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Originally published In Press as doi:10.1074/jbc.M506700200 on July 6, 2005

J. Biol. Chem., Vol. 280, Issue 36, 31708-31713, September 9, 2005
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A Role for Gz in Pancreatic Islet {beta}-Cell Biology*

Michelle E. Kimple{ddagger}, Andrew B. Nixon{ddagger}, Patrick Kelly{ddagger}§, Candice L. Bailey{ddagger}, Kenneth H. Young¶, Timothy A. Fields¶, and Patrick J. Casey{ddagger}||

From the {ddagger}Department of Pharmacology and Cancer Biology and Department of Pathology, Duke University, Medical Center, Durham, North Carolina 27710

Received for publication, June 20, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucose-stimulated insulin secretion and {beta}-cell growth are important facets of pancreatic islet {beta}-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein Gz and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic {beta}-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G{alpha}z impacts are intact in {beta}-cells, and G{alpha}z activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) {beta}-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G{alpha}i family members are not involved in this process in this {beta}-cell line. Indeed, overexpression of a selective deactivator of G{alpha}z, the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G{alpha}z expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) {beta}-cell line couples to Gz predominantly and perhaps even exclusively. These data provide the first evidence for Gz signaling in pancreatic {beta}-cells, and identify an endogenous receptor-mediated signaling process in {beta}-cells that is dependent on G{alpha}z function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pancreatic islet {beta}-cells are the sole producers of insulin in the body and thus play an important homeostatic role in the secretion of insulin in response to glucose and other secretagogues. Normally, the release of insulin from {beta}-cells induces the peripheral tissues to take up available glucose from the plasma. Disregulation of this process can lead to the development of Type 2 diabetes, wherein the {beta}-cells are unable to compensate for peripheral tissue insulin resistance, either through inadequate {beta}-cell function and/or a decrease in {beta}-cell mass (1).

Certain hormones such as norepinephrine, somatostatin, galanin, and E prostaglandins decrease the secretion of insulin from {beta}-cells in response to glucose (2). Pretreatment of pancreatic islet preparations with pertussis toxin (PTX)1 alleviates the inhibitory effects of some of these hormones, leading to the original description of PTX as islet-activating protein (3). PTX catalyzes the ADP-ribosylation of a cysteine residue near the C terminus of nearly all G{alpha} subunits of the G{alpha}i subfamily (4), suggesting that the inhibition of glucose-stimulated insulin secretion (GSIS) is mediated by G{alpha}i proteins. However, several reports indicate a lack of effect, or only a partial effect, of PTX pretreatment on inhibition of insulin secretion in islets (5–8). This phenomenon of PTX insensitivity has been suggested to be because of insufficient PTX or too short a period of exposure (2), although an alternative explanation is that certain GPCRs are coupled to a PTX-insensitive G protein. The finding that the PTX-insensitive G{alpha}i family member G{alpha}z (9) might be expressed in pancreatic islet cells (10) opened the possibility that such signaling pathways exists in {beta}-cells.

Clues to potential unique functions of G{alpha}z have emerged from yeast two-hybrid screens that identified proteins that selectively interact with constitutively active G{alpha}z (11–14). One of the best characterized G{alpha}z effectors is Rap1GAP, a GTPase activating protein (GAP) for a member of the Ras family of small G proteins, Rap1. The physical interaction between G{alpha}z and Rap1GAP blocks both the ability of regulators of G protein signaling (RGS) proteins to stimulate the intrinsic GTP hydrolysis activity of G{alpha}z and the ability of activated G{alpha}z to inhibit adenylyl cyclase (12). In cell-based studies, activated G{alpha}z is able to recruit Rap1GAP from the cytosol to the plasma membrane. Through this process, activation of G{alpha}z inhibits Rap1 activation and accompanying ERK phosphorylation and differentiation of PC12 cells that is normally induced by treatment with either a cAMP analogue or nerve growth factor (15). Based on these findings, a model has been proposed in which receptor-mediated activation of Gz results in recruitment of Rap1GAP to the plasma membrane, where it can effectively down-regulate Rap1 signaling processes (15). Other effectors of G{alpha}z that were discovered through yeast two-hybrid screens include RGSZ1, a G{alpha}z-selective RGS protein (13) and the transcriptional cofactor Eya2 (11, 14).

In the present study we demonstrated that G{alpha}z and members of a unique G{alpha}z signaling axis are expressed in pancreatic {beta}-cells and {beta}-cell-derived lines. In addition, utilizing three separate methods to perturb G{alpha} signaling, we establish that G{alpha}z is required for PGE1-mediated inhibition of GSIS. These data strongly suggest that Gz has a physiological role in important facets of {beta}-cell biology.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Anti-GFP, G{alpha}12, Rap1, myc-tag, HA-tag, and G{alpha}z primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-G{alpha}z antibody P-961 (16) was also used in certain experiments, as indicated in the respective figure legends. Anti-Rap1GAP was a gift of Paul Polakis, Genentech, anti-pan-G{alpha}i was a gift of Tom Gettys, Pennington Biomedical Research Institute, and anti-RGSZ1 was a gift of Elliot Ross, UT-Southwestern. siRNA oligonucleotides used were a control luciferase plasmid sequence (Dharmacon, Lafayette, CO) and rat G{alpha}z-specific oligonucleotides (Ambion, Austin, TX) as stated in the figure legends.

