Mol. Cells 2016; 39(10): 756-761
Published online October 28, 2016
https://doi.org/10.14348/molcells.2016.0183
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: res1e272684@yahoo.co.jp
We have identified 88 interactor candidates for human growth hormone (GH) by the yeast two-hybrid assay. Among those, we focused our efforts on carboxypeptidase E (CPE), which has been thought to play a key role in sorting prohormones, such as pro-opiomelanocortin (POMC), to regulated secretory vesicles. We found that CPE co-localizes with and interacts with GH in AtT20 pituitary cells. Downregulation of CPE led to decreased levels of GH secretion, consistent with involvement of CPE in GH sorting/secretion. Our binding assay
Keywords AtT20, carboxypeptidase E, growth hormone, pituitary, secretion
Endocrine cells have specialized features to secrete peptide hormones. It has been known that these cells have two distinct pathways for hormone secretion: the constitutive and regulated pathway (Burgess and Kelly, 1987). These pathways have been extensively characterized for prohormones (hormones) such as pro-opiomelanocortin (POMC) and insulin (Moore et al
The structures reminiscent of the POMC-sorting motif have been identified for other prohormones or propeptides, including proenkephalin, proinsulin and pro-BDNF (Cool et al
In the present work, we searched for interactors of human GH by the yeast two-hybrid method, and identified a number of interactor candidates, including carboxypeptidase E. We then showed that GH and CPE, expressed in AtT20 mouse pituitary cells or in
For yeast two-hybrid screening, we used bait and prey vectors that we constructed ourselves (A. Mizutani et al
Yeast cells containing the GH bait plasmid and the prey library yeast cells were mated, and were plated on his-selective medium. Note that on this selective medium, only His-plus diploid cells can grow irrespective of the presence of the bait and prey plasmid. The His-plus colonies were observed under a microscope (BX61; Olympus Co.) for red and cyanic fluorescence, and only entirely red and cyanic colonies (i.e., colonies that grew on the selective plate dependently on both the bait and prey plasmids) were picked. The picked cells were successively streaked for three times on the same histidine-minus selective medium and also on the non-selective medium, containing histidine, to verify bait- and prey-dependency. The clones that went through the procedure were subjected to PCR amplification and sequence determination.
AtT20 cells were maintained in DMEM/F12 (Invitrogen) supplemented with 10% horse serum and 2.5% fetal bovine serum. For transient expression, plasmids were transfected with Lipofectamine 2000 (Invitrogen). Cells at 36-h and 40-h post transfection were used for microscopic and immunoprecipitation analyses, respectively. The cells for microscopic observation were fixed with 4% paraformaldehyde prior to acquiring images on a BZ-9000 microscope (KEYENCE). For RNA interference experiments, siRNA against mouse CPE or a control siRNA (Bonac Co.) was transfected into AtT20 cells with Lipofectamine 2000 at a final concentration of 75 nM. At 18 hours post siRNA transfection, cells were transfected by the GH-HA expression plasmid. At 28 h post GH-HA transfection, the media was changed to fresh media, the cells were incubated for 3 h, and the media and cells were harvested for protein analyses.
For immunoprecipitation with transfected AtT20 cells, equal amounts of protein extracts were incubated with anti-HA beads (Roche Diagnostics) in a Tris-based pH7.5 buffer, composed of 50 mM Tris pH7.5, 150 mM NaCl, 1 mM EDTA, 15% Glycerol, 0.1% IGEPAL CA-630, 1 mM dithiothreitol and Complete protease inhibitor (Roche Diagnostics) at 4°C for 2 h. Immune complexes were fractionated on a SDS-polyacrylamide gel, followed by immunoblotting. The blots were probed with anti-Flag antibody (Sigma-Aldrich Corp.). The signals were detected by chemiluminescence (ECL-Plus and ECL-prime; GE Healthcare) and LAS1000 imager (Fujifilm). For bacterial expression of proteins, BL21 (DE3) was used. The GH and CPE proteins were purified with Strep-Tactin Sepharose (Qiagen). Degrees of purity of different proteins can be seen in Supplementary Fig. B. For interaction assays, purified GST-GH or control GST proteins were incubated with glutathione Sepharose (GE Healthcare) for 1 h at 4°C, and the beads were washed by a MES-based pH5.5 buffer, composed of 50 mM MES (pH5.5), 120 mM NaCl, 5 mM KCl and 0.1% IGEPAL CA-630 (Cool et al., 1997). The CPE proteins, mixed with 10-times volume of the pH5.5 buffer, were added to the washed glutathione beads, incubated at 23°C for 30 min and then at 4°C for 1 h. The bound proteins were analyzed by Western blot with anti-Flag antibody or by SYPRO Ruby staining, following SDS-polyacrylamide gel electrophoresis. Detailed procedures are described in Supplementary Materials and Methods. Note that immunoprecipitation and GST pull-down experiments were performed multiple times, generating consistent results.
We screened human growth hormone (GH) interaction partners by the yeast two-hybrid screening system, which we developed in our laboratory (A. Mizutani et al.; a through description of the system to be published elsewhere). The human GH coding sequence, fused to the DNA-binding domain of GAL4 or POU2F2, was used as bait (Fig. 1A). Bait cells and prey library cells were mated, and were plated on a selective medium, where only His+ diploid cells can grow. On the his selective media, true positive colonies, growth of which should depend on the presence of both bait and prey plasmid, are expected to be entirely red and cyanic (see an example of positive cells shown in Fig. 1B). We were, therefore, able to identify rare red/cyanic positive colonies on the primary selective plates, where most colonies were not entirely red or entirely cyanic and were judged to be false positives.
We screened ten million of mated cells for GAL-GH and for POU-GH bait, and analyzed 135 red/cyanic positive clones in total. Sequence analyses of these clones led to identification of 88 yeast two-hybrid GH interactors (Supplementary Table). Two factors, OTUB1 and PDHB, were previously identified as GH interactors (Ewing et al
We first confirmed the GH-CPE interaction in our two-hybrid assay. As shown in Fig 1B, the fluorescent assay indicated that essentially all the colonies exhibited red and cyanic fluorescence when grown on the his-selective medium, showing that His-plus phenotype relies on both the POU-GH and TAD-CPE plasmids. The results were further confirmed by a more conventional growth assay and a LacZ assay, depicted in Fig. 1C, showing that growth under his selection and induction of
We analyzed intracellular localization of GH and CPE in AtT20 mouse pituitary cells. mVenus and mCherry proteins when expressed in AtT20 were distributed throughout the entire cells (Fig. 2A; top panels). In contrast, when fused to GH and CPE, mVenus and mCherry fluorescence showed punctuated distribution and were mostly co-localized densely at the tips of cell processes (Fig. 2A; bottom panels). These localization patterns are indicative of GH- and CPE-fusions localizing to RSV (Rivas and Moore, 1989).
We tested if CPE can interact with GH in immunoprecipitation assay using AtT20 cells. The 3×Flag-tagged CPE was co-expressed with HA-tagged or non-tag GH, and the cell extracts were subjected to immunoprecipitation with anti-HA antibody beads. The results of Western blot analyses of input extracts and immunoprecipitates are shown in Fig. 2B. The results showed that a significant amount of CPE-3×Flag was co-immunoprecipitated with GH-HA (Fig. 2B; 1st lane). When non-tag GH was subjected to the same analysis, only a small amount of CPE-3×Flag was detected (Fig. 2B; 2nd lane). Western blot analyses of input extracts showed that expression levels of CPE-3×Flag are similar between the GH-HA and non-tag GH control sample (Fig. 2B; see panel on the right). The results indicated that CPE and GH, when ectopically expressed in AtT20 cells, interact with each other. Because we were not able to reproduce CPE-GH interaction by mixing them
It has been previously reported that some substitution mutations of CPE are detrimental to its ability to bind to POMC (Zhang et al., 1999). Two of these mutations (i.e., R255S and K260A), however, did not show any appreciable effect on the ability of CPE to bind to GH (see the results for CPE 1-434 and CPE 218-434, shown to the left and to the right, respectively, in Fig. 3B). The result suggested that GH interacts with CPE in a different manner than POMC.
