targeting of CgA to the SGs in PC12 cells, and forced CgA to the constitutive secretory pathway (Fig. 6Go).

Since Loh et al. (6) proposed CPE as a sorting receptor to SGs, there have been differing views regarding the sorting mechanism of SG cargo proteins, namely, the sorting receptor hypothesis, exemplified by CPE, and the selective granin aggregation hypothesis (2, 3, 27). Especially, the sorting mechanism of CgA had been in confusion because the sorting of CgA was not disturbed in Neuro-2a cells depleting CPE (38). Our group (29) and Taupenot et al. (36) proposed a nonessential role of an N-terminal disulfide loop between Cys17 and Cys38 in the sorting of CgA to the SGs, which was previously thought to be essential for it (3, 5). Although the N-terminal loop of CgA and CgB has been shown to bind to the inositol (1,4,5)-triphosphate receptor (39, 40), the (1,4,5)-triphosphate receptor distributes largely in the endoplasmic reticulum, with some in the mitochondria, nucleus, plasma membrane, and SG membrane (41), suggesting that this receptor is not a primary determinant for the sorting of granins to the SGs. In the present study, by contrast, we demonstrated that the CgA constructs lacking the disulfide loop can target to the SGs in PC12 cells as well as in other endocrine cells as long as the SgIII-binding domain is included (19, 29). Glombik et al. (42) demonstrated that the disulfide loop of CgB mediates membrane binding in the TGN, and does so at a 5-fold higher efficiency, if two loops are present in the reporter protein in the PC12 cells. But the specificity of SG membranes to bind the disulfide loop has not been determined. Tooze et al. (27) emphasized the importance of the lipid raft in the SG-budding membrane of the TGN where regulated secretory proteins accumulate and proposed that the disulfide loop could function by mediating interactions between monomers of regulated secretory proteins and/or binding directly to putative loop receptors in the TGN. However, the loop receptors have not been characterized yet.

SgIII binds a constant amount of CgA 41–109, whereas it binds increasing amounts of full-length CgA 1–444, suggesting that the CgA sequence before CgA 40 or after CgA 110 is essential to make CgA aggregates (Fig. 7Go). CgA 1–40 was reported to make homodimer with the N-terminal disulfide loop between Cys17 and Cys38 (30). However, to make a multimer, other regions on the CgA sequence appear to be essential for forming the aggregates (43). Nevertheless, SgIII has the capacity for binding both monomers and multimers of CgA. Our studies indicate that a small quantity of SgIII is enough to target a large quantity of CgA in aggregates to the SGs. Although SgIII is able to bind peptide hormones such as POMC (7) and adrenomedullin in this study (Fig. 8BGo), a small quantity of SgIII would be insufficient as a hormone carrier to SGs, and a large quantity of CgA seems to function as a hormone carrier in PC12 cells. Because several peptide hormones are reportedly contained in the SGs of PC12 cells (44, 45, 46), we screened candidate hormones by Northern blot and noted that adrenomedullin is highly abundant. Adrenomedullin is known to be involved in the regulation of various physiological functions such as hypotensive action, bronchodilation, diuresis, water intake, aldosterone production, endothelin synthesis, and ACTH secretion (46). Adrenomedullin is synthesized in most tissues of the body (47) and secreted by many different cell types including vascular endothelial cells (48), smooth muscle cells (49), fibroblasts (50), macrophages (51), and astrocytes (52). Although adrenomedullin is secreted by constitutive exocytosis from nonendocrine cells, it is released by regulated exocytosis along with catecholamines upon stimulation of adrenal chromaffin cells (46). CgA is a major soluble protein in the core of catecholamine-storage granules (5). Consistently, adrenomedullin was localized with CgA and SgIII in the SGs and coprecipitated in an increasing manner with CgA aggregates when increasing doses of adrenomedullin were mixed with a constant dose of CgA for ultracentrifugation (Fig. 8CGo). Furthermore, siRNA-directed SgIII knockdown decreased the retention of CgA and adrenomedullin in the cells (Fig. 9CGo), suggesting that CgA and adrenomedullin are released through a constitutive secretory pathway. Thus, we propose that SgIII is essential for targeting CgA and adrenomedullin to SGs and tethering them to the SG membranes. We emphasize that the sorting function of SgIII for CgA is a common mechanism in the SG biogenesis in endocrine cells including PC12 cells. Furthermore, a small quantity of SgIII is enough to target a large quantity of CgA aggregates together with adrenomedullin to SGs in PC12 and perhaps in adrenal chromaffin cells. In terms of the two conflicting hypotheses; the receptor-mediated sorting hypothesis and the selective granin aggregation hypothesis for the SG formation, both hypotheses seem to be consistent. We suggest that the sorting function of SgIII for CgA to SGs and the involvement of adrenomedullin to CgA aggregates are coupled in a coordinating manner for the functional SG formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of Fusion Proteins Using Bacterial Expression Vectors
GST-fused granins were made using pGEX-KG (53). The rat SgIII, CgA, and 7B2 fragments were as follows: SgIII 23–471, SgIII 374–471, SgIII {Delta}(187–373), CgA 1–444, CgA {Delta}(41–109), and 7B2 27–210. The GST-fusion proteins were expressed in BL21(DE3) and were purified on glutathione beads.

Antibodies
The following antibodies were used: mouse monoclonal antibody to FLAG (Sigma Chemical Co., St. Louis, MO), GST (Sigma), rabbit polyclonal antiserum to SgII (54), mouse monoclonal antibody to SgII (Abcam, Cambridge, MA), fluorescein isothiocyanate (FITC)-labeled antimouse IgG (Jackson ImmunoResearch, West Grove, PA), Texas Red-labeled antirabbit IgG (Jackson ImmunoResearch), Alexa488-labeled anti-goat IgG (Molecular Probes, Eugene, OR), rabbit polyclonal anti-SgIII antibody (SgIII 373–471) (19), rabbit polyclonal anti-CgA antibody (CgA 426–438) (30), rabbit polyclonal anti-CgA antibody (Y291; peptide antibody against CgA 94–130) (Yanaihara Institute Inc., Shizuoka, Japan), mouse monoclonal anti-CgA antibody (Lab Vision Corp., Fremont, CA), mouse monoclonal anti-CPE antibody (Research Diagnostics, Inc., Flanders, NJ), mouse monoclonal anti-{alpha}-tubulin antibody (Sigma), sheep polyclonal anti-rat TGN38 antibody (Serotec, Oxford, UK), and goat polyclonal anti-rat adrenomedullin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Cell Culture
Rat pheochromocytoma-derived PC12 cells, kindly presented by Dr. Regis B. Kelly, were maintained in DMEM supplemented with 10% fetal bovine serum and 10% horse serum with 5 ng/ml NGF. Rat insulinoma-derived-INS-1 cells, kindly presented by Dr. Claes B. Wollheim, were maintained in DMEM supplemented with 10% fetal bovine serum (55).

In Vitro Binding of a Large Form of SgIII with CgA
For solubilization of the PC12 cells and INS-1 cells, we used a buffer containing 0.1 M NaCl, 1% Triton X-100, 2 mM EGTA, and a protease inhibitor cocktail (1 µg/ml each of aprotinin, leupeptin, and pepstatin A and 0.4 mM phenylmethylsulfonyl fluoride). The buffer was adjusted to pH 5.5 by 50 mM 2-[N-morpholino]ethanesulfonic acid (MES) or to pH 7.4 by 20 mM HEPES. Soluble extracts were incubated for 12 h at 4 C with GST-CgA 1–444, GST-CgA {Delta}(41–109), or GST alone immobilized onto glutathione beads under continuous rotation. The beads were then pelleted by centrifugation. The proteins bound to the GST fusion proteins were subjected to 10% SDS-PAGE for immunoblotting.

Northern Blot Analysis
Total RNAs (10 µg) from the rat tissues and cell lines were electrophoresed on an agarose gel and then blotted onto a membrane. The blot was hybridized with radiolabeled cDNAs encoding rat SgIII 1–471, proprotein convertase 1/3 (PC1/ 3) 1–752, proprotein convertase 2 (PC2) 1–502, cathepsin L 1–334, β-NGF 1–241, NT-3 1–258, NPY 1–98, enkephalin 1–269, adrenomedullin 1–185, and β-actin 35–116.

