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Manuscript M0:08062 revised 10-29-2000

Targeting of Membrane Proteins to the Regulated Secretory Pathway in Anterior Pituitary Endocrine Cells

Rajaâ El Meskini, Gregory J. Galano, Ruth Marx, Richard E. Mains, Betty A. Eipper*

Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Ave, Farmington CT 06030-3401USA.

Key Words: secretory granules, internalization, amidation, peptide, cytoskeleton, adenovirus

Correspondence: *Betty A. Eipper, Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Ave, Farmington CT 06030-3401. Tel: 860-679-8898; Fax: 860-679-1885; e-mail: eipper@uchc.edu

Abbreviations: PHM, peptidylglycine α-hydroxylating monooxygenase; PAL, peptidyl-α-hydroxyglycine α-amidating lyase.

Acknowledgments: We gratefully acknowledge Lixian Jin and Kate Deanehan for help with tissue culture and Marie Bell for general laboratory assistance. We thank Dr. Victor May for critically reading this manuscript. This work was supported by National Institutes of Health Grant DK-32949.

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 1, 2000 as Manuscript M008062200 by guest on M

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Abstract

Unlike the neuroendocrine cell lines widely used to study trafficking of soluble and membrane proteins to secretory granules, the endocrine cells of the anterior pituitary are highly specialized for the production of mature secretory granules. Therefore we investigated the trafficking of three membrane proteins in primary anterior pituitary endocrine cells. PAM, an integral membrane protein essential to the production of many bioactive peptides, is cleaved and enters the regulated secretory pathway even when expressed at levels 40-fold higher than endogenous levels. Myc-TMD/CD, a membrane protein lacking the lumenal, catalytic domains of PAM, is still stored in granules. Secretory granules are not the default pathway for all membrane proteins, since Tac accumulates on the surface of pituitary endocrine cells. Over-expression of PAM is accompanied by a diminution in its endoproteolytic cleavage and in its BaCl2-stimulated release from mature granules. Since internalized PAM/PAM-antibody complexes are returned to secretory granules, the endocytic machinery of the pituitary endocrine cells is not saturated. As in corticotrope tumor cells, expression of PAM or myc-TMD/CD alters the organization of the actin cytoskeleton. PAM-mediated alterations in the cytoskeleton may limit maturation of PAM and storage in mature granules.

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Introduction

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme involved in the posttranslational processing of many prohormones and neuropeptides. PAM catalyzes the formation of α-amidated peptides from peptide precursor molecules with a COOH-terminal glycine. In neurons and endocrine cells, biologically active peptides are stored in secretory granules which undergo regulated release in response to external stimuli. Localized in secretory granules of many neural and endocrine tissues, PAM is one of a small number of posttranslational processing enzyme occurring naturally in soluble and membrane forms (1-4). For this reason, we have used PAM to investigate the trafficking of soluble and membrane proteins into secretory granules (5;6).

Soluble and membrane PAM are targeted to the regulated secretory pathway in different ways (5;6). By stably expressing wild type and mutant PAM in AtT-20 corticotrope tumor cells, protein domains containing trafficking information were identified (7-9). The two catalytic domains of PAM can be expressed independently and both are efficiently targeted to secretory granules. In contrast, membrane PAM is predominantly localized in the trans-Golgi network (TGN) at steady state. The small amount of membrane PAM on the cell surface is rapidly internalized and accumulates in the TGN region (7). The cytosolic domain of PAM contains information necessary for the trafficking of this membrane protein within the secretory pathway (7). Membrane proteins lacking the cytosolic domain are less extensively cleaved by secretory granule endoproteases, accumulate on the plasma membrane, and fail to undergo internalization. A marker protein lacking both catalytic domains of PAM, consisting only of the PAM signal sequence followed by its transmembrane/cytosolic domain, is highly localized to the TGN region of AtT-20 cells (10); appending the cytosolic domain of PAM to Tac, a plasma membrane protein, rerouted Tac to the TGN (11). The cytosolic tail of PAM interacts with proteins that affect cytoskeletal organization and expression of membrane PAM alters the organization of the actin cytoskeleton, perhaps explaining the ability of membrane PAM to affect secretory granule biogenesis (12).

The endocrine cells of the anterior pituitary are highly specialized for the production of peptide hormones, their storage and regulated release from mature secretory granules (4). Our initial studies of PAM trafficking in anterior pituitary endocrine cells identified similarities and clear differences in the trafficking of endogenous PAM in primary cells and exogenous PAM in AtT-20 tumor cells. As in AtT-20 cells, the endogenous PAM in pituitary cells is subjected to endoproteolytic cleavage, generating soluble PHM and PAL. In both cases, PAM processing products are subjected to regulated exocytosis. Unlike transfected AtT-20 cells, immunostaining and subcellular fractionation identifies PAM mainly in pituitary secretory granules. Anterior pituitary cells rapidly internalize membrane PAM from the cell surface, but unlike AtT-20 cells, massive accumulation of retrieved PAM in the TGN region is not observed in primary pituitary cells.

In order to elucidate the trafficking of membrane proteins in endocrine cells and understand the distinctly different steady state localization of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines, we investigated the trafficking of three membrane proteins in primary anterior pituitary: membrane PAM (PAM-1); a truncated version of PAM with only a small lumenal epitope (Myc-TMD/CD); and the interleukin 2 receptor α-

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chain (Tac), normally a T-lymphocyte plasma membrane protein (13;14). We used recombinant adenovirus to over-express membrane PAM or myc-TMD/CD in anterior pituitary cells; Tac was expressed in the same cells by transfection. Using immunofluorescent staining and subcellular fractionation, we show that the transmembrane and cytosolic domains of PAM are sufficient to target the protein to the secretory granules. In parallel, the capacity of pituitary endocrine cells to cleave and store greatly increased amounts of PAM has been assessed. Although over-expression of PAM alters the organization of the actin cytoskeleton, neither the regulated secretion or the endocytic machinery is affected in anterior pituitary cells.

