Corticotropin-releasing factor binding protein enters the regulated secretory pathway in neuroendocrine cells and cortical neurons

Neuropeptides 45 (2011) 273–279

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Corticotropin-releasing factor binding protein enters the regulated secretory pathway in neuroendocrine cells and cortical neurons Elías H. Blanco 1, Juan Pablo Zúñiga 1, María Estela Andrés, Alejandra R. Alvarez, Katia Gysling ⇑ Millenium Nucleus in Stress and Addiction, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago 8330025, Chile

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Article history: Received 23 November 2010 Accepted 7 May 2011 Available online 31 May 2011 Keywords: Corticotropin releasing hormone (CRH) Release Sorting CRH-BP Binding protein Neuropeptides Regulated secretory pathway

a b s t r a c t Corticotropin releasing factor binding protein (CRF-BP) is a 37 kDa glycoprotein that binds CRF with high affinity. CRF-BP controls CRF levels within plasma during human pregnancy. It has also been shown that CRF-BP is expressed in various brain nuclei. Main actions that have been proposed for brain CRF-BP are either decreasing available CRF or facilitating CRF ligand-induced activation of CRF-R2 receptors. For both actions, it is necessary the release of CRF-BP from CRF-BP expressing neurons. However, the secretion mode of CRF-BP is currently unknown. We used heterologous expression of CRF-BP-Flag in PC12 cells and in primary culture of rat cortical neurons to study CRF-BP secretion mode. We observed that CRFBP-Flag immunoreactivity presents the typical cytoplasmatic punctuate pattern that has been described for neuropeptides and proteins that enter the regulated secretory pathway in PC12 cells. Quantitative analysis of double immunofluorescence confocal images showed that CRF-BP-Flag colocalizes with secretogranin II, marker of secretory granules, both in PC12 and in primary-cultured rat neurons. Furthermore, CRF-BP-Flag is released from PC12 cells upon high K+-depolarization. Thus, our results show that CRF-BP is efficiently sorted to the regulated secretory pathway in two cellular contexts, suggesting that the extracellular levels of CRF-BP in the central nervous system depends on neuronal activity. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction One of the most important adaptations in mammals ensuring survival is the stress response. The corticotropin releasing factor (CRF) system is composed of CRF, key actor of the stress response, described in 1981 (Vale et al., 1981). CRF binds to CRF-R1 and CRFR2 receptors (Dautzenberg and Hauger, 2002). In addition, a soluble glycoprotein, Corticotropin Releasing Factor Binding Protein (CRF-BP), that binds CRF with high affinity was isolated from human plasma during pregnancy (Behan et al., 1989). Three other neuropeptides, urocortin, urocortin 2 and urocortin 3 with high affinity for one or both CRF receptors have been added to the CRF system (Vaughan et al., 1995; Lewis et al., 2001; Reyes et al., 2001), but only urocortin binds CRF-BP with high affinity (Lewis et al., 2001). CRF-BP is a 37 kDa glycoprotein which contains a single glycosylation site on asparagine 204 (Suda et al., 1989; Potter et al., 1991; Behan et al., 1993) that was originally associated to the binding of CRF. However, recently it has been shown that this glycosylation is not necessary for CRF binding (Huising et al., 2008). CRF-BP was first described as a circulating peripheral factor that binds CRF ⇑ Corresponding author. Tel.: +562 3542654; fax: +562 3542660. 1

E-mail address: [email protected] (K. Gysling). These authors contributed equally to this work.

0143-4179/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2011.05.002

regulating the levels of free CRF, and thus regulating the hypothalamus–pituitary–adrenal axis during gestational stages (Behan et al., 1989; Perkins and Linton, 1995). CRF-BP was first considered as a passive CRF buffer; however, other roles for CRF-BP have been also proposed (reviewed by Westphal and Seasholtz, 2006). It has been shown that CRF-BP facilitates the CRF ligand-induced activation of CRF-R2 receptors in rat midbrain (Ungless et al., 2003; Wang et al., 2007). In addition, immunohistochemical and in situ hybridization studies have shown the discrete expression of CRF-BP in the rat brain. Neocortex, hippocampus and amygdaloid complex are main areas of CRF-BP expression (Potter et al., 1992). Electron microscopy studies have shown that CRF-BP is localized in a neuropeptide-like fashion in isocortex and in vesicle-like structures in the bed nucleus of the stria terminalis (Peto et al., 1999). Thus, both physiological and anatomical evidence suggest that CRF-BP could have an active neuropeptide-like role in the brain. However, there are not available studies of CRF-BP subcellular sorting to support its putative neuropeptide-like role. We hypothesized that CRF-BP is sorted into secretory granules, hallmark of the regulated secretory pathway. To test this hypothesis, we studied the secretion mode of CRF-BP using heterologous expression of human CRF-BP (Huising et al., 2008) in the PC12 pheochromocytome cell line. PC12 cells are an endocrine cell line that has been extensively used to study the regulated secretory pathway (Chanat