Cell Culture—The rat insulinoma line Ins-1 and the selected glucose-responsive Ins-1(832/13) subline were obtained from Chistopher Newgard at the Sarah W. Stedman Nutrition and Metabolism Center at Duke University. Cells were maintained in RPMI 1640 medium containing 10% fetal calf serum, 10 mM Hepes, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM {beta}-mercaptoethanol. The mouse insulinoma line, Min6, was obtained from Dr. J. Miyazaki (University of Tokyo, Tokyo, Japan). Min6 cells were maintained in Dulbecco's modified Eagle's medium containing 15% heat-inactivated fetal calf serum, 10 mM Hepes, and 50 µM {beta}-mercaptoethanol. The rat pancreatic exocrine line Panc-1 was obtained from the Cell Culture Facility at the Duke University Comprehensive Cancer Center. Panc-1 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. All cells were kept at 37 °C in a humidified atmosphere containing 95% air and 5% CO2 and passaged after reaching 80–90% confluence.

Immunohistochemistry—Tissues were fixed in 10% neutral buffered formalin and then processed into paraffin blocks using standard protocols (17). Briefly, 5-µm sections of formalin-fixed, paraffin-embedded tissue were placed on positively charged slides and permitted to dry, followed by removal of paraffin. Endogenous peroxidase activity was quenched, and sections were then hydrated, followed by incubation with a 5% goat serum solution. Sections were subjected to heat-induced epitope retrieval (17) by steaming in 100 mM glycine, pH 3.0. Sections were next incubated with Background Buster® (Innovex Biosciences, Richmond, CA) to reduce background staining and then with anti-glucagon, anti-insulin, or anti-G{alpha}z antisera (Santa Cruz Biotechnology). Bound antibodies were detected by incubation with biotinylated goat anti-rabbit antisera (Vector Laboratories, Burlingame, CA) followed by RTU Vectastain ABC reagent (Vector Laboratories, Burlingame, CA) and finally by horseradish peroxidase-labeled streptavidin (Jackson ImmunoResearch, West Grove Park, PA). Visualization of bound immune complex was performed by using an horseradish peroxidase substrate. Hematoxylin counterstain (Fisher Scientific, Pittsburgh, PA) was used, followed by traditional dehydration, clearing, and mounting.

Immunoblot Analysis—Adherent cells were washed in phosphate-buffered saline and lysed by scraping with a rubber policeman and sonicating in a Tris-EDTA solution. In some cases, cytosol and membrane fractions were separated by ultracentrifugation for 5 min at 200,000 x g. Protein content was determined by the Bradford method (18) (Bio-Rad Protein Assay, Bio-Rad). Unless otherwise indicated, 40 µg of total cellular protein was separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with the appropriate primary antibody, followed by incubation with an anti-rabbitor anti-mouse-horseradish peroxidase conjugate (Amersham Biosciences). Horseradish peroxidase was detected using Western Lightning® chemiluminescence reagent (PerkinElmer).

Generation of Recombinant Retroviruses and Selection of Stable Cell Lines—The cDNA encoding wild-type G{alpha}z was obtained from the UMR cDNA Resource Center (University of Missouri, Rolla, MO). The G{alpha}z coding region was subcloned into the EcoRI-NotI sites of the retroviral shuttle vector pLPCX (Clontech). pLPCX-G{alpha}z and pVSV-G were cotransfected into the pantropic packaging cell line GP2 293 using cesium chloride precipitation according to manufacturer's protocol. Viral medium was filtered and concentrated 48 h after transfection. Concentrated viral medium was mixed with 5 µg/ml Polybrene and used to infect Ins-1(832/13) cells. The infection medium was removed after 24 h and replaced with growth medium. Stable transformants were selected 24 h later with 2 µg/ml puromycin for 14 days.

Generation of Recombinant Adenoviruses and Infection of Ins-1(832/13) Cells—cDNAs encoding G{alpha}z(QL), G{alpha}i2(QL), G{alpha}12(QL), RGS2, p115 RhoGAP, and RGSZ1 were obtained from the UMR cDNA resource center. The respective coding regions were subcloned into the adenoviral shuttle vector pAdTrack-CMV (Stratagene, La Jolla, CA). pAdTrack-CMV constructs were linearized with PmeI and electroporated into BJ5183-AD-1 cells (Stratagene). Recombinant adenoviral plasmids were purified from isolated colonies and transfected into HEK 293 cells (American Type Culture Collection, Manassas, VA) using Lipofectamine (Invitrogen). Viral plaques formed between 7–10 days posttransfection, and viral lysates were used to serially infect HEK 293 cells for large scale viral preparations.

Ins-1(832/13) cells were plated at 2.5–3 x 105 cells/well in 12-well tissue culture plates. Cells were infected for 1 h with an empirical titer of virus that resulted in nearly 100% infection as determined by GFP expression. Cells were assayed for cAMP generation and/or insulin secretion 24 h postinfection.