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Materials and Methods

Primary anterior pituitary cell cultures. Cultures were prepared as described (4). Anterior pituitaries were dissected from the neurointermediate lobes of 5 to 20 adult male Sprague-Dawley rats (175-200 g; Charles River, Wilmington, MA) and dissociated with collagenase (Worthington Biochemical Corporation Lakewood NJ), hyaluronidase (Sigma, St Louis, MO) and benzonase (EM Science, Germany), followed by trypsin (Sigma). This procedure consistently produced 1.5 x 106 cells/anterior pituitary. The dissociated cells were plated on protamine-coated culture wells in DMEM-F12 supplemented with 10 % fetal clone III bovine serum (Hyclone, Logan, UT) and 10 % Nu-Serum IV (Collaborative Research, Bedford, MA) for the first 24 h and then maintained in the same medium containing 10 µM cytosine arabinoside (Sigma).

Recombinant adenovirus constructs. Where indicated, anterior pituitary cells were infected with 2 x 103 plaque-forming units/ml of recombinant adenoviruses (V) 72 h after plating. Recombinant adenoviruses have been described previously (15): PAM-1V encodes full length rat PAM-1 (nucleotides 293-3245); POMCV encodes mouse POMC (nucleotides 1-920); myc-TMD/CDV encodes a chimeric protein described previously (10) in which the PAM signal (amino acids 1-26; nucleotides 298-375) and the pro-domain (amino acids 27-35; nucleotides 376-420) were fused in frame to myc epitope followed by the transmembrane domain (TMD) and cytoplasmic domain (CD) (amino acids 858-976; nucleotides 2871-3225) of PAM. For recombinant adenovirus constructions, cDNAs were subcloned into the virus shuttle vector (pAdLox) as described previously for PAM-1V and POMCV (15). HEK293-CRE8 cells were then co-transfected with the shuttle vector and purified Ψ5 adenoviral DNA to produce recombinant adenovirus. The next day, the virus-containing cell culture medium was replaced with serum-containing medium; for all experiments the cells were used 48 h after infection, but cells were viable for at least 2 weeks after infection. pCDM.Tac expression vector, kindly provided by Dr. Juan Bonifacino (Cell Biology and Metabolism Branch, National Institutes of Health) was transiently transfected in anterior pituitary cells using GenePorter (Gene Therapy Systems) following the manufacturer’s protocols. Mouse AtT-20 corticotrope tumor cells expressing PAM-1 (AtT-20 PAM-1) were grown in DMEM-F12 supplemented with 10 % fetal clone III bovine serum, 10 % Nu-Serum IV and G418 as previously described (6).

Secretion experiments and analysis. For each secretion experiment, pituitary cell cultures were initially rinsed for three 30 min periods in complete serum free medium (CSFM)/BSA: DMEM/F12 with 1 µg/ml insulin, 0.1 µg/ml transferrin and 0.1 mg/ml fatty acid-free BSA. Following this equilibration period, medium was collected for two 1 h periods of basal secretion (1st basal, 2nd basal) followed by a 1 h period of stimulated secretion using either 1 mM BaCl2 or 1 µM phorbol 12-myristate 13-acetate (PMA) (Sigma) diluted in CSFM/BSA (4;16). After the collected media were centrifuged to remove cell debris, a mixture of protease inhibitors was added (4) and the samples were stored at -80 oC. Cells were scraped into 20 mM Na (N-Tris[hydroxymethyl]methyl-2-aminoethansulfonic acid) (NaTES)/10 mM mannitol, 1 % Triton X-100, pH 7.4 (TMT) containing protease inhibitors and used for PHM or PAL assays or for Western blot analysis. Rat anterior pituitary tissue was homogenized in the same buffer. Before use, samples were frozen and thawed three times and centrifuged for 5 min to remove debris.

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PHM and PAL assays. PHM and PAL activities were measured in cell extracts or media as previously described using α-N-acetyl-Tyr-Val-Gly and α-N-acetyl-Tyr-Val-OH-Gly substrates, respectively (17). Samples were assayed in duplicate and reactions were carried out for 1.5 h. PHM and PAL specific activity is expressed as picomoles of product formed per hour (Units) per microgram of protein, or as per cent of the corresponding total enzyme activity in the cell extract.

ACTH radioimmunoassay. Anterior pituitary cells were extracted in 5 N acetic acid containing protease inhibitors; the soluble material was lyophilized, dissolved in immunoassay buffer, centrifuged to remove insoluble material and stored frozen. Assays were performed on media and cell extracts using antibody Kathy (1:20,000) and [125I]ACTH(1-39) (NEN Life Science). Antiserum Kathy only recognizes POMC products in which the COOH-terminal end of ACTH(1-39) is exposed (18).

Subcellular fractionation. Cultures prepared from 10 anterior pituitaries were harvested at 4oC in 10 volumes of homogenization buffer containing 0.32 M sucrose, 10 mM Tris-HCl, pH 7.4, and protease inhibitors, passed 6 times through a 26-gauge needle, and then 12 times through a ball-bearing homogenizer (H&Y Enterprises, Redwood City, CA) (8). Cell debris (P1) was removed by centrifugation at 800 x g for 5 min. The resulting supernatant (S1) was separated into a P2 pellet and soluble fraction (S2) by centrifugation at 20,000 x g for 30 min (2). The P2 pellet largely enriched in secretory granules was resuspended in homogenization buffer and fractionated further on a discontinuous sucrose gradient. Resuspended P2 was layered onto a density gradient consisting of 200 µl each of 0.4 M, 0.6 M, 0.8 M and 1.0 M sucrose, 350 µl each of 1.2 M, 1.4 M and 1.6 M sucrose, and 200 µl of 2.0 M sucrose; this gradient was designed to keep the densest secretory granules from pelleting. Gradients were centrifuged for 2 h at 120,000 g; 150 µl fractions were collected from the top of the gradient and proteins in an equal fraction of each sample were analyzed. Finally, a P3 pellet and a cytosolic fraction (S3) were obtained following centrifugation of S2 at 350,000 x g (2;4).

