Calnuc plays a role in dynamic distribution of Gαi but not Gβ subunits and modulates ACTH secretion in AtT-20 neuroendocrine secretory cells
© Lin et al; licensee BioMed Central Ltd. 2009
Received: 04 February 2009
Accepted: 25 March 2009
Published: 25 March 2009
In AtT-20 cells ACTH secretion is regulated by both Ca2+ and G proteins. We previously demonstrated that calnuc, an EF-hand Ca2+ binding protein which regulates Alzheimer's β-amyloid precursor protein (APP) biogenesis, binds both Ca2+ as well as Gα subunits. Here we investigate calnuc's role in G protein-mediated regulation of ACTH secretion in AtT-20 neuroendocrine secretory cells stably overexpressing calnuc-GFP. Similar to endogenous calnuc, calnuc-GFP is mainly found in the Golgi, on the plasma membrane (PM), and associated with regulated secretion granules (RSG). By deconvolution immunofluorescence, calnuc-GFP partially colocalizes with Gαi1/2 and Gαi3 at the PM and on RSG. Cytosolic calnuc(ΔSS)-CFP with the signal sequence deleted also partially colocalizes with RSG and partially cosediments with Gαi1/2 in fractions enriched in RSG. Overexpression of calnuc-GFP specifically increases the distribution of Gαi1/2 on the PM whereas the distribution of Gβ subunits and synaptobrevin 2 (Vamp 2) is unchanged. Overexpression of calnuc-GFP or cytosolic calnuc(ΔSS)-CFP enhances ACTH secretion two-fold triggered by mastoparan or GTPγS but does not significantly affect glycosaminoglycan (GAG) chain secretion along the constitutive pathway or basal secretion of ACTH. Calnuc's facilitating effects on ACTH secretion are decreased after introducing anti-Gαi1/2, Gαi3, Gβ or calnuc IgG into permeabilized cells but not when Gα12 or preimmune IgG is introduced. The results suggest that calnuc binds to Gα subunits on the Golgi and on RSG and that overexpression of calnuc causes redistribution of Gαi subunits to the PM and RSG, indicating that calnuc plays a role in dynamic distribution of only Gα but not Gβ subunits. Thus calnuc may connect G protein signaling and calcium signaling during regulated secretion.
Calnuc (nucleobindin) [1, 2], an EF-hand Ca2+ binding protein, was previously reported to bind Ca2+ and several Gα subunits in vivo [3, 4]. Calnuc is unusual in that it is found both within the Golgi lumen and in the cytoplasm . We previously demonstrated that the luminal pool of calnuc constitutes of an agonist-releasable Ca2+ store in the Golgi , and regulates Alzheimer's β-amyloid precursor protein (APP) biogenesis , whereas cytoplasmic calnuc binds several Gα subunits [3, 7, 8].
Transport along the regulated secretory pathway and exocytosis of secretion granules involves vesicular trafficking, fusion of secretory granules with the plasma membrane (PM), followed by release of granule contents. Regulated secretion is stimulated by Ca2+  and heterotrimeric G proteins, including several Gα and Gβγ subunits [10–12]. Among these, Gαi3 was found to facilitate histamine release from mast cells , noradrenaline release from adrenal chromaffin cells , and adrenocorticotropic hormone (ACTH) secretion from AtT-20 cells . The recent discovery that corticotrophin releasing hormone (CRH) and vasopressin (VP) regulate ACTH secretion via binding to the Type 1 CRH receptor and the V1b receptor, which are G protein coupled receptors (GPCRs), verifies the regulation of ACTH secretion by G proteins . Moreover, several G proteins have been found on intracellular membranes as well as on the PM. Gαi3 is associated with Golgi membranes as well as at the PM [17, 18], and Gαi1/2 is found on secretory vesicles [14, 19, 20].