Cyclic AMP Immunoassay—Ins-1(832/13) cells were grown to 100% confluence in 12-well tissue culture plates. On the day of the assay the cells were washed once with a 3 mM glucose-containing Hepes-buffered saline solution (HBSS) (114 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4,20mM Hepes, pH 7.2, 25.5 mM NaHCO3,10mM CaCl2, 0.2% bovine serum albumin) and then preincubated for 2 h in fresh 3 mM glucose-HBSS. Next, 1 ml of 3 mM glucose- or 15 mM glucose-HBSS was added to each well for 2 h. All wells were treated with 0.2 mM isobutylmethylxanthine for 20 min prior to and during the stimulation period. At the end of the stimulation time, the HBSS was removed, the cells were washed once in phosphate-buffered saline, and cells were lysed in 300 µl of lysis reagent (cAMP Biotrack® Enzymeimmunoassay System, Amersham Biosciences). Determinations of cAMP in each sample were performed in duplicate according to manufacturer's protocol. Protein content was used to normalize the cAMP assay results.

Glucose-stimulated Insulin Secretion Assays—Ins-1(832/13) cells were grown to 100% confluence in 12-well tissue culture plates. The day before the GSIS assay, selected cells were treated with 100 ng/ml PTX (EMD Biosciences, San Diego, CA) or infected with recombinant adenoviruses as noted in the figure legends. Confluent Ins-1(832/13) cells were preincubated in 3 mM glucose-HBSS as described above for cAMP assays. Next, 1 ml of 3 mM glucose- or 15 mM glucose-HBSS was added to each well for 2 h. In certain cases GPCR agonists UK14,304 (Sigma-Aldrich) or PGE1 (EMD Biosciences) were added during the stimulation period. Following stimulation, the secretion medium was removed and assayed for insulin content (Coat-A-Count® [125I]insulin radioimmunoassay, Diagnostic Products Corp., Los Angeles, CA). The remaining cells were washed once in phosphate-buffered saline and lysed by scraping and sonicating in Tris-EDTA buffer. The protein concentration was used to normalize the insulin assay results. Insulin secretion was represented as fold stimulation, which is the ratio of insulin secretion in 15 mM to that in 3 mM glucose.

siRNA-mediated Gene Silencing—Ins-1(832/13) cells were grown to 50–75% confluence in 10-cm dishes. Cells were trypsinized, and 5 x 106 cells were electroporated with 200 pmol of annealed siRNA oligonucleotide using the Amaxa Nucleofector® (Amaxa Inc., Gaithersburg, MD), solution T, program T20. Each reaction was split between 4 wells of a 12-well plate. The GSIS assay was performed 48 h postelectroporation.

Statistical Analyses—Data were analyzed using GraphPad Prism v4 (GraphPad Software Inc., San Diego, CA). Data are given as the mean ± S.E. and compared by one- or two-way analysis of variance, followed by the relevant post-test to determine p values. A probability of p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
There are two reports of G{alpha}z protein expression in rat pancreatic islets of Langerhans (10, 19), but neither the specificity nor the relevance of this expression was determined. To confirm that G{alpha}z is expressed in islets and to delineate its expression pattern, an immunohistochemical study was undertaken. Mouse pancreases were formalin-fixed and immunostained for G{alpha}z, glucagon (to identify the {alpha}-cells), or insulin (to identify the {beta}-cells). Confirming the previous report (10), G{alpha}z is expressed in both the periphery and interior of the islet (Fig. 1A), suggesting expression in both the {alpha}-cells and {beta}-cells, respectively (Fig. 1, B and C). In a separate immunohistochemical experiment, we sought to demonstrate the specificity of G{alpha}z expression in islets. Again, G{alpha}z expression was readily detected in the periphery and interior of the islet (Fig. 1D), but all staining was lost when the G{alpha}z antibody was preincubated with the G{alpha}z-specific peptide to which the antibody was raised (Fig. 1E). Furthermore, G{alpha}z expression was readily detected by immunoblot analysis of isolated rat islets (Fig. 1F).



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  		FIG. 1.
G{alpha}z is specifically expressed in pancreatic islet cells and {beta}-cell-derived lines. A, expression of G{alpha}z in pancreatic islets. Mouse pancreases were sectioned and incubated with G{alpha}z antisera. G{alpha}z is expressed in the glucagon-secreting {alpha}-cells at the periphery of the islet (B,as demonstrated by glucagon staining) and the insulin-secreting {beta}-cells at the interior of the islet (C, as demonstrated by insulin staining). D, duplicate experiment to A. E, preincubation of G{alpha}z antisera with a G{alpha}z-specific blocking peptide completely abrogates G{alpha}z staining in the islet, confirming that the G{alpha}z staining in D is specific. F, cell lysates from isolated rat islets and Panc-1, Min6, Ins-1, and Ins-1(832/13) cells were prepared, and proteins were separated by SDS/PAGE, and immunoblot analysis was performed with antibodies against G{alpha}z, Rap1GAP, or Rap1. Two G{alpha}z antibodies that recognize different epitopes were used, Santa Cruz Biotechnology (SC) and P-961 (961). G{alpha}z and Rap1GAP expression is restricted to islets and {beta}-cell-derived lines and is essentially absent from the exocrine-derived Panc-1 cells.