Western blot analysis. Samples were fractionated by 10 or 12 % SDS-PAGE, and subjected to Western blot analysis using rabbit polyclonal antisera to PHM (JH1761, 1:1000), exon A (JH629, 1:1000) and PAL (JH471, 1:1000) or mouse monoclonal antibodies to the cytosolic domain of PAM (6E6, 1:50) (15;19)(4), myc epitope (9E10 hybridoma, 1:50) (10)and synaptobrevin-2 (VAMP-2, 1: 5000) (Synaptic Systems) (20). Proteins were visualized using the Enhanced Chemiluminescence kit (Amersham Lifescience) (21).

Immunofluorescent staining. PAM, myc-TMD/CD, POMC, TGN38, and VAMP-2 were detected in anterior pituitary cells using indirect immunofluorescence. Cells (200,000 cells/well of a 4-well slide) were fixed with 4 % paraformaldehyde in PBS (50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) for 30 min, permeabilized with 0.075 % Triton X-100 and blocked with 2 mg/ml BSA in PBS for 1 h at room temperature. Cells were incubated overnight at 4oC with rabbit polyclonal antibodies against PAM exon A (JH629), PAM CD (JH571) or TGN38 (JH1481) (19), and monoclonal antibodies to myc (10), PAM-CD (6E6) and ACTH (Novocastra Labs) (15), synaptobrevin-2 (VAMP-2) (Synaptic Systems) (20), γ-adaptin (AP-1) (Transduction Laboratories) (22) and α-adaptin (AP-2) (Transduction Laboratories) (23). Permeabilized or non-permeabilized anterior pituitary cells expressing membrane Tac protein were stained using a monoclonal antibody 7G7 (AMAC, Westbrook, ME) directed against the lumenal domain of Tac. The antigen–antibody complexes were visualized using FITC-conjugated goat anti-rabbit

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IgG (Caltag, San Francisco, CA) or Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). To detect filamentous actin, cells were stained with FITC-phalloidin (0.1 µg/ml; Sigma). Cells were viewed with a Zeiss Axioskop microscope (Carl Zeiss, Thornwood, NY) and photographed with a Micromax CCD camera (Princeton Instruments, Princeton, NJ) or a Spot RT camera (Diagnostic Instruments, Sterling Heights, MI).

Antibody internalization experiments. Primary pituitary cells were incubated in CSFM/BSA containing a 1:500 dilution of rabbit polyclonal antiserum to PAM exon A (JH629) for 20 min at 37oC and either prepared immediately for immunofluorescence staining or chased in antibody-free medium for 1 or 2 h at 37oC (4;9). To determine whether the endocytosis of PAM were affected by the binding of bivalent PAM antibody, control experiments were performed using monovalent Fab fragments. We generated Fab fragments by digestion of IgG prepared from PAM exon A antiserum with immobilized papain as described by the manufacturer (Pierce Chemical Company, Rockford, IL), the Fab fraction was separated from non-digested IgG and Fc fragments using immobilized protein A-Sepharose (Sigma Immunochemicals, St. Louis, MO). The purity of the Fab fragments was verified by SDS-PAGE. Primary pituitary cells were incubated with exon A antibody or Fab fragments prepared from the same serum for 20 min at 37 oC and then harvested or chased in the absence of antibody for additional time periods, as described above.

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Results

Adenovirus mediated increase in PAM expression in anterior pituitary cells.

To evaluate the level of protein expression achieved following infection of primary cells with adenovirus, PHM and PAL specific activities were compared in PAM-1V infected pituitary cells, non-infected pituitary cells (4), anterior pituitary tissue and stably transfected AtT-20 PAM-1 cells (Fig.1, upper). PHM and PAL specific activities increase 40- to 50 fold after PAM-1V infection, reaching levels substantially higher than in adult rat anterior pituitary or stably transfected AtT-20 PAM-1 cells (Fig.1, upper). Specific activities in PAM-1V infected pituitary cells are about 2-fold higher than in adult rat atrium, the richest source of PAM in the body (24).

In order to assess the fraction of cells expressing virally encoded PAM, we used indirect immunofluorescence (Fig.1, bottom). Virally encoded PAM-1 is expressed at varying levels in essentially all of the primary pituitary cells (Fig.1A and B). Consistent with the 50-fold increase in PHM and PAL specific activities, endogenous PAM is not visualized under the same conditions (Fig.1D and E). Since these cultures contain very few non-endocrine cells (4), almost all of the virally encoded PAM is expressed in cells proficient at producing secretory granules. Punctate staining for virally encoded PAM is observed throughout the cytosol of most of the endocrine cells (Fig. 1C). The vesicular staining pattern observed for virally encoded PAM mimicks the pattern observed previously for endogenous PAM in pituitary cells (4).

Subcellular localization of virally encoded PAM in anterior pituitary cell culture.

To evaluate better the subcellular localization of PAM, PAM-1V infected pituitary cultures were simultaneously visualized for PAM (Fig.2A) and TGN38 (Fig.2B), a trans-Golgi network marker (25). PAM (in red) is localized to vesicular structures distributed throughout the cell, while TGN38 (in green) is highly localized to a compact, reticular structure in the perinuclear region of each cell (Fig.2B, arrows). Very little PAM is identified in the TGN region (Fig. 2C). In contrast, double immunofluorescent staining of PAM-1V infected pituitary cells for PAM (Fig.2D) and VAMP-2 (Fig.2E), a secretory granule marker (20), reveals a largely overlapping distribution. As with endogenous anterior pituitary PAM (4), overlaying the PAM and VAMP-2 images (Fig.2F, PAM in green and VAMP-2 in red) reveals a range of colors, with some structures enriched in PAM and others in VAMP-2. Despite massive adenovirus mediated over-expression, membrane PAM is largely localized to vesicular structures with no accumulation on the cell surface or in the TGN area.

Localization of myc-TMD/CD in anterior pituitary cells.