We have previously reported that calnuc is associated with regulated secretion granules (RSG)  and binds to Gαi3 in the Golgi . In addition, we hypothesized that calnuc might modulate regulated secretion by virtue of its ability to bind Gαi3 and Ca2+. To obtain direct evidence for the role of calnuc in the regulation of G protein mediated ACTH secretion we overexpressed calnuc-GFP in AtT-20 cells. We report here that overexpressed calnuc-green fluorescent protein (GFP) partially codistributes with Gαi1/2 as well as Gαi3 on the cytoplasmic surface of regulated secretory granules (RSG), facilitates ACTH secretion triggered by the G protein activators GTPγS or mastoparan and causes redistribution of Gαi subunits by increasing Gαi1/2 on the PM and Gαi3 on RSG. Thus calnuc, the only protein demonstrated to bind both Ca2+ and Gα subunits , appears to play an important role in regulation of G protein and Ca2+-related signaling events in endocrine cells.
Distribution of Endogenous Calnuc in AtT-20 Cells and in Cells Stably Overexpressing Calnuc-GFP or Calnuc (ΔSS)-CFP
When calnuc(ΔSS)-CFP with the signal sequence deleted which is located in the cytoplasm  is expressed and viewed by live cell imaging, it is seen to be distributed throughout the cytoplasm (Fig. 1C). However, when fixed cells are permeabilized before fixation (to release cytosolic calnuc), immunostained with an anti-GFP IgG, and examined by immunofluorescence and deconvolution analysis, calnuc(ΔSS)-CFP is also seen to be associated with RSG based on colocalization with ACTH (Fig. 1G–I). The findings with this mutant suggest that some of the cytosolic calnuc binds to the cytoplasmic surface of RSG. Thus calnuc appears to be located both inside RSG as well as bound to the cytoplasmic surface of RSG membranes.
Distribution of Gαi Subunits in Parental (NT) AtT-20 Cells and Those Stably Expressing Calnuc-GFP and Calnuc(ΔSS)-CFP
Calnuc-GFP partially colocalizes with Gαi3 in the Golgi (Fig. 3G–I) and partially co-distributes with Gαi1/2 (Fig. 3J–L) and Gαi3 (Fig. 3G–I) on RSG and the PM. In cells stably expressing cytosolic calnuc(ΔSS)-cyan fluorescent protein (CFP), the majority of the Gαi1/2 and membrane associated calnuc colocalize on RSG (Fig. 3M–O). Both Gαi1/2 and calnuc(ΔSS)-CFP are concentrated on the cytoplasmic surface of RSG and the PM. Thus, based on deconvolution analysis of our immunofluorescence results, it is evident that 1) both Gαi1/2 and Gαi3 are found on RSG where they partially colocalize with ACTH, and 2) in cells expressing calnuc-GFP the distribution of Gαi subunits along the PM and RSG is enhanced.
Distribution of ACTH, Calnuc and Its Mutants as Well as G Protein Subunits in Membrane vs Cytosolic Fractions from Parental and Stably Transfected AtT-20 Cells
Overexpression of Calnuc-GFP Does Not Affect Constitutive Secretion of GAG Chains or Basal Secretion of gACTH and Its Precursors in AtT-20 Cells
Overexpression of Calnuc-GFP Increases ACTH Secretion from AtT-20 Cells Stimulated by Mastoparan or GTPγS and the Effect Is G protein-dependent
Next we investigated whether the effects of calnuc on ACTH secretion is G protein dependent by introducing affinity purified antibodies against Gαi, Gβ or calnuc into the cytoplasm of calnuc-GFP cells. ACTH secretion triggered by mastoparan from cells pre-treated with anti-calnuc, anti-Gα1/2, anti-Gαi3 and anti-GβIgG decreased by 42%, 33%, 50%, and 42% respectively, compared to non-treated AtT-20 cells or those treated with preimmune IgG (Fig. 6B). No significant change in ACTH secretion was seen from non-stimulated calnuc-GFP cells or those pre-treated with either anti-Gα12 as a negative control or calnuc preimmune IgG. Together the results obtained with mastoparan treatment and antibody inhibition support the conclusion that calnuc as well as G protein subunits stimulate regulated secretion of ACTH in AtT-20 cells.