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and Huttner, 1991; Chanat et al., 1993; Gorr et al., 1999; Taupenot et al., 2002; Chen et al., 2005; Courel et al., 2008). We also studied the subcellular localization of CRF-BP in primary cultures of rat cortical neurons. Our results show, for the first time, that CRF-BP readily enters the regulated secretory pathway in two different cellular contexts. 2. Materials and methods

cells were incubated with either Rabbit polyclonal anti secretogranin II (SgII, Abcam) 1/200 as marker of secretory granules, or Rabbit polyclonal anti Giantin (Abcam) 1/500 to immune detect Golgi; or Rabbit polyclonal anti Calnexin (Sigma) 1/200 to immuno detect ER. Cells were washed and incubated for 1 h with the following secondary antibodies: Donkey anti Rabbit Alexa Fluor 488 (green) and Donkey anti Mouse Alexa Fluor 594 (red) (Invitrogen™). Cells were washed and mounted with Dako mounting media.

2.1. Cell culture conditions

2.4. Confocal microscopy

PC12 cells were grown in 100 mm plates with Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% Horse Serum (Gibco), 5% Fetal Bovine Serum (Gibco) and 1% Penicillin– Streptomycin–Glutamine 100 (Gibco) at 37° C 10% CO2.

Immunofluorescence images were captured with a confocal microscope (OlympusÒ, Fluoview 1000) and Fluoview v6.0 software. The images were digitally obtained with a 100 objective (N.A 1.4 oil) and using a sequential mode of laser scanning. In the conditions used each pixel corresponds to 38 nm. All the images were obtained without Gain and with Offset Zero. Staking pictures are composed of 26 z-planes with a Z-step of 80 nm per cell. Captured images were processed with ImageJ software (Rasband, WS, ImageJ, NIH, http://rsb.info.nih.gov/ij). Deconvolution and colocalization analysis were made with ‘‘Iterative Deconvolve 3D’’ and ‘‘JaCoP’’ plugins, respectively (Bolte and Cordelières, 2006).

2.2. DNA transfection and construction of expression vectors For cell transfection, the construct pcDNA3.1/CRF-BP-Flag, kindly provided by Dr. Wylie Vale (Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, CA), was amplified in Escherichia coli DH5a (Invitrogen) and purified by the AxyPrep Plasmid Miniprep Kit (Axygen Biosciences). The pcDNA3.1/CRFBP construct was previously successfully used to overexpress CRF-BP by Dr. Vale’s group (Huising et al., 2008). Twenty-four hrs before transfection, 50,000 cells per well were cultured on round coverslips pre-treated with 50 lg/mL of poli-L-lysine (Sigma). Five hundred nanograms of plasmid DNA were transfected with Lipofectamine 2000Ò (Invitrogen™) according to manufacturer’s instructions. Precursor of human CART peptide, including the signal peptide (MESSRVRLLPLLGAALLLMLPLLGTRA) and the signal peptide fragment were obtained by PCR using specific oligonucleotide primers incorporating an XhoI restriction site followed by a Kozak translation initiation consensus sequence (GCCACC-ATG) at the 50 end, and a BamHI restriction endonuclease site at the 30 end. pCR/hCART vector served as a template [kindly donated by Dr. Patrick Keller (Keller et al., 2006). The amplified fragments were purified, digested with XhoI and BamHI and subcloned in frame into the same sites of the mammalian expression vectors for the enhanced fluorescence variant of GFP (EGFP), pEGFP-N3 (Clontech), under the control of the cytomegalovirus promoter to produce CART-EGFP and SP-EGFP chimeras. Previously, EGFP was mutated (A206K) using PCR-driven overlap extension and pEGFPN3 as the template (Heckman and Pease, 2007) to work with a monomeric version of EGFP (Zacharias et al., 2002). The truncated form of chromogranin A (DN_CgA-Flag) without the fragment 41109 corresponding to the sorting domain of chromogranin A described by Hosaka et al. (2002), was a kindly donated by Dr. Hosaka and was used as control of constitutive secretion. All the constructs were verified by restriction analysis and nucleotide sequence analysis. In addition, the expression of each chimera tagged to GFP was confirmed by immunoblotting of total cell lysate of transfected PC12 cells using a purified polyclonal rabbit anti-GFP (Santa Cruz, SC-9996). The expression of the transfected CRF-BP-Flag construct was confirmed by immunoblotting of total cell lysate of transfected PC12 cells using Anti-Flag (1:500; Stratagene) and anti-CRF-BP (1:100; Santa Cruz) antibodies. 2.3. Immunofluorescence Forty-eight hours after transfection, cells were fixed with 4% PFA for 30 min at room temperature and permeabilized with 0.2% Triton X 100/2.5% BSA for 30 min at room temperature. Cells were incubated for 2 h at room temperature with Mouse Anti-Flag M2 Antibody (Stratagene) 1/1000 and, after washing with PBS,