We used siRNA against CPE to test the effect of acute downregulation of CPE on GH expression and/or secretion in AtT20 cells. The cells were sequentially transfected by anti-CPE siRNA (or by a control siRNA) and then by the GH-HA plasmid. The amounts of GH secreted in the media were analyzed by Western blot analyses, together with cellular levels of CPE, GH and beta-actin (Fig. 4A). The results showed that anti-CPE siRNA significantly reduced CPE expression and also the amounts of GH secreted in the media. In contrast, effects of the siRNA were very limited, if any, on the levels of cellular GH. In AtT20 cells under these conditions, therefore, CPE plays a critical role in the GH sorting/secretion process. Because the presence of serum results in induction of GH secretion by several folds (Fig. 4B), we assume that most of the siRNA effects that we observed are due to decreased induction of GH secretion by serum.
We identified 88 GH interactor candidates in the two-hybrid assay. Some of them are supposed to be localized to nucleus or to mitochondria, and they are presumed to be false positives; they do not interact with GH in mammalian cells in spite of that they do so in the yeast two-hybrid assay. We focused our efforts on CPE in the present work. There are, however, other interactor candidates that attracted our attention. In addition to OTUB1 and PDHB, which were previously identified as GH interactors in a high-throughput biochemical screening (Ewing et al
Carboxypeptidase activity of CPE is often associated with proteolytic processing of prohormones such as POMC and proinsulin (Davidson and Hutton, 1987; Hook and Loh, 1984). The CPE protein, furthermore, is thought to play an important role in targeting prohormones to RSV as a receptor, presumably through direct interaction with sorting motifs of prohormones (Cool et al., 1997; Zhang et al., 1999). On the other hand, GH does not require proteolytic activity of CPE for its processing. We have shown here, nevertheless, that CPE co-localizes with and interacts with GH in AtT20 cells. The interaction was also reproduced
In our GH-CPE binding assay
The role of CPE in the hormone sorting and secretion pathway has been intensively studied for POMC and proinsulin, using CPE-mutant mice, or using pituitary or neuroblast cells with siRNA against CPE (Cawley et al., 2012 and references therein). With some complications due to residual activity of the CPE missense mutant and a mechanism compensatory to CPE defects, for example (Cawley et al
Mol. Cells 2016; 39(10): 756-761
Published online October 31, 2016 https://doi.org/10.14348/molcells.2016.0183
Copyright © The Korean Society for Molecular and Cellular Biology.
Akiko Mizutani1,2,*, Hidetoshi Inoko1, and Masafumi Tanaka1
1Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan, 2Faculty of Health and Medical Science, Teikyo Heisei University, Higashi-Ikebukuro, Toshima, Tokyo 170-8445, Japan
Correspondence to:*Correspondence: res1e272684@yahoo.co.jp
We have identified 88 interactor candidates for human growth hormone (GH) by the yeast two-hybrid assay. Among those, we focused our efforts on carboxypeptidase E (CPE), which has been thought to play a key role in sorting prohormones, such as pro-opiomelanocortin (POMC), to regulated secretory vesicles. We found that CPE co-localizes with and interacts with GH in AtT20 pituitary cells. Downregulation of CPE led to decreased levels of GH secretion, consistent with involvement of CPE in GH sorting/secretion. Our binding assay
Keywords: AtT20, carboxypeptidase E, growth hormone, pituitary, secretion
Endocrine cells have specialized features to secrete peptide hormones. It has been known that these cells have two distinct pathways for hormone secretion: the constitutive and regulated pathway (Burgess and Kelly, 1987). These pathways have been extensively characterized for prohormones (hormones) such as pro-opiomelanocortin (POMC) and insulin (Moore et al
The structures reminiscent of the POMC-sorting motif have been identified for other prohormones or propeptides, including proenkephalin, proinsulin and pro-BDNF (Cool et al
In the present work, we searched for interactors of human GH by the yeast two-hybrid method, and identified a number of interactor candidates, including carboxypeptidase E. We then showed that GH and CPE, expressed in AtT20 mouse pituitary cells or in
For yeast two-hybrid screening, we used bait and prey vectors that we constructed ourselves (A. Mizutani et al
Yeast cells containing the GH bait plasmid and the prey library yeast cells were mated, and were plated on his-selective medium. Note that on this selective medium, only His-plus diploid cells can grow irrespective of the presence of the bait and prey plasmid. The His-plus colonies were observed under a microscope (BX61; Olympus Co.) for red and cyanic fluorescence, and only entirely red and cyanic colonies (i.e., colonies that grew on the selective plate dependently on both the bait and prey plasmids) were picked. The picked cells were successively streaked for three times on the same histidine-minus selective medium and also on the non-selective medium, containing histidine, to verify bait- and prey-dependency. The clones that went through the procedure were subjected to PCR amplification and sequence determination.