Calnuc Does Not Regulate G protein Activity
Calnuc-GFP Localizes in Both Light and Heavy Membrane Fractions
Overexpression of Calnuc Affects Distribution of Gαi Subunits
Overexpression of Calnuc-GFP Increases Distribution of Gαi1/2 on the PM in AtT-20 Cells
Calnuc, the first protein identified that binds to both Ca2+ and heterotrimeric G proteins, was previously shown to be localized both in the Golgi and in the cytoplasm . Our previous studies established that cytoplasmic calnuc specifically interacts with several Gα subunits in vivo as shown by both co-immunoprecipitation [3, 4] and FRET analysis  and that calnuc binds the C-terminal α5-helix region of Gαi3 through its EF-hand Ca2+-binding region .
In this study we focused on defining the role of calnuc in G protein dependent activation of ACTH secretion and used GTPγS or mastoparan [15, 23], a receptor mimetic activator of Gi/o subunits, to trigger ACTH secretion. We found that overexpression of calnuc increased nearly two-fold ACTH secretion induced by mastoparan or GTPγS compared to non-transfected cells. The fact that no differences were seen between calnuc-GFP (located both within organelles along the secretory pathway and in the cytoplasm) and calnuc lacking its signal sequence (located exclusively in the cytoplasm) together with our finding that ACTH secretion is reduced when anti-calnuc antibodies are introduced into permeabilized cells demonstrate that it is the cytosolic pool that is responsible for calnuc's effects on secretion. By immunofluorescence we found that some of the calnuc(ΔSS)-CFP is associated with the cytoplasmic side of RSG, indicating that this association must occur by protein-protein interaction between calnuc and a binding protein found on the cytoplasmic surface of the RSG membrane.
Using antibodies against Gαi2 or Gαi3 we were able to similarly impair ACTH secretion, but antibodies against Gαi12 were without effect because mastoparan does not activate Gα12. We found that mastoparan increases the initial rate of GTPγS binding to Gαi3 four-fold compared to Gαi3 alone or in the presence of calnuc. Mastoparan does so by unwinding the α5 helix of Gαi1 which is highly homologous to Gαi3 . Although mastoparan and calnuc have the same binding site in the α5 helix of Gαi3, it appears that mastoparan and calnuc have different mechanisms regarding regulation of ACTH secretion because unlike mastoparan, calnuc can not regulate G protein activity but affects G protein distribution.
We also investigated the effects of overexpression of calnuc-GFP on G protein distribution. Our immunofluorescence results demonstrate that in AtT-20 cells stably expressing calnuc-GFP, calnuc-GFP partially colocalizes with Gαi1/2 as well as Gαi3 on the PM and Golgi membranes which is similar to what we reported earlier in EcR-CHO cells overexpressing calnuc and Gαi3-GFP  or in COS-7 cells overexpressing calnuc-GFP and Gαi3-YFP visualized by FRET . Here we show (see Fig. 2C) that in non-transfected AtT-20 cells Gαi1/2 is found mainly on secretory granules (RSG) but not the PM which is consistent with reports by others on chromaffin cells [14, 19] and rat melanotrophs . However, in AtT-20 cells overexpressing calnuc the distribution of Gαi1/2 is shifted in that more is found on the PM and on RSG based on results obtained by both immunofluorescence (Fig. 2D) and cell fractionation (Figs. 9, 10). When calnucΔSS-GFP lacking the calnuc signal sequence is overexpressed in the cytoplasm, the majority of Gαi1/2 was found to colocalize with cytosolic calnuc on RSG. These results suggest that there is a dynamic distribution of Gαi between the cytoplasm, the PM, and membranes of subcellular compartments such as the Golgi.
Calnuc resembles several other proteins that are also localized in two pools [30, 31]. We established that calnuc is distributed both in the Golgi lumen and in the cytoplasm in numerous cell lines [3, 7]. Recently, NEFA [32, 33] a protein that has high homology to calnuc and also has a signal sequence, has also been found both within the Golgi lumen and in the cytoplasm . Several mechanisms have been proposed to explain the dual localization of the same protein [30, 31, 35, 36]. It has been suggested that under some conditions the signal sequence may be masked by binding proteins other than SRP [35, 36]. The detailed mechanism for generating two pools of calnuc remains to be elucidated.