2.5. Colocalization analysis Quantification of fluorescence colocalization was done using the method described by Van Steensel et al. (1996). Twenty-six z-plane images per cell (PC12 and primary cortical neurons) obtained from at least three independent experiments were processed for each data. In the case of neurites, single image from six neurite segments were analyzed. Cross correlation function (CCF) of dual labeling images was calculated by shifting the green image over a distance of DX pixels in the X-direction with respect to the red image or vice versa, with 20 pixels 6 DX 6 +20 pixels. These values are plotted as the function of DX (pixel shift). For each value of DX, the Pearson’s correlation coefficient was calculated. 2.6. CRF-BP-Flag secretion assay For this assay, we use the calcium saline medium (CaSB) described by Courel et al. (2008). A batch of transfected PC12 cells were incubated in CaSB (150 mm NaCl, 5 mm KCl, 2 mm CaCl2, and 10 mm HEPES, pH 7.4) for 30 min to measure basal secretion. In order to determine induced-secretion, after the 30 min basal period, PC12 cells were incubated in 55 mM K+-CaSB. To avoid change in osmolarity, we replaced equimolar amount of KCl by NaCl. Thereafter, PC12 cells were incubated in this high K+ medium for the same time period of 30 min used for basal levels. To quantify basal and stimulated release of CRF-BP-Flag the respective supernatants were collected, cleared by centrifugation (5 min, 1000 rpm, 4 °C), and concentrated using reverse phase Sep-Pak C-18 silica cartridges (Strata, Phenomenex). Thereafter, proteins were separated by SDS–PAGE on 12% polyacrylamide gels and transferred onto nitrocellulose sheets (BioRad). Membranes were blocked with 5% non-fat milk prepared in PBS 1 for 2 h at room temperature and incubated with Mouse Anti-Flag M2 Antibody (Stratagene) 1:200 at 4 °C over night with constant agitation. Membranes were washed with 0.05% Tween 20 in PBS. After washing, membranes were incubated for 2 h with Anti Mouse (1:2000) IgG conjugated with peroxidase (Jackson). After incubation, membranes were washed and exposed to a chemiluminescent substrate (SuperSignal, Thermo Scientific) for band detection. We used Image J software to quantify the 37 kDa band intensities.

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Fig. 1. Expression and immunoblotting of CRF-BP in PC12 cells transfected with the pcDNA3.1/CRF-BP-Flag. Lysates of PC12 cells transfected with pcDNA3.1/CRF-BPFlag were subjected to Western blot. Lanes 1 and 2 were incubated with anti CRF-BP antibody and lanes 3 and 4 with anti-Flag antibody. Arrowhead depicted the CRFBP-Flag 37 kDa band. Upper bands are unspecific.

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neurons. Rats were obtained from the Animal Care Facility of the Faculty of Biological Sciences, Catholic University of Chile. All procedures were approved by the bio-ethic committee of the Faculty of Biological Sciences, Catholic University of Chile and were performed in strict accordance with the guidelines published in the ‘‘NIH Guide for the Care and Use of Laboratory Animals’’ and with the principles presented in the ‘‘Guidelines for the Use of Animals in Neuroscience Research’’ by the Society for Neuroscience. Cortical neurons were seeded in polylysine-coated round coverslips in 6 well plaques and maintained in Neurobasal medium (GIBCO) supplemented with B27 (Invitrogen, Carlsbad, CA). To inhibit glial proliferation, 2 lM cytosine arabinoside was added on the first day. The transfection was performed with Lipofectamine (Invitrogen) after 4 days of in vitro growing. Twenty-four hrs after transfection, cells were fixed with 4% PFA in PBS for 30 min. The immunodetection protocol was the same employed with PC12 cells. 3. Results

2.7. Primary culture of rat cortical neurons

3.1. Expression of CRF-BP in PC12 cells transfected with the pcDNA3.1/ CRF-BP-Flag

Cortex from Sprague–Dawley rats at embryonic day 18 were dissected and primary cultures of rat cortical neurons were prepared as described by Alvarez et al. (2004) for hippocampal

We tested whether human CRF-BP is adequately expressed in PC12 cells transfected with the pcDNA3.1/CRF-BP-Flag vector. As can be seen in Fig. 1, the transfection of this vector expressed

Fig. 2. Subcellular distribution of CRF-BP-Flag. PC12 cells were transfected with the CRF-BP-Flag expression vector and incubated in basal medium for 48 h. Aldehyde-fixed cells were subjected to double immunofluorescence protocols using Flag, SgII, Giantin and Calnexin antibodies. Alexa 594 (red) second antibody was used to visualize CRF-BPFlag and Alexa 488 (green) second antibody was used to visualize the endogenous subcellular markers SgII, Giantin and Calnexin. Cells were examined by deconvolution microscopy. (A1–C1) CRF-BP-Flag immunofluorescence; (A2–C2) imunofluoresecence for each of the three subcellular markers. (A3–C3) Merging of CRF-BP-Flag imunofluorescence with the respective subcellular marker. (A4–C4) Colocalization analysis of CRF-BP-Flag with SgII (A4), Giantin (B4) and Calnexin (C4). CCFs for colocalization of CRF-BP-Flag with SgII, Giantin and Calnexin were obtained from a total of 14, 11 and 13 cells, respectively. Scale bars: A–C = 2 lm; zoom = 0.2 lm.