The routing of integral membrane PAM involves contributions from the two lumenal, catalytic domains and from the COOH-terminal cytoplasmic domain (11). In order to evaluate the importance of the transmembrane and cytoplasmic domains of PAM in trafficking in anterior pituitary endocrine cells, we generated a recombinant adenovirus encoding myc-TMD/CD (Fig. 3). In this chimeric protein (Fig.3A), the PAM signal and NH2-terminal regions allow the myc epitope tag, which precedes the transmembrane (TMD) and cytoplasmic (CD) domains of PAM, to adopt a lumenal location (10).

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Primary anterior pituitary cells were infected with adenovirus encoding myc-TMD/CD and the chimeric protein was visualized simultaneously using antisera to PAM-CD and myc (Fig.3B and C). With both antisera, vesicular staining distributes throughout the cytosol of all of the pituitary cells (Fig.3B and C). Antisera to the lumenal domain (Fig. 3C, myc) and to the cytoplasmic domain (Fig. 3B, PAM-CD) exhibited identical patterns. Simultaneous visualization of myc-TMD/CD and TGN38 (Fig.3D and E) demonstrated distinctly different patterns; myc-TMD/CD does not accumulate in the TGN area (Fig. 3F). In contrast, simultaneous visualization of myc-TMD/CD and VAMP-2 (Fig. 3G and H) suggests that myc-TMD/CD, like intact PAM-1, is largely localized to secretory granules (Fig. 3I).

Western blot analysis of extracts of cultures infected with the myc-TMD/CD virus identified a single 23 kDa protein that is recognized by antibodies to myc and to the cytosolic domain of PAM (Fig. 4A). To explore further the trafficking of myc-TMD/CD in anterior pituitary cells, we used differential centrifugation followed by sucrose gradient fractionation (Fig. 4B and C). The majority of the myc-TMD/CD is recovered in the P2 pellet (2)(Fig.4B). Visualization of VAMP-2 demonstrated the presence of secretory granules in the P2 fraction (Fig.4B). The P2 pellet was further fractionated on a sucrose gradient (Fig. 4C). Fractions 9 to 11 are enriched in myc-TMD/CD and in VAMP-2, confirming the localization of myc-TMD/CD to secretory granules. A similar subcellular localization is observed for the majority of the endogenous anterior pituitary PAM (4) and for virally encoded PAM-1 expressed in anterior pituitary cells (data not shown). Anterior pituitary endocrine cells commit a significant percentage of their total protein synthesis to production of secretory granules (26;27). Therefore we considered the possibility that secretory granule localization might represent the default trafficking pathway in these cells. To address this issue, we transfected anterior pituitary cultures with Tac, the interleukin-2 receptor α chain, a membrane protein that normally resides on the surface of T-lymphocytes (13;14). Tac was previously shown to accumulate on the surface of AtT-20 cells (11). Immunofluorescent staining of non-permeabilized pituitary cells, using an antibody against the lumenal domain of Tac, demonstrates that a significant amount of the expressed protein is present on the cell surface (Fig. 4D). The localization of Tac is distinctly different from that of PAM-1 or myc-TMD/CD, which are not visualized without permeabilization (11). Thus the transmembrane/cytosolic domain of PAM is sufficient to direct the protein to secretory granules in pituitary endocrine cells. The ability of myc-TMD/CD to direct trafficking to secretory granules is not apparent in AtT-20 cells because both myc-TMD/CD and membrane PAM are largely localized to the TGN area of AtT-20 cells at steady state.

Processing of over-expressed PAM-1 in anterior pituitary cells.

Despite the high levels of expression achieved using adenovirus, anterior pituitary endocrine cells appear to be able to accommodate the exogenous PAM in vesicular structures (4). To investigate the ability of pituitary cells to cleave the increased amounts of PAM, we used Western blot analysis (Fig. 5). Consistent with the dramatic increase in enzyme activity, analysis of the same amount of protein from infected vs. non-infected cells fails to visualize endogenous PAM when the exposure times are restricted to yield a non-saturated signal for the infected cells; therefore, longer exposures are shown for non-infected cultures. In PAM-1V infected pituitary cells, intact PAM-1 (125 kDa) is processed into membrane PAL (70 kDa), soluble PAL (50 kDa) and soluble PHM (45 kDa) (Fig.5A). Production of these proteins requires cleavage within exon

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A and between PAL and the transmembrane domain. As previously described (4), PAM-1 derived proteins of similar mass are identified in non-infected cell extracts (Fig.5A).

To clarify the identity of the cleavage products, antisera to PHM, PAL and CD were also used (Fig.5B). Again, longer exposures of blots from non-infected cells are shown to facilitate comparison of the products of endogenous PAM processing (primarily PAM-2 and PAM-3, with small amounts of PAM-1) and virally expressed PAM-1 processing. The PHM and PAL antibodies recognize PAM-1 and PHM, or PAM-1, mPAL and sPAL, respectively. The CD antibody identifies PAM-1 along with mPAL and a 27 kDa protein containing the transmembrane and cytosolic domains of PAM (Fig.5B). Despite the high level of expression, substantial amounts of virally encoded PAM-1 are cleaved into products resembling the endogenous products (4) (Fig.5B). The increased fraction of protein recovered as intact PAM-1 or membrane PAL in virally infected cells suggests that the cleavage capacity of the cells becomes limiting.

Basal and stimulated secretion of PAM proteins from PAM-1V infected pituitary cells.

To evaluate the ability of pituitary endocrine cells to store increased amounts of PAM in the regulated secretory pathway, we compared the basal and stimulated secretion of PHM activity from PAM-1V infected and non-infected cultures (Fig.6A). PHM secretion was examined during two sequential basal collection periods before stimulation with BaCl2 or PMA (4). Data are expressed in units (Fig. 6A, left) and as a percentage of cell content of PHM activity (Fig. 6A, right). Basal secretion of PHM activity increases about 10–fold when cell content of enzyme rose 40–fold in response to the PAM-1V infection. Over-expression of PAM-1 did not result in increased basal secretion of PHM activity when expressed as a percent of cell content per hour (0.55 ± 0.1 % vs. 1 ± 0.1 % of cell content per hour, infected vs. non-infected, respectively) (Fig. 6A, right). Over-expression of membrane PAM does not lead to increased basal secretion of enzyme.