Regulated exocytosis has been extensively investigated to date in AtT-20 and other cells [37–40]. It has become clear that it requires multiple steps that need to be controlled in time and space. Ca2+ and heterotrimeric G proteins have been shown to affect exocytosis either separately or in synergy depending on the cell type. In AtT-20 cells exocytosis is dependent on both Ca2+ and heterotrimeric G proteins [12, 41]. We found that calnuc binds both Ca2+ and heterotrimeric G proteins and is found on secretory vesicles which places it in a strategic location to serve as a vesicle bound controller of regulated secretion.
In summary, in this study we demonstrate that overexpression of calnuc, a Ca2+ and heterotrimeric G protein binding protein, results in redistribution of both Gαi1/2 on the PM and Gαi3 on RSG, indicating that calnuc plays a role in dynamic distribution of only Gα but not Gβ subunits. Calnuc, which binds to Gα subunits on the vesicles, modulates G protein activator triggered ACTH secretion by redistributing Gαi1/2 and Gαi3.
Polyclonal rabbit IgG (F-5059) against recombinant full length calnuc was generated and affinity purified as previously described . Affinity purified rabbit anti-calnuc IgG raised against the C-terminal 14 amino acids of rat/mouse calnuc (EQPPVLPQLDSQHL) or human calnuc (LLERLPEVEVPQHL) was obtained from AVIVA System Biology Corp. (San Diego, CA). Polyclonal antibodies against ACTH (UV16) and monoclonal antibody (mAb) against synaptobrevin 2 (Vamp 2) (69.1) were provided by Drs. J.D. Castle (University of Virginia, Charlottesville, VA) and P. DeCamilli (Yale University, New Haven, CT), respectively. Affinity purified IgG against Gαi1/2 (AS) and Gαi3 (EC) were gifts from Drs. Teresa Jones and A. Spiegel (NIDDK). Polyclonal anti-Gβ antibody (T-20) (Santa Cruz) which recognizes all Gβ subunits and CFP cDNA were provided by Drs. P. Insel and R. Tsien (University of California, San Diego), respectively. MAbs against ACTH and GFP were purchased from Novacastra Laboratories (Burlingame, CA) and Clontech Laboratories (Palo Alto, CA), respectively. Highly cross-adsorbed Alexa Fluor® 488 or 594-conjugated F(ab')2 fragments of goat anti-mouse or goat anti-rabbit IgG (H+L) were from Molecular Probes (Eugene, OR). Affinity purified goat anti-mouse and goat anti-rabbit IgG (H+L) conjugated to horseradish peroxidase were from Bio-Rad (Hercules, CA). Supersignal chemiluminescent reagent was purchased from Pierce (Rockford, IL). All chemicals were obtained from Sigma except as indicated.
AtT-20/D-16v pituitary cells were cultured in DME medium (high glucose) supplemented with 10% (v/v) horse serum, 2.5% (v/v) FCS (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate. Cells were used as 80% confluent monolayers for transfection and subsequently selected and maintained in the same culture medium containing 0.25 mg/ml G418 sulfate (Calbiochem, La Jolla, CA).
Establishment of AtT-20 Cells Stably Overexpressing Calnuc-GFP and Calnuc(ΔSS)-CFP
AtT-20 cells were transfected with GFP, calnuc-GFP or calnuc(ΔSS)-CFP (lacking a signal sequence) cDNAs cloned in the pcDNA3 vector, followed by G418 selection (0.75 mg/ml) for 2–3 wk as previously described . Cells were subsequently sorted by FACS (Ex/Em: 488/530 ± 15) (FACSVantage SE, Beckton Dickson, San Jose, CA) in the Flow Cytometry Core Facility, UCSD Cancer Center. The highest expressors (0.12% of the positive cells) were collected and maintained in media containing 0.25 mg/ml G418. Selection by FACS sorting was repeated 3 times until 100% of the cells were positive for calnuc-GFP or calnuc(ΔSS)-CFP.
Immunofluorescence and deconvloting analysis
Fluorescence images were collected from live AtT-20 cells stably overexpressing calnuc-GFP, calnuc(ΔSS)-CFP or GFP alone using a Zeiss Axiophot equipped with a FITC-filter (Ex/Em: 485/510).