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CRF-BP. Both anti-CRF-BP (lane 1) and anti-Flag (lane 3) antibodies labeled a band of the expected molecular size for CRF-BP (ffi37 kDa). A faint band of 37 kDa was also observed in nontransfected PC12 cells that could correspond to endogenous CRF-BP, as it has been previously described (Chatzaki et al., 2002). Additional bands corresponded to unspecific immunoreactivity from antibodies used.

respectively. We found poor or no evident colocalization between CRF-BP and Giantin (Fig. 2B3), and between CRF-BP-Flag and Calnexin (Fig. 2C3). These observations indicate that once synthesized, CRF-BP is efficiently transported between ER and Golgi, and from the trans-Golgi network to the secretory granules. 3.4. Colocalization analysis shows high degree of colocalization of CRFBP with SgII and exclusion from ER

3.2. CRF-BP colocalizes with endogenous SgII in PC12 cells Human CRF-BP-Flag was detected by Flag-immunoreactivity and its sub-cellular localization was analyzed by confocal microscopy. CRF-BP-Flag was detected in the cytoplasm of PC12 cells presenting a punctuate pattern with a random distribution (Fig. 2A1), characteristic of the sub-cellular distribution of proteins that follow the regulated secretory pathway (Taupenot et al., 2002). To confirm that CRF-BP-Flag is sorted to the regulated secretory pathway, we compare its subcellular distribution with the distribution of endogenous SgII (Taupenot et al., 2003). Both, CRF-BP-Flag (Fig. 2A1) and SgII (Fig. 2A2) immunofluorescence showed the classical punctuate pattern of proteins and neuropeptides sorted to secretory granules of the regulated secretory pathway. Merging of both the CRF-BP-Flag and SgII labeling shows colocalization (Fig. 2A3). 3.3. CRF-BP colocalizes poorly with Golgi and none with endoplasmic reticulum (ER) markers To further confirm that CRF-BP is sorted to the regulated secretory pathway, we compared the colocalization of CRF-BP-Flag with Giantin and Calnexin. Giantin is a protein of the golgin family that resides at the Golgi, attached to its membrane by a C-terminal anchor domain (Rosing et al., 2007) and it has been used as specific marker of Golgi’s apparatus. Calnexin is a lectin that mediates protein folding in the ER (Myhill et al., 2008) and it has been used as specific marker of ER. As expected, Giantin-ir (Fig. 2B1) and Calnexin-ir (Fig. 2C1), presented the characteristic Golgi and ER pattern,

We quantified the degree of colocalization between the immunofluorescence of CRF-BP-Flag and those of different subcellular markers. Pearson’s coefficient obtained from merged composite microphotographs is the approach most commonly used to quantify colocalization between two labels (Chen et al., 2005; Taupenot et al., 2002; Van Steensel et al., 1996). Even though this approach is useful, it should be used with caution to analyze colocalization in small organelles such as secretory granules, as saturated images could overscore the degree of colocalization. To avoid overscore induced by saturated images, we applied the method used by Van Steensel et al. (1996) to study colocalization of two proteins in cell nuclei. This method gives the Pearson’s coefficient (CCF value at X = 0) and allows to discriminate between a truly and random overlapping. As shown in Fig. 2A4, the quantitative analysis of merged CRFBP-Flag and SgII labels yielded a ‘‘bell shape’’ curve indicating colocalization of CRF-BP-Flag with SgII, with a CCF (at X = 0) of 0.46 ± 0.04, further supporting the observation presented in Fig. 2A3. In contrast, when CRF-BP-Flag and Giantin labeling were analyzed, a flat bell shape tending to be parallel to the X axis was generated indicating a marginal colocalization of both labels (Fig. 2B4). In the case of Calnexin and CRF-BP-Flag, the analysis yielded an inverted bell shape curve indicating exclusion behavior between both labels (Fig. 2C4). CCF (at X = 0) obtained for CRF-BPFlag colocalization with ER and Golgi markers were 0.13 ± 0.02 and 0.12 ± 0.01, respectively; indicating low degree of colocalization. Thus, the quantitative analysis confirmed that CRF-BP-Flag colocalizes with SgII, a marker of secretory granules.

Fig. 3. CRF-BP-Flag colocalizes with CART-EGFP. PC12 were cotransfected with the CRF-BP-Flag expression vector and an expression vector for the full length precursor of CART fused to EGFP (CART-EGFP) or for the signal peptide fragment of proCART (SP-EGFP). Aldehyde-fixed cells were subjected to immunofluorescence protocol using Flag antibody and Alexa 594 (red) as second antibodies. CART-EGFP and SP-EGFP were visualized by EGFP auto-fluorescence (green). Cells were examined by deconvolution microscopy. (A1) CART-EGFP fluorescence. (B1) SP-EGP fluoresecence. (A2 and B2) CRF-BP-Flag immunofluorescence. (A3 and B3) Merging of CRF-BP-Flag with CART-EGFP or SP-EGFP. (A4 and B4) Colocalization analysis of CRF-BP-Flag with CART-EGFP and SP-EGFP, respectively. CCFs were obtained from 5 cells in each case. Scale bar = 2 lm.

