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Endocrinology Vol. 141, No. 3 947-952
Copyright © 2000 by The Endocrine Society

ARTICLES

Role of G Protein-Coupled Receptor Kinases in Glucose-Dependent Insulinotropic Polypeptide Receptor Signaling1

Chi-Chuan Tseng and Xiao-Ying Zhang

Section of Gastroenterology, Boston Veterans Administration Medical Center, and Boston University School of Medicine, Boston, Massachusetts 02118

Address all correspondence and requests for reprints to: Chi-Chuan Tseng, M.D., Ph.D., Section of Gastroenterology, Boston University School of Medicine, Boston, Massachusetts 02118.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of class II G protein-coupled receptors. Recent studies have suggested that desensitization of the GIPR might contribute to impaired insulin secretion in type II diabetic patients, but the molecular mechanisms of GIPR signal termination are unknown. Using HEK L293 cells stably transfected with GIPR complementary DNA (L293-GIPR), the mechanisms of GIPR desensitization were investigated. GIP dose dependently increased intracellular cAMP levels in L293-GIPR cells, but this response was abolished (65%) by cotransfection with G protein-coupled receptor kinase 2 (GRK2), but not with GRK5 or GRK6. ß-Arrestin-1 transfection also induced a significantly decrease in GIP-stimulated cAMP production, and this effect was greater with cotransfection of both GRK2 and ß-arrestin-1 than with either alone. In ßTC3 cells, expression of GRK2 or ß-arrestin-1 attenuated GIP-induced insulin release and cAMP production, whereas glucose-stimulated insulin secretion was not affected. GRK2 and ß-arrestin-1 messenger RNAs were identified by Northern blot analysis to be expressed endogenously in ßTC3 and L293 cells. Overexpression of GRK2 enhanced agonist-induced GIPR phosphorylation, but receptor endocytosis was not affected by cotransfection with GRKs or ß-arrestin-1. These results suggest a potential role for GRK2/ß-arrestin-1 system in modulating GIP-mediated insulin secretion in pancreatic islet cells. Furthermore, GRK-mediated receptor phosphorylation is not required for endocytosis of the GIPR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOSE-DEPENDENT insulinotropic polypeptide (GIP) is one of the major mediators in the regulation of nutrient-dependent insulin release from pancreas (1, 2, 3). GIP is released postprandially from the small intestine into the circulation; in addition, specific G protein-coupled receptors for GIP have been demonstrated on pancreatic islet ß-cells (4, 5). Several studies have been performed in the past to define the role of GIP in the pathophysiology of noninsulin-dependent diabetes mellitus (type 2). Despite elevated serum GIP levels, diabetic patients were found to have diminished GIP-mediated insulinotropic effects (6, 7, 8). These results suggest that elevated serum GIP levels in diabetic patients might induce desensitization of the GIP receptor (GIPR) on the pancreatic islet cells and that this mechanism could contribute to impaired insulin secretion (9).

Although the precise mechanisms for the decline in the insulinotropic activity of GIP in diabetic patients are unknown, agonist-induced desensitization of G protein-coupled receptors is a well documented phenomenon. Desensitization may involve one or both of the protein families, the regulators of G protein signaling (RGSs) or G protein-coupled receptor kinases (GRKs). RGS proteins have recently been demonstrated to act as GTPase-activating protein and, upon agonist stimulation, have bound and decreased the half-life of the activated G{alpha}-subunit (10, 11). Our laboratory has shown that RGS2 inhibited GIP-stimulated cAMP production in GIPR complementary DNA (cDNA)-transfected human embryonal kidney cells (L293) as well as GIP-mediated insulin release in ßTC3 cells, suggesting a regulatory role for RGS2 in GIP-induced desensitization (12).

In addition to RGS, some receptors are phosphorylated by GRKs, and this results in uncoupling them from interaction with G protein-coupled receptors (13). This mechanism has been extensively studied in the ß2-adrenergic receptor (ß2AR). Upon agonist stimulation, ß2AR is phosphorylated, which permits ß-arrestin to bind the receptor, and this results in disassociation of G protein from the receptor (14). Whether this mechanism plays a role in GIP-mediated desensitization has not been examined previously. In this report, we have investigated the role of GRKs/ß-arrestin in desensitization of the GIPR on ßTC3 cell and L293 cell stably expressed GIPR cDNA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Human embryonic kidney L293 cells stably expressed GIPR cDNA (L293-GIPR) were cultured in MEM, and ßTC3 cells (gift from S. Efrat) were cultured in DMEM medium containing 1 mM glucose at 37 C in a 95% air-5% CO2 atmosphere. Both media were supplemented with 10% FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin. The experiments were performed in ßTC3 cells during passages 62–70.