For immunofluorescence, cells were fixed in 2% paraformaldehyde in phosphate buffer and permeabilized as previously described . They were then incubated with 0.1 μg polyclonal rabbit anti-calnuc, anti-ACTH, anti-Gαi1/2(AS), and anti-Gαi3(EC), or anti-GFP mAb at room temperature for 1 h, followed by incubation with Alexa Fluor 488 or 594-conjugated anti-rabbit or anti-mouse F(ab')2. Specimens were examined with a Zeiss Axiophot microscope equipped for epifluorescence.
For deconvolution analysis of immunofluorescence results, images were collected with an Applied Precision DeltaVision imaging system (Issaquah, WA) coupled to a Zeiss S100 fluorescence microscope (Carl Zeiss; Thornwood, NY). Cross-sectional images of cells were obtained with 150-nm step width to optimize reconstruction of the center plane image. Deconvolution was done on a Silicon Graphics Octane® visual workstation (SGI, Mountain View, CA) equipped with Delta Vision reconstruction software.
Assessment of Constitutive Secretion of Glycosaminoglycans (GAG) from AtT-20 Cells
Non-transfected AtT-20 cells or those transfected with calnuc-GFP were pretreated with 0.5 mM xyloside at 37°C for 30 min and subsequently pulse-labeled with [35S] sulfate (150 μCi/ml) (ICN Biomedicals) for 5 min as described . Labeled GAG chains secreted into the medium at selected intervals from 15 min to 2 h were precipitated with cetylpyridinium chloride (CPC). Samples were collected by vacuum filtration and counted by liquid scintillation as previously described .
Immunoprecipitation of Metabolically Labeled ACTH and its Precursors
Parental AtT-20 cells or cells stably expressing calnuc-GFP were pulse-labeled  with [35S]Met (0.5 mCi/ml) (NEN® Life Science Products, Boston, MA) for 20 min at 37°C and subsequently chased in unlabeled DMEM for 2 h. ACTH released into the medium was immunoprecipitated with anti-ACTH IgG and protein A beads (Cytelligen Corp., San Diego, CA), followed by separation on 10–20% Tris-Tricine gels (Bio-Rad) and autoradiography with Kodak film .
ACTH Release from Permeabilized AtT-20 Cells
The permeabilization protocol used followed that described previously by others . In brief, stably transfected or non-transfected AtT-20 cells were plated on tissue culture plates (5 × 104/well) for 48 h, and subsequently rinsed with 0.1% BSA in DMEM, followed by permeabilization buffer (20 mM digitonin, 137 mM NaCl, 2.7 mM KCl, 5.6 mM glucose, 1 mg/ml BSA, 20 mM Hepes, pH 7.2), plus either 10 μM mastoparan (Neosystem Laboratoire, Strasbourg, France) or 100 μM GTPγS (Roche Molecular Biochemicals, Indianapolis, IN) at 37°C for 15 min [13, 15, 43]. To introduce antibodies into cells, affinity purified anti-calnuc (F-5059), anti-Gαi1/2 (AS), anti-Gαi3 (EC) or anti-GβIgG (30 μg/ml) were added and incubated with cells in permeabilization buffer at 37°C for 10 min according to a published protocol , followed by addition of 10 μM mastoparan for 15 min. Media were collected, and cells were lysed in 0.5% Triton X-100 at 4°C for 30 min, followed by centrifugation (14,000 × g for 5 min). ACTH in supernatants of both the medium and cell lysate were assessed by enzyme-linked immunosorbent assay (ELISA) using ACTH (Rat) EIAH kits (Peninsula Laboratories, San Carlos, CA) with a Vmax Kinetic Microplate Reader (λ = 450 nm) (Molecular Devices, Sunnyvale, CA). ACTH secretion was plotted as percent of total ACTH (secreted ACTH in supernatants vs secreted + intracellular ACTH). The results from each experiment were subjected to statistic analysis, and the final plotted results (mean ± SD) represent the average of values obtained in indicated separate experiments performed in either duplicate or triplicate as shown in figure legend.