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3.5. CRF-BP colocalizes with CART peptide To further probe that CRF-BP-Flag is sorted similarly to a typical neuropeptide, its subcellular distribution was compared with that of cocaine and amphetamine regulated transcript (CART) neuropeptide. PC12 cells were cotransfected with CRF-BP-Flag and an expression vector encoding the precursor of CART fused to EGFP (CART-EGFP). As can be seen in Fig. 3, CART-EGFP (Fig. 3A1) and CRF-BP-Flag (Fig. 3A2) yielded a similar punctuate pattern with high colocalization (Fig. 3A3). As a control, CRF-BP-Flag distribution was compared with that of the signal peptide of CART fused to EGFP (SP-EGFP). As can be seen in Fig. 3B1–B3, CRF-BP-Flag colocalized poorly with SP-EGFP which showed a perinuclear subcellular pattern. The quantitative analysis yielded a ‘‘bell shape’’ curve for the merging of CRF-BP-Flag and CART-EGFP (Fig. 3A4). As expected, the merging of CRF-BP-Flag with SP-EGFP yielded a flat bell shape curve indicating poor colocalization of CRF-BP with SP-EGFP (Fig. 3B4). Both CCF (at X = 0) were significantly different (0.48 ± 0.04 vs 0.19 ± 0.01; p < 0.01 by Mann–Whitney test). Interestingly, it is possible to observe secretory vesicles that were stained only for either CRF-BP-Flag or CART-EGFP (Fig. 3), indicating a degree of segregation for the sorting of both molecules. 3.6. CRF-BP secretion is induced by exposure to 55 mM-K+ An important parameter to establish whether a protein is sorted to the regulated secretory pathway is to show that its secretion is stimulus-dependent. In order to demonstrate that CRF-BP-Flag release is stimulus-dependent, PC12 cells transfected with the pcDNA3.1/CRF-BP-Flag were first incubated in CaSB and thereafter in 55 mM K+-CaSB for the same time period. As can be seen in Fig. 4 A, when PC12 cells were incubated in CaSB no basal secretion of CRF-BP-Flag was detected. In contrast, when PC12 cells were incubated in 55 mM K+-CaSB for the same time period, inducedsecretion of CRF-BP-Flag was detected in two independent secretion assays (Fig. 5A). As a control we overexpressed a truncated form of chromogranin A (DN_CgA-Flag) that lack its sorting domain (Hosaka et al., 2002). As expected, DN_CgA-Flag was not detected in both normal and 55 mM K+ media (Fig. 5A). In order to control whether the constructs were adequately expressed, we determined the amount of CRF-BP-Flag and of DN_CgA-Flag present in the incubation medium in which transfected cells were maintained during the 48 h previous to the secretion assay (Fig. 5B). As can be seen in Fig. 5B, both CRF-BP-Flag and of DN_CgA-Flag were detected in the extracellular media in which respective transfected cells were maintained during the 48 h previous the secretion assays, indicating that they were adequately expressed. In order to control the specificity of the Flag antibody, we analyze the incubation medium in which transfected cells with the empty pCDNA3.1 were maintained. In this case no Flag-like immunoreactivity was observed (Fig. 4B). 3.7. CRF-BP colocalizes with endogenous SgII in primary cultures of rat cortical neurons We have also studied CRF-BP-Flag sorting in rat cortical neurons. Immunofluorescence of endogenous SgII (Fig. 5A1 and B1) and of transfected CRF-BP-Flag (Fig. 5A2 and B2) showed punctuate patterns throughout the cytoplasm and neurites of transfected cortical neurons. The merging of both labels showed CRF-BP-Flag is partially stored with SgII (Fig. 5A3 and B3). The quantitative analysis of the merging of CRF-BP-Flag and SgII labeling yielded a bell shape curve in the cell body (Fig. 5A4) with a CCF (at X = 0) of 0.40 ± 0.03; and in neurites (Fig. 5B4) with a CCF (at X = 0) of 0.74 ± 0.05. Taken together, the data indicate that CRF-BP-Flag colocalizes with SgII in cortical neurons.

Fig. 4. High K+-induced secretion of CRF-BP-Flag. PC12 cells transfected with either CRF-BP-Flag or DN_CgA-Flag expression vectors were incubated for 30 min in CaSB (basal secretion) and thereafter for additional 30 min in 55 mM K+-CaSB (stimulated secretion). CRF-BP-Flag and DN_CgA-Flag secreted in the respective media were analyzed by Western blotting using anti Flag antibody (a-Flag). (A) Immunoblot showing secretion assays for pCDNA3.1/CRF-BP-Flag (dish A and B, two independent assays) and for pCDNA3.1/DN_CgA-Flag (dish C, 1 assay). () Basal and (+) stimulated secretion of the respective protein. (B) Immunoblot showing the amount of CRF-BP-Flag (dish A and B) and DN_CgA-Flag (dish C) present in the incubation media in which respective transfected cells were maintained, during the 48 h previous to the secretion assays. Dish D corresponds to the 48 h incubation medium of PC12 cells transfected with the empty pCDNA3.1 vector.