Secretion was stimulated with Ba2+, which mimics the effect of Ca2+ on regulated exocytosis (28), or with PMA, which acts via protein kinase C to stimulate a cascade of protein phosphorylations and induce Ca2+-independent release (29). BaCl2 stimulates secretion from mature granules, while PMA stimulates Ca2+-independent exocytosis from both immature and mature pools of vesicles (30). BaCl2 is more effective at stimulating secretion of PHM activity from non-infected cells (3.5 ± 0.5–fold; 4.5 ± 0.5 % of cell content/h) than from infected cells (2.5 ± 0.5–fold over basal; 1.3 ± 0.2 % of cell content/h). In contrast, PMA is at least as effective on infected cells (6 ± 0.4 % of cell content/h) as on non-infected cells (5 ± 0.5 % of cell content/h). PMA stimulates PHM secretion from infected cells 10-fold while secretion from non-infected cells is only stimulated 4-fold. The ability to stimulate secretion demonstrates the presence of a significant amount of virally encoded PAM in the regulated secretory pathway.

The PAM proteins secreted during basal and challenge periods were subjected to Western blot analysis using antisera to PHM (Fig.6B). Under basal conditions, soluble, bifunctional PAM proteins of 110 and 105 kDa are secreted along with 45 kDa PHM (Fig.6B). Addition of BaCl2 or PMA stimulated secretion of all three PAM proteins. As expected from the enzyme assays, PMA was a more potent secretagogue than BaCl2. Secretion of the 105 and 110 kDa PAM proteins was more responsive to PMA than to BaCl2. This result suggests that both

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bi- and mono-functional PAM proteins derived from PAM-1 processing are stored in the secretory granules of PAM-1V infected pituitary cells.

Effect of exogenous PAM-1 expression on pituitary hormone secretion.

Over-expression of PAM-1 in AtT-20 cells impairs the regulated secretion of the endogenous peptide hormone, ACTH (16). Given the extremely high levels of PAM expression achieved by infection of primary pituitary cells, we wanted to determine whether secretion of endogenous hormone were affected. Secretion of GH from somatotropes and ACTH from corticotropes was evaluated under basal and stimulated conditions (Fig.7A and B). Based on Western blot analysis, BaCl2 is as effective at stimulating infected and non-infected somatotropes (Fig.7A). PMA is a more effective GH secretagogue than BaCl2, with no differences observed between infected and non-infected cells. PMA is more effective than BaCl2 (10-fold vs. 2-fold) at stimulating ACTH secretion, with no differences observed between infected and non-infected cells (Fig.7B). Unlike the situation in AtT-20 corticotrope tumor cells, over-expression of PAM-1 does not impair regulated secretion from primary somatotropes or corticotropes.

Internalization of exogenous PAM-1 in anterior pituitary cells.

The steady state localization of PAM reflects a balance of fluxes between different subcellular compartments. Internalization of membrane PAM from the cell surface is an important component of PAM trafficking and can be monitored by incubating live cells with antiserum to a lumenal domain of PAM. In PAM-1 transfected AtT-20 cells, PAM internalized from the plasma membrane accumulates in the TGN region(7). In contrast, endogenous PAM internalized from the surface of primary pituitary cells is distributed to vesicular structures dispersed throughout the cell (4). In order to determine if over-expression of PAM in anterior pituitary cells overloads the sorting machinery and alters trafficking of membrane PAM in the endocytic pathway, we studied PAM internalization in PAM-1V infected pituitary cells.

Pituitary cells expressing virally encoded PAM were incubated with exon A antiserum for 20 min, washed and either harvested or chased for 1 h or 2 h. The Exon A antiserum binds to PAM-1 on the cell surface and the PAM/PAM antibody complex can then undergo endocytosis. The internalized PAM/PAM-antibody complex is visualized using a fluorescent second antibody. Localization of the internalized PAM/PAM-antibody complexes was then compared to the steady state distribution of various organelle markers. Substantial amounts of PAM/PAM-antibody complex are internalized from the surface of PAM-1V infected pituitary cells (Fig.8). The signal is much more robust than that observed from non-infected cells. The intensity of the signal obtained from the internalized PAM/PAM-antibody complexes remains constant for hours of chase, suggesting that the internalized complexes are not degraded or rapidly re-secreted. Identical internalization of PAM-1 proteins was observed when pituitary cells were incubated with monovalent Fab fragments prepared from the same exon A antiserum (not shown). Thus, the internalization of PAM from the surface of pituitary cells was not caused by the binding of bivalent antibodies.

After the pulse incubation with the exon A antibody, PAM/PAM antibody complexes are present in small vesicles of uniform size that are distributed throughout the cytosol (Fig. 8A).

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These PAM/PAM-antibody complexes are often co-localized with AP-2, an early endosomal marker (23;31) (Fig.8B) . After 1 h of chase, internalized PAM/PAM-antibody complexes are in a more heterogeneous collection of vesicles predominantly in the TGN area (Fig. 8E). These vesicles co-localize with γ-adaptin AP-1, part of the adaptor complex required for the assembly of clathrin-coated buds from the Golgi (31)(32) (Fig.8F). Although internalized PAM/antibody complexes are observed in the TGN area, no accumulation is observed. After a 2 h chase, the PAM/PAM-antibody containing vesicular structures are coincident with secretory granules visualized by antiserum to VAMP-2 (20) (Fig.8G and H). Over-expression of PAM in anterior pituitary cells does not appear to overload the endocytic machinery; internalization of PAM-1 is similar in infected and non-infected cells (4).

Effect of membrane protein over-expression on filamentous actin organization in anterior pituitary cells.