Cell transfection
L293-GIPR and ßTC3 cells were transfected with GRK2, GRK5, GRK6, ß-arrestin-1 (cDNAs provided by J. L. Benovic, Thomas Jefferson University, Philadelphia, PA), or pCDNA3 cDNA (as control) using the Lipofectamine method according to the manufacturer’s protocol (Life Technologies, Inc., Gaithersburg, MD). Briefly, cells were seeded in a 12-well plate (105 cells/well) and cultured overnight in the presence of medium with 10% FBS. For transfection, DNA was diluted into serum-free medium, and Lipofectamine (4 µl/well) was added and incubated at room temperature for 15 min to allow DNA-liposome complexes to form. During this 15-min period, cells were rinsed twice with serum-free medium and then incubated with 1 ml DNA-liposome for 5 h. After incubation, 1 ml medium supplemented with 20% FBS was added, and the cells were incubated for an additional 48 h before analysis. In some experiments, cells were cotransfected with pCMV-ßgal plasmid to determine transfection efficiency, and cell lysates were analyzed for the expression of transfected GRK2 or ß-arrestin-1 protein (antisera obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

cAMP assay
GIP-stimulated cAMP production was examined in L293-GIPR and ßTC3 cells. After transfection, cells were first washed with PBS and then incubated with 500 µl medium and 100 µM isobutylmethylxanthine, followed by the appropriate concentrations of GIP. Cells were incubated for 10 min at 37 C and extracted with 500 µl cold absolute ethanol, followed by freeze-thawing. The lysed cells were collected, and the cAMP levels were measured by RIA (cAMP assay kit, Amersham Pharmacia Biotech, Arlington Heights, IL).

Insulin secretion
For the measurement of insulin release, experiments were carried out in Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) containing 129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.0 mM CaCl2, 1.2 mM MgSO4, 10 mM HEPES, and 0.1% BSA. Glucose concentrations are stated in the individual experiments. Two days after transfection, ßTC3 cells were washed twice with freshly made glucose-free KRB buffer and incubated with the same buffer for 10 min. After removal of the incubation buffer, cells were washed with glucose-free KRB buffer again and exposed for 30 min to KRB buffer containing the test reagents (5 mM glucose or/and GIP). Samples of the incubation medium were collected, centrifuged at 4 C to remove cell debris, and stored at -20 C for insulin measurement (rat insulin RIA kit, Linco Research, Inc., St. Charles, MO).

Northern blot hybridization analysis
Total RNA from ßTC3 and L293 cells was extracted using the acid/phenol method of Chomczynski and Sacchi (15). Northern blot hybridization analysis was performed using stringent conditions [42 C, 50% (vol/vol) formamide/5 x sodium saline citrate (SSC); 1 x SSC = 0.15 M NaCl and 0.15 M sodium citrate, pH 7.2]. Ten micrograms of total RNA were denatured in gel-running buffer [0.04 M 3-(N-morpholino)propanesulfonic acid, 10 mM sodium acetate, 0.5 mM EDTA (pH 7.5), 50% formamide, and 6% formaldehyde]. The RNA was then electrophoresed on a 1.5% agarose/6% formaldehyde gel. The integrity of the extracted RNA was determined by the visualization of 28S and 18S ribosomal RNA bands with ethidium bromide staining. After electrophoresis at 10 V/cm, the RNA was transferred from the gel to a Duralon-UV filter by capillary action, as described by the manufacturer (Stratagene, La Jolla, CA). Hybridization was then performed using the RGS2 and ß-arrestin-1 cDNAs that were radiolabeled with [32P]deoxy-CTP, using the Klenow fragment of DNA polymerase I and random oligonucleotides as primers (Promega Corp., Madison, WI). The blots were prehybridized for 2 h at 42 C in 5 x SSC, 10 x Denhardt’s solution, 50% (vol/vol) formamide, 50 mM NaPO4, 1% SDS (BRL, Rockville, MD), and 10 mg/ml herring sperm DNA (Sigma, St. Louis, MO). The filters were then hybridized at 42 C for 16–24 h in 5 x SSC, 1 x Denhardt’s solution, 50% formamide, 20 mM NaPO4, 0.5% SDS, and herring sperm DNA at 20 µg/ml and approximately 107 cpm probe/100-cm2 filters. After hybridization, blots were washed once at room temperature in 1 x SSC and 1% SDS for 15 min, once at room temperature in 0.5 x SSC and 0.5% SDS for 15 min, twice at room temperature in 0.1 x SSC and 0.1% SDS for 15 min, and once at 50 C in 0.1 x SSC and 0.1% SDS for 30 min. Autoradiograms were developed after exposure to Kodak BioMax MS film (Eastman Kodak Co., Rochester, NY) for 12–96 h at -70 C using a Cronex intensifying screen (DuPont, Wilmington, DE). The hybridization signals were quantified by laser densitometry and integration of the autoradiographic images.