 
We next sought to confirm that G{alpha}z is expressed in {beta}-cell-derived lines that are often used to assess {beta}-cell function in vitro. We obtained several {beta}-cell-derived lines, including Min6, Ins-1, and Ins-1(832/13). Although Min6 and Ins-1 cells are established {beta}-cell lines, each has a fairly low GSIS response compared with intact islets (20). In contrast, Ins-1(832/13) cells are a derivative of the Ins-1 cell line with robust GSIS of a magnitude similar to that of normal islets (7–10-fold responses to stimulatory glucose) (21–23). Lysates from each cell type were normalized for protein content and analyzed by immunoblotting for the members of the best described G{alpha}z-specific signaling pathway: G{alpha}z, Rap1GAP, and Rap1 (12, 15). Interestingly, both G{alpha}z and Rap1GAP were expressed in {beta}-cell-derived lines and in isolated islets but not the exocrine pancreatic Panc-1 cells (Fig. 1F). These findings are consistent with specific G{alpha}z expression in the cells of the pancreatic islet.

Activation of Gi subfamily members by a number of GPCR ligands can inhibit various {beta}-cell functions, including glucose-stimulated cAMP production and insulin secretion (24). To assess a potential role for Gz in these processes, we first measured glucose-stimulated cAMP production in Ins-1(832/13) infected with mutationally active (QL (25)) forms of G{alpha}z, G{alpha}i2, or G{alpha}12 (Fig. 2A). Expression of G{alpha}z(QL) and G{alpha}i2(QL) significantly inhibited cAMP production, whereas, as expected, expression of G{alpha}12(QL) had no effect (Fig. 2B). We next assessed the ability of Gz to impact GSIS. As was seen with cAMP production, both G{alpha}z(QL) and G{alpha}i2(QL) significantly inhibited GSIS in Ins-1(832/13) cells, whereas G{alpha}12(QL) had no effect (Fig. 2C). These findings indicate that G{alpha}z can function to inhibit adenylyl cyclase in {beta}-cells similar to other G{alpha}i proteins (24).



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  		FIG. 2.
Impact of expression of mutationally active G{alpha} subunits on cAMP production and GSIS in Ins-1(832/13) cells. A, expression of activated G{alpha} subunits in Ins-1(832/13) cells. Ins-1(832/13) cells were infected with adenoviruses expressing GFP, G{alpha}z(QL), G{alpha}i2(QL), or G{alpha}12(QL). Cell lysates were normalized for protein content and immunoblot analysis performed to confirm expression of respective constructs. B, cAMP production in Ins-1(832/13) cells expressing the indicated constructs was measured after a 2-h incubation in high glucose buffer. Data are the mean ± S.E. of one representative experiment repeated three times. C, GSIS in Ins-1(832/13) cells expressing the indicated constructs was measured after a 2-h incubation in low or high glucose buffer and is displayed as the ratio of insulin secretion in high glucose to low glucose buffer, normalized to control (control fold stimulation = 5.06 ± 1.33) Data are the mean ± S.E. of at least three independent experiments. For both B and C, data were compared by one-way analysis of variance followed by followed by Dunnett's test post hoc; *, p < 0.05; **, p < 0.01.

 
As noted above, members of a unique G{alpha}z signaling axis, G{alpha}z, Rap1GAP, and Rap1, are present in pancreatic islet {beta}-cells. To initially assess whether this signaling axis is functional in {beta}-cells, we expressed G{alpha}z(QL) in Ins-1(832/13) cells and tested its ability to cause a translocation of Rap1GAP from the cytosol to the membrane, a function that is specific to activated G{alpha}z, and not other G{alpha}i subfamily members (15). Cells expressing either G{alpha}z(QL) or G{alpha}i2(QL) (Fig. 3A) were disrupted and subjected to ultracentrifugation to obtain membrane and cytosol fractions and then immunoblotted for Rap1GAP. Importantly, G{alpha}z(QL) but not G{alpha}i2(QL) caused a significant translocation of Rap1GAP from the cytosol to the membrane fraction of Ins-1(832/13) cells (Fig. 3B). These results indicate that the G{alpha}z-Rap1GAP axis is intact in this {beta}-cell line.

We next assessed whether endogenous GPCRs in In-1(832/13) cells could in fact transmit signals through Gz. The {alpha}2A-adrenergic receptor has already been shown to be capable of signaling through Gz in other cell types (15, 26, 27) and has a clear role in the inhibition of GSIS (5, 28–30). Another possible candidate for an endogenous Gz-coupled receptor in {beta}-cells is the E prostanoid receptor because of the reported PTX-insensitive inhibition of {beta}-cell function by PGE1 and PGE2 (8). Indeed, treatment of Ins-1(832/13) expressing wild-type G{alpha}z with either the {alpha}2A-adrenoreceptor agonist UK14,304 or the E prostanoid receptor agonist PGE1 resulted an increase of Rap1GAP in the membrane fraction of the cells (Fig. 3C). Because Rap1GAP translocation is a function that is attributed to activated G{alpha}z but not G{alpha}i2 ((15) and Fig. 2B), these results provided the impetus to study the effects of these GPCR pathways in a biologically relevant model.