AtT20 cells were maintained in DMEM/F12 (Invitrogen) supplemented with 10% horse serum and 2.5% fetal bovine serum. For transient expression, plasmids were transfected with Lipofectamine 2000 (Invitrogen). Cells at 36-h and 40-h post transfection were used for microscopic and immunoprecipitation analyses, respectively. The cells for microscopic observation were fixed with 4% paraformaldehyde prior to acquiring images on a BZ-9000 microscope (KEYENCE). For RNA interference experiments, siRNA against mouse CPE or a control siRNA (Bonac Co.) was transfected into AtT20 cells with Lipofectamine 2000 at a final concentration of 75 nM. At 18 hours post siRNA transfection, cells were transfected by the GH-HA expression plasmid. At 28 h post GH-HA transfection, the media was changed to fresh media, the cells were incubated for 3 h, and the media and cells were harvested for protein analyses.
For immunoprecipitation with transfected AtT20 cells, equal amounts of protein extracts were incubated with anti-HA beads (Roche Diagnostics) in a Tris-based pH7.5 buffer, composed of 50 mM Tris pH7.5, 150 mM NaCl, 1 mM EDTA, 15% Glycerol, 0.1% IGEPAL CA-630, 1 mM dithiothreitol and Complete protease inhibitor (Roche Diagnostics) at 4°C for 2 h. Immune complexes were fractionated on a SDS-polyacrylamide gel, followed by immunoblotting. The blots were probed with anti-Flag antibody (Sigma-Aldrich Corp.). The signals were detected by chemiluminescence (ECL-Plus and ECL-prime; GE Healthcare) and LAS1000 imager (Fujifilm). For bacterial expression of proteins, BL21 (DE3) was used. The GH and CPE proteins were purified with Strep-Tactin Sepharose (Qiagen). Degrees of purity of different proteins can be seen in Supplementary Fig. B. For interaction assays, purified GST-GH or control GST proteins were incubated with glutathione Sepharose (GE Healthcare) for 1 h at 4°C, and the beads were washed by a MES-based pH5.5 buffer, composed of 50 mM MES (pH5.5), 120 mM NaCl, 5 mM KCl and 0.1% IGEPAL CA-630 (Cool et al., 1997). The CPE proteins, mixed with 10-times volume of the pH5.5 buffer, were added to the washed glutathione beads, incubated at 23°C for 30 min and then at 4°C for 1 h. The bound proteins were analyzed by Western blot with anti-Flag antibody or by SYPRO Ruby staining, following SDS-polyacrylamide gel electrophoresis. Detailed procedures are described in Supplementary Materials and Methods. Note that immunoprecipitation and GST pull-down experiments were performed multiple times, generating consistent results.
We screened human growth hormone (GH) interaction partners by the yeast two-hybrid screening system, which we developed in our laboratory (A. Mizutani et al.; a through description of the system to be published elsewhere). The human GH coding sequence, fused to the DNA-binding domain of GAL4 or POU2F2, was used as bait (Fig. 1A). Bait cells and prey library cells were mated, and were plated on a selective medium, where only His+ diploid cells can grow. On the his selective media, true positive colonies, growth of which should depend on the presence of both bait and prey plasmid, are expected to be entirely red and cyanic (see an example of positive cells shown in Fig. 1B). We were, therefore, able to identify rare red/cyanic positive colonies on the primary selective plates, where most colonies were not entirely red or entirely cyanic and were judged to be false positives.