GIPR binding and internalization
GIP (5 mg) was iodinated by the chloramine-T method and was purified with C18 cartridges (Sep-Pak, Millipore Corp., Milford, MA) using an acetonitrile gradient of 30–45%, as described previously (12). The specific activity of the radiolabeled peptide was usually about 10–50 mCi/mg. Aliquots were lyophilized and reconstituted in binding buffer at 4 C to a concentration of 106 cpm/100 ml. Binding studies were performed by suspending dissociated L293-GIPR cells transfected with GRKs or ß-arrestin-1 cDNA (3 x 106 cells/ml) in binding buffer consisting of 138 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 2.6 mM CaCl2, 10 mM HEPES, 10 mM glucose, and 1% BSA (fraction V, protease free; Sigma). Binding was performed at room temperature in the presence of 106 cpm/ml [125I]GIP. Nonsaturable binding was determined by the amount of radioactivity associated with the cells when incubated in the presence of 10-6 M GIP. Specific binding was defined as the difference between counts in the absence and the presence of unlabeled peptide. Internalization of GIP-R was measured as the percentage of [125I]GIP resistant to an acid wash (16). Specifically, dissociated cells were incubated with [125I]GIP for 30 min at 37 C in the binding buffer, after which cell samples were added to 10 vol 0.2 M acetic acid (pH 2.5) containing 0.5 M NaCl for 5 min at 4 C or to a similar volume of binding buffer. Results were expressed as the percentage of bound [125I]GIP that was internalized.

Receptor phosphorylation
Whole cell phosphorylation assays was carried out using L293-GIPR cells transiently expressing GRKs. For these studies, transfected cells in monolayer were washed twice with PBS, placed in fresh serum-free medium containing 0.3 mCi/ml [32P]orthophosphate (Amersham Pharmacia Biotech) and incubated for 3 h at 37 C in a 5% CO2 atmosphere. Cells were stimulated with 100 nM GIP or 0.9% NaCl (control) for 15 min. Incubation was terminated by placing plates on ice and washing five times with ice-cold PBS. Membranes were prepared by scraping with a rubber policeman in an ice-cold hypotonic lysis buffer (10 mM Tris, pH 7.4, at 4 C and 5 mM EDTA) supplemented with phosphatase inhibitors (10 mM sodium pyrophosphate and 10 mM NaF) and protease inhibitors (10 mg/ml soybean trypsin inhibitor, 10 mg/ml benzamidine, and 5 mg/ml leupeptin), followed by centrifugation at 40,000 x g. Crude membrane pellets were resuspended in the above lysis buffer, sonicated for 15 s, and repelleted by centrifugation at 40,000 x g. For solubilization, membrane fractions were suspended in PBS with 1% Triton X-100, 0.05% SDS, 1 mM EDTA, 1 mM EGTA, and the phosphatase and protease inhibitors mentioned above and stirred for 2 h at 4 C. Unsolubilized material was separated and discarded by centrifugation at 40,000 x g. GIPRs were purified via immunoprecipitation with antiserum directed against the N-terminal extracellular domain of GIPR (17) (provided by Timothy Kieffer, University of Alberta, Edmonton, Canada). Solubilized material was first incubated with preimmune serum (1:200) and protein A-Sepharose 6MB beads (Sigma) for 1 h, and the beads were removed by centrifugation. The supernatant was then incubated with GIPR antiserum (1:200) and 50 µl protein A-Sepharose 6MB beads overnight. Beads complexed to immunoprecipitated material were washed five times with 1 ml ice-cold solubilized buffer, resuspended in SDS sample buffer, sonicated for 5 min, and centrifuged, after which supernatant containing equivalent amounts of protein was subjected to 10% SDS-PAGE. Gels were dried and exposed using Kodak BioMax MS film for 6 h at -70 C, using a Cronex intensifying screen. Incorporated 32P was analyzed by densitometry scanning using a video imaging device and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