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  		FIG. 3.
Mutationally active G{alpha}z and receptor activation target Rap1GAP to the membrane. A, expression of activated G{alpha} subunits in Ins-1(832/13) cells. Ins-1(832/13) cells were infected with adenoviruses expressing GFP, G{alpha}z(QL), G{alpha}i2(QL), or G{alpha}12(QL). Cell lysates were normalized for protein content, and immunoblot analysis was performed to confirm expression of respective constructs. B, Rap1GAP translocation to the membrane of Ins-1(832/13) cells expressing activated G{alpha}z. Ins-1(832/13) cells expressing the indicated constructs were lysed by sonication, and membrane fractions were separated by ultracentrifugation. Lysate and membrane samples were normalized for protein content, separated by SDS/PAGE, and an immunoblot analysis for Rap1GAP was performed. C, UK14,304 or PGE1 treatment significantly increases membrane-associated Rap1GAP in Ins-1(832/13) cells. Ins-1(832/13) cells stably expressing wild-type G{alpha}z were stimulated with vehicle (ctrl), 10 µM UK14,304 (UK), or 10 µM PGE1 and lysed by sonication, and membrane were fractions separated by ultracentrifugation. Lysate and membrane samples were normalized for protein content and separated by SDS/PAGE, and an immunoblot analysis for Rap1GAP was performed. Both UK14,304 and PGE1 significantly increased the amount of Rap1GAP in the membrane fraction as compared with vehicle treatment. The immunoblot shown is from a single experiment that is representative of three such experiments.

 
To discern possible Gz-specific effects on {beta}-cell biology, Ins-1(832/13) cells were treated with PTX to irreversibly inactivate all other G{alpha}i subfamily members save G{alpha}z (data not shown). Following PTX treatment, GSIS assays were performed with and without UK14,304 or PGE1 addition. Although UK14,304 strongly inhibited GSIS, this inhibition was significantly PTX-sensitive (Fig. 4A), suggesting that although the {alpha}2A-adrenoreceptor can couple to Gz in Ins-1(832/13) cells (Fig. 3B) it may predominantly signal through other G{alpha}i subfamily members. Strikingly, however, the inhibition of GSIS by PGE1 was completely insensitive to PTX pretreatment (Fig. 4A), suggesting that the E prostanoid receptor in Ins-1(832/13) cells signals predominantly though Gz.

To test the hypothesis that the PGE1 receptor signals predominantly through Gz, we made use of regulator of G protein signaling (RGS) proteins, which can be used to selectively inhibit specific subtypes of heterotrimeric G proteins (31). Ins-1(832/13) cells were infected with adenoviruses encoding the RGS domains of RGSZ1, RGS2, or p115 RhoGEF, which selectively inhibit the PTX-insensitive Gz (13, 32), Gq (33), and G12 proteins (34, 35), respectively. GSIS was measured in Ins-1(832/13) cells expressing these RGS proteins in the absence or presence of PGE1. Only the RGS domain of RGSZ1 attenuated the ability of PGE1 to inhibit GSIS (Fig. 4B), strongly implicating a Gz-specific signaling pathway from the E prostanoid receptor to the secretion apparatus.



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  		FIG. 4.
PGE1-mediated inhibition of effect on GSIS is dependent on G{alpha}z function. A, PGE1-mediated inhibition of GSIS is PTX-insensitive. Ins-1(382/13) cells were treated with 100 ng/ml PTX to irreversibly inactivate all G{alpha}i family members save G{alpha}z. GSIS was measured in Ins-1(832/13) cells after a 2-h incubation in low or high glucose (glc) buffer, with and without 10 µM PGE1 or 10 µM UK14,304, and is displayed as the ratio of insulin secretion in high glucose to low glucose buffer, normalized to control (control fold stimulation = 5.44 ± 0.96 (ctrl) and 10.83 ± 3.12 (+PTX)). Data are the mean ± S.E. of at least three independent experiments with three replicates each. B, PGE1 mediated inhibition of GSIS is blocked by expression of the RGS domain of RGSZ1. GSIS in Ins-1(832/13) cells expressing the indicated constructs (inset) was measured after a 2-h incubation in low or high glucose buffer with and without 10 µM PGE1. GSIS is displayed as the ratio of insulin secretion in high glucose to low glucose buffer, normalized to control (control fold stimulation = 5.18 ± 0.36 (GFP), 4.81 ± 0.41 (RGSZ1), 3.58 ± 0.49 (RGS2), and 3.61 ± 0.30 (p115 RGS)). Data are the mean ± S.E. of at least two independent experiments with three replicates each. C, PGE1-mediated inhibition of GSIS is blunted by knock-down of G{alpha}z expression. GSIS in Ins-1(832/13) cells treated with siRNA oligonucleotides targeting a control luciferase sequence (siLuc) or a G{alpha}z-specific sequence (siG{alpha}z) (inset) was measured after a 2-h incubation in low or high glucose buffer, with and without 10 µM PGE1. GSIS is displayed as the ratio of insulin secretion in high glucose to low glucose buffer, normalized to control (control fold stimulation = 3.46 ± 0.04 (siLuc) and 2.18 ± 0.07 (siG{alpha}z)). Data are the mean ± S.E. of at least two independent experiments with at least two replicates each. In A, B, and C, data were compared by two-way analysis of variance followed by Bonferroni's test post hoc; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns = not significant.