The cytoskeleton, along with many cytosolic proteins, governs secretory granule formation, maturation, translocation and exocytosis (33)(34;35). PAM-1 interacts, via its cytosolic domain, with proteins that play a powerful role in cytoskeletal organization(12;36). In AtT-20 cells, expression of PAM-1 alters the organization of the actin cytoskeleton (12;16). Using FITC-phalloidin, we studied the distribution of filamentous actin in pituitary endocrine cells expressing PAM-1 or myc-TMD/CD (Fig.9). For comparison, filamentous actin staining was evaluated in non-infected cells and in POMC-V infected pituitary cultures. Non-infected (Fig.9A, B) or POMCV (Fig.9C, D) infected cells show organized patches of filamentous actin throughout the cytosol, with more intense signal at the margins of the cell immediately beneath the plasma membrane (Fig.9B and D).

In contrast, PAM-1V infected endocrine cells (Fig.9E, F) exhibit a different pattern of filamentous actin. The patches of filamentous actin are gone and filamentous actin is no longer concentrated under the plasma membrane (Fig.9F). In some PAM-1V infected cells, filamentous actin localizes to the TGN region of the cell, overlapping regions enriched in PAM protein (Fig.9F). Filamentous actin staining in cells expressing myc-TMD/CD (Fig.9G and H) is indistinguishable from staining in cells expressing PAM-1, and quite distinct from the pattern in non-infected and POMCV-infected cells.

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Discussion

The single most striking difference between the behavior of endogenous PAM in primary pituitary cells and exogenous PAM in AtT-20 cells is the steady state localization of the protein (4). In pituitary cells, as in hypothalamic neurons and cultured atrial myocytes, PAM is concentrated in secretory granules (3;37). When integral membrane PAM proteins are expressed in the neuroendocrine AtT-20 cell line, much of the protein is found in the vicinity of the Golgi apparatus (4;19). At the immunoelectron microscopic level, a significant amount of overlap is observed between transfected PAM and endogenous TGN38, with PAM expression enriched in the more distal compartments of the TGN (19).

To explore trafficking to secretory granules in cells equipped to assemble a large number of secretory granules, we expressed three membrane proteins in primary anterior pituitary cells. Using the adenovirus system, PAM-1 is expressed at varying levels in essentially all of the primary pituitary cells. Following infection, PAM enzyme activity, both PHM and PAL, is 40- to 50-fold higher than in adult rat anterior pituitary or stably transfected AtT-20 PAM-1 cells. Adenoviral mediated PAM expression in pituitary is high, but only double the physiological level of PAM in adult rat atrium (24). The over-expressed membrane PAM is localized primarily to vesicular structures, with very little PAM in the TGN region and no accumulation of PAM on the cell surface. This distribution stands in sharp contrast to the TGN accumulation observed in AtT-20 PAM-1 cells (19). The largely overlapping PAM and VAMP-2 localization, with variable ratio of PAM and VAMP-2, is consistent with current models of vesicle biogenesis, with continuous removal of membrane proteins and lumenal content via clathrin coated vesicles (38;39).

Our earlier studies in AtT-20 cells clearly identified routing determinants in both the lumenal and cytosolic domains of membrane PAM (8;9;16;19). Expressed independently, both lumenal domains are efficiently stored in secretory granules (4;40). Studies in AtT-20 cells indicated that the cytosolic domain of membrane PAM contains endocytic trafficking information, but a role for cytosolic signals in granule entry was not apparent. PAM mutants truncated so that they lack most of the cytosolic domain are less extensively cleaved by secretory granule endoproteases, are localized on the plasma membrane, and fail to undergo internalization (19). Like intact PAM, myc-TMD/CD, lacking both catalytic (lumenal) domains, is highly localized to the TGN region of AtT-20 cells (10) and appending the cytosolic domain of PAM to Tac reroutes Tac to the TGN (11). In contrast, myc-TMD/CD is enriched in the secretory granules of anterior pituitary endocrine cells. This result suggests the presence of granule targeting signals in the transmembrane and/or cytosolic domain of PAM.

AtT-20 cells contain far fewer secretory granules than pituitary endocrine cells (18;41). In many systems, lumenal proteins enter vesicular traffic derived from the TGN by default (42). Sorting of regulated secretory proteins is then accomplished passively, by removal from immature granules of a subset of protein components. Therefore we considered the possibility that secretory granules are a default pathway for membrane proteins in pituitary cells. However, Tac, which accumulates on the cell surface when expressed in a variety of endocrine and non-endocrine cells (11;13), also localizes to the plasma membrane of anterior pituitary endocrine cells. Therefore, pituitary secretory granules do not represent a default pathway, and the granule

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localization of myc-TMD/CD indicates that it contains a secretory granule routing signal. P-selectin is a platelet and endothelial cell granule membrane protein. In AtT-20 cells, exogenous P-selectin is targeted to secretory granules by signals in its cytoplasmic tail and transmembrane domain (43-45). Recently, Cutler et al. (1999) demonstrated that the same cytoplasmic signal is required for the appearance of P-selectin in immature and mature dense core vesicles as well as synaptic-like microvesicles in PC12 cells (46).

Anterior pituitary endocrine cells show an immense storage capacity for exogenous PAM. Remarkably, over-expression of PAM-1 in pituitary cells actually decreases the basal secretion of PHM activity as a percentage of cell content. Although high levels of expression do not lead to PAM accumulation on the cell surface, there is clearly some limitation to PAM proteolytic processing, with intact membrane PAM accounting for a higher percentage of the PAM protein in infected cells than in non-infected cells. Secretion of PHM is stimulated by both BaCl2 and PMA. Secretion of PHM from the BaCl2 responsive compartment is limited in pituitary cells over-expressing PAM-1 (1.3 % of cell content/h vs. 4.5 % from non-infected cells). Assuming that BaCl2 is stimulating Ca2+-dependent exocytosis from mature granules (28;47), these data suggest that access to mature granules is limited upon over-expression of membrane PAM. BaCl2 stimulated release of GH and ACTH occurs normally following over-expression of PAM, indicating that mature granules form normally. Access of PAM to mature granules seems to be impaired.