Statistics
Results are expressed as the mean ± SE. The statistical tests were either one- or two-way ANOVA and were performed using SigmaStat statistical software for Windows (Jandel Scientific, San Rafael, CA). P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of GRK/ß-arrestin-1 on GIP-stimulated cAMP production
The transfection efficiency of this in vitro system was determined by cotransfecting with pCMV-ßgal plasmid and staining for ß-galactosidase expression in some experiments. In general, transfection efficiency was similar with different cDNAs, but was higher in L293 cells than in ßTC3 cells. The percentage of cells staining positively for ß-galactosidase after transfection was estimated to be 40% for L293 cells and 20% for ßTC3 cells, and these results were consistent with a moderate increase in GRK2 or ß-arrestin-1 protein in transfected ßTC3 cells (~3- and 2.5-fold increases over endogenous protein level).

In our previous studies we have shown that L293 cells expressing GIPR increased intracellular cAMP levels when stimulated with GIP (12). To examine the effect of GRKs, GIP-induced cAMP production was examined in L293-GIPR cells, and the cAMP levels were measured 10 min after agonist stimulation when the maximal response occurred (12). As illustrated in Fig. 1Go, GIP-stimulated cAMP production in L293-GIPR cells was dose dependent, with a maximal effect seen at 10-7 M GIP (control). Cotransfection of GRK5 or GRK6 cDNA (Fig. 1Go, GRK5 and GRK6) did not affect cAMP levels, whereas GRK2 coexpression suppressed maximal GIP-stimulated cAMP production by about 65% (Fig. 1Go, GRK2). This inhibitory effect was not due to the interference of receptor expression by transfection, as all transfected cells exhibited similar receptor numbers (data not shown).



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  		Figure 1. Inhibition of GIP-stimulated cAMP production by GRK2. L293-GIPR cells were transfected with pCDNA3 (CONTROL), GRK2 (GRK2), GRK5 (GRK5), or GRK6 (GRK6) cDNAs. At 48 h, cells were exposed to the indicated concentrations of GIP for 10 min, when the maximal response occurred. Each data point represents the mean ± SE of at least three separate experiments, with each value determined in duplicate.

 
The classical example of GRK-mediated receptor desensitization was demonstrated by ligand interaction with G protein-coupled receptor and phosphorylation of receptor by GRKs. Phosphorylation of the receptor subsequently induced a conformational change in the receptor that permitted ß-arrestin binding to the receptor and prevented it from activating G proteins (18). To determine whether ß-arrestin-1 is involved in the GIPR desensitization pathway, ß-arrestin-1 cDNA was transfected to L293-GIPR cells. Similar to the results shown in Fig. 1Go, GRK2, but not GRK6, transfection induced a significant decrease in cAMP levels (Fig. 2Go). Overexpression of ß-arrestin-1 in L293-GIPR cells resulted in a small, but statistically significant, decrease (28 ± 10%) in GIP-stimulated cAMP production (Fig. 2Go, BARR1). This inhibition was abolished when a dominant negative mutant of ß-arrestin-1 (BARR1-V53D) was expressed. Cotransfection of ß-arrestin-1 with GRK2 further inhibited GIP-induced cAMP generation (75 ± 12% decrease).



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  		Figure 2. Effect of GRKs/ß-arrestin-1 transfection on maximal cAMP production mediated by GIP. L293-GIPR cells were transfected with pCDNA3 (CONTROL), GRK2 (GRK2), GRK6 (GRK6), ß-arrestin-1 (BARR1), dominant negative ß-arrestin mutant V53D (BARR1-V53D), or a combination of GRK2 and ß-arrestin-1 (BARR1+GRK2) cDNAs. At 48 h, cell were exposed to 10-7 M GIP for 10 min. Each data point represents the mean ± SE of four separate experiments, with each value determined in duplicate. *, P < 0.05; **, P < 0.01 (compared with control).