 
To confirm the requirement for G{alpha}z in PGE1-mediated inhibition of GSIS, we employed a RNA interference approach targeting G{alpha}z expression. siRNA oligonucleotides targeting the G{alpha}z transcript or a control luciferase sequence were introduced into Ins-1(832/13) cells. This strategy resulted in a decrease in G{alpha}z expression of ~90% after 48 h (Fig. 4C, inset). This knock-down of G{alpha}z expression significantly attenuated the ability of PGE1 to inhibit GSIS (Fig. 4C). Taken together, the PTX insensitivity of the PGE1 effect on GSIS, the ability of the RGS domain of RGSZ1 to completely block the inhibition of GSIS by PGE1, and the effect of knockdown of G{alpha}z expression demonstrate that the endogenous receptor for PGE1 in the Ins-1(832/13) {beta}-cell line is coupled predominantly, if not solely, to Gz.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
G{alpha}z, the PTX-insensitive member of the G{alpha}i subfamily of heterotrimeric G protein {alpha}-subunits, was first identified in 1988 (36, 37), yet still relatively little is known about its function in vivo. Functional roles for G{alpha}z have been demonstrated in the nervous system and platelets (9). There is increasing evidence that G{alpha}z is expressed and/or functional in other tissues (10, 38–41), but many of these results are still preliminary. In this study, we provide the first compelling evidence that G{alpha}z is both present and functional in pancreatic {beta}-cells. Furthermore, although receptor coupling to Gz has been previously studied in reconstituted systems or in G{alpha}z-null mice (9, 26, 42), direct evidence for endogenous receptor coupling to this G protein has remained elusive. Hence, the finding in this study that endogenous Gz can couple to the endogenous E prostanoid receptor in a pancreatic {beta}-cell line to inhibit GSIS is an important advance in our understanding of the potential physiological roles of this unique member of the Gi subfamily.

The {alpha}2A-adrenoreceptor was previously shown to couple to Gz in neuronal model PC12 cells (15) and in platelets (26). Furthermore, {alpha}2A-adrenoreceptor stimulation causes an increase in membrane-localized Rap1GAP in Ins-1(832/13) cells expressing wild-type G{alpha}z (Fig. 3B). However, the inhibition of GSIS by the {alpha}2A-adrenoreceptor agonist UK14,304 is PTX-sensitive in Ins-1(832/13) cells, suggesting that the endogenous receptor predominantly couples to other Gi subfamily members (Fig. 4A). This data is supported by the PTX-sensitive inhibition of GSIS by other {alpha}2-adrenoreceptor agonists in isolated islets or {beta}-cell-derived lines (43, 44) and emphasizes the caution that must be used when interpreting experiments studying coupling of receptors to G proteins in reconstituted systems.

The results described in our experiments from three different modes of perturbing G{alpha} signaling, PTX treatment, RGS overexpression, and siRNA-mediated knock-down, lead to a single conclusion: that the endogenous E prostanoid receptor in Ins-1(832/13) cells selectively couples to Gz. E prostanoids, along with norepinephrine, somatostatin, and galanin, are classic physiological inhibitors of GSIS (45). Exogenous PGE1 and PGE2 inhibit the acute insulin response, i.e. the spike in insulin following a glucose challenge (46). E prostanoids also have a physiological role in islet homeostasis. PGE2 is produced by the cyclooxygenase-catalyzed metabolism of arachidonic acid in normal pancreatic islets and acts in an autocrine/paracrine fashion to modulate cell growth and hormone secretion (47). Furthermore, some nonsteroidal anti-inflammatory drugs, which are cyclooxygenase inhibitors, tend to improve the acute insulin response in diabetics (46–48). As EP2 and EP4 receptors mainly couple to Gs, whereas EP1 receptors couple to Gq subfamily members (49), an EP3 receptor splice variant is the most likely link between E prostanoids and Gz. Supporting this, the EP3 mRNA is the predominant mRNA subtype expressed in rat islets (48).

There exists a tendency to classify G{alpha}z as simply a G{alpha}i subfamily member that is insensitive to PTX. However, although the similarities between G{alpha}z and other G{alpha}i proteins cannot be ignored, the limited tissue distribution of G{alpha}z (9), its unusual biochemical properties (16), and the identification of G{alpha}z-specific effectors such as Rap1GAP, RGSZ1, and Eya2 support a unique role for G{alpha}z. Our results have led to the first described role(s) for Gz signaling in pancreatic {beta}-cells and provide a greater understanding of the importance that individual G proteins play in {beta}-cell functions.