PMA stimulated release of a comparable percentage of enzyme content/h from non-infected and PAM-1 infected pituitary cultures (5 ± 0.5 % and 6 ± 0.4 % respectively). PMA stimulates protein kinase C, inducing Ca2+-independent exocytosis via phospholipase D, which is known to activate secretory vesicle budding from the TGN (29;48). Thus PMA is able to affect secretion at earlier steps of the secretory pathway, including immature as well as mature granules (diagramed in Fig. 10). PAM-1 over-expression in pituitary endocrine cells results in a dramatic expansion of the PAM content of the immature and mature pools of granules (Fig. 10); basal secretion from infected cells is stimulated 10-fold by PMA while basal secretion from non-infected cells is stimulated only 4-fold. The limited endoproteolytic cleavage of virally expressed PAM also suggests accumulation in immature granules. PMA stimulated GH and ACTH secretion normally following over-expression of PAM-1.

The cytosolic tail of PAM interacts with proteins involved in cytoskeletal organization and expression of membrane PAM or myc-TMD/CD alters regulated secretion in AtT-20 cells, perhaps by affecting the actin cytoskeleton (12;16). Similarly, virally over-expressed PAM and myc-TMD/CD affect filamentous actin organization in anterior pituitary cells (Fig. 10). In contrast, over-expression of the soluble POMC precursor is without effect on the actin cytoskeleton, ruling out non-specific effects of adenovirus-mediated expression. PAM-1 and myc-TMD/CD infected cells exhibit an absence of punctate actin aggregates, instead exhibiting diffuse staining for filamentous actin and some concentration of filamentous actin in the TGN region (Fig. 10). The accumulation of filamentous actin in the TGN area may be involved in the accumulation of membrane PAM in immature granules following over-expression.

As observed for endogenous anterior pituitary PAM and for PAM-1 transfected AtT-20 cells, robust internalization of PAM/PAM-antibody complexes from the plasma membrane occurs under basal conditions. In both cell systems, internalized PAM/PAM-antibody

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complexes are initially present in small uniform vesicles distributed all over the cell. Later a more heterogeneous collection of intracellular vesicular structures is observed. PAM retrieved from the surface of anterior pituitary cells passes through an early endosomal compartment recognized by antisera to the plasma membrane adaptor, AP-2 (23;31) (Fig. 10). At later times, the recycled PAM/antibody complex is more localized with AP-1(31;32). By 2h, internalized PAM largely colocalizes with VAMP-2 in secretory granules (20). As for endogenous PAM, the enzyme internalized from the surface of pituitary endocrine cells was never collected in the TGN region (4). Therefore, over-expression of membrane PAM in anterior pituitary endocrine cells does not overload the endocytic sorting machinery, and membrane PAM retrieved from the pituitary cell surface has rapid access to secretory granules. In the same context, it has been demonstrated that VAMP-2 stained synaptic vesicles in neuroendocrine PC12 cells form by budding from tubular extensions of sorting endosomes; granule proteins together with the transferrin receptor are delivered to the early endosomes, where they are sorted into synaptic-like microvesicles and recycling vesicles (49). In the current study we propose targeting of recycled membrane PAM from the pituitary cell surface to the secretory granules via the endosomal compartment.

In summary, we propose a model of PAM trafficking in primary pituitary cells where the transmembrane and/or cytosolic domains of PAM is sufficient to target the protein to secretory granules (Fig.10). PAM proteins leaving immature secretory granules largely go to mature granules, with a smaller amount of PHM and the bifunctional enzyme undergoing constitutive-like secretion (4). Little membrane PAM reaches the cell surface, and that which does is rapidly internalized via the endosomal compartment for recycling towards the TGN or toward basal secretion (Fig.10A). Upon over-expression, PAM proteins accumulate in immature secretory granules (Fig.10B). The expression of membrane PAM affects cytoskeletal organization, probably via interactor proteins that recognize the CD of PAM, resulting in a delay of secretory granule maturation and an alteration of PAM processing. Over-expression of membrane PAM does not lead to PAM accumulation on the cell surface, nor does it overload the endocytic pathway.

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Figure Legends

Fig.1: PAM-1 over-expression in primary anterior pituitary cells. After 3 days in culture, primary anterior pituitary cells were infected with recombinant adenovirus encoding PAM-1; infected and non-infected cultures were analyzed 2 days later. (Upper) Duplicate samples of pituitary cultures, adult anterior pituitary (AP) or stably transfected AtT-20 PAM-1 cells were extracted for measurement of PHM and PAL activities. Data are mean ± SD (n = 4). (Lower) Cultures were fixed and permeabilized and PAM was visualized using antibody to exon A and FITC-tagged goat anti-rabbit IgG. (A, B) PAM-1V infected cells and (D, E) non-infected cells were visualized under identical conditions; immunofluorescence (A, D) and the corresponding phase-contrast image (B, E). At higher magnification (C), the vesicular nature of the PAM staining (Ves) is apparent, as is the unstained cell nucleus (N).

Fig.2: Subcellular localization of exogenous PAM-1 in primary pituitary cells. (A, B and C) Primary pituitary cells infected with the PAM-1 virus were simultaneously visualized with mouse monoclonal antibody to PAM-CD (A, arrow heads) and rabbit polyclonal antibody to TGN38 (B, arrows). The primary antibodies were then visualized with fluorescently tagged secondary antisera (PAM, red; TGN38, green, in C). (D, E and F) Cells were simultaneously visualized with antisera to PAM (exon A) and VAMP-2 (D and E, arrows). Primary antibodies were visualized with fluorescently tagged secondary antisera (PAM, green; VAMP-2, red). (F) Higher magnification images were superimposed; areas visualized by both antibodies appear yellow (arrows), vesicles stained only by VAMP-2 antibody are indicated by lines and those stained only by PAM antibody by arrow heads. The magnification was the same for A, B, C, D and E.