Immunoprecipitations
PC12 and INS-1 cells grown in 100-mm dishes were solubilized in 1 ml 50 mM MES (pH 5.5) containing 0.15 M NaCl, 10 mM CaCl2, 1% Nonidet P-40, and the protease inhibitor cocktail. After removal of the insoluble materials by centrifugation, soluble extracts (1 mg total protein) were incubated with 10 µl diluted antisera (1:100) for 1 h at 4 C. Then 30 µl of a 50% (wt/vol) slurry of protein A or protein G-Sepharose was added, and the mixture was further incubated for 12 h at 4 C under continuous rotation. One fifth of the precipitated immunocomplexes underwent 10% SDS-PAGE for immunoblotting.

Light Microscopy Analysis
For light microscopy, we isolated pituitaries and adrenals from eight rats that were killed by cervical dislocation under light ether anesthesia. The pituitaries and adrenals were quickly removed, cut into small pieces, and processed as described previously (56).

Expression of FLAG-Tagged Constructs
The FLAG-tagged constructs (Stratagene, La Jolla, CA) were made in the pcDNA3 (Invitrogen, Carlsbad, CA) as follows: 1) CgA 1–444, 2) CgA 1–112, 3) CgA {Delta}(41–109) deleting CgA 41–109, 4) CgA {Delta}(17–38) deleting CgA 17–38, 5) SgIII 1–471, 6) SgIII {Delta}(187–373) deleting SgIII 187–373, and 7) SgIII {Delta}(40–186) deleting SgIII 40–186. PC12 cells were cultured on eight-well Lab-Tek chamber slides, and then the DNA constructs were introduced to the cells with Lipofectamine 2000 reagents (Invitrogen).

Deglycosylation of SgIII
PC12 and INS-1 cells were cultured in the absence or presence of various amounts of tunicamycin to inhibit N-glycosylation. The N-linked oligosaccharides of SgIII were also removed by enzymatic deglycosylation. Briefly, PC12 and INS-1 cells were lysed with 200 mM sodium phosphate (pH 8.6), 1% Nonidet P-40, 1% sodium dodecyl sulfate, and 2% 2-mercaptoethanol. The cell extracts were heated at 100 C for 10 min and then incubated with 1 U/ml glycopeptidase F (Wako, Osaka, Japan) at 37 C for the indicated time. Samples were subjected to 7.5% SDS-PAGE for immunoblotting with the anti-SgIII polyclonal antibody. The cell extracts were further subjected to treatment with TFMS, which cleaves glycosidically linked sugar chains, as described elsewhere (57).

Purification of SgIII from PC12 Cells
PC12 cells were harvested for extraction of SgIII in 50 mM MES-NaOH (pH 5.5), 1 mM EGTA, 0.1 M NaCl, 1% (vol/vol) Triton X-100, and the protease inhibitor cocktails. The extract was chromatographed first on DEAE-Sepharose fast flow (Amersham Pharmacia Biotech, Piscataway, NJ). The SgIII-rich fraction was eluted with a linear gradient of NaCl from 0.05–1 M in 20 mM 1-methylpiperagine-HCl (pH 5.3). After desalting by dialysis, the fraction was rechromatographed on DEAE-Sepharose fast flow. SgIII was eluted with a linear gradient of NaCl from 0.05–0.5 M in 20 mM Tris-HCl (pH 8.0). The SgIII-rich faction was dialyzed and subjected to Macro-Prep High S support (Bio-Rad, Hercules, CA) chromatography. The column was eluted with a linear gradient from 0.05 M NaCl in 20 mM citric acid (pH 2.9), to 0.05 M NaCl in 20 mM HEPES (pH 7.4). The SgIII-rich fraction was dialyzed to remove salts and was then evaporated to dryness.

Determination of N-Terminal Amino Acid Sequence
An aliquot of the SgIII fraction from the PC12 cells was subjected to SDS-PAGE, and proteins were then transferred to a polyvinylidene difluoride membrane. The membrane was subsequently stained with Coomassie brilliant blue R-250. The band of about 75–80 kDa was excised from the membrane, and the N-terminal amino acid sequence was determined by a gas-phase amino acid sequence analyzer (Procise 492; Applied Biosystems, Foster City, CA), as described elsewhere (58).