4. Discussion Our results show that CRF-BP is efficiently sorted to the regulated secretory pathway in PC12 cells and in rat cortical neurons. In both PC12 and rat cortical neurons, transfected CRF-BP-Flag colocalizes with endogenous SgII, marker of the regulated secretory pathway. CRF-BP-Flag also colocalizes with overexpressed CART-EGFP, but not with a construct bearing only the signal peptide of CART fused to EGFP which should sort EGFP to the constitutive secretory pathway (Taupenot et al., 2002). Furthermore, CRF-BP is secreted upon high K+-induced depolarization. Altogether, CRF-BP has a neuropeptide-like behavior being sorted to the secretory granules and released upon stimulation. It has been reported the expression of CRF-BP in PC12 cells (Chatzaki et al., 2002). However, the PC12 cells used in present study express only marginal amounts of CRF-BP and therefore it is not possible to study the sorting of native CRF-BP Thus, we took advantage of the overexpression of CRF-BP tagged to Flag. Overexpression could be inducing alterations in cellular functioning. However, this approach has been successfully used to study the sorting of numerous proteins and neuropeptides (Hosaka et al.,

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Fig. 5. CRF-BP-Flag colocalizes with SgII in rat primary cortical neurons. (A1 and B1) endogenous SgII immunoflorescence in neuronal soma and neurites, respectively. (A2 and B2) CRF-BP-Flag immunofluorescence. (A3 and B3) Merging of both labels at the level of somas and neurites, respectively. Arrowheads indicated some secretory granules with both stain. (A4) CCFs were obtained from 10 neuronal somas and from six neurite segments. Scale bar: 2 lm.

2002; Courel et al., 2008). It allows comparing different proteins and their truncated constructs bearing fluorescent tags to quantify them such as HA, Flag, and others. A good example of such study is the paper by Hosaka et al. (2002). In their study, it was shown that the domain 49-109 of chromogranin A is the sorting domain responsible for accessing the regulated pathway. K+-induced exocytosis from PC12 cells transfected with either CRF-BP-Flag or the truncated form of chromogranin A, without its sorting domain (Hosaka et al., 2002) secreted only overexpressed CRF-BP-Flag and not DN_CgA-Flag, further proving that CRF-BP is readily secreted through the regulated secretory pathway. To better analyze the degree of colocalization avoiding overscore induced by saturated images, we applied the method designed by Van Steensel et al. (1996) to analyze the colocalization of gluco- and mineralo-corticoid receptors in nuclear compartments of hippocampal neurons. The Van Steensel’s analysis of CRF-BP-Flag and SgII immunofluorescence generated a bell shape curve and a CCF at X = 0 of 0.46 ± 0.04 confirming the high degree of colocalization between CRF-BP-Flag and SgII. This degree of colocalization observed for CRF-BP-Flag and endogenous SgII is similar to that found by Courel et al. (2008) for the colocalization between exogenous SgII and endogenous chromogranin B in PC12 cells, two proteins that are specific hallmarks of secretory granules. It is worth to mention that the Flag octapeptide has been validated as an excellent tag to study the sorting pathway of other proteins such as chromogranin A (Hosaka et al., 2002) or secretogranin III (Hosaka et al., 2002; Han et al., 2008). Flag allows a good visualization of the subcellular localization without interfering with the sorting itself. In addition, we have shown that the presence of Flag in the CRF-BP expression vector does not interfere with the expression of CRF-BP. The inverted bell shape observed for colocalization between CRF-BP-Flag and Calnexin, ER marker, indicates that CRF-BP-Flag and Calnexin present an exclusion sorting behavior. The exiguous bell shape curve observed for colocalization between CRF-BP-Flag and Giantin, a Golgi marker, indicates that CRF-BP and Giantin present a marginal and most probably random colocalization. Therefore, exhaustive analysis of colocalization indicates that CRF-BP is significantly sorted into secretory granules. In addition, our results validate the suitability of the method described by

Van Steensel et al. (1996) to quantify and analyze the behavior of proteins sorted into the regulated secretory pathway. Interestingly, partial segregation of the labeling for CRF-BP-Flag and CART-EGFPm was observed. Sobota et al. (2006) have studied the eventual segregation of cargo proteins and have proposed that segregation occurs during the process of granule maturation. The strong colocalization between CRF-BP-Flag and endogenous SgII observed in both somas and neurites of cortical neurons indicate that CRF-BP-Flag is also co-stored with SgII in secretory granules of cortical neurons. Thus, our results indicate that CRF-BP has a significant ability to enter into the regulated secretory pathway in two different cellular contexts. In contrast, in a previous ultrastructural study it was shown that CRF-BP was sorted into the regulated secretory pathway mainly in the bed nucleus of stria terminalis and less frequently in the cortex; meanwhile diffuse labeling was observed in cortex and other brain regions (Peto et al., 1999). Further studies are needed to explain the apparent contradiction between our results and this previous ultrastructural study. As proposed by Peto et al. (1999), it is tempting to speculate that part of the diffuse labeling they observed could be an artifact of preembedding immunolabeling methods. The secretion assay clearly showed that CRF-BP-Flag is secreted upon stimulation with high K+-medium. Interestingly, a previous report showed a significant increase of extracellular CRF-BP and CRF-BP mRNA from rat cortical neurons incubated for 48 h in the presence of IBMX/Forskolin (Behan et al., 1995). It was suggested that the higher extracellular CRF-BP observed in the presence of IBMX/Forskolin was due to an increased rate of CRF-BP synthesis. It has been shown that cAMP induces the release of the content of secretory granules (Burgess et al., 1985; Moore and Kelly, 1985). Thus, the increase in secretion of CRF-BP observed by Behan et al. (1995) could be also due to a cAMP-mediated increase in regulated secretion and not only an increase in CRF-BP synthesis. It has been reported that CRF-BP is associated to cell membranes (Behan et al., 1996). This evidence seems contradictory with the present evidence showing that CRF-BP is sorted to secretory granules and released upon stimulation. We cannot discard that part of CRF-BP may remain attached to vesicle membranes. However, our results showing that CRF-BP overexpressed in PC12 cells is secreted to the extracellular medium after a high-K+ stimulus