   FOOTNOTES
 
* This work was supported in part by Grant GM55717 from the National Institutes of Health (to P. J. C.), National Institutes of Health Clinical Scientist Development Award DK62833 (to T. A. F.), and National Institutes of Health training Grants DK07568 and DK067799 (to M. E. K.), and GM19663 (to A. B. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Duke University Medical School Alumni Scholarship. Back

|| To whom correspondence should be addressed. Tel.: 919-613-8613; Fax: 919-613-8642; E-mail: casey006{at}mc.duke.edu.

1 The abbreviations used are: PTX, pertussis toxin; PGE, prostaglandin E; QL, designating mutationally activated form of a G{alpha} subunit; HA, hemagglutinin; RGS, regulator of G protein signaling; GAP, GTPase-activating protein; HBSS, Hepes-buffered saline solution; GSIS, glucose-stimulated insulin secretion; GPCR, G protein-coupled receptor; ERK, extracellular signal-related kinase; GFP, green fluorescent protein; siRNA, small interfering RNA. Back


   ACKNOWLEDGMENTS
 
We thank the members of the Casey laboratory, especially Missy Infante, for general laboratory assistance. We also thank members of the Christopher Newgard laboratory, especially Hans Hohmeier, Danhong Lu, and Thomas Becker, for productive discussions. We also thank Christopher Newgard for critical review of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lingohr, M. K., Buettner, R., and Rhodes, C. J. (2002) Trends Mol. Med. 8, 375-384[CrossRef][Medline] [Order article via Infotrieve]
  2. Sharp, G. W. (1996) Am. J. Physiol. 271, C1781-C1799[Medline] [Order article via Infotrieve]
  3. Katada, T., and Ui, M. (1979) J. Biol. Chem. 254, 469-479[Abstract/Free Full Text]
  4. Moss, J., and Vaughan, M. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 303-379[Medline] [Order article via Infotrieve]
  5. Hillaire-Buys, D., Gross, R., Roye, M., Ribes, G., and Loubatieres-Mariani, M. M. (1992) Eur. J. Pharmacol. 218, 359-362[CrossRef][Medline] [Order article via Infotrieve]
  6. Seaquist, E. R., Neal, A. R., Shoger, K. D., Walseth, T. F., and Robertson, R. P. (1992) Diabetes 41, 1390-1399[Abstract]
  7. Ribalet, B., and Eddlestone, G. T. (1995) J. Physiol. 485, 73-86[Abstract/Free Full Text]
  8. Sjoholm, A. (1996) Biochim. Biophys. Acta 1313, 106-110[Medline] [Order article via Infotrieve]
  9. Fields, T. A., and Casey, P. J. (1997) Biochem. J. 321, 561-571[Medline] [Order article via Infotrieve]
  10. Skoglund, G., Basmaciogullari, A., Rouot, B., Marie, J. C., and Rosselin, G. (1999) J. Endocrinol. 162, 31-37[Abstract]
  11. Fan, X., Brass, L. F., Poncz, M., Spitz, F., Maire, P., and Manning, D. R. (2000) J. Biol. Chem. 275, 32129-32134[Abstract/Free Full Text]
  12. Meng, J., Glick, J. L., Polakis, P., and Casey, P. J. (1999) J. Biol. Chem. 274, 36663-36669[Abstract/Free Full Text]
  13. Glick, J. L., Meigs, T. E., Miron, A., and Casey, P. J. (1998) J. Biol. Chem. 273, 26008-26013[Abstract/Free Full Text]
  14. Embry, A. C., Glick, J. L., Linder, M. E., and Casey, P. J. (2004) Mol. Pharmacol. 66, 1325-1331[Abstract/Free Full Text]
  15. Meng, J., and Casey, P. J. (2002) J. Biol. Chem. 277, 43417-43424[Abstract/Free Full Text]
  16. Casey, P. J., Fong, H. K., Simon, M. I., and Gilman, A. G. (1990) J. Biol. Chem. 265, 2383-2390[Abstract/Free Full Text]
  17. Werner, M., Von Wasielewski, R., and Komminoth, P. (1996) Histochem. Cell Biol. 105, 253-260[CrossRef][Medline] [Order article via Infotrieve]
  18. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  19. Zigman, J. M., Westermark, G. T., LaMendola, J., and Steiner, D. F. (1994) Endocrinology 135, 31-37[Abstract]
  20. Ulrich, A. B., Schmied, B. M., Standop, J., Schneider, M. B., and Pour, P. M. (2002) Pancreas 24, 111-120[CrossRef][Medline] [Order article via Infotrieve]
  21. Hohmeier, H. E., Mulder, H., Chen, G., Henkel-Rieger, R., Prentki, M., and Newgard, C. B. (2000) Diabetes 49, 424-430[Abstract]
  22. Schisler, J. C., Jensen, P. B., Taylor, D. G., Becker, T. C., Knop, F. K., Takekawa, S., German, M., Weir, G. C., Lu, D., Mirmira, R. G., and Newgard, C. B. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7297-7302[Abstract/Free Full Text]