Immunostaining
PC12 cells were fixed with 4% paraformaldehyde and then permeabilized with high-salt TPBS (0.01 M sodium phosphate buffer; 0.5 M NaCl; 0.1% Tween 20, pH 7.3) containing 0.1% Triton X-100. The cells were incubated with a mixture of rabbit anti-SgII antibody (1:2000), mouse monoclonal anti-FLAG antibody (1:400), rabbit anti-CgA antibody (Y291; 1:500), goat anti-adrenomedullin antibody (1:200), and sheep anti-TGN38 antibody for 18 h at 4 C. For the second antibody reaction, the cells were incubated for 1 h at 20 C with a mixture of FITC-labeled antimouse IgG, Texas Red-labeled antirabbit IgG, Alexa488-labeled antigoat IgG, and Texas Red-labeled antisheep IgG. The coverslips were mounted in 90% glycerol (vol/vol in PBS) containing 0.1% p-phenylenediamine dihydrochloride (Sigma). Stained cells were observed with a laser scanning confocal microscope (LSM5Pascal; Carl Zeiss Co., Ltd., Oberkochen, Germany).

Pulse-Chase Experiment
We transfected FLAG-tagged SgIII {Delta}(40–186) and HA-tagged CgA with either a 2:1 [1.2 µg pcDNA SgIII {Delta}(40–186)-FLAG, 0.6 µg pcDNA CgA-HA, and 2.6 µg pcDNA3] or 10:1 [4.0 µg pcDNA SgIII {Delta}(40–186)-FLAG and 0.4 µg pcDNA CgA-HA] ratio to PC12 cells in a six-well (35-mm diameter) plate. Empty pcDNA vector was supplemented to adjust a total DNA amount for transfection. The PC12 cells were radiolabeled with 0.2 mCi [35S]methionine/cysteine (Amersham Pharmacia Biotech) for 2 h. After radiolabeling, the medium was changed to DMEM with or without 10 mM carbachol stimulation for 1 h. Cell extracts and culture media were immunoprecipitated with the anti-HA antibody and subjected to SDS-PAGE for fluorography. The 35S signals of blots were recorded using BAS-1800II (Fujifilm, Tokyo, Japan).

In Vitro Aggregation-Binding Assay
Recombinant SgIII and CgA were purified, as described elsewhere (7). Briefly, SgIII 23–471, CgA 1–444, CgA 41–109, and adrenomedullin 21–182 were placed in the pGEX-6P-1 (Amersham), and GST-fusion proteins were produced in the BL21(DE3) strain and then purified on glutathione beads. To remove a GST fragment from the GST-fusion protein, GST-fused SgIII, CgA, and adrenomedullin were digested with pReScission protease (Amersham). Each reaction mixture was centrifuged at 3000 x g for 10 min to obtain SgIII 23–471, CgA 1–444, CgA 41–109, or adrenomedullin 21–182. To examine the self-aggregation property of the four fragments, SgIII 23–471 (0.1–1 µg/ml), CgA 1–444 (0.1–1 µg/ml), CgA 41–109 (0.1–1 µg/ml), or adrenomedullin 21–182 (0.1–1 µg/ml with or without 1 µg/ml CgA), each fragment was incubated in 50 mM MES (pH 5.5), 0.1 M NaCl, 10 mM CaCl2 for 2 h at 37 C and then centrifuged at 100,000 x g for 30 min at 4 C. To prevent nonspecific binding of proteins to the polycarbonate ultracentrifuge tubes, 0.01% Triton X-100 was included in the mixture (59). Pellets (aggregated protein precipitates) and the supernatant fraction of SgIII or CgA proteins were directly subjected to SDS-PAGE. Immunoreactive (ir) adrenomedullin was measured by RIA (Phoenix Pharmaceuticals, Inc., Belmont, CA).

To examine SgIII binding to aggregated forms of CgA, we made the three GST-fusion proteins GST-SgIII, GST-SgIII {Delta}(187–373), and GST-7B2. They were expressed in the BL21 strain by the pGEX-KG. GST-fusion proteins were detached from glutathione beads by reduced glutathione. GST-SgIII was incubated in 50 mM MES (pH 5.5), 0.1 M NaCl, 10 mM CaCl2, 0.01% Triton X-100 for 2 h at 37 C with either CgA 1–444 or CgA 41–109, as indicated in Fig. 7BGo. As a control, GST-SgIII {Delta}(187–373) or GST-7B2 was incubated with increasing amounts of CgA 1–444. Each GST-fusion protein was precipitated by the anti-GST antibody and protein G-Sepharose 4 Fast Flow (Amersham). One tenth of the precipitates were subjected to SDS-PAGE and immunoblotting for visualization.

