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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.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
-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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
, 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. 1, GRK5 and GRK6) did not affect cAMP levels, whereas
GRK2 coexpression suppressed maximal GIP-stimulated cAMP production by
about 65% (Fig. 1, 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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, GRK2, but not GRK6, transfection induced a significant decrease in cAMP
levels (Fig. 2). 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. 2, 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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, 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. 3, 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. 3, GRK2 and BARR1). Although overexpression of
ß-arrestin-1 appeared to induce a small decrease in
glucose-stimulated insulin release (Fig. 3, 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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) or ß-arrestin-1 (Fig. 4B) 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. 4, 3.5-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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, 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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, 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Footnotes |
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Received September 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits. Cell 86:445–452[CrossRef][Medline]
subunits. Nature 383:172–175[CrossRef][Medline]
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