 
Attenuation of GIP-stimulated cAMP generation and insulin release in ßTC3 cells
Previous reports from this laboratory demonstrated that preincubation of ßTC3 cells with GIP significantly decreased insulin release in response to subsequent GIP stimulation (12). To examine the effect of overexpression of GRKs on insulin release, ßTC3 cells were transfected with GRK2, ß-arrestin-1, or pCDNA-3 (as control) cDNA. Transfected cells were grown in the medium containing 1 mM glucose for 48 h. After washing with KRB buffer, cells were incubated with one of the three buffers (KRB without glucose, KRB with 5 mmol/liter glucose, or KRB with 5 mmol/liter glucose and 10-7 M GIP) for 30 min. The concentration of GIP was chosen for its maximal insulin stimulatory effect reported from this laboratory previously (12). Media were collected for insulin measurement, cells were scraped, and intracellular cAMP levels were determined as described above. In pCDNA-3-transfected control cells, the presence of glucose induced a 2.05 ± 0.15-fold increase in insulin release (Fig. 3Go, control, middle bar). The addition of GIP further stimulated insulin release and resulted in a 2.90 ± 0.15-fold increase over basal insulin secretion (Fig. 3Go, control, black bar). This incretin effect of GIP was significantly reduced in GRK2- or ß-arrestin-1-transfected cells, whereas the glucose-stimulated insulin secretion was not affected by GRK2 transfection (Fig. 3Go, GRK2 and BARR1). Although overexpression of ß-arrestin-1 appeared to induce a small decrease in glucose-stimulated insulin release (Fig. 3Go, BARR1), the difference was not statistically significant (P = 0.13, compared with control). Furthermore, the inhibitory effect of GRK2 and ß-arrestin-1 on insulin release was consistent with the down-regulation of cAMP generation observed in these cells. In the presence of glucose, GIP stimulation resulted in an increase in cAMP over the control level (1.84 ± 0.35 vs. 0.35 ± 0.10 pmol/105 cells; P < 0.05), and these increases were significantly reduced in GRK2- or ß-arrestin-1-transfected cells with cAMP levels of 0.84 ± 0.22 and 1.05 ± 0.30 pmol/105 cells, respectively.



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  		Figure 3. Attenuation of GIP-stimulated insulin release by GRK2 and ß-arrestin-1 in ßTC3 cells. ßTC3 cells were transfected with pCDNA3 (CONTROL), GRK2 (GRK2), or ß-arrestin-1 (BARR1) cDNA. Insulin release was determined in cells exposed to KRB buffer without glucose (basal; open bar), KRB buffer with 5 mM glucose (gray bar), or KRB buffer with 5 mM glucose and 10-7 M GIP (black bar) for 30 min. The results are expressed as the fold increase over the basal state and as the mean ± SE of four separate experiments. *, P < 0.05; **, P < 0.01 (compared with basal).

 
Expression of GRK2 and ß-arrestin-1 in ßTC3 and L293 cells
RNAs from ßTC3 and L293 cells were examined for the presence of GRK2 (Fig. 4AGo) or ß-arrestin-1 (Fig. 4BGo) messenger RNA (mRNA) by Northern blot analysis, with a randomly labeled fragment of GRK2 or ß-arrestin-1 cDNA as a probe. As shown in Fig. 4Go, 3Go.5-kb GRK2 and 3.0-kb ß-arrestin-1 mRNAs were detected in both ßTC3 and L293 cells. By Western blot analysis, both GRK2 and ß-arrestin-1 proteins were also expressed in ßTC3 and L293 cells (data not shown).



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  		Figure 4. RNA blot-hybridization analysis of GRK2 and ß-arrestin-1 mRNAs from L293 and ßTC3 cells. Total cellular RNA (10 µg) was loaded, electrophoresed, transferred to a Duralon-UV filter, and hybridized to the 32P-labeled GRK2 or ß-arrestin-1 DNA. The size standard (in kilobases) is indicated at the left.