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suggest that at least a proportion of CRF-BP is in the releasable pool. Experiments deleting CRF-BP specific domains should help to reveal its sorting mechanism into the regulated secretory pathway. In conclusion, our results together with the available evidence strongly suggest that CRF-BP is readily sorted to the regulated secretory pathway in PC12 cells and neurons, and released upon stimulation. The functional implications of this neuropeptide-like behavior of CRF-BP should be further studied. Acknowledgements This work was supported by Grants from FONDECYT No. 1070340 and from Millenium Science Initiative MSI No. P06/008F. Elias Blanco was supported by CONICYT and PUC (VRAID) graduate student fellowships. References Alvarez, A.R., Sandoval, P.C., Leal, N.R., Castro, P.U., Kosik, K.S., 2004. Activation of the neuronal c-Abl tyrosine kinase by amyloid-beta-peptide and reactive oxygen species. Neurobiol. Dis. 17326, 336. Behan, D.P., Linton, E.A., Lowry, P.J., 1989. Isolation of the human plasma corticotrophin-releasing factor-binding protein. J. Endocrinol. 122, 23–31. Behan, D.P., Potter, E., Sutton, S., Fischer, W., Lowry, P.J., Vale, W.W., 1993. Corticotropin-releasing factor-binding protein. A putative peripheral and central modulator of the CRF family of neuropeptides. Ann. NY Acad. Sci. 697, 1–8. Behan, D.P., Maciejewski, D., Chalmers, D., De Souza, E.B., 1995. Corticotropin releasing factor binding protein (CRF-BP) is expressed in neuronal and astrocytic cells. Brain Res. 698, 259–264. Behan, D.P., Cepoi, D., Fischer, W.H., Park, M., Sutton, S., Lowry, P.J., Vale, W.W., 1996. Characterization of a sheep brain corticotropin releasing factor binding protein. Brain Res. 709, 265–274. Bolte, S., Cordelières, F.P., 2006. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232. Burgess, T.L., Craik, C.S., Kelly, R.B., 1985. The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer. J. Cell Biol. 101, 639–645. Chanat, E., Huttner, W.B., 1991. Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network. J. Cell Biol. 115, 1505–1519. Chanat, E., Weiss, U., Huttner, W.B., Tooze, S.A., 1993. Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways. EMBO J. 12, 2159–2168. Chatzaki, E., Margioris, A.N., Gravanis, A., 2002. Expression and regulation of corticotropin-releasing hormone binding protein (CRH-BP) in rat adrenals. J. Neurochem. 80, 81–90. Chen, Z.Y., Ieraci, A., Teng, H., Dall, H., Meng, C.X., Herrera, D.G., Nykjaer, A., Hempstead, B.L., Lee, F.S., 2005. Sortilin controls intracellular sorting of brainderived neurotrophic factor to the regulated secretory pathway. J. Neurosci. 25, 6156–6166. Courel, M., Vasquez, M.S., Hook, V.Y., Mahata, S.K., Taupenot, L., 2008. Sorting of the neuroendocrine secretory protein secretogranin II into the regulated secretory pathway: role of N- and C-terminal alpha-helical domains. J. Biol. Chem. 283, 11807–11822. Dautzenberg, F.M., Hauger, R.L., 2002. The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol. Sci. 23, 71–77. Gorr, S.U., Huang, X.F., Cowley, D.J., Kuliawat, R., Arvan, P., 1999. Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins. Am. J. Physiol. 277, C121–C131. Han, L., Suda, M., Tsuzuki, K., Wang, R., Ohe, Y., Hirai, H., Watanabe, T., Takeuchi, T., Hosaka, M., 2008. A large form of secretogranin III functions as a sorting receptor for chromogranin A aggregates in PC12 cells. Mol. Endocrinol. 22, 1935–1949. Heckman, K.L., Pease, L.R., 2007. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat. Protoc. 2, 924–932. Hosaka, M., Watanabe, T., Sakai, Y., Uchiyama, Y., Takeuchi, T., 2002. Identification of a chromogranin A domain that mediates binding to secretogranin III and