GTPγS Binding Assay
Purified recombinant His6-Gαi3  and His6-calnuc  were prepared as described previously and GTPγS binding was assessed as described by others . Mastoparan (100 μM) or His6-calnuc (1 μM) was incubated with 2 μM [35S]GTPγS (6000 cpm/pmol, NEN® Life Science Products)  in the reaction buffer containing 10% glycerol, 1 mM DTT, 1 mM EDTA, 0.1 mM MgCl2, and 50 mM Hepes, pH 8.0. Reactions were started by addition of 200 nM His6-Gαi3 and incubated at 30°C for 5–60 min. At each time point, 50 μl of the reaction mixture were collected, diluted with ice-cold dilution buffer (160 mM NaCl, 0.2 mM GTP, 1 mM EDTA, and 20 mM Hepes, pH 8.0). Samples were collected on nitrocellulose filters by vacuum filtration. Filters were washed with ice cold washing buffer (25 mM MgCl2, 100 mM NaCl, and 25 mM Tris, pH 8.0). Bound radioactivity was determined by liquid scintillation counting. Total bound radioactivity never exceeded 2% of the total radioactivity.
Membrane (100,000 g pellets) and cytosolic fractions (100,000 g supernatants) were prepared by centrifugation of postnuclear supernatants from AtT-20 cells and analyzed by immunoblotting and ECL .
Light membranes containing PM and Golgi membranes were separated from heavy fractions containing ER and granules by centrifugation on discontinuous sucrose gradients using protocols similar to those previously published with minor modifications [3, 5]. Briefly, postnuclear supernatants (PNS) prepared from AtT-20 cells were loaded on the top of sucrose step gradient containing 0.2, 0.4, 0.6, 1.0, 1.4, and 1.8 M in 1 mM Tris-HCl, pH 7.5, and centrifuged at 55,000 × g (SW60Ti rotor) for 2 h at 4°C. After centrifugation, 12 fractions were collected from the bottom, followed by centrifugation at 100,000 × g for 1 h. The resultant pellets were solubilized in Laemmli sample buffer and the solubilized proteins were separated on 10% Tris-Glycine or 10–20% Tris-Tricine gels (Bio-Rad) and analyzed by immunoblotting.
To separate PM from Golgi membranes sucrose gradient flotation  was applied as previously described  with minor modifications. In brief, postnuclear supernatants from AtT-20 cells were resuspended in 1.0 ml 1.3 M sucrose, followed by overlay with 0.5 ml of 1.2 M, 1.5 ml of 1.14 M, 0.5 ml of 0.99 M and 0.9 M sucrose, followed by centrifugation at 170,000 × g (SW60Ti rotor) for 15 h at 4°C. Eight fractions were collected by centrifugation (100,000 × g for 1 h), and membrane pellets were analyzed by SDS-PAGE followed by immunoblotting. Quantification of each band was performed by densitometry using Scan Analysis software (Biosoft, Cambridge, UK). Fraction density was determined using a digital refractometer (ABBE Mark II) (Cambridge Instruments, Buffalo, NY) as described .
Alzheimer's β-amyloid precursor protein
regulated secretion granules
post nucleus supernatants
deleting signal sequence
glycosylated adrenocorticotropic hormone
green fluorescent protein
cyan fluorescent protein
We thank Drs. J.D. Castle, P. DeCamilli, T. Jones, A. Spiegel, R Tsien for gifts of reagents. We are grateful to Drs A. Desai and K. Oegema (Ludwig Institute for Cancer Research, UCSD) for the use of the deconvolution microscopes as well as to D. Young (Flow Cytometry Core Facility, John and Rebecca Moores Cancer Center, UCSD) for technical assistance in FACS sorting. We thank Dr L. Duan (Aviva System Biology, San Diego, CA) for his help in generating anti-human or mouse calnuc C-terminal peptides antibodies.TF is supported by a Ramón y Cajal contract from the Spanish Ministry of Science and Technology and by grants from the European Union (MIRG-CT-2006-026702), the Instituto de Salud Carlos III (FIS PI052270) and the Medical Foundation of the Mutua Madrileña. This work was supported by the National Institutes of Health grants CA100768 and DK17780 to MGF.
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