 
GIPR internalization and the GRK/ß-arrestin paradigm
To determine whether GRK/ß arrestin paradigm influenced GIPR internalization, we evaluated agonist-induced receptor internalization in L293-GIPR cells overexpressing GRKs or ß-arrestin-1. Internalization of GIP-R was measured as the percentage of [125I]GIP resistant to an acid wash on L293-GIPR cells, and data were presented at 30 min when maximal endocytosis of the GIPR occurred (time-course data not shown). As demonstrated in Fig. 5Go, upon agonist stimulation, 46 ± 8% of surface GIPR was internalized after 30 min (control). The extent of receptor internalization was not affected by coexpression of GRKs, ß-arrestin-1, or ß-arrestin-1-V53D. To exclude the possibility that the current observation is a cell-specific event, similar studies were performed in COS-7-transfected cells, and the results remained unchanged (data not shown). Moreover, the rate of receptor internalization was unaffected by GRKs/ß-arrestin-1 transfection when experiments were performed at 15 or 45 min after agonist exposure (data not shown).



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  		Figure 5. Effect of GRKs/ß-arrestin-1 on internalization of GIPRs on L293 cells. L293-GIPR cells were transfected with pCDNA3 (CONTROL) and GRKs/ß-arrestin-1 cDNA as indicated on the abscissa. Transfected L293-GIPR cells were incubated with [125I]GIP, and internalization was measured after 30 min. Internalization of GIPR was assessed as nonacid-extractable radioligand uptake and was expressed as a percentage of total binding. Each data point represents the mean ± SE of three separate experiments, with each value determined in duplicate.

 
GRK2 phosphorylates GIPRs
To examine whether GRK could phosphorylate the GIPR, experiments were performed by measuring basal and agonist-stimulated receptor phosphorylation in L293-GIPR cells transfected with vector (pCDNA3) or GRK2. As shown in Fig. 6Go, when expressed in L293 cells, GRK2 is capable of enhancing phosphorylation of the GIP-activated GIPR. In contrast, GRK2 has little effect on phosphorylation of the GIPR in the basal state. Densitometric analysis of GIP-induced phosphorylation signals revealed a 2.10 ± 0.35-fold increase in GRK2- over vector-transfected cells. These findings are consistent with the known property of the GRKs that they prefer the agonist-occupied receptor as a substrate (14).



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  		Figure 6. Effect of GRK2 transfection on agonist-induced phosphorylation of the GIPR in L293 cells. L293-GIPR cells were transiently transfected with vector (pCDNA3) or GRK2 as indicated. Transfected cells were metabolically labeled with 32P for 3 h and then incubated with or without 100 nM GIP for 15 min. Lysates were then prepared, and equal amounts of GIPR were immunoprecipitated as described in Materials and Methods. Autoradiograms of the dried gels were obtained using Kodak BioMax MS film. Only the relevant portions of the autoradiograms of a representative experiment are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Receptor desensitization is defined as the refractory state of receptors to subsequent agonist stimulation in the face of continuous agonist exposure. It is a phenomenon commonly to many G protein-coupled receptors and has been shown to be mediated by a number of separate mechanisms, including receptor internalization, down-regulation, or G protein uncoupling. The mechanism of receptor desensitization is well studied in the ß2AR (13, 14, 18). Upon agonist stimulation, the intracellular portion of the ß2AR was phosphorylated by GRKs, which allowed ß-arrestins to bind to the receptor, thereby preventing the receptor from activating G proteins (13, 14, 18). Similar mechanisms were described in other G protein-coupled receptors, including muscarinic M1, M3, and M5 receptors (19), as well as the receptors for substance P (20), endothelin A and B (21), thrombin (22), and GnRH (23).

GIPR belongs to the family of class II G protein-coupled, seven-transmembrane receptors (24, 25). This group consists of receptors for glucagon, secretin, calcitonin, PTH, vasoactive intestinal polypeptide, and pituitary adenylyl cyclase-activating peptide. These receptors lack many of the structure signature sequences present in the ß-adrenergic receptor family (class I) of G protein-coupled receptors (24). As described above, GRKs appear to play an important role in desensitization of the ß-adrenergic receptor; their function in GIPR signaling is unknown. In the current report, overexpression of GRK2 or ß-arrestin-1 significantly repressed GIP-stimulated cAMP production in a heterologous transfected cell system, indicating that the GRK/ß-arrestin paradigm regulates GIPR signaling. The presence of GRK2 and ß-arrestin-1 mRNAs in the L293 and ßTC3 cells further support the involvement of the GRK/ß-arrestin system in desensitization of the GIPR. Furthermore, GIPR endocytosis is unaffected by overexpression of GRKs or ß-arrestin-1 DNA, indicating that receptor internalization plays a minor role in the desensitization process.