279

targeting to secretory granules in pituitary cells and pancreatic beta-cells. Mol. Biol. Cell 13, 3388–3399. Huising, M.O., Vaughan, J.M., Shah, S.H., Grillot, K.L., Donaldson, C.J., Rivier, J., Flik, G., Vale, W.W., 2008. Residues of corticotropin releasing factor-binding protein (CRF-BP) that selectively abrogate binding to CRF but not to urocortin 1. J. Biol. Chem. 283, 8902–8912. Keller, P.A., Compan, V., Bockaert, J., GiacobinoJP, Charnay., Bouras, C., Assimacopolus-Jeanet, F., 2006. Characterization and localization of CART binding sites. Peptides 27, 1328–1334. Lewis, K., Li, C., Perrin, M.H., Blount, A., Kunitake, K., Donaldson, C., Vaughan, J., Reyes, T.M., Gulyas, J., Fischer, W., Bilezikjian, L., Rivier, J., Sawchenko, P.E., Vale, W.W., 2001. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc. Natl Acad. Sci. USA 98, 7570–7575. Moore, H.P., Kelly, R.B., 1985. Secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways. J. Cell Biol. 101, 1773–1781. Myhill, N., Lynes, E.M., Nanji, J.A., Blagoveshchenskaya, A.D., Fei, H., Carmine Simmen, K., Cooper, T.J., Thomas, G., Simmen, T., 2008. The subcellular distribution of calnexin is mediated by PACS-2. Mol. Biol. Cell 19, 2777–2788. Perkins, A.V., Linton, E.A., 1995. Placental corticotrophin-releasing hormone: there by accident or design? J. Endocrinol. 147, 377–381. Peto, C.A., Arias, C., Vale, W.W., Sawchenko, P.E., 1999. Ultrastructural localization of the Corticotropin-releasing factor-binding protein in rat brain and pituitary. J. Comp. Neurol. 413, 241–254. Potter, E., Behan, D.P., Fischer, W.H., Linton, E.A., Lowry, P.J., Vale, W.W., 1991. Cloning and characterization of the cDNAs for human and rat corticotropin releasing factor-binding proteins. Nature 349, 423–426. Potter, E., Behan, D.P., Linton, E.A., Lowry, P.J., Sawchenko, P.E., Vale, W.W., 1992. The central distribution of a corticotropin-releasing factor (CRF)-binding protein predicts multiple sites and modes of interaction with CRF. Proc. Natl Acad. Sci. USA 89, 4192–4196. Reyes, T.M., Lewis, K., Perrin, M.H., Kunitake, K.S., Vaughan, J., Arias, C.A., Hogenesch, J.B., Gulyas, J., Rivier, J., Vale, W.W., Sawchenko, P.E., 2001. Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc. Natl Acad. Sci. USA 98, 2843– 2848. Rosing, M., Ossendorf, E., Rak, A., Barnekow, A., 2007. Giantin interacts with both the small GTPase Rab6 and Rab1. Exp. Cell Res. 313, 2318–2325. Sobota, J.A., Ferraro, F., Bäck, N., Eipper, B.A., Mains, R.E., 2006. Not all secretory granules are created equal: partitioning of soluble content proteins. Mol. Biol. Cell 17, 5038–5052. Suda, T., Sumitomo, T., Tozawa, F., Ushiyama, T., Demura, H., 1989. Corticotropinreleasing factor-binding protein is a glycoprotein. Biochem. Biophys. Res. Commun. 165, 703–707. Taupenot, L., Harper, K.L., Mahapatra, N.R., Parmer, R.J., Mahata, S.K., O’Connor, D.T., 2002. Identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin A. J. Cell Sci. 115, 4827–4841. Taupenot, L., Harper, K.L., O’Connor, D.T., 2003. The chromogranin-secretogranin family. N. Engl. J. Med. 348, 1134–1149. Ungless, M.A., Singh, V., Crowder, T.L., Yaka, R., Ron, D., Bonci, A., 2003. Corticotropin-releasing factor requires CRF binding protein to potentiate NMDA receptors via CRF receptor 2 in dopamine neurons. Neuron 39, 401–407. Vale, W., Spiess, J., Rivier, C., Rivier, J., 1981. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and betaendorphine. Science 213, 1394–1397. Van Steensel, B., van Binnendijk, E.P., Hornsby, C.D., van der Voort, H.T., Krozowski, Z.S., de Kloet, E.R., van Driel, R., 1996. Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons. J. Cell Sci. 109, 787–792. Vaughan, J., Donaldson, C., Bittencourt, J., Perrin, M.H., Lewis, K., Sutton, S., Chan, R., Turnbull, A.V., Lovejoy, D., Rivier, C., Rivier, J., Sawchenko, P.E., Vale, W., 1995. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378, 287–292. Wang, B., You, Z.B., Rice, K.C., Wise, R.A., 2007. Stress-induced relapse to cocaine seeking: roles for the CRF(2) receptor and CRF-binding protein in the ventral tegmental area of the rat. Psychopharmacology 193, 283–294. Westphal, N.J., Seasholtz, A.F., 2006. CRH-BP: the regulation and function of a phylogenetically conserved binding protein. Front Biosci. 11, 1878– 1891. Zacharias, D., Violin, J., Newton, A., Tsien, R., 2002. Partitioning of lipid-modified monomeric GFPs into membrane microdoamins of live cells. Science 296, 913–916.