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Endocrinology Vol. 140, No. 3 1132-1140
Copyright © 1999 by The Endocrine Society

ARTICLES

Pancreatic Glucagon-Like Peptide-1 Receptor Couples to Multiple G Proteins and Activates Mitogen-Activated Protein Kinase Pathways in Chinese Hamster Ovary Cells1

Chahrzad Montrose-Rafizadeh, Pavel Avdonin, Michael J. Garant, Buel D. Rodgers, Sutapa Kole, Huan Yang, Michael A. Levine, William Schwindinger and Michel Bernier

Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health (C.M.-R., M.J.G., B.D.R., S.K., H.Y., M.B.), Baltimore, Maryland 21224; the Institute of Developmental Biology, Russian Academy of Science (P.A.), Moscow, Russia; and the Department of Medicine, Johns Hopkins University (M.A.L., W.S.), Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Chahrzad Montrose-Rafizadeh, Ph.D., Eli Lilly & Co., Lilly Corporate Center, Drop Code 1543, Indianapolis, Indiana 46285. E-mail: montrose{at}lilly.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs{alpha}, Gq/11{alpha}, and Gi1,2{alpha}, but not Gi3{alpha}. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046–38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046–38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046–38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCAGON-LIKE peptide-1 (GLP-1) is an incretin hormone that is released from epithelial cells of the gastrointestinal mucosa after food intake. GLP-1 produces multiple physiological effects, including enhancement of glucose-mediated insulin secretion in pancreatic ß-cells, increased glucose metabolism in cultured adipocytes stimulated with insulin, inhibition of food intake, and modulation of gastric acid secretion (1, 2, 3, 4, 5, 6). In addition, GLP-1 stimulates the expression of genes encoding insulin, GLUT-1, GLUT-2, hexokinase I, and glucokinase in pancreatic cells (7, 8, 9). The initial step in GLP-1 action requires binding of the hormone to the GLP-1 receptor, which belongs to the superfamily of G protein-coupled receptors (GPCRs) (10). GPCRs interact with and promote the dissociation of heterotrimeric G proteins into G{alpha}-GTP and Gß{gamma} subunits, each of which may then associate with effector molecules (11, 12). Activation of the GLP-1 receptor results in stimulation of adenylyl cyclase, leading to an increase in intracellular cAMP and PKA activation in the pancreas (13, 14, 15, 16). Treatment with an anti-Gs{alpha} antibody inhibits the ability of GLP-1 to increase cAMP levels in parietal cell membranes (6). Moreover, GLP-1 stimulation of COS cells overexpressing the GLP-1 receptor results in increased intracellular Ca2+ via release from intracellular stores, concomitant with phospholipase C activation (17). Similarly, GLP-1 treatment of Chinese hamster ovary (CHO) cells that express both the cloned rat GLP-1 receptor and the human insulin receptor (CHO/GLPR) results in increased cAMP levels and intracellular Ca2+ (18, 19). Taken together, these findings support the idea that the GLP-1 receptor might associate with multiple G proteins. However, no information is available about the identity of G proteins coupled to the GLP-1 receptor.

Mitogen-activated protein (MAP) kinases are cytoplasmic serine/threonine kinases that transmit extracellular signals to the nucleus via phosphorylation of transcription factors and other signaling molecules (20, 21). MAP kinase pathways are activated by a number of extracellular stimuli, including growth factors that interact with cell surface receptor tyrosine kinases (20). A number of receptors that couple to heterotrimeric G proteins have recently been shown to stimulate the activity of two members of the MAP kinase superfamily, which includes the extracellular signal-regulated kinase (ERK) (22, 23, 24) and p38 MAP kinase (25). Whether the two different classes of receptor (GPCRs and receptor tyrosine kinases) require distinct signaling pathways in MAP kinase activation remains unclear.

The objective of this study was 3-fold: 1) to characterize the G{alpha} proteins that interact with the cloned GLP-1 receptor in CHO/GLPR cells, 2) to examine whether ligand-stimulated GLP-1 receptor activates ERK and p38 MAP kinase, and 3) to determine the possible cross-talk between the GLP-1 receptor and the insulin receptor toward MAP kinase activation in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
CHO/GLPR cells were generated as described previously (18, 19). In brief, CHO cells overexpressing the human insulin receptor (CHO/HIR) were cotransfected with plasmids containing the gene for hygromycin resistance and the full-length rat GLP-1 receptor complementary DNA (a gift from Dr. B. Thorens, University of Lausanne, Lausanne, Switzerland) under the control of the simian virus 40 promoter. Cells were selected by culture in the presence of hygromycin (700 µg/ml). As controls, CHO/HIR cells were transfected with the corresponding hygromycin plasmid alone. Cells were grown in Ham’s F-12 medium containing 10% FCS and maintained in a humidified air incubator with 5% CO2.

Binding of GLP-1 and insulin
Cells were seeded on 12-well dishes and grown to confluence. Four hours before the start of the experiment, culture medium was changed to serum-free medium. For insulin binding, cells were washed twice with 0.5 ml ice-cold buffer A [10 mM HEPES (pH 7.6), 120 mM NaCl, 1.2 mM MgSO4, 1 mM EDTA, 15 mM sodium acetate, and 10 mM glucose] and then incubated overnight at 4 C with 0.5 ml buffer A containing 1% BSA plus various concentrations of insulin (0.06–30 nM) and 30,000 cpm [125I]insulin (Amersham, Arlington Heights, IL). The next day, the supernatant was discarded, and cells were washed three times with 0.5 ml ice-cold Dulbecco’s PBS and lysed with 0.5 ml 0.5 N NaOH-0.1% SDS for 10 min at room temperature. For GLP-1 binding, serum-starved cells were washed twice with 0.5 ml binding buffer B [10 mM Tris (pH 7.6), 120 mM NaCl, 1.2 mM MgSO4, 15 mM sodium acetate, and 5 mM KCl] and then incubated with 0.5 ml buffer B containing 2% BSA, 500 U/ml aprotinin, 10 mM glucose, various concentrations of GLP-1 (0.03–30 nM), and 30,000 cpm [125I]GLP-1 (Amersham). After an overnight incubation at 4 C, the supernatant was discarded, and the cells were washed three times and lysed as indicated above. The radioactivity was measured using a ICN Apec Series {gamma}-counter (Costa Mesa, CA).

Membrane preparation
Confluent cells that had been cultured on 150-cm2 dishes were placed in serum-free Ham’s F-12 for 2 h at 37 C. Cells were then washed once with 10 ml ice-cold buffer C [10 mM HEPES (pH 7.5), 0.25 M sucrose, and 5 mM EDTA] and scraped into 5 ml ice-cold buffer C containing 20 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.2 mM phenylmethylsulfonylfluoride (PMSF), and the cell suspension was centrifuged at 600 x g for 10 min at 4 C. The pellets obtained from 10 dishes were homogenized on ice in 10 ml chilled buffer C containing protease inhibitors using a Polytron (Brinkmann Instruments, Inc., Westbury, NY) at setting 7 and twice for 15 sec each time. The homogenate was centrifuged at 12,000 x g for 20 min at 4 C, and the clarified supernatant was centrifuged at 100,000 x g for 1 h at 4 C. The membrane pellet was reconstituted in buffer D [50 mM HEPES (pH 7.5), 0.25 M sucrose, 1 mM MgCl2, 1 mM dithiothreitol (DTT), 20 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.2 mM PMSF], homogenized using a Dounce homogenizer, and stored in aliquots at -80 C.

Preparation of [32P]GTP-azidoanilide
[32P]GTP-azidoanilide was synthesized as previously described (26). After purification, [32P]GTP-azidoanilide was divided into aliquots, dried on a speed vacuum centrifuge, and stored at -20 C until use.

Labeling and immunoprecipitation of membrane-associated G proteins
Aliquots of purified membranes (70–100 µg protein) were centrifuged at 8000 x g for 15 min, after which pellets were resuspended in buffer E [50 mM HEPES (pH 7.5), 5 mM MgCl2, 30 mM KCl, 1 mM benzamidine, and 0.1 mM EDTA] and incubated for 10 min at 25 C with 5–10 µCi [32P]GTP-azidoanilide in the absence or presence of different concentrations of GLP-1. The reaction was stopped by incubation on ice for 1 min, and the samples were centrifuged at 8000 x g for 10 min at 4 C. Pellets were resuspended in buffer E containing 20 mM glutathione and irradiated for 10 min at 4 C using UV light at 254 nm (100-watt lamp at a distance of 10 cm).

The immunoprecipitation of G protein {alpha}-subunits was performed as described previously (27). The photolabeled membranes were precipitated at 8000 x g for 15 min at 4 C, and the pellets were solubilized in 40 µl 2% SDS at room temperature, followed by the addition of 130 µl buffer F [10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5% SDS, 1% deoxycholate, 1% Triton X-100, 0.2 mM PMSF, and 10 µg/ml aprotinin]. The samples were centrifuged at 8000 x g for 10 min at 4 C to remove insoluble material. The clarified supernatants (160 µl) were transferred to tubes containing 5–10 µl polyclonal rabbit antisera raised against Gs, Gq, Gi1,2, and Gi3 (1 mg/ml protein; New England Nuclear Corp., Boston, MA). After an overnight incubation at 4 C with constant rotation, 40 µl reconstituted protein A-Sepharose (10% suspension in buffer F) were added to each sample, and the incubation was continued for another 2 h at 4 C with rotation. Thereafter, the immune complexes were recovered by centrifugation and subjected to a series of washes as followed: two washes of 1 ml each containing 50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.5% SDS, and 600 mM NaCl; one wash of 1 ml containing 100 mM Tris (pH 7.4), 300 mM NaCl, and 10 mM EDTA; and one wash of 1 ml containing 20 mM Tris (pH 7.4) and 1 mM EDTA. The immune pellets were resuspended in Laemmli sample buffer and boiled for 10 min. An aliquot (20 µl) of the samples was subjected to SDS-PAGE under reducing conditions (28). To separate the Gi1,2{alpha} protein isoforms, eluted materials were electrophoresed in gels containing 9% acrylamide and 6 M urea. Gels were dried, and band intensity was quantified by electronic autoradiography using a Packard Instant Imager (Packard Instruments, Meriden, CT). Antibodies against Gs{alpha} and Gq{alpha} are specific and do not cross-react with other G{alpha} proteins (29, 30). Although some weak cross-reactivity has been shown previously (29) between antibodies for Gi1,2{alpha} and Gi3{alpha}, we did not observe any significant cross-reactivity, as evident by the absence of contaminating bands (Figs. 1Go, B and C, and 3). The protein concentration was determined by the method of Bradford (31).



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  		Figure 1. Coupling of the GLP-1 receptor to various G protein {alpha}-subunits. A–C, Membranes from CHO/GLPR cells were preincubated in the absence or presence of the indicated concentrations of GLP-1 for 10 min at 25 C in the presence of 5–10 µCi [32P]GTP-azidoanilide before irradiation. The degree of GTP cross-linking in various G{alpha} subunits (Gs, Gq/11,, Gi1,2, and Gi3) was determined as described in Materials and Methods. D, Membranes from CHO/GLPR cells (GLPR) or vector-transfected CHO/HPH cells (HPH) were preincubated in the presence or absence of 1 µM GLP-1 for 10 min at 25 C in the presence of 5–10 µCi [32P]GTP-azidoanilide before irradiation. After immunoprecipitation with Gi1,2{alpha} antibody, the immune pellets were electrophoresed on a polyacrylamide gel containing 6 M urea to better separate Gi1{alpha} and Gi2{alpha}. The degree of GTP cross-linking in Gi1{alpha} and Gi2{alpha} is shown.

 
Western blot analysis
Cells were lysed directly in Laemmli sample buffer containing 5% 2-mercaptoethanol, and 1 mM orthovanadate, and equal amounts of proteins from each sample were electrophoresed on 4–12% polyacrylamide gels (Novex, San Diego, CA) under reducing conditions and then transferred by electrotransfer onto polyvinylidene difluoride membranes. The membranes were incubated in blocking buffer [20 mM Tris (pH 7.5), 137 mM NaCl, and 0.1% Tween-20 (TBS-T) with 5% (wt/vol) nonfat dry milk] overnight at 4 C. The membranes were then incubated with primary antibody (1:1,000 to 1:10,000 dilution) in TBS-T containing 1% nonfat dry milk for 1 h at room temperature or overnight at 4 C. After a series of washes in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Amersham) for 1 h at room temperature. The immunoblots were visualized using the ECL chemiluminescence detection system (Amersham), and the signals were quantified by laser densitometry (ImageQuant, Molecular Dynamics, Inc., Sunnyvale, CA). The polyclonal anti-MKK3 and phospho-specific anti-MKK3/MKK6 antibodies were obtained from New England Biolabs, Inc. (Beverly, MA). The polyclonal anti-p38 MAP kinase antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and rat anti-ERK1/ERK2 antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Phospho-specific anti-Erk antibody (32) was obtained from Promega Corp. (Madison, WI).

Determination of MAP kinase activity
MAP kinase activity was determined in anti-ERK1/ERK2 immunoprecipitates as described by Kole et al. (33). p38 MAP kinase activity was determined as recently reported (34) with some modifications. Confluent cells grown in 35-mm dishes were treated with various factors, as described in the figure legends, and lysed at 4 C for 15 min in 0.5 ml lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 10 mM ß-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM NaF, 1% Triton X-100, 1 mM orthovanadate, 0.15 mM Pefabloc SC, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 10 nM okadaic acid, 10 nM tautomycin, and 10 nM cypermethrin. Equal amounts of protein from clarified lysates were immunoprecipitated with anti-p38 MAP kinase antibody for 90 min at 4 C, after which protein G/protein A-agarose (Oncogene Research Products, Cambridge, MA) was added for another 90 min. The immune complexes were washed twice in lysis buffer, then twice in lysis buffer containing 0.5 M NaCl, followed by two additional washes with kinase assay buffer [20 mM HEPES (pH 7.4), 1 mM EGTA, 25 mM ß-glycerophosphate, 20 mM MgCl2, and 0.2 mM orthovanadate]. The kinase reaction was performed by resuspending the pellet in 20 µl kinase assay buffer containing 0.1 mg/ml recombinant glutathione-S-transferase (GST)-activating transcription factor-2 (ATF-2) fusion protein (gift from Dr. Roger J. Davis, University of Massachusetts, Worcester, MA), 25 µg/ml cAMP-dependent protein kinase inhibitor peptide (Upstate Biotechnology, Lake Placid, NY), 20 µM [{gamma}-32P]ATP (10 cpm/fmol), and 1 mM DTT for 15 min at 22 C. The reaction was stopped by the addition of Laemmli sample buffer, and the samples were electrophoresed using 4–12% SDS-polyacrylamide gels under reducing conditions. Gels were dried and subjected to autoradiography at -70 C.

Statistical methods
Data are expressed as the mean ± SEM, and differences between groups were analyzed by two-tailed Student’s t test or ANOVA. Significant differences were determined when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stable expression of rat GLP-1 receptor in CHO/GLPR cells
We obtained seven independent CHO cell lines in which [125I]GLP-1 binding was significantly above the basal levels. Of these clones, the cell line with the highest binding activity, designated CHO/GLPR, was used for this study. CHO/GLPR cells specifically bound [125I]GLP-1 in a saturable manner. Scatchard analysis revealed that the dissociation constant (Kd) and the maximal binding were 4.9 ± 1.6 nM (mean ± SEM; n = 4) and 112,000 ± 18,000 GLP-1 receptor sites/cell, respectively. Similar to parental CHO/HIR cells, the Kd and binding capacity for [125I]insulin were 1.2 ± 0.7 nM and 166,000 ± 36,000 receptor sites/cell, respectively, in CHO/GLPR cells.

Functional coupling of GLP-1 receptor with G proteins
The interaction of GLP-1 receptor with specific G proteins was determined by incorporation of a radiolabeled nonhydrolyzable GTP analog into various G{alpha} proteins present in CHO/GLPR cell membranes. [32P]GTP incorporation into Gs{alpha} and Gq/11{alpha} (Fig. 1AGo) as well as Gi1,2{alpha} (Fig. 1BGo) increased after GLP-1 stimulation. In contrast, GLP-1 did not affect GTP incorporation into Gi3{alpha} (Fig. 1CGo). This experiment was repeated several times using various concentrations of GLP-1, and the compilation of the results is presented in Fig. 2Go. GLP-1 caused a concentration-dependent increase in G{alpha} protein labeling, with maximal responses at 10 nM for Gs{alpha} and 100 nM for Gq/11{alpha} and Gi1,2{alpha}. Concentrations of GLP-1 as high as 1 µM failed to stimulate Gi3{alpha} labeling over the levels seen in untreated controls. To assess whether the effect of GLP-1 on Gi1,2{alpha} protein activation was via the transfected GLP-1 receptor and not due to other endogenous receptors expressed in CHO cells, we analyzed the effect of GLP-1 on Gi1,2{alpha} protein coupling in membranes from CHO/HIR cells transfected with empty vector (CHO/HPH cells). As shown in Fig. 1DGo, GLP-1 increased labeling of Gi1,2{alpha} protein only in CHO cells transfected with GLP-1 receptor. In addition, GLP-1 had no effect on intracellular cAMP (18) or intracellular calcium (data not shown) in vector-transfected cells, suggesting that the effects in CHO/GLPR cells are specific for GLP-1 receptor. Our data indicate that GLP-1 receptors are functionally coupled to distinct G{alpha} subunits, which is consistent with our recent study showing that GLP-1 increases cAMP and intracellular Ca2+ levels in CHO/GLPR cells (18, 19). The coupling to Gi1,2{alpha} protein was also significant, but somewhat small, and it could be due to the overexpression of GLP-1 receptor in CHO cells.



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  		Figure 2. Concentration dependency of G protein coupling to the GLP-1 receptor. The counts per min of [32P]GTP-azidoanilide incorporation into G{alpha} subunits was quantified by electronic autoradiography using a Packard Instant Imager. Values represent the fold increase over values in the untreated control group (mean ± SEM; n = 3–6). *, **, and ***, P < 0.05, 0.01, and 0.001, respectively, compared with the untreated group.

 
Additional experiments were performed to determine the relative expression of Gs{alpha}, Gq/11{alpha}, Gi1,2{alpha}, and Gi3{alpha} proteins in total cell extracts from CHO/GLPR cells, parental CHO/HIR cells, and RIN1046–38 cells, an insulinoma cell line containing endogenous pancreatic GLP-1 receptors (35). Figure 3Go shows a representative Western blot analysis of G{alpha} protein levels in these three different cell lines. Our results indicate that the four G protein {alpha}-chains were expressed in all three cell lines. Although RIN1046–38 cells showed levels of Gs{alpha} and Gq/11{alpha} proteins that were identical to those from CHO/GLPR cells, this insulinoma cell line contained higher levels of Gi3{alpha} than Gi2{alpha}. The pattern of G{alpha} protein expression in CHO/GLPR cells was similar to that of CHO/HIR cells, which indicates that overexpression of the GLP-1 receptor did not change levels of G{alpha} protein in CHO/GLPR cells.



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  		Figure 3. Immunodetection of G{alpha} subunits. CHO/GLPR, CHO/HIR, and RIN1046–38 cells were lysed, and equal amounts of total cell proteins were fractionated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The G{alpha} subunits (indicated to the right) were detected by immunoblotting using specific polyclonal antibodies. Molecular mass markers are shown on the left.

 
Activation of p38 MAP kinase
p38 MAP kinase was recently shown to play a key role in glucose-mediated induction of insulin gene expression (36). As p38 MAP kinase can be activated by G protein-coupled receptors (25, 37), we were interested in determining whether ligand-stimulated GLP-1 receptors could stimulate p38 MAP kinase in CHO/GLPR cells. p38 MAP kinase activity was measured in an immunoprecipitation-based kinase assay that uses recombinant GST-ATF-2 fusion protein as the substrate for phosphorylation (37).

A representative experiment is shown in Fig. 4Go, upper panel. The autoradiograms of the dried gels from several experiments were quantified by scanning densitometry, and values are expressed as the fold increase over that in untreated cells (Fig. 4Go, lower panel). Treatment of CHO/GLPR cells with 10 nM GLP-1 for 30 min increased activity 2.5-fold over the basal value (P < 0.01). Similarly, 1-h treatment with arsenite (0.4 mM) led to activation of p38 MAP kinase, in agreement with earlier findings by Liu and collaborators (38). In contrast to the effect of GLP-1, 10 µM forskolin or 1 mM 8-bromo-cAMP had no effect on ATF-2 phosphorylation (1.07 ± 0.08-fold; n = 4). We next measured the degree of stimulation of p38 MAP kinase in RIN1046–38 insulinoma cells and found a significant 2-fold increase in activity produced by GLP-1 (P < 0.05; Fig. 5AGo). Similarly, increasing glucose concentrations in the medium from 5 to 11 mM increased p38 activity by 1.3-fold (n = 2), which is in accord with previously published data (36). These results suggest that activation of GLP-1 receptors generates signals necessary for the stimulation of p38 MAP kinase in GLP-1-responsive cells.



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  		Figure 4. Effect of GLP-1 on p38 MAP kinase activity. CHO/GLPR cells were treated without (-) or with (+) 10 nM GLP-1 for 30 min or with 400 µM arsenite for 1 h. The cells were lysed, and p38 MAP kinase activity was determined in an immunoprecipitation-based kinase assay using recombinant GST-ATF-2 fusion protein as the substrate. A representative autoradiogram is shown (upper panel). The results of densitometric analyses of ATF-2 phosphorylation are represented as fold induction over untreated cells (lower panel). Values are the mean ± SEM (n = 7). **, P < 0.01 compared with untreated cells.

 


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  		Figure 5. GLP-1 responsiveness in RIN 1046–38 insulinoma cells. A, RIN 1046–38 cells were preincubated in 5 mM glucose for 2 h and then treated in the absence (lanes 1–3) or presence (lanes 4–6) of 10 nM GLP-1 for 30 min or in the presence of 400 µM arsenite for 1 h (lane 7), followed by measurement of p38 MAP kinase activity. A representative autoradiogram is shown (upper panel). The autoradiograms were quantitated by scanning densitometry, and the results were expressed as the fold stimulation over the control value (lower panel). Values are the mean ± SEM (n = 5). *, P < 0.05 compared with untreated cells. B, RIN 1046–38 cells were incubated with 10 nM GLP-1 for the times indicated and lysed, and immunoblot analysis was performed on whole cell lysates using antiphosphospecific ERK1/ERK2 antibody or antibody against ERK1/ERK2, phosphospecific MKK3/MKK6, or MKK3 proteins. The blots were developed by enhanced chemiluminescence, and the signals were quantitated by laser densitometry. This experiment was repeated twice with similar results.

 
Next, we studied the effect of ligand-stimulated insulin receptor activation on p38 MAP kinase activity in CHO/GLPR cells. A 15-min incubation with 100 nM insulin resulted in an increase in p38 MAP kinase activity of 1.84 ± 0.3-fold over the basal value (mean ± SEM; n = 7; P < 0.05; Fig. 6AGo). As GLP-1 can also stimulate the p38 MAP kinase pathway, the possibility exists that GLP-1 and insulin may have additive effects. To test this hypothesis, CHO/GLPR cells were pretreated without or with 10 nM GLP-1 for 2 h followed by the addition of 100 nM insulin for 15 min. Under these experimental conditions, GLP-1 increased insulin-stimulated values by 1.96 ± 0.4-fold (P < 0.05; n = 10) over the effect of insulin alone. These results suggest a possible convergence of signaling events between the insulin receptor tyrosine kinase and the G protein-coupled GLP-1 receptor. Treatment of CHO/GLPR cells with GLP-1 for 2 h did not alter the affinity and binding capacity for [125I]insulin (data not shown). Furthermore, neither autophosphorylation nor the tyrosine kinase activity of the insulin receptor was affected by GLP-1 (data not shown).



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  		Figure 6. Effects of bacterial toxins on GLP-1- and/or insulin-mediated increases in p38 MAP kinase activity. A, CHO/GLPR cells were incubated in the absence or presence of 10 nM GLP-1 for 2 h, followed by the addition of 100 nM insulin for 15 min. The cells were lysed, and p38 MAP kinase activity was determined as described in Fig. 4Go. Data represent the mean ± SEM (n = 7–10). *, P < 0.05; ***, P < 0.001. B, CHO/GLPR cells were pretreated without and with 10 µg/ml CTX or 0.5 µg/ml PTX for 20 h before the addition of 10 nM GLP-1 where indicated. Two hours later, insulin (100 nM) was added for 15 min where indicated. Results are the means of two experimental values and represent the fold increase over values in untreated control cells.

 
To characterize the G proteins that transduce the stimulatory action of GLP-1 on p38 MAP kinase activity, the effects of pertussis toxin (PTX) and cholera toxin (CTX) were tested (Fig. 6BGo). Pretreatment of CHO/GLPR cells with 10 µg/ml CTX for 20 h completely blocked the GLP-1-stimulated increase in p38 activity, whereas PTX pretreatment at 0.5 µg/ml had no effect. Similarly, the additive effects of GLP-1 and insulin on p38 activation were not affected by PTX, but were blocked by CTX pretreatment. These effects were not due to uneven p38 protein recovery in the immune complexes (data not shown). Control experiments confirmed that CTX treatment was able to reduce the increase in intracellular cAMP mediated by GLP-1, and that PTX treatment blocked the signal induced by lysophosphatidic acid, a G{alpha}i-coupled receptor ligand (data not shown).

Activation of MKK3/MKK6
MKK3 and MKK6, the upstream regulators of p38 MAP kinase, are dual specificity kinases whose activities depend on serine and threonine phosphorylation (39, 40). As no information is currently available regarding MKK3/MKK6 activation by GLP-1, we performed an immunoblot analysis of their phosphorylation status using a phosphospecific anti-MKK3/MKK6 antibody. GLP-1 incubation increased MKK3/MKK6 phosphorylation in CHO/GLPR cells (Fig. 7AGo, upper panel) and in RIN 1046–38 insulinoma cells (Fig. 5BGo, third panel) by 3.4 ± 0.6 (mean ± SEM; n = 4; P < 0.05) and 3.4 ± 0.1 (n = 3; P < 0.05) fold over the basal value, respectively. Pretreatment of CHO/GLPR cells with CTX had a modest effect on basal MKK3/MKK6 phosphorylation levels, but fully blocked the stimulatory effect of GLP-1. In contrast, PTX treatment did not affect GLP-1-stimulated MKK3/MKK6 activation. Reprobing the blot with anti-MKK3 antibody revealed similar quantities of MKK3 protein loaded on the gel (Fig. 7AGo, lower panel).



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  		Figure 7. Effects of bacterial toxins on GLP-1 and/or insulin-induced dual phosphorylation of MKK3/MKK6. CHO/GLPR cells were pretreated without and with 10 µg/ml CTX or 0.5 µg/ml PTX for 20 h, followed by a 2-h incubation with 10 nM GLP-1 (A). Insulin was then added at 100 nM (B) for 15 min where indicated. Immunoblot analysis was performed on whole cell lysates using antiphosphospecific MKK3/MKK6 antibody (upper panels) or an antibody against MKK3 protein (lower panels). This experiment was repeated twice with comparable results.

 
Phosphorylation of MKK3/MKK6 was increased 3-fold over the basal value by a 15-min incubation with 100 nM insulin in CHO/GLPR cells (Fig. 7BGo, upper panel). The activation of MKK3/MKK6 by insulin was not affected after preincubation with PTX (lanes 5 and 6 vs. lanes 1 and 2) or CTX (lanes 9 and 10 vs. lanes 1 and 2). GLP-1 increased insulin-stimulated values by 1.6-fold over the effect of insulin alone (Fig. 7BGo, lane 4 vs. 2). Determination of the effect of bacterial toxins on the activation of the MKK3/MKK6 pathway mediated by insulin plus GLP-1 indicated that CTX inhibited the enhancing role of GLP-1. By comparison, MKK3/MKK6 phosphorylation levels stimulated by insulin and GLP-1 were not affected by pretreatment with PTX.

Activation of ERK
ERK activity was first measured in an immunoprecipitation-based assay in which the addition of GLP-1 to CHO/GLPR cells led to a 1.7 ± 0.1-fold increase in 32P incorporation in myelin basic protein compared with that in unstimulated cells (mean ± SEM; n = 5; P < 0.01). Similar results were obtained when the levels of the active form of ERK1 and ERK2 were determined by Western immunoblotting with phospho-specific ERK antibody (32). Treatment of CHO/GLPR cells (Fig. 8AGo, upper panel) and RIN 1046–38 insulinoma (Fig. 5BGo, first panel) with 10 nM GLP-1 resulted in 1.5- and 2.7-fold increases in ERK dual phosphorylation, respectively. This enhancing effect of GLP-1 in CHO/GLPR cells was abolished in the presence of CTX, but not in the presence of PTX. Reprobing the blot with anti-ERK antibody revealed similar quantities of ERK protein loaded on the gel (Fig. 8AGo, lower panel). Control experiments showed that similar to the effect on p38 MAP kinase activation, simply raising cAMP levels by incubation of cells with 1 mM 8-bromo-cAMP did not enhance ERK tyrosine phosphorylation (n = 2).



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  		Figure 8. Effects of bacterial toxins on GLP-1 and/or insulin-induced ERK dual phosphorylation. CHO/GLPR cells were pretreated without and with 10 µg/ml CTX or 0.5 µg/ml PTX for 20 h, followed by a 2-h incubation with 10 nM GLP-1 (A). Insulin was then added at 100 nM (B) for 15 min where indicated. Immunoblot analysis was performed on whole cell lysates using antiphosphospecific ERK1/ERK2 antibody (upper panels) or an antibody against ERK1/ERK2 protein (lower panels). This experiment was repeated twice with comparable results.

 
Figure 8BGo (upper panel) shows that ERK phosphorylation was increased 14-fold over the basal level by a 15-min incubation with 100 nM insulin in CHO/GLPR cells. Activation of the ERK pathway by insulin was not affected after preincubation with PTX (lanes 5 and 6 vs. lanes 1 and 2) or CTX (lanes 9 and 10 vs. lanes 1 and 2). GLP-1 increased insulin-stimulated values by 2-fold over insulin alone (Fig. 8BGo, lane 4 vs. 2). Determination of the effect of bacterial toxins on the activation of ERK by insulin plus GLP-1 indicated that CTX inhibited the enhancing role of GLP-1. By comparison, ERK phosphorylation levels stimulated by insulin and GLP-1 were not affected by pretreatment with PTX.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In previous work, it has been reported that the rat GLP-1 receptor possesses the characteristic structure of the GPCR superfamily (10). The established signal transduction linked to GLP-1 receptor is an increase in intracellular cAMP in ß-cells (13). However, GLP-1 is also able to elicit phospholipase C activation (17) and increases in intracellular free calcium levels (17, 19) when the GLP-1 receptor is overexpressed in heterologous systems. A similar observation was made with other members of this glucagon-secretin receptor family, including receptors for glucagon, secretin, PTH, and calcitonin (41). This indicates that GLP-1 action might be mediated either via a direct coupling of the GLP-1 receptor with different G proteins or via the activation of a single G protein that modulates various functions through different subunits (42). In that regard, G protein ß{gamma}-subunits from Gs or Gi have been shown to activate pathways such as phospholipase C (43, 44), phosphatidylinositol 3'-kinase (45, 46), and MAP kinase (22). In this study we showed that the cloned pancreatic GLP-1 receptor is able to couple directly to different G protein {alpha}-subunits, including Gs{alpha}, Gq/11{alpha}, and, to a certain extent, Gi1,2{alpha}, but not Gi3{alpha}, in CHO/GLPR cells. The ability of the GLP-1 receptor to couple with several G proteins might be a function of overexpression; however, it remains to be seen whether such diversity in coupling exists in other cell types expressing GLP-1 receptors endogenously. In pancreatic ß-cells, the rise in intracellular Ca2+ caused by GLP-1 appears to be mediated by either activation of plasma membrane calcium channels (14, 47, 48) or a mechanism involving Ca2+-induced calcium release (49). The fact that Gq/11{alpha} and Gs{alpha} contents were identical in CHO/GLPR cells and RIN1046–38 cells supports the idea that GLP-1 signal transduction is not dictated by G protein availability in different cell types. However, as we did not assess the presence of multiple G protein coupling in pancreatic ß-cells, the possibility still exists that they are present only in CHO cells overexpressing GLP-1 receptor.

It has been reported that p38 MAP kinase might be involved in glucose-induced insulin gene expression in MIN6 insulinoma cells (36). In addition, the p38 MAP kinase has been shown to have an important role in various cellular functions, including the phosphorylation of transcription factors (50), the control of apoptosis (51, 52, 53), and cell cycle-mediated events (54). In mammalian cells, p38 MAP kinase is activated by endotoxins, cytokines, and environmental stresses. Very recently, Yamauchi and collaborators (25) identified novel activators of p38 MAP kinase, and they proposed that GPCRs, such as those for Gq/11{alpha}-coupled m1 muscarinic acetylcholine receptors, Gi{alpha}-coupled m2 muscarinic acetylcholine receptors, and Gs{alpha}-coupled ß-adrenergic receptors, activate p38 MAP kinase via Gß{gamma} subunits. In this study, we show that the cloned GLP-1 receptor triggers the activation of p38 MAP kinase. Neither forskolin nor 8-bromo-cAMP could mimic the enhancing effect of GLP-1 on p38 MAP kinase activity, which suggests that increased cAMP levels are not responsible for triggering this GLP-1-mediated response. Of interest, GLP-1 also induces the phosphorylation/activation of MKK3/MKK6, two direct upstream activators of p38 MAP kinase (37, 39, 40). p38 MAP kinase and MKK3/MKK6 followed a similar activation pattern, consistent with the sequential activation of MKK3/MKK6 and p38 MAP kinase in GLP-1-stimulated cells.

Other studies have found that GLP-1 induces activation and tyrosine phosphorylation of 42- and 44-kDa ERKs in INS-1 cells (23). Our results show that in CHO/GLPR cells, insulin and GLP-1 both increase ERK tyrosine phosphorylation. However, insulin was much more potent than GLP-1. This is in sharp contrast to the relative effects of insulin and GLP-1 on p38 activation, where similar levels of activation were observed. This result argues that the rise in intracellular cAMP levels mediated by GLP-1 might attenuate Raf1 kinase activity (55), thereby inhibiting ERK activity. Alternatively, cAMP might enhance the expression and activity of dual specificity Thr/Tyr phosphatases, a class of enzymes that directly and specifically regulate MAP kinase family members (56) through enhanced tyrosine and threonine dephosphorylation. In support of this view, Kusari and collaborators (57) observed a significant increase in MKP-1 expression with a concomitant decrease in ERK activity by cAMP derivatives.

To elucidate the role of heterotrimeric G proteins in GLP-1-induced MAP kinase cascade activation, we compared the effects of bacterial toxins on the signals mediated by GLP-1. CTX and PTX exerted different inhibitory effects on the GLP-1 signals. Induction of ERK and p38 MAP kinase activities by GLP-1 was abolished totally by CTX, but was not affected by PTX. As a simple rise of intracellular cAMP after incubation with 8-bromo-cAMP was unable to activate ERK or p38 MAP kinase, these observations suggest the possibility that Gß{gamma} associated with Gs protein could be involved in GLP-1-induced ERK and p38 MAP kinase activation.

To analyze further the pathway that links the GLP-1 receptor to the MAP kinase activation, we explored the role of receptor tyrosine kinase activation. It is well established that the insulin receptor tyrosine kinase mediates 42- and 44-kDa ERK activation in a Ras-dependent manner (58). Furthermore, insulin induces a second class of MAP kinases, the JNK/SAPK, in Rat1/HIRc cells (59). However, it is not yet clear whether insulin can enhance p38 MAP kinase activity. On the one hand, insulin was found to have no effect on p38 MAP kinase activity in muscle (60) while causing a marked inhibition in PC12 cells and Rat1 fibroblasts (52). On the other hand, insulin has recently been reported to induce p38 MAP kinase activity in hepatoma cells (61). These controversial observations raise the possibility that insulin’s ability to modulate p38 MAP kinase may be related to the cell type studied. Thus, we assessed the ability of insulin to induce MKK3/MKK6 and p38 MAP kinase activation in CHO/GLPR cells that express human insulin receptors. Insulin elevated both activities severalfold. Further stimulation was observed with the combination of insulin and GLP-1, which might indicate that the GLP-1 receptor uses many of the same intermediates involved in insulin receptor signaling. The potential of bacterial toxins to regulate the insulin signal differed from that of GLP-1; insulin signaling was insensitive to CTX or PTX, whereas GLP-1 action was fully blocked by CTX. These observations suggest that toxin-sensitive G proteins play a minor role in the insulin-mediated increase in MAP kinase activity in CHO cells.

In conclusion, we have directly demonstrated the coupling of activated GLP-1 receptor to multiple G proteins by GTP-azidoanilide photoaffinity labeling. Although coupling to Gi1,2{alpha} or Gq{alpha} may be important for some of the actions of GLP-1, stimulation of the MAP kinase pathways seems to require functional coupling between the activated GLP-1 receptor and the Gs{alpha} subunit. Lastly, the ability of insulin to enhance MKK3/MKK6, p38 MAP kinase, and ERK activities is augmented by GLP-1 pretreatment. Our findings provide new insight into the mechanisms of GLP-1 action and indicate that modulation of GLP-1 receptor function may have important effects on cellular responsiveness to insulin.


   Acknowledgments
 
We thank Drs. Bernard Thorens and Roger Davis for providing the cloned GLP-1 receptor and the recombinant GST-ATF-2, and Mrs. Lisa Adams for expert technical assistance.


   Footnotes
 
1 This work was supported by Russian Foundation for Fundamental Research Grant 96–04-49921 (to P.A.). Back

Received May 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nathan DM, Schreiber E, Fogel H, Mojsov S, Habener JF 1992 Insulinotropic action of glucagon-like peptide-1 (7–37) in diabetic and nondiabetic subjects. Diabetes Care 15:270–276[Abstract]
  2. Montrose-Rafizadeh C, Egan JM, Roth J 1994 Incretin hormones regulate glucose-dependent insulin secretion in RIN 1046–38 cells: mechanisms of action. Endocrinology 135:589–594[Abstract]
  3. Egan JM, Montrose-Rafizadeh C, Wang Y, Bernier M, Roth J 1994 Glucagon-like peptide-1 (7–36) amide (GLP-1) enhances insulin-stimulated glucose metabolism in 3T3–L1 adipocytes: one of several potential extrapancreatic sites of GLP-1 action. Endocrinology 135:2070–2075[Abstract]
  4. Fehmann HC, Goke R, Goke B 1995 Cell and molecular biology of the incretin hormones glucagon-like peptide-1 and glucose-dependent insulin releasing polypeptide. Endocr Rev 16:390–410[CrossRef][Medline]
  5. Tang-Christensen M, Larsen PJ, Goke R, Fink-jensen A, Jessop DS, Moller M, Sheikh SP 1996 Central administration of GLP-1-(7–36) amide inhibits food and water intake in rats. Am J Physiol 271:R848–R856
  6. Schmidtler J, Dehne K, Offermanns S, Rosenthal W, Classen M, Schepp W 1994 Stimulation of rat parietal cell function by histamine and GLP-1-(7–36) amide is mediated by Gs{alpha}. Am J Physiol 266:G775–G782
  7. Fehmann HC, Habener JF 1992 Insulinotropic hormone glucagon-like peptide-I (7–37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma BTC-1 cells. Endocrinology 130:159–166[Abstract]
  8. Wang Y, Egan JM, Raygada M, Nadiv O, Roth J, Montrose-Rafizadeh C 1995 Glucagon-like peptide-1 affects gene transcription and messenger ribonucleic acid stability of components of the insulin secretory system in RIN 1046–38 cells. Endocrinology 136:4910–4917[Abstract]
  9. Wang Y, Perfetti R, Greig NH, Holloway HW, DeOre KA, Montrose-Rafizadeh C, Elahi D, Egan JM 1997 Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats. J Clin Invest 99:2883–2889[Medline]
  10. Thorens B 1992 Expression cloning of the pancreatic B cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci USA 89:8641–8645[Abstract/Free Full Text]
  11. Wess J 1997 G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J 11:346–354[Abstract]
  12. Rebois RV, Warner DR, Basi NS 1997 Does subunit dissociation necessarily accompany the activation of all heterotrimeric G proteins? Cell Signalling 9:141–151[CrossRef][Medline]
  13. Gefel D, Hendrich GK, Mojsov S, Habener JF, Weir GC 1990 Glucagon-like peptide 1 analogs: effects on insulin secretion and adenosine 3',5' cyclic monophosphate formation. Endocrinology 126:2164–2168[Abstract]
  14. Lu M, Wheeler MB, Leng X-H, Boyd III AE 1993 The role of the free cytosolic calcium level in ß-cell signal transduction by gastric inhibitory polypeptide and glucagon-like peptide 1 (7–37). Endocrinology 132:94–100[Abstract]
  15. Ding WG, Renstrom E, Rorsman P, Buschard K, Gromada J 1997 Glucagon-like peptide I and glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat alpha-cells by a protein kinase A-mediated mechanism. Diabetes 46:792–800[Abstract]
  16. Fehmann HC, Strowski M, Goke B 1994 Interaction of glucagon-like peptide-1 (7–37) and somastostatin-14 on signal transduction and proinsulin gene expression in ßTC-1 cells. Metabolism 43:787–792[CrossRef][Medline]
  17. Wheeler MD, Lu M, Dillon JS, Leng X-H, Chen C, Boyd AE 1993 Functional expression of the rat glucagon-like peptide-I receptor: evidence for coupling to both adenylyl cyclase and phospholipace C. Endocrinology 133:57–62[Abstract]
  18. Montrose-Rafizadeh C, Yang H, Rodgers BD, Beday A, Pritchette LA, Eng J 1997 High potency antagonists of the pancreatic glucagon-like peptide-1 receptor. J Biol Chem 272:21201–21206[Abstract/Free Full Text]
  19. Montrose-Rafizadeh C, Yang H, Wang Y, Roth J, Montrose MH, Adams LG 1997 Novel signal transduction and peptide specificity of glucagon-like peptide receptor in 3T3–L1 adipocytes. J Cell Physiol 172:275–283[CrossRef][Medline]
  20. Cobb MH, Goldsmith EJ 1995 How MAP kinases are regulated. J Biol Chem 270:14843–14846[Free Full Text]
  21. Davis RJ 1995 Transcriptional regulation by MAP kinases. Mol Reprod Dev 42:459–467[CrossRef][Medline]
  22. Faure M, Voyno-Yasenetskaya TA, Bourne HR 1994 cAMP and ß{gamma} subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells. J Biol Chem 269:7851–7854[Abstract/Free Full Text]
  23. Frodin M, Sekine N, Roche E, Filloux C, Prentki M, Woolheim CB, Van Obberghen E 1995 Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting ß-cell line, INS-1. J Biol Chem 270:7882–7889[Abstract/Free Full Text]
  24. Kubota A, Yamada Y, Yasuda K, Someya Y, Ihara Y, Kagiomoto S, Watanabe R, Kuroe A, Ishida H, Seino Y 1997 Gastric inhibitory polypeptide activates MAP kinase through the wortmannin-sensitive and -insensitive pathways. Biochem Biophys Res Commun 235:171–175[CrossRef][Medline]
  25. Yamauchi J, Nagao M, Kaziro Y, Itoh H 1997 Activation of p38 mitogen-activated protein kinase by signaling through G protein-coupled receptors. Involvement of Gß{gamma} and G{alpha}q/11 subunits. J Biol Chem 272:27771–27777[Abstract/Free Full Text]
  26. Schwindinger WF, Fredericks J, Watkins L, Robinson H, Bathon JM, Pines M, Suva LJ, Levine MA 1998 Coupling of the PTH/PTHrP receptor to multiple G-proteins: Direct demonstration of receptor activation of Gs, Gq/11 and Gi(1) by [{alpha}-32P]GTP-{gamma}-azidoanilide photoaffinity labeling. Endocrine 8:201–209[CrossRef][Medline]
  27. Allgeier A, Offermanns S, Van Sande J, Spicher K, Schultz G, Dumont JE 1994 The human thyrotropin receptor activates G-proteins Gs and Gq/11. J Biol Chem 269:13733–13735[Abstract/Free Full Text]
  28. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  29. Simons WF, Goldsmith PK, Codina J, Unson CG, Spiegel, AM 1989 Gi2 mediates {alpha}2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G{alpha} C-terminal antibodies. Proc Natl Acad Sci USA 86:7809–7813[Abstract/Free Full Text]
  30. Shenker A, Goldsmith P, Unson CG, Spiegel AM 1991 The G protein coupled to thromboxane A2 receptor in human platelets is a member of the novel Gq family. J Biol Chem 266:9309–9313[Abstract/Free Full Text]
  31. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254
  32. Schaefer EM, Jarvis BW 1997 A comparison of western blot techniques using phosphospecific antibody detection of protein kinase activation. Promega Notes 64:26–30
  33. Kole HK, Garant MJ, Kole S, Bernier M 1996 A peptide-based protein-tyrosine phosphatase inhibitor specifically enhances insulin receptor function in intact cells. J Biol Chem 271:1430–14307[Abstract/Free Full Text]
  34. Heidenreich KA, Kummer JL 1996 Inhibition of p38 mitogen-activated protein kinase by insulin in cultured fetal neurons. J Biol Chem 271:9891–9894[Abstract/Free Full Text]
  35. Montrose-Rafizadeh C, Wang Y, Janczewski AM, Henderson TE, Egan JM 1997 Overexpression of glucagon-like peptide-1 receptor in an insulin-secreting cell line enhances glucose responsiveness. Mol Cell Endocrinol 130:109–117[CrossRef][Medline]
  36. Macfarlane WM, Smith SB, James RF, Clifton AD, Doza YN, Cohen P, Docherty K 1997 The p38/reactivating kinase mitogen-activated protein kinase cascade mediates the activation of the transcription factor insulin upstream factor 1 and insulin gene transcription by high glucose in pancreatic ß-cells. J Biol Chem 272:20936–20944[Abstract/Free Full Text]
  37. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ 1995 Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270:7420–7426[Abstract/Free Full Text]
  38. Liu Y, Guyton KZ, Gorospe M, Xu Q, Lee JC, Holbrook NJ 1996 Differential activation of ERK, JNK/SAPK and p38/CSBP/RK MAP kinase family members during the cellular response to arsenite. Free Radic Biol Med 21:771–781[CrossRef][Medline]
  39. Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B, Davis RJ 1996 MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol 16:1247–1255[Abstract]
  40. Han J, Lee JD, Jiang Y, Li Z, Feng L, Ulevitch RJ 1996 Characterization of the structure and function of a novel MAP kinase kinase (MKK6). J Biol Chem 271:2886–2891[Abstract/Free Full Text]
  41. Gudermann T, Kalkbrenner F, Schultz G 1996 Diversity and selectivity of receptor-G protein interaction. Annu Rev Pharmacol Toxicol 36:429–459[Medline]
  42. Clapham DE, Neer EJ 1993 New roles for G-protein ß{gamma}-dimers in transmembrane signalling. Nature 365:403–406[CrossRef][Medline]
  43. Smrcka AV, Sternweis PC 1993 Regulation of purified subtypes of phosphatidylinositol-specific phospholipase C ß by G protein {alpha} and ß{gamma} subunits. J Biol Chem 268:9667–9674[Abstract/Free Full Text]
  44. Iiri T, Hamma Y, Ohoka Y, Robishaw JD, Katada T, Bourne HR 1995 Potentiation of Gi-mediated phospholipase C activation by retinoic acid in HL-60 cells: possible role of G{gamma}2. J Biol Chem 270:5901–5908[Abstract/Free Full Text]
  45. Stephens L, Smrcka A, Cooke FT, Jackson TR, Sternweis PC, Hawkins PT 1994 A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein ß{gamma} subunits. Cell 77:83–93[CrossRef][Medline]
  46. Thomason PA, James SR, Casey PJ, Downes CP 1994 A G-protein ß{gamma}-subunit-responsive phosphoinositide 3-kinase activity in human platelet cytosol. J Biol Chem 269:16525–16528[Abstract/Free Full Text]
  47. Holz IV GG, Leech CA, Habener JF 1993 Activation of a cAMP-regulated Ca2+ signaling pathway in pancreatic ß-cells by the insulinotropic hormone glucagon-like peptide-1. J Biol Chem 270:17749–17757[Abstract/Free Full Text]
  48. Zawalich WS, Rasmussen H 1990 Control of insulin secretion: a model involving Ca2+, cAMP and diacylglycerol. Mol Cell Endocrinol 70:119–137[CrossRef][Medline]
  49. Gromada J, Dissing S, Bokvist K, Renstrom E, Frokjaer-Jensen J, Wulff BS, Rorsman P 1995 Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting BTC3-cells by enhancement of intracellular calcium mobilization. Diabetes 40:767–774
  50. Han J, Jiang Y, Li Z, Kravchenko VV, Ulevitch RJ 1997 Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 386:296–299[CrossRef][Medline]
  51. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME 1995 Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326–1331[Abstract/Free Full Text]
  52. Kummer JL, Rao PK, Heidenreich KA 1997 Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase. J Biol Chem 272:20490–20494[Abstract/Free Full Text]
  53. Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi M, Matsumoto K, Miyazono K, Gotoh Y 1997 Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275:90–94[Abstract/Free Full Text]
  54. Lavoie JN, L’Allemain G, Brunet A, Muller R, Pouyssegur J 1996 Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem 271:20608–20616[Abstract/Free Full Text]
  55. Burgering BM, Pronk GJ, van Weeren PC, Chardin P, Bos JL 1993 cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J 12:4211–4220[Medline]
  56. Keyse SM 1995 An emerging family of dual specificity MAP kinase phosphatases. Biochim Biophys Acta 1265:152–160[Medline]
  57. Kusari AB, Byon J, Bandyopadhyay D, Kenner KA, Kusari J 1997 Insulin-induced mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) attenuates insulin-stimulated MAP kinase activity: a mechanism for the feedback inhibition of insulin signaling. Mol Endocrinol 11:1532–1543[Abstract/Free Full Text]
  58. Skolnik EY, Batzer A, Li N, Lee CH, Lowenstein E, Mohammadi M, Margolis B, Schlessinger J 1993 The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science 260:1953–1955[Abstract/Free Full Text]
  59. Miller BS, Shankavaram UT, Horney MJ, Gore AC, Kurtz DT, Rozenzweig SA 1996 Activation of cJun NH2-terminal kinase/stress-activated protein kinase by insulin. Biochemistry 35:8769–8775[CrossRef][Medline]
  60. Goodyear LJ, Chang PY, Sherwook DJ, Dufresne SD, Moller DE 1996 Effects of exercise and insulin on mitogen-activated protein kinase signaling pathways in rat skeletal muscle. Am J Physiol 1996 271:E403–E408
  61. Sutherland C, Tebbey PW, Granner DK 1997 Oxidative and chemical stress mimic insulin by selectively inhibiting the expression of phosphoenolpyruvate carboxykinase in hepatoma cells. Diabetes 46:17–22[Abstract]




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