To examine whether adrenomedullin binds to SgIII, CgA, or 7B2, GST constructs fused with SgIII 23–471, CgA 1–444, or 7B2 27–210 (50 ng each) were immobilized onto glutathione beads. The beads were incubated with PC12 cell extract (1 ml, including 821-1216 pg adrenomedullin) in 50 mM MES (pH 5.5), 0.1 M NaCl, 10 mM CaCl2, 0.01% Triton X-100 for 2 h at 4 C under continuous rotation. After centrifugation, precipitates were detached from glutathione beads by reduced glutathione and divided into two fractions, supernatants and glutathione beads. Adrenomedullin was measured for the two fractions by RIA.

siRNA Constructs
The siRNAs were synthesized against rat SgIII by Ambion (Austin, TX). Maximal knockdown efficiency was achieved with 305si (sense, CGAUGCCGAUUCAACUAAAtt, and antisense, UUUAGUUGAAUCGGCAUCGtc), 306si (sense, GCUUGGAAAGGAGAACGAAtt, and antisense, UUCGUUCUCCUUUCCAAGCcg), and 307si (sense, CAAAGAAGAUUACGACCUUtt, and antisense, AAGGUCGUAAUCUUCUUUGtt). PC12 cells were transfected with 0.3 µg pEGFP-N2 (Invitrogen) and 20 pmol each of SgIII siRNA in eight-well Lab-Tek chamber slides using Lipofectamine 2000 (Invitrogen). For control, Ambion’s Silenser Select Negative Control no. 1 (nonspecific siRNA) was used.

Single-Cell RT-PCR
Single-cell RT-PCR was performed as described previously (60). Briefly, cytoplasmic content was aspirated from GFP-expressing PC12 cells by a patch pipette. The content was expelled into a tube with 2.75 µl of a solution containing random hexamers (5 µM at the final concentration; Roche Diagnostics); pdT15 (final 5 µM; Roche Diagnostics, Mannheim, Germany); dATP, dCTP, dGTP, and dTTP (final 0.5 mM each; Sigma); and 10 U of ribonuclease inhibitor (Promega, Madison, WI). Reverse transcription was performed by adding Sensiscript reverse transcriptase (0.5 µ;, QIAGEN, Valencia, CA) for 6 h at 37 C in a final volume of 10 µl. One microliter of RT product was then amplified with the rat SgIII primers (upper primer, 5-ccggaaaaacccacaagcaggac-3, 1 µM; lower primer, 5-gagctcctcaaagtcatcgtc-3, 1 µM), rat CgA primers (upper primer, 5-atgcgctcctccgcggctttggc-3, 1 µM; lower primer, 5-ctggtgtcgaaggatggagagg, 1 µM), rat adrenomedullin primers (upper primer, 5-atgaagctggtttccatcacc-3, 1 µM; lower primer, 5-gagcccaagagtctgggtcgggac-3, 1 µM), or rat SgII primers (upper primer, 5-cttccttccagcgaaaccagctgc, 1 µM; lower primer, 5-tgagctctctgccaagtgac-3, 1 µM) in 25 µl reaction using KOD DNA polymerase (Toyobo, Osaka, Japan). The PCR program was as follows: initially 95 C for 2 min, 55 C for 2 min, and 68 C for 1 min followed by 35 PCR cycles of 95 C for 1 min, 55 C for 1 min, and 68 C for 30 sec using GeneAmp PCR System 9600 (Applied Biosystems), yielding 190-, 218-, 205-, and 227-bp fragments for SgIII, CgA, adrenomedullin, and SgII, respectively.


   ACKNOWLEDGMENTS
 
We thank Ms. M. Kosaki and Ms. M. Hosoi for their technical support.


   FOOTNOTES
 
This work was supported by Japan Diabetes Foundation (to M.H.), Japan Society for the Promotion of Science (to L.H.), and Grants-in-Aid and the Global COE Program from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 15, 2008

Abbreviations: CgA, Chromogranin A; CPE, carboxypeptidase E; DEAE, diethylaminoethyl; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; MES, 2-[N-morpholino]ethanesulfonic acid; NGF, nerve growth factor; NPY, neuropeptide Y; NT-3, neurotrophin 3; POMC, proopiomelanocortin; SG, secretory granule; SgII, secretogranin II; siRNA, small interfering RNA; TFMS, trifluoromethanesulfonic acid; TGN, trans-Golgi network.

Received for publication January 8, 2008. Accepted for publication May 9, 2008.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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