  23. Lu, D., Mulder, H., Zhao, P., Burgess, S. C., Jensen, M. V., Kamzolova, S., Newgard, C. B., and Sherry, A. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2708-2713[Abstract/Free Full Text]
  24. Robertson, R. P., Seaquist, E. R., and Walseth, T. F. (1991) Diabetes 40, 1-6[Abstract]
  25. Masters, S. B., Miller, R. T., Chi, M. H., Chang, F. H., Beiderman, B., Lopez, N. G., and Bourne, H. R. (1989) J. Biol. Chem. 264, 15467-15474[Abstract/Free Full Text]
  26. Yang, J., Wu, J., Kowalska, M. A., Dalvi, A., Prevost, N., O'Brien, P. J., Manning, D., Poncz, M., Lucki, I., Blendy, J. A., and Brass, L. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9984-9989[Abstract/Free Full Text]
  27. Kammermeier, P. J., Davis, M. I., and Ikeda, S. R. (2003) Mol. Pharmacol. 63, 183-191[Abstract/Free Full Text]
  28. Rodriguez-Pena, M. S., Collins, R., Woodard, C., and Spiegel, A. M. (1997) Endocrine 7, 255-260[Medline] [Order article via Infotrieve]
  29. Schmidt, A., Hescheler, J., Offermanns, S., Spicher, K., Hinsch, K. D., Klinz, F. J., Codina, J., Birnbaumer, L., Gausepohl, H., and Frank, R. (1991) J. Biol. Chem. 266, 18025-18033[Abstract/Free Full Text]
  30. Scheltdorf, M., and Mest, H. J. (2002) Naunyn-Schmiedeberg's Arch. Pharmacol. 366, 204-208[CrossRef][Medline] [Order article via Infotrieve]
  31. Kozasa, T., and Ye, R. D. (2004) Methods Mol. Biol. 237, 153-167[Medline] [Order article via Infotrieve]
  32. Wang, J., Ducret, A., Tu, Y., Kozasa, T., Aebersold, R., and Ross, E. M. (1998) J. Biol. Chem. 273, 26014-26025[Abstract/Free Full Text]
  33. Heximer, S. P., Watson, N., Linder, M. E., Blumer, K. J., and Hepler, J. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14389-14393[Abstract/Free Full Text]
  34. Arai, K., Maruyama, Y., Nishida, M., Tanabe, S., Takagahara, S., Kozasa, T., Mori, Y., Nagao, T., and Kurose, H. (2003) Mol. Pharmacol. 63, 478-488[Abstract/Free Full Text]
  35. Meigs, T. E., Juneja, J., Demarco, C. T., Stemmle, L. N., Kaplan, D. D., and Casey, P. J. (2005) J. Biol. Chem. 280, 18049-18055[Abstract/Free Full Text]
  36. Matsuoka, M., Itoh, H., Kozasa, T., and Kaziro, Y. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5384-5388[Abstract/Free Full Text]
  37. Fong, H. K., Yoshimoto, K. K., Eversole-Cire, P., and Simon, M. I. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3066-3070[Abstract/Free Full Text]
  38. Maghazachi, A. A., Al-Aoukaty, A., Naper, C., Torgersen, K. M., and Rolstad, B. (1996) J. Immunol. 157, 5308-5314[Abstract]
  39. Jiang, M., Pandey, S., Tran, V. T., and Fong, H. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3907-3911[Abstract/Free Full Text]
  40. Kabouridis, P. S., Waters, S. T., Escobar, S., Stanners, J., and Tsoukas, C. D. (1995) Mol. Cell Biochem. 144, 45-51[CrossRef][Medline] [Order article via Infotrieve]
  41. Varrault, A., Pena, M. S., Goldsmith, P. K., Mithal, A., Brown, E. M., and Spiegel, A. M. (1995) Endocrinology 136, 4390-4396[Abstract]
  42. Ho, M. K., and Wong, Y. H. (2001) Oncogene 20, 1615-1625[CrossRef][Medline] [Order article via Infotrieve]
  43. Lang, J., Nishimoto, I., Okamoto, T., Regazzi, R., Kiraly, C., Weller, U., and Wollheim, C. B. (1995) EMBO J. 14, 3635-3644[Medline] [Order article via Infotrieve]
  44. Laychock, S. G., and Bilgin, S. (1987) FEBS Lett. 218, 7-10[CrossRef][Medline] [Order article via Infotrieve]
  45. Cheng, H., Straub, S. G., and Sharp, G. W. (2003) Am. J. Physiol. 285, E287-E294
  46. Robertson, R. P. (1983) Annu. Rev. Med. 34, 1-12[CrossRef]
  47. Robertson, R. P. (1998) Diabetes 47, 1379-1383[Abstract/Free Full Text]
  48. Tran, P. O., Gleason, C. E., Poitout, V., and Robertson, R. P. (1999) J. Biol. Chem. 274, 31245-31248[Abstract/Free Full Text]
  49. Negishi, M., Sugimoto, Y., and Ichikawa, A. (1995) Biochim. Biophys. Acta 1259, 109-119[Medline] [Order article via Infotrieve]

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M. E. Kimple, J. W. Joseph, C. L. Bailey, P. T. Fueger, I. A. Hendry, C. B. Newgard, and P. J. Casey
G{alpha}z Negatively Regulates Insulin Secretion and Glucose Clearance
J. Biol. Chem., February 22, 2008; 283(8): 4560 - 4567.
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