Fig.3: Myc-TMD/CD localization in anterior pituitary cells. (A) Diagram showing myc-TMD/CD relative to PAM-1. (B through I) Primary pituitary cells infected with the myc-TMD/CD virus were simultaneously visualized with two antisera. (B and C) Myc-TMD/CD protein was localized using antisera to PAM-CD (FITC) and to myc (Cy3). Myc-TMD/CD (D; arrowheads) was visualized simultaneously with TGN38 (E, arrows); the superimposed images are shown in F. Myc-TMD/CD (G) was visualized simultaneously with VAMP-2 (H). The superimposed images (I) demonstrate PAM/VAMP co-localization (yellow, arrows). The scale bar for B through H is shown in B.

Fig.4: Biochemical analysis of pituitary cells expressing myc-TMD/CD. (A) Extracts of anterior pituitary cultures infected with the myc-TMD/CD virus were subjected to Western blot analysis and visualized with antisera to myc and PAM-CD. Anterior pituitary cultures infected with the myc-TMD/CD virus were subjected to differential centrifugation (B) and the secretory granule enriched 20,000 x g pellet (P2) was further fractionated on discontinuous sucrose density gradients (C) (2). Myc-TMD/CD was identified by Western blot analysis using antibody to myc and secretory granules were identified using a VAMP-2 antibody. (D and E) Anterior pituitary cells were transfected with vector encoding Tac. Transfected Tac was visualized by immunofluorescent staining of fixed, non-permeabilized (D) or permeabilized cells (E) using a monoclonal antibody to Tac (11).

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Fig.5: Exogenous PAM-1 processing in anterior pituitary cultures. The major cleavage products of PAM-1 are diagrammed (A, left side; apparent molecular masses in kDa are indicated in italics); TMD, transmembrane domain; CD, cytosolic domain; empty lollipop, N-linked oligosaccharide; filled lollipop, O-linked oligosaccharide. Equal amounts of protein from PAM-1V infected and non-infected cultures extracted with TES-mannitol/TX-100 were subjected to Western blot analysis. PAM proteins were visualized with antisera to exon A (A), PHM, PAL or PAM-CD (B). The molecular weight markers are shown. Since the PAM proteins in non-infected cultures were barely visible under conditions optimized for analysis of infected cultures, longer exposures are shown for these samples (indicated by dashed lines).

Fig.6: Basal and stimulated secretion of PHM activity and PAM protein from PAM-1V infected pituitary cells. Infected and non-infected cultures were incubated in basal medium for two sequential 1 h periods, then exposed to either 1 mM BaCl2 or 1 µM PMA for 1 h. Cultures were extracted in TMT. (A) PHM activity measured in duplicate samples of medium was expressed as units of product formed per microgram of protein (left) and as per cent of cell content of enzyme activity (right). Data are the mean ± SD for four cultures for each secretagogue; where error estimates are small, error bars are not visible. Similar results were obtained in three additional independent experiments. (B) Equal amounts of basal and stimulated media from PAM-1V infected cells were analyzed by Western blot and probed with antiserum to PHM.

Fig.7: Over-expression of PAM-1 does not impair regulated secretion. The secretion experiments described in Fig.6 were subjected to additional analysis. (A) GH secretion was evaluated by Western blot analysis of medium samples. (B) ACTH secretion was determined by radioimmunoassay. Similar results were obtained in three additional complete experiments.

Fig.8: Internalization of PAM ectodomain antibody under basal conditions. PAM-1V infected anterior pituitary cells were incubated with exon A rabbit antibody for 20 min and either harvested (Pulse 20 min A-D) or allowed to internalize PAM-antibody complexes for 1 h or 2 h at 37 oC (E-H). Fixed, permeabilized cells and immunostained using monoclonal antibodies to various subcellular markers, AP-2 for early endosome; AP-1 for immature granules; and VAMP-2 for secretory granules. The internalized PAM-antibody complex (in green) and the different markers (in red) were visualized as described in Methods. Arrows indicate PAM containing vesicles; arrow heads show areas of yellow staining when internalized PAM co-localizes with one of the subcellular marker.

Fig.9: Effect of PAM-1 over-expression on filamentous actin in anterior pituitary cells. Non-infected pituitary cells (A and B), POMCV (C and D), PAM-1V (E and F) and myc-TMD/CDV (G and H) infected cells were stained using the PAM exon A antiserum (A and E), an ACTH (C) or PAM-CD (G) monoclonal antibody and FITC-phalloidin. Cells were photographed under identical conditions. Filamentous actin staining throughout the cytosol is indicated by arrows; areas with an intense filamentous actin signal around the cell edges (arrow heads) or in the TGN region (asterisk) are indicated.

Fig.10: Trafficking of membrane PAM in primary pituitary cells. (A) Directed by targeting signals encoded by both the lumenal and the cytosolic domains, endogenous PAM proteins exit immature secretory granules (ISG) and have unlimited access to mature secretory granules

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(MSG), which are stored for days; very little newly synthesized PAM enters endosomes for recycling to the TGN or basal secretion (CLV, constitutive like vesicle). Very little PAM is on the cell surface at steady state and PAM appearing on the plasma membrane (PM) is rapidly endocytosed. (B) When over-expressed in primary pituitary cells, PAM basal secretion is not impaired. PAM proteins accumulate in the immature secretory granules affecting the actin cytoskeleton via PAM-CD interactors, which delay the maturation of secretory granules and entry into the BaCl2 stimulable pool of vesicles. Therefore, PAM endoproteolytic cleavage is less efficient. No PAM accumulation is observed on the cell surface and PAM proteins reaching the cell surface are retrieved via the endosomal compartment and directed to the secretory pathway.

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MSG

PM

PMAPMA + BaCl2

PMAPMA + BaCl2

Actin filaments

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Rajaa El Meskini, Gregory J. Galano, Ruth Marx, Richard E. Mains and Betty A. EipperPituitary Endocrine Cells

Targeting of Membrane Proteins to the Regulated Secretory Pathway in Anterior

published online November 1, 2000J. Biol. Chem. 

  10.1074/jbc.M008062200Access the most updated version of this article at doi:

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