From the Howard Hughes Medical Institute, Departments of Cell Biology and Medicine, Divisions of Gastroenterology and Cardiology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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The secretin receptor is a member of a structurally distinct class of G protein-coupled receptors designated as Class II. The molecular mechanisms of secretin receptor signal termination are unknown. Using transiently transfected HEK 293 cells expressing the secretin receptor, we investigated its mechanisms of desensitization. Binding of [125I]-secretin to plasma membranes of receptor-expressing cells was specific, with a Kd of 2 nM. Secretin evoked an increase in cellular cAMP with an EC50 of 0.4 nM. The response was maximal by 20 min and desensitized rapidly and completely. Immunoprecipitation of a functional, N-terminal epitope-tagged secretin receptor was used to demonstrate agonist-dependent receptor phosphorylation, with an EC50 of 14 nM. Pretreatment with protein kinase A or C inhibitors failed to alter secretin-stimulated cAMP accumulation. G protein-coupled receptor kinases (GRKs) are known to be involved in the desensitization of Class I G protein-coupled receptors; therefore, the effect of cotransfection of GRKs on secretin-stimulated cAMP signaling and phosphorylation was evaluated. GRKs 2 and 5 were the most potent at augmenting desensitization, causing a 40% reduction in the maximal cAMP response to secretin. GRK 5 also caused a shift in the EC50 to the right (p < 0.05). GRK 4 and GRK 6 did not alter dose-dependent signaling, and GRK 3 was intermediate in effect. Receptor phosphorylation correlated with desensitization for each GRK studied, whereas second messenger-dependent kinase phosphorylation appeared to be less important in secretin receptor signal termination.
We demonstrate agonist-dependent secretin receptor phosphorylation coincident with profound receptor desensitization of the signaling function in HEK 293 cells, suggesting a role for receptor phosphorylation in this paradigm. Although GRK activity appears important in secretin receptor desensitization in HEK 293 cells, protein kinases A and C appear to play only a minor role. These results demonstrate that the GRK-arrestin system regulates Class II G protein-coupled receptors.
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INTRODUCTION |
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The gastrointestinal hormone secretin stimulates pancreatic water
and bicarbonate secretion, leading to neutralization of acidic chyme in
the intestine. Secretin also plays a role in gastric acid release and
intestinal motility. Secretin exerts these effects by binding to
specific heptahelical membrane receptors and activating the
heterotrimeric G protein, Gs, leading to elevation of
cellular cAMP levels. Receptor sequence analysis has divided G
protein-coupled receptors
(GPCRs)1 into subfamilies: I)
rhodopsin/-adrenergic, II) secretin/glucagon, and III) the
metabotropic glutamate receptor families (1, 2). The secretin/glucagon
receptor subfamily comprises a structurally distinct class of
receptors, designated Class II. This group consists of receptors for
secretin, glucagon, calcitonin, parathyroid hormone, pituitary adenylyl
cyclase-activating peptide, vasoactive intestinal polypeptide, and
others. These receptors lack many of the structural signature sequences
present in the prototypic rhodopsin/
-adrenergic receptor family of
GPCR (Class I) (1).
GPCR signaling is dynamically regulated. The rapid process by which
GPCR-mediated signals are attenuated is termed desensitization. Although the mechanisms of desensitization have been well characterized for the -adrenergic receptor family, the mechanisms regulating Class
II GPCR signal transduction are largely unknown. Typically, signal
termination occurs via two distinct pathways (2). Mechanisms that
modulate only stimulated GPCRs are termed homologous desensitization. Receptor phosphorylation by G protein-coupled receptor kinases (GRKs)
is believed to be a major component of this rapid diminished responsiveness for Class I receptors (3, 4). Other modes of
signal attenuation that involve second messengerdependent
protein kinases acting on both active and unstimulated receptors are
termed heterologous desensitization (2, 5). Both protein kinase A and
protein kinase C have been shown to play a role in this mode of
desensitization (2). With the cloning of the secretin receptor (6), it
is now possible to investigate the molecular basis of its
desensitization.
Previous work on the secretin receptor has been hampered by lack of appropriate biochemical tools to demonstrate specific receptor protein phosphorylation. Recently, Ozcelebi et al. (7) demonstrated a secretin-stimulated phosphorylated protein that migrated at 57,000-62,000 on SDS-polyacrylamide gel electrophoresis, consistent with the predicted molecular weight of the secretin receptor. This band was not present when a C-terminal-truncated mutant secretin receptor was stimulated with agonist. In contrast, Holtmann et al. (8) studied a mutant secretