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Endocrinology Vol. 147, No. 11 5294-5302
Copyright © 2006 by The Endocrine Society

Decreased Glucagon Responsiveness by Bile Acids: A Role for Protein Kinase C{alpha} and Glucagon Receptor Phosphorylation

Tadashi Ikegami, Lada Krilov, Jianping Meng, Bhumika Patel, Kelli Chapin-Kennedy and Bernard Bouscarel

Gastroenterology Research Laboratory, Department of Biochemistry and Molecular Biology (T.I., L.K., J.M., B.P., K.C., B.B.) and Department of Medicine (B.B.), The George Washington University, Washington, D.C. 20037

Address all correspondence and requests for reprints to: Bernard Bouscarel, Ph.D., D.Sc., Gastroenterology Research Laboratory, The George Washington University Medical Center, 2300 I Street, Northwest, 523 Ross Hall, Washington, D.C. 20037. E-mail: bbouscarel{at}mfa.gwu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dihydroxy bile acids like chenodeoxycholic acid (CDCA) induce heterologous glucagon receptor desensitization. We previously demonstrated that protein kinase C (PKC) was activated by certain bile acids and mediated the CDCA-induced decrease in glucagon responsiveness. The aim of the present study was to explore the role of PKC in the phosphorylation and desensitization of the glucagon receptor by CDCA. Desensitization was evaluated by measuring adenylyl cyclase activity. Receptor phosphorylation was assayed by metabolic labeling with [{gamma}-32P] ATP. Protein kinase C (PKC) translocation and activation was visualized by fluorescence microscopy. CDCA decreased cAMP production induced by glucagon in a dose-dependent manner without affecting cAMP synthesis through stimulation of either stimulatory GTP-binding protein (Gs) by NaF or adenylyl cyclase by forskolin. The CDCA-induced inhibition of adenylyl cyclase activity was potentiated by the phosphatase inhibitor, okadaic acid. The desensitizing effect of CDCA was bile acid-specific and was significantly reduced in the presence of PKC inhibitors and after PKC down-regulation by phorbol 12-myristate 13-acetate. CDCA increased glucagon receptor phosphorylation more than 3-fold at concentrations as low as 25 µM. Furthermore, CDCA significantly stimulated human recombinant PKC{alpha} autophosphorylation in vitro, as well as PKC{alpha} translocation to the plasma membrane and phosphorylation in vivo at concentrations as low as 25 µM. CDCA also stimulated PKC{delta} translocation to the perinuclear region. Activated PKC{alpha}, PKC{zeta}, and to a lesser extent, PKC{delta}, phosphorylated the glucagon receptor in vitro. This study demonstrates that certain bile acids, such as CDCA, stimulate phosphorylation and heterologous desensitization of the glucagon receptor, involving at least PKC{alpha} activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GLUCAGON RECEPTOR belongs to the B family of the G protein-coupled receptors (GPCRs) and is closely related to glucagon like peptide-1, secretin, vasoactive intestinal peptide, and gastric inhibitory polypeptide receptors (1). Glucagon plays a key role in the regulation of hepatic cellular function, including cell proliferation, protein, and DNA synthesis as well as glucose metabolism (2). In addition, glucagon has been shown to regulate bile acid metabolism (synthesis, uptake, and efflux) (3).

Loss of responsiveness, referred to as desensitization, is an integral part of the adaptive regulatory mechanism to prevent GPCR overstimulation and to ensure precise spatiotemporal regulation of signal transduction (4). Desensitization is subclassified as "homologous" when the receptor-specific agonists are responsible for the decreased responsiveness, and "heterologous" when the decreased receptor responsiveness involves an agonist-independent pathway (5). Desensitization of GPCRs has been observed in various diseases, for a variety of GPCRs, and has been associated with a defect in transmission of the hormonal signal from the receptor to the adenylyl cyclase, including alteration of receptor number and/or affinity and phosphorylation state, as well as the expression level, and/or activity and phosphorylation state of the associated G protein.

Previously, we have reported that dihydroxy bile acids were the most potent bile acids to inhibit stimulated cAMP production not only in hepatocytes (6, 7, 8) but also in cells of nonhepatic origin, such as human dermal fibroblasts (9).

The protein kinase C (PKC) family consists of related serine/threonine protein kinases. Based on structural properties and cofactor requirements, the family members have been subclassified into the conventional (c)PKCs (PKC{alpha}, PKCßI, PKCßII, and PKC{gamma}), the novel (n)PKCs (PKC{delta}, PKC{epsilon}, PKC{theta}, and PKC{eta}) and the atypical (a)PKCs (PKC{zeta}, PKCµ, and PKC{iota}) PKCs. These PKC isoforms are differentially expressed and regulated (10). Although PKC{alpha}, PKC{delta}, and PKC{zeta} are ubiquitous, others, like PKC{theta} and PKC{gamma} are almost exclusively expressed in hematopoietic T cells and brain, and adrenal tissue, respectively (11).

Activation of classic and novel PKC isoforms, such as PKC{alpha}, PKCß, PKC{delta}, and PKC{epsilon}, by various agents, including diacylglycerol (DAG) and phorbol 12-myristate 13-acetate (PMA) results in the redistribution of the PKC enzymes from the cytosol to various cellular domains in a subtype-dependent manner (10, 12). Once activated, PKC is rapidly down-regulated by proteolytic degradation at a PKC isoform-specific rate (13, 14).

Although the aforementioned PKC isoforms have been implicated in the regulation of various cellular functions, including cell proliferation and differentiation, as well as GPCR activity, the mechanism of action of PKCs is not always clear (10, 12). The mechanism of the PKC-mediated attenuation of the ß-adrenergic GPCR signaling response has been particularly well studied and includes direct phosphorylation of the receptor, the G protein, or the adenylyl cyclase (15, 16). Studies from our and other laboratories have suggested that the classic PKCs were involved in reducing the glucagon-mediated cAMP production by uncoupling the glucagon receptor from the stimulatory G protein (7, 17). Although PKC activation has been implicated in the bile acid inhibitory action, the mechanisms of PKC activation, as well as those responsible for the decreased glucagon response remain to be clarified. Therefore, the present study was undertaken to further address the mechanism(s) responsible for the bile acid-induced glucagon receptor desensitization. The involvement of specific PKC isoforms was investigated. The phosphorylation of the glucagon receptor was studied both in vitro, using glucagon receptor protein expressed in bacteria and human recombinant PKCs, as well as in vivo, using glucagon receptor transfected into HEK293 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[{alpha}32P]ATP, Hyperfilm, and ECL detection kit were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Chenodeoxycholic acid (CDCA) was supplied by Dr. Falk Pharma GmbH (Freiburg, Germany), whereas taurocholic acid (TCA) was purchased from Steraloids (Wilton, NH). The pET23b plasmid, BL21(DE3)pLysS bacteria and His-tag purification kit were purchased from Novagen (Madison, WI). Human recombinant PKCs were from Pan Vera LLC (Madison, WI). PKC{alpha}-YFP and PKC{delta}-YFP plasmids were kindly provided by Dr. R. Kubitz (Heinrich-Heine University, Dusseldorf, Germany). The anti-glucagon receptor antibody (ST-18) was a generous gift from Dr. T. Sakmar (Rockefeller University, New York, NY). Affinity-purified polyclonal rabbit anti-PKC{alpha} antibody was from Life Technologies, Inc. (Frederick, MD). Affinity-purified polyclonal rabbit anti-PKCß2, {delta}, {epsilon}, and {zeta} antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-PKC{alpha} (Ser657) antibody was from Upstate (Lake Placid, NY). Rabbit horseradish peroxidase (HRP)-labeled antimouse antibody was from Miles Scientific (Neperville, IL). PMA, dequalinium chloride (DECA), and GF109203X (Gö6850, bisindolylmaleimide I) were purchased from Calbiochem (San Diego, CA). Monoclonal mouse anti-ß-actin antibody was purchased from Sigma (St. Louis, MO). Other chemicals were of the highest purity available.

Cell culture and transfection
Rat glucagon receptor cDNA (1.5 kb)-transfected human embryonic kidney 293 cells (HEK-GR) were established and subcloned as described previously (18, 19). The cells were maintained in DMEM (Mediatech Inc., Herndon, VA) under standard conditions. For fluorescence microscopy experiments, HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions.

Isolation of membrane fraction from HEK293 cells and adenylyl cyclase activity assay
The membranes from the HEK293 clones were prepared as previously described (19). The supernatant was centrifuged at 50,000 rpm for 90 min on a two-phase 10% and 44.5% sucrose gradient. The plasma membrane fraction was detected at the interphase. Plasma membrane proteins (10 µg) were incubated with the bile acid in the presence and absence of 100 nM okadaic acid for 30 min at 30 C in a buffer containing 0.4 mM [{alpha}-32P]ATP (1–4 x 106 cpm) in 50 mM HEPES (pH 8.0), 0.2 mM EGTA, 5 mM MgCl2, 0.1 mM cAMP, 8.5 mM creatine phosphate, and 50 U/ml creatine phosphokinase. Glucagon, NaF, or forskolin was added for an additional 15 min. The reaction was stopped by adding a solution containing 1% SDS, 4 mM ATP, and [3H]cAMP used as internal standard, and the samples were maintained at 95 C for 5 min. Adenylyl cyclase activity was measured as previously described by Solomon et al. (20) using a combination of Dowex and Alumina columns.

Immunoblotting, phosphorylation, and deglycosylation of PKC and/or the glucagon receptor
The total and phosphorylated expression level of the PKC was determined by immunoblotting using specific antibodies. The phosphorylation and deglycosylation of the plasma membrane glucagon receptor was studied in the presence of [{gamma}-32P]ATP (1–5 cpm/fmol) for 30 min and N-glycosidase F (Biolab, Cambridge, MA) for up to 3 h, respectively. Autoradiograms were visualized with a STORM phosphoimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Deglycosylation of the glucagon receptor was confirmed by immunoblotting.

Fluorescence microscopy
HEK293 cells were seeded onto 35-mm glass-bottom dishes (MatTek, Ashland MA) 24 h after transfection with PKC{alpha}-YFP or PKC{delta}-YFP plasmids. The following day, the cells were washed twice and the media was replaced with serum-free and phenol-red-free DMEM. The cells were treated with either 50 µM CDCA or 200 nM PMA. During the course of the experiment, the culture dishes were kept on a heated microscope stage. Images were collected at 30-sec intervals using an Olympus IX-81 microscope (x60 objective). For phospho-PKC{alpha} detection, the cells were seeded onto poly-L-lysine-coated coverslips in 35-mm dishes and starved for 1 h before treatment with PMA or CDCA for 15 min. After treatment, the cells were washed with PBS, fixed in 3.7% paraformaldehyde for 10 min, and incubated in 8% BSA/PBS (blocking buffer) for 1 h. The anti-phospho-PKC{alpha} primary antibody was diluted in blocking buffer and incubated with the fixed cells overnight at 4 C, followed by a 45-min incubation with an antirabbit-Alexa fluor 568 (Molecular Probes) secondary antibody. The cells were then washed and mounted on slides using Mowiol. The coverslips were processed and the data was analyzed as described above.

In vitro phosphorylation of the histidine-tagged glucagon receptor by recombinant PKC isoforms
The rat glucagon receptor cDNA was inserted into a pET23b plasmid and expressed in BL21 (DE3)pLysS according to the manufacturer’s instructions (Novagen). The glucagon receptor was purified using an His-tag purification kit (Novagen) and incubated with the human recombinant PKC ({alpha}, {delta}, and {zeta}) isoforms in HEPES buffer (pH 7.4) containing 0.3% Triton X-100, phosphatidyl-serine (PS), and diolein, sonicated for 30 sec at 4 C, and further incubated in the presence of [{gamma}-32P]ATP (1–5 cpm/fmol) for 10 min at 30 C. The proteins were separated by SDS-PAGE, transferred onto a nitrocellullose membrane, and the intensity of the phosphorylated bands was quantified using ImageQuant software (Molecular Dynamics). The specific activity of the human recombinant PKC isoforms tested was the following: PKC{alpha} 2120 nmol per milligram of protein per minute; PKC{delta} 2300 nmol per milligram of protein per minute and PKC{zeta} 1548 nmol per milligram of protein per minute.

Statisitical analysis
Except as otherwise indicated, results were expressed as mean ± SE. The statistical significance of the mean differences was determined by either one-way ANOVA or Student’s paired t test using Prism software (Graphpad Software Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Study of the regulation of adenylyl cyclase activity by bile acids in HEK cells expressing the glucagon receptor
Previously, we have reported that dihydroxy bile acids were able to attenuate glucagon-induced cAMP production in isolated hepatocytes without affecting the number of glucagon receptors (7). Therefore, using plasma membranes isolated from glucagon receptor stably transfected HEK cells, we studied the site of action of the bile acid. As shown in Fig. 1Go, glucagon, NaF, and forskolin, which stimulate the glucagon receptor, the G protein, and the adenylyl cyclase, respectively, increased cAMP formation. Furthermore, 50 µM CDCA attenuated glucagon-stimulated adenylyl cyclase activity by 40 ± 8% but had no effect on adenylyl cyclase activity stimulated by either NaF or forskolin (Fig. 1Go). These data suggest that the effect of the bile acid is at the level of either the glucagon receptor or the coupling of the receptor and G protein.


Figure 1
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  		FIG. 1. Comparative effect of bile acid on the stimulation of adenylyl cyclase by either glucagon, NaF, or forskolin. Isolated plasma membrane proteins (10 µg) from HEK293 cell 3-8D clone were preincubated with 50 µM CDCA for 30 min in a buffer containing 0.4 mM [{alpha}-32P]ATP (1–4 x 106 cpm) in 50 mM HEPES (pH 8.0), 0.2 mM EGTA, 5 mM MgCl2, 0.1 mM cAMP, 8.5 mM creatine phosphate, and 50 U/ml creatine phosphokinase. The samples were further incubated for 15 min with 100 nM glucagon (Glu), 10 µM NaF, and 10 µM forskolin (FK). The reaction was stopped by maintaining the samples at 95 C for 5 min and the addition of 1% SDS, 4 mM ATP. [3H]cAMP was used as internal control. The activity of adenylyl cyclase (AC) was determined by measuring the formation of cAMP from [{alpha}-32P]ATP as described under Materials and Methods. The results are the Mean + SE from three different experiments and were expressed as the percentage of basal determined in the absence of any agent. *, Significantly different from control; P < 0.05.

 
In the presence of 100 nM of the phosphatase inhibitor, okadaic acid, the inhibitory effect of CDCA was augmented by around 2-fold (Fig. 2AGo). However, under these conditions, okadaic acid alone did not significantly affect the glucagon-induced cAMP production. This augmented effect of the bile acid was not observed when the nonactive phosphatase inhibitor norokadaic acid (100 nM), was tested (Fig. 2AGo). This inhibitory effect was bile acid-specific, because it was not observed with the trihydroxy bile acid TCA even in the presence of the phosphatase inhibitor (Fig. 2AGo). These results are in agreement with our previous findings in both hepatocytes and dermal fibroblasts (7, 9). Moreover, the presence of okadaic acid augmented the inhibitory effect of CDCA (10–100 µM) on glucagon-induced cAMP production with a significance of P < 0.05 at CDCA concentrations equal to or greater than 25 µM (Fig. 2BGo). Based on these results, and unless otherwise indicated, okadaic acid was used in the subsequent experiments.


Figure 2
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  		FIG. 2. Effect of okadaic acid and norokadaic acid on the regulation of glucagon-induced adenylyl cyclase activity by bile acid. A, Plasma membrane proteins (10 µg) were incubated with 50 µM of either CDCA (CD) or TCA (TC), in the presence and absence of 100 nM okadaic acid (OA) or norokadaic acid (NOA) for 30 min at 30 C. The adenylyl cyclase activity was determined 15 min later after the addition of 100 nM glucagon (Glu) as described in Fig. 1Go and Materials and Methods. The results are the mean + SE from three different experiments and were expressed as the percentage of control determined in the presence of glucagon alone. Although not shown, okadaic acid did not affect either the basal or glucagon-induced adenylyl cyclase activity. B, Plasma membrane proteins (10 µg) were incubated with increasing concentrations (1–100 µM) of CDCA and in the presence and absence of 100 nM okadaic acid for 30 min at 30 C. The adenylyl cyclase activity was determined as described above. The results are the mean + SE from three different experiments and were expressed as the percentage of inhibition of cAMP production induced by glucagon alone. *, Significantly different from control; P < 0.05. **, Significantly different from control; P < 0.01. +, Significantly different from CDCA alone; P < 0.05.

 
CDCA and PMA induce PKC{alpha} and PKC{delta} translocation
Having established that CDCA inhibits the glucagon response with a phosphorylation-dependent mechanism, we next explored the possible mediators of this inhibitory effect. Phorbol esters activate specific PKCs and induce their translocation from the cytosol to specific cellular domains. HEK293 cells were transfected with plasmids encoding PKC{alpha}-YFP and PKC{delta}-YFP, respectively. A time-dependent PKC{alpha}-YFP translocation from the cytoplasm to the plasma membrane in response to 200 nM PMA and 50 µM CDCA was observed in live cells by fluorescence microscopy (Fig. 3AGo). PMA-induced translocation was rapid and maximum by 5 min, whereas CDCA-induced translocation was delayed and reached a maximum at about 10–15 min of stimulation. Furthermore, this increased translocation was associated with an increased colocalization of PKC{alpha}-YFP and phospho-PKC{alpha} at the plasma membrane measured 15 min after stimulation (Fig. 3CGo). Similarly, CDCA induced a predominant accumulation of PKC{delta}-YFP in the perinuclear domain, most probably in the Golgi complex with a maximum accumulation 20–25 min after stimulation (Fig. 3BGo). PKC{delta} translocation is observed not only with CDCA but also with PMA, with kinetics similar to what we have observed in human dermal fibroblasts (21). These results suggest that CDCA induces PKC translocation to the plasma membrane with preference for PKC{alpha} over PKC{delta}, thereby potentially facilitating glucagon receptor and PKC{alpha} interaction. To further support PKC{alpha} translocation and activation by CDCA, we measured the cytoplasmic and membrane PKC{alpha} expression and phosphorylation levels by immunoblotting using specific antibodies. The results confirm that 50 µM CDCA stimulates PKC{alpha} accumulation in the plasma membrane by over 2-fold over that of control with a 4-fold increase in PKC{alpha} phosphorylation (Fig 3DGo). Under the same conditions, PMA induced PKC{alpha} membrane translocation and phosphorylation by over 3- and 10-fold respectively (Fig 3DGo).


Figure 3
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  		FIG. 3. Effect of CDCA on PKC{alpha} and PKC{delta} translocation. HEK293 cells transfected with PKC{alpha}-YFP (A) or PKC{delta}-YFP (B) were treated with either 50 µM CDCA or 200 nM PMA, and images were collected every 20 sec over a 30-min period. PMA was used as a positive control. The cells were maintained on a heated microscope stage throughout the experiment. C, The PKC{alpha}-YFP cells were seeded onto poly-L-lysine-coated coverslips in 35-mm dishes and starved for 1 h before treatment with either CDCA or PMA for 15 min. After treatment, the cells were washed with PBS, fixed in 3.7% paraformaldehyde for 10 min, and incubated in 8% BSA/PBS (blocking buffer) for 1 h. The anti-phospho-PKC{alpha} primary antibody was diluted in blocking buffer and incubated with the fixed cells overnight at 4 C, followed by a 45-min incubation with an antirabbit-Alexa fluor 568 secondary antibody. The cells were then washed and mounted on slides using Mowiol. Images are representative of three independent experiments. Images were acquired using a fluorescence microscope (Olympus IX81) and a x60 objective. D, Cells were treated for 10 min with or without 200 nM PMA, 50 µM CDCA, and the membrane (M) and cytosolic (C) fractions were loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose membranes. The immunoblots were probed with antibodies specific for total PKC{alpha}, phospho-PKC{alpha}, or ß-actin. The results were quantified by densitometric scanning using STORM and ImageQuant software, normalized against the respective ß-actin optical density signals and expressed between parentheses as fold increase over the respective control. The arbitrary value for control was set at 1. The blots are representative of three independent experiments.

 
Role of PKC in the attenuation of bile acid-stimulated adenylyl cyclase activity
We have detected the presence of several PKC isoforms, including PKC{alpha}, PKCßII, PKC{delta}, PKC{zeta}, and PKC{epsilon} in the HEK-GR cells. These isoforms are both differently expressed and localized (Fig. 4AGo). Treatment with 1 µM PMA for 24 h resulted in the down-regulation of the PKC{alpha} and PKC{zeta} isoforms by 50 and 35%, respectively (Fig. 4Go, A and B). Under these conditions, down-regulation of PKC{delta} was not significant, whereas PKCßII and PKC{epsilon} were not down-regulated at all.


Figure 4
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  		FIG. 4. PKC protein expression in HEK293 clone 3-8D after exposure of the cells to PMA. A, HEK-GR cells were incubated for 24 h in the absence (PMA–) and presence (PMA+) of 1 µM PMA. Total and membrane cellular proteins (10–20 mg/ml) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with specific polyclonal antibodies against PKC{alpha}, PKCßII, PKC{delta}, PKC{epsilon}, PKC{zeta}, as well as ß-actin and HRP-labeled secondary antibody. The nitrocellulose membrane was exposed to Amersham-Hyperfilm diagnostic film. B, The results representing the membrane fraction were quantified by densitometric scanning using STORM and ImageQuant software and normalized against the respective ß-actin optical density signals and expressed in percent of the respective control. The results are the mean + SE from three different experiments. *, Significantly different from respective control (PMA–); P < 0.05.

 
To investigate the involvement of PKC in glucagon receptor desensitization, HEK-GR cells were preincubated with or without 1 µM PMA for 24 h, followed by treatment with either 50 µM CDCA or 100 nM PMA to attenuate adenylyl cyclase activity (Fig. 5AGo). These experiments were performed in the presence of okadaic acid. The ability of 10 µM forskolin or 100 nM glucagon to stimulate cAMP production was not affected by 24 h PMA treatment. However, both the PMA and CDCA inhibitory effects on adenylyl cyclase activity were completely abolished after PMA treatment for 24 h (Fig. 5AGo). Similarly, the bile acid effect was significantly reduced (P < 0.05) when either DECA or GF109203X, two specific PKC inhibitors, were tested (Fig. 5BGo). GF109203X inhibits predominantly the cPKC and nPKC isoforms whereas DECA is more specific for PKC{alpha} and PKCß. Due to the higher specificity of these inhibitors, and DECA in particular, for the cPKC isoforms, these results underscore the importance of at least PKC{alpha} in mediating the attenuation of cAMP production by CDCA and PMA.


Figure 5
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  		FIG. 5. Effect of PKC down-regulation on the bile acid-induced inhibition of adenylyl cyclase activation. A, The cells were incubated in the absence (PMA–) and presence (PMA+) of 1 µM PMA for 24 h, followed by incubation with 50 µM CDCA or 100 nM PMA for 30 min before the adenylyl cyclase activity was determined 15 min after the addition of 100 nM glucagon (Glu). Forskolin (FK) was used to assess the maximum adenylyl cyclase (AC) activation. The results are the mean + SE from three experiments and are expressed as percentage of control determined in the presence of glucagon alone. B, Plasma membrane proteins were incubated with either 10 µM DECA or 0.05 and 5 µM of GF109203X (GF) for 15 min and further incubated with and without 50 µM CDCA for 15 min before determining the adenylyl cyclase activity after the addition of 100 nM glucagon for 15 min. The adenylyl cyclase activity was assessed as described under legend of Fig. 1Go and Materials and Methods. The results were expressed as either the percentage of control determined in the presence of glucagon alone (A) or the percentage of CDCA inhibition (B). The results are the mean + SE from three different experiments. *, Significantly different from glucagon alone; P < 0.05. +, Significantly different from CDCA alone; P < 0.05.

 
Stimulation of phosphorylation of the glucagon receptor by bile acids and PKC
Previously, it has been reported that the decreased response of the ß-adrenergic receptor (desensitization) was due, at least in part, to receptor phosphorylation (22). Therefore, we studied the potential phosphorylation of the glucagon receptor by CDCA and PMA, as well as glucagon, in vitro using isolated plasma membranes (Fig. 6AGo). In these experiments, the glucagon receptor was deglycosylated by N-glycosidase F before protein separation. The results indicate that 250 µM CDCA and 100 nM PMA stimulated glucagon receptor phosphorylation to a similar extent of over 4-fold of control and to a greater level than that of glucagon (Fig. 6BGo). Furthermore, the increased glucagon receptor phosphorylation by approximately 3-fold was observed at a CDCA concentration as low as 25 µM and was maximal at 250 µM (Fig. 6BGo).


Figure 6
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  		FIG. 6. Effect of glucagon, bile acid, and PMA on the glucagon receptor phosphorylation. A, The cell plasma membranes (40 µg) were incubated without (0 h) and with N-glycosidase F (N-GF) for 1 or 3 h and then separated on a SDS-PAGE and the glycosylated glucagon receptor (G-GR) or its deglycosylated form (GR) were detected by immunoblotting (WB). B, Cell plasma membranes were incubated with either increasing concentrations of CDCA (25–250 µM), 100 nM PMA, or 100 nM glucagon and in the presence of [{gamma}-32P]ATP (0.5 mCi/1 mM) for 30 min at 30 C. After stopping the reaction by washing and centrifuging the membranes, the samples were incubated for 2 h at 37 C with N-glycosidase F. The proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane, exposed to a phosphorscreen, and the phospholabeling (PL) was visualized using STORM and ImageQuant software. Representative experiments of at least three, respectively, are shown. The values expressed between parentheses are fold increase over control. The arbitrary value for control was set at 1.

 
To further investigate whether PKC isoforms phosphorylate the glucagon receptor directly, the His-GR protein was expressed in bacteria and partially purified using a His-tag purification column. When the receptor was incubated in vitro with recombinant PKC{alpha} in the presence of 100 nM PMA and PS, the phosphorylation of the glucagon receptor was increased in glucagon receptor concentration-dependent manner (Fig. 7AGo). No glucagon receptor phosphorylation was observed in the absence of PKC{alpha}. Furthermore, we observed autophosphorylation of the PKC{alpha} induced by PMA. Autophosphorylation of PKC{alpha} was also observed in vitro under the same conditions and upon CDCA stimulation (21).


Figure 7
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  		FIG. 7. Effect of the PKC{alpha} on His-tag glucagon receptor phosphorylation. A, Different concentrations (0.5–2 µg) of His-tagged glucagon receptor (His-GR) were incubated with [{gamma}-32P] ATP, and PS in the absence and presence of recombinant PKC{alpha} and 100 nM PMA for 30 min at 30 C. After stopping the reaction by the addition of an excess amount of cold ATP, the proteins were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, exposed to a phosphorscreen, and visualized (STORM; Molecular Dynamics). B, His-tagged glucagon receptor was incubated with [{gamma}-32P] ATP, PS, and diolein, as well as recombinant PKC{alpha} in the presence of either glucagon (100 nM), PMA (100 nM), or CDCA (100 µM) for 30 min at 30 C. After stopping the reaction by the addition of an excess amount of cold ATP, the proteins were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, exposed to a phosphorscreen, and visualized (STORM, Molecular Dynamics). C, Different concentrations (1–2 µg) of His-tagged glucagon receptor (His-GR) were incubated with [{gamma}-32P] ATP, PS, and diolein in the presence of the recombinant PKC{alpha}, PKC{zeta}, and PKC{delta} isoforms with a respective specific activity of 2120, 1548, and 2300 nmol phosphate per milligram of protein per minute for 30 min at 30 C. The amount of each PKC isoform used was first adjusted to a comparable activity level. After stopping the reaction by the addition of an excess amount of cold ATP, the proteins were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, exposed to a phosphorscreen, and visualized as described above. All the blots were also probed with a specific polyclonal antibody against PKC{alpha} and HRP-labeled secondary antibody. The nitrocellulose membrane was exposed to Amersham-Hyperfilm diagnostic film and analyzed by densitometric scanning using the ImageQuant software (Molecular Dynamics). A representative experiment of at least three. The values expressed between parentheses are fold increase over control. The arbitrary value for control was set at 1.

 
Next, we compared the PKC{alpha}-dependent increased His-GR phosphorylation by either 100 nM glucagon, 100 nM PMA, or 100 µM CDCA (Fig. 7BGo). The presence of PKC{alpha} alone did not result in any GR phosphorylation. However, all three reagents increased glucagon receptor phosphorylation. Glucagon, PMA, and CDCA stimulated glucagon receptor phosphorylation by 14-, 55-, and 10-fold, respectively. Although glucagon was found to increase the translocation and activation of PKC{alpha} in live cells, PMA and CDCA triggered glucagon receptor phosphorylation even in the absence of glucagon (Fig. 7BGo). This is in agreement with previous reports that heterologous GPCR desensitization does not require ligand occupancy.

When different PKC isoforms were tested at concentrations that provide a similar level of activity, we observed that, whereas PKC{alpha} and PKC{zeta} significantly stimulated glucagon receptor phosphorylation, a 5-fold higher concentration of PKC{delta} was necessary to induce phosphorylation of the glucagon receptor. The extent of protein phosphorylation by PKC{zeta} was independent of the glucagon receptor concentration (Fig. 7CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study extends the previous findings by us and others demonstrating that PKC stimulation results in the uncoupling of the glucagon receptor and Gs and leading to a decreased glucagon-induced adenylyl cyclase activity and cAMP production (7, 17). The novel findings of the present study are that the glucagon receptor is phosphorylated when the glucagon receptor-expressing cells or the glucagon receptor proteins are incubated with CDCA. However, the phosphorylation of the receptor by bile acid requires PKC{alpha}, whereas PKC{delta} and PKC{zeta} cannot be completely excluded at the present time.

The glucagon receptor, a 485-amino acid protein, is a member of a large family of seven-helix transmembrane GPCRs. The glucagon receptor is expressed in various tissues, including liver (23). Using antibodies raised against this receptor, as well as glucagon receptor mutants, certain amino acid residues have been shown to be important in the interaction of the receptor with glucagon and with the Gs (18, 24, 25). The carboxyl terminus, as well as the second and third intracellular loop domain of the glucagon receptor (18), play an important role in the coupling of the receptor to the G protein.

Heterologous receptor desensitization, which can occur in the absence of agonist binding, represents both a negative feedback inhibition and a means of cross-talk between receptors. This type of desensitization is mediated by second messenger-activated protein kinases, such as PKC and PKA. Several examples of PKC-mediated desensitization within the glucagon receptor family have been reported. Indeed, PMA promotes heterologous desensitization of glucagon-like peptide-2 (26), vasoactive intestinal peptide (27), pituitary adenylate cyclase-activating polypeptide (28), and PTH receptor (29). In our previous study, we have reported that the PKA-mediated glucagon receptor desensitization was insignificant in HEK293 cells stably expressing the glucagon receptor (19). Furthermore, the inhibitory effect of the bile acid, CDCA, on the glucagon response persisted in the presence of H89, a PKA-specific inhibitor, suggesting that PKA is not involved in the bile acid-induced glucagon receptor desensitization (21). These data support previous findings suggesting that glucagon receptor desensitization was PKA independent (30). We have found in the present study that the inhibitory effect of CDCA and PMA on glucagon response was diminished in the presence of the cPKC inhibitors GF109203X and DECA, or after the down-regulation by PMA, underscoring the importance of at least PKC{alpha} in the hormonal desensitization by bile acids. We propose that long-term incubation with PMA inhibited at least PKC{alpha} activation and its associated role in the reduction of cAMP production by CDCA. It is worthwhile to mention that, after 24-h treatment of the cells with PMA, the glucagon receptor and the stimulatory G protein could already be uncoupled due to glucagon receptor phosphorylation, which could explain to a certain extent the lack of inhibitory effect of CDCA. Although speculative, this would further support the similar effect of CDCA and PMA on lowering the glucagon responsiveness.

In the HEK293 model, we have detected the presence of the cPKCs (PKC{alpha} and PKCß2), which require calcium, DAG, and PS for activation, the nPKC (PKC{delta}, and PKC{epsilon}), which are calcium-independent, and aPKC{zeta}, which depends only on PS for activation. Furthermore, although the PKC{alpha} and PKC{zeta} were mostly cytosolic, PKCß was associated mainly with the plasma membrane. PKC{delta} was evenly distributed into these two fractions. Certain of these PKC isoforms, including PKC{alpha} and PKC{delta}, have been reported to be activated by bile acids through several proposed mechanisms. Those include mimicking the effect of PS, increasing DAG synthesis, stabilizing DAG in the plasma membrane, and increase calcium mobilization or direct activation (for review see Ref. 3). Although still somewhat speculative, the results of the present study support either a direct activation or increased sensitivity of PKC for DAG by the bile acid or even an indirect activation possible through an increased DAG synthesis, because the PKC-induced glucagon receptor phosphorylation did not require any addition of either PS or DAG in the plasma membrane preparation. To study the role of specific PKC isoforms in the inhibitory effect of bile acids on the glucagon response, we attempted to down-regulate mainly the cPKCs and nPKCs by 24-h exposure of the HEK cells to 1 µM PMA. Among the DAG/PMA binding isoforms studied, PKC{alpha} and PKC{delta} were down-regulated, whereas PKC{epsilon} and PKCß2 were not affected. A lesser PKC{epsilon} than PKC{delta} down-regulation by PMA was also observed in human dermal fibroblasts (21). Differential down-regulation of PKC isoforms by PMA (feedback regulation) has been previously observed (31, 32, 33). Interestingly, PKC{zeta}, which does not bind DAG/PMA, was also down-regulated by PMA, providing support for an indirect enzyme regulation (34).

The dose-dependent activation of PKCs, and PKC{alpha} in particular, by CDCA is relevant for the bile acid concentrations reached at least under cholestatic conditions (35). Previous studies from this laboratory have implicated PKC{alpha} and ßII in the modulation of glucagon-induced cAMP synthesis by bile acids (7). This effect was mimicked in hepatocytes isolated from cholestatic hamsters induced by ligation of the common bile duct (8). Furthermore, stimulated cAMP production was inhibited by bile acids and by PMA in dermal fibroblasts (9) and this inhibitory effect was diminished in the presence of the PKC inhibitor, staurosporine, in both fibroblasts and hepatocytes (7, 9). These findings and the results of our present study suggest that the PKC-dependent mechanism of the regulatory effect of bile acids is not cell type specific.

The present study has addressed the mechanism(s) responsible for the bile acid-induced attenuation of cAMP production. Bile acids have been proposed to decrease cAMP production by acting on the following sites: adenylyl cyclase, G protein and the GPCR. PKC activation has been shown to alter the functional status of Gs in rat hepatocytes causing the desensitization of hepatic adenylyl cyclase (36). Furthermore, Zimmermann et al. (37) have found that PKC{alpha} can alter both the activity of adenylyl cyclase isoforms and their responsiveness to G protein regulation. PKC{alpha} inhibits ß-adrenergic receptor-induced cAMP production involving phosphorylation of the receptor (15) or the G protein (38). Therefore, at least through PKC{alpha} activation, dihydroxy bile acids could induce phosphorylation of the glucagon receptor, the Gs, or adenylyl cyclase, resulting in a decreased cAMP production. However, the bile acid did not directly affect the stimulation of both the G protein and the adenylyl cyclase. Furthermore, the bile acid effect, but not that of either glucagon or forskolin on adenylyl cyclase activation was abrogated after prolonged incubation of the cells with PMA. These combined results suggest that the target of CDCA is the glucagon receptor, similar to what was reported by us in isolated hepatocytes (7). The present study demonstrates the ability of CDCA to activate PKC{alpha} and PKC{delta} and stimulate their translocation to the plasma membrane. This supports previous reports showing that: tauro- (T) ursodeoxycholic acid (UDCA) selectively induces PKC{alpha} translocation to the plasma membranes of HEPG2 cells (39); glycol- (G) CDCA induces translocation of PKC{alpha}, PKC{delta}, and PKC{epsilon} to the hepatic membranes (40); DCA induced PKCßI and PKC{alpha} membrane translocation in fibroblasts (41) and in colonic epithelial cells (42). To our knowledge, this is the first study to demonstrate a CDCA-induced translocation of PKC{alpha} and PKC{delta} to the plasma membrane and perinuclear domain, respectively. However, in HEK293 cells, a detectable amount of PKC{alpha} was already translocated to the membrane and phosphorylated under the basal state. Nevertheless, the addition of CDCA further increased the PKC{alpha} membrane association and phosphorylation. Translocation of PKC{alpha} to the plasma membrane could facilitate the interaction of this kinase with the glucagon receptor, and therefore, the phosphorylation of the latter by the former. Although PKC{delta} translocation by CDCA is shown as well, PKC{delta} translocated predominantly to the perinuclear region so it is likely to interact with glucagon receptor located in the plasma membrane. The present study establishes that PKC{alpha} phosphorylates the glucagon receptor in the presence of either PMA or CDCA in vitro. Additionally, the phosphorylation of the glucagon receptor by CDCA in vivo was also predominately cPKC-dependent and was observed even in the absence of glucagon. PKC{zeta} was considered to play a lesser role in the regulation of the glucagon receptor, at least in vitro, based on the fact that the 2-fold increase in glucagon receptor concentrations (see Fig. 7CGo) did not lead to an increased phosphorylation by PKC{zeta}, suggesting that the phosphorylation observed was glucagon receptor concentration independent.

Phosphorylation is a common mechanism of GPCR desensitization, and several putative PKC consensus sites have been proposed in the intracellular region of the glucagon receptor (30, 43). Furthermore, the combined results from the present study that 1) the PKCs involved are PMA-sensitive, 2) that inhibitors of the classic PKC isoforms can block the CDCA-induced GR phosphorylation, and 3) that recombinant PKC{alpha} is more potent than either PKC{delta} or PK{zeta} to phosphorylate GR led us to suggest that PKC{alpha} could be the predominant PKC isoform involved in CDCA-mediated GR phosphorylation. To our knowledge, this is the first direct demonstration of glucagon receptor phosphorylation by PKC{alpha}, both in vivo and in vitro. However, this study does not preclude the possibility that other PKC isoforms beside PKC{alpha} could be involved in glucagon receptor desensitization. Other kinases, including GPCR kinases (GRKs) could also be involved, but their involvement in the glucagon receptor phosphorylation has not been confirmed to date. Furthermore, GRK action requires ligand occupancy and, therefore, is not important in heterologous desensitization. However, in vivo, certain GRKs can be activated by PKC and, therefore, PKC could potentiate homologous glucagon receptor desensitization as well.

Similar to what was undertaken in the present study, several reports have demonstrated that the GPCRs expressed in Escherichia coli, are functional. Those include the serotonin 5HT4(a) receptor, the cannabinoid receptor, and the neurotensin receptor (44, 45, 46). However, one has to be cautious in the data interpretation because it remains possible that appropriate folding of the receptor, as well as proper ligand binding may not have been achieved with the glucagon receptor expressed in E. coli due to either or both lack of co- and posttranslational modifications, such as glycosylation and palmitoylation, as well as absence of the proper plasma membrane molecular environment.

We are aware that other mechanisms including destabilization of the GPCR-G protein complex could be responsible for the reduced GPCR response by bile acids. Jones and Garrison (47) have reported that bile acids can destabilize the interaction of the ß and {gamma} subunits of various G proteins in a dose-dependent manner. However, whereas the authors observed a destabilizing effect with 0.5% (~12 mM) of cholic acid, no effect was observed at concentrations less than 0.1% (~2.5 mM). Thus, with the bile acid concentrations used in our study (1–250 µM), destabilization of the GPCR complex by bile acids remains an unlikely mechanism. Indeed, we have reported that bile acid-induced inhibition of hepatic cAMP production was observed with dihydroxy bile acid concentrations as low as 5 µM for UDCA and 10 µM for CDCA (7). Furthermore, our observed effect was bile acid specific: whereas UDCA and CDCA were the most potent, the taurine conjugate of cholic acid, as well as ursocholic acid were ineffective in lowering cAMP production even at 0.5–1 mM concentrations (7).

In conclusion, this study is the first to suggest that, although dihydroxy bile acids activate different PKC isoforms, at least PKC{alpha} is involved in the bile acid-induced glucagon receptor phosphorylation and desensitization.


   Acknowledgments
 
We thank Lauren Epstein and Julie Zastrow for their skillful technical assistance.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK46954 and DK56108.

First Published Online August 17, 2006

Abbreviations: CDCA, Chenodeoxycholic acid; DAG, diacylglycerol; DECA, dequalinium chloride; GPCR, G protein-coupled receptor; GRK, GPCR kinase; Gs, stimulatory GTP-binding protein; HRP, horseradish peroxidase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidyl-serine; TCA, taurocholic acid; UDCA, ursodeoxycholic acid.

Received April 20, 2006.

Accepted for publication August 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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