We screened ten million of mated cells for GAL-GH and for POU-GH bait, and analyzed 135 red/cyanic positive clones in total. Sequence analyses of these clones led to identification of 88 yeast two-hybrid GH interactors (Supplementary Table). Two factors, OTUB1 and PDHB, were previously identified as GH interactors (Ewing et al
We first confirmed the GH-CPE interaction in our two-hybrid assay. As shown in Fig 1B, the fluorescent assay indicated that essentially all the colonies exhibited red and cyanic fluorescence when grown on the his-selective medium, showing that His-plus phenotype relies on both the POU-GH and TAD-CPE plasmids. The results were further confirmed by a more conventional growth assay and a LacZ assay, depicted in Fig. 1C, showing that growth under his selection and induction of
We analyzed intracellular localization of GH and CPE in AtT20 mouse pituitary cells. mVenus and mCherry proteins when expressed in AtT20 were distributed throughout the entire cells (Fig. 2A; top panels). In contrast, when fused to GH and CPE, mVenus and mCherry fluorescence showed punctuated distribution and were mostly co-localized densely at the tips of cell processes (Fig. 2A; bottom panels). These localization patterns are indicative of GH- and CPE-fusions localizing to RSV (Rivas and Moore, 1989).
We tested if CPE can interact with GH in immunoprecipitation assay using AtT20 cells. The 3×Flag-tagged CPE was co-expressed with HA-tagged or non-tag GH, and the cell extracts were subjected to immunoprecipitation with anti-HA antibody beads. The results of Western blot analyses of input extracts and immunoprecipitates are shown in Fig. 2B. The results showed that a significant amount of CPE-3×Flag was co-immunoprecipitated with GH-HA (Fig. 2B; 1st lane). When non-tag GH was subjected to the same analysis, only a small amount of CPE-3×Flag was detected (Fig. 2B; 2nd lane). Western blot analyses of input extracts showed that expression levels of CPE-3×Flag are similar between the GH-HA and non-tag GH control sample (Fig. 2B; see panel on the right). The results indicated that CPE and GH, when ectopically expressed in AtT20 cells, interact with each other. Because we were not able to reproduce CPE-GH interaction by mixing them
It has been previously reported that some substitution mutations of CPE are detrimental to its ability to bind to POMC (Zhang et al., 1999). Two of these mutations (i.e., R255S and K260A), however, did not show any appreciable effect on the ability of CPE to bind to GH (see the results for CPE 1-434 and CPE 218-434, shown to the left and to the right, respectively, in Fig. 3B). The result suggested that GH interacts with CPE in a different manner than POMC.
We used siRNA against CPE to test the effect of acute downregulation of CPE on GH expression and/or secretion in AtT20 cells. The cells were sequentially transfected by anti-CPE siRNA (or by a control siRNA) and then by the GH-HA plasmid. The amounts of GH secreted in the media were analyzed by Western blot analyses, together with cellular levels of CPE, GH and beta-actin (Fig. 4A). The results showed that anti-CPE siRNA significantly reduced CPE expression and also the amounts of GH secreted in the media. In contrast, effects of the siRNA were very limited, if any, on the levels of cellular GH. In AtT20 cells under these conditions, therefore, CPE plays a critical role in the GH sorting/secretion process. Because the presence of serum results in induction of GH secretion by several folds (Fig. 4B), we assume that most of the siRNA effects that we observed are due to decreased induction of GH secretion by serum.
We identified 88 GH interactor candidates in the two-hybrid assay. Some of them are supposed to be localized to nucleus or to mitochondria, and they are presumed to be false positives; they do not interact with GH in mammalian cells in spite of that they do so in the yeast two-hybrid assay. We focused our efforts on CPE in the present work. There are, however, other interactor candidates that attracted our attention. In addition to OTUB1 and PDHB, which were previously identified as GH interactors in a high-throughput biochemical screening (Ewing et al
Carboxypeptidase activity of CPE is often associated with proteolytic processing of prohormones such as POMC and proinsulin (Davidson and Hutton, 1987; Hook and Loh, 1984). The CPE protein, furthermore, is thought to play an important role in targeting prohormones to RSV as a receptor, presumably through direct interaction with sorting motifs of prohormones (Cool et al., 1997; Zhang et al., 1999). On the other hand, GH does not require proteolytic activity of CPE for its processing. We have shown here, nevertheless, that CPE co-localizes with and interacts with GH in AtT20 cells. The interaction was also reproduced
In our GH-CPE binding assay
The role of CPE in the hormone sorting and secretion pathway has been intensively studied for POMC and proinsulin, using CPE-mutant mice, or using pituitary or neuroblast cells with siRNA against CPE (Cawley et al., 2012 and references therein). With some complications due to residual activity of the CPE missense mutant and a mechanism compensatory to CPE defects, for example (Cawley et al
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