As stated above, GIP is one of the incretins modulating nutrient-dependent insulin release from the pancreas. To further examine the role of GRKs in the regulation of GIPR function, similar studies were performed in ßTC3 cells. The ßTC3 cells originally arose from a lineage of transgenic mice expressing an insulin-promoted simian virus 40 T antigen hybrid oncogene in pancreatic ß-cells, were shown to possess GIPR, and released insulin in response to glucose and/or GIP stimulation (12). In the current study, overexpression of GRK2 or ß-arrestin-1 in ßTC3 cells significantly reduced the insulin stimulatory effect of GIP, but not that of glucose. These results support the findings from the heterologous transfected cell system indicating the role of GRKs in regulating desensitization of the GIPR. Furthermore, glucose-stimulated insulin release was not affected by GRK, suggesting that the involvement of GRK/ß-arrestin system was specific to the signaling pathway of the GIPR.

Although GRK1 is known to express primarily in the retina and the pineal (26, 27), GRK2 is distributed ubiquitously. GRK2 is known to be involved in phosphorylation of numerous receptors, including angiotensin AT1 (28), GnRH (23), and follitropin (29) receptors. Moreover, the arrestin family is comprised of at least four proteins, two of which are expressed in the retina (30); the others, including ß-arrestin-1 and -2, are widely distributed in mammalian tissues (18, 19). Recent studies have suggested that there is little substrate specificity for ß-arrestin-1 and -2 among various seven-transmembrane receptors (31). In addition, arrestins are found to bind preferentially to phosphorylated receptors (31). The latter may account for the moderate effect of ß-arrestin-1 transfection on GIP-stimulated cAMP production observed in the current report. Moreover, coexpression of GRK2 and ß-arrestin-1 produced a greater inhibition on cAMP production than either alone, also supporting this idea. In our study, the presence of GRK2 and ß-arrestin-1 mRNA messages in the islet ß-cell line and their effects on GIP-stimulated insulin release suggest a potential role of GRK/ß-arrestin in the GIP-mediated desensitization of the pancreatic islet ß-cells. Whether other GRKs or arrestins are involved in the same process warrants further investigation.

Previous studies in the ß2AR demonstrated that internalization of the ß2AR was due in part to trafficking of the receptor via the clathrin-coated pit pathway, which is mediated by receptor phosphorylation (32). Our findings that overexpression of GRK2 enhances agonist-induced GIPR phosphorylation but does not affect GIPR internalization suggest that agonist-stimulated GIPR internalization occurs by a mechanism that is not dependent on receptor phosphorylation. Similar findings were observed in the PTH and GnRH receptors (33, 34). In those receptors, agonist stimulation results in receptor phosphorylation by GRKs, but this phosphorylation is not required for receptor endocytosis. Hence, the mechanism underlying GIPR internalization may be distinct from that proposed for the ß2AR as well as other class I G protein-coupled, seven-transmembrane receptors. Alternately, receptor phosphorylation and internalization might be mediated by different GRKs and/or at different domains of the receptor, as observed on the follitropin receptor (35). Whether this phenomenon occurs in GIPR requires further examination.

The precise association between GIPR desensitization and abnormal insulin secretion in type 2 diabetic patients is not clear. Our laboratory has previously shown that elevated serum GIP levels reduced GIP-stimulated insulin release in the rat (9). Moreover, hyperglycemia has been shown to be a stimulatory factor on GIP secretion (36, 37). It is likely that increased levels of GIP in the serum of diabetic patients induces chronic desensitization of the GIPRs on the islet ß-cells, and that this mechanism could contribute to abnormal insulin secretion. Previously, we demonstrated an essential role of RGS2 in the signaling pathway of the GIPR (12). In the present report we show that GRK2 also plays a significant role in mediating desensitization of the GIP function. These hypotheses, which are based on in vitro observations, may not represent in vivo mechanisms. More studies are needed to establish the role of the GRK/ß-arrestin system in GIP-induced desensitization.


   Footnotes
 
1 This work was supported by USPHS Grant DK-52186 (to C.-C.T.). Back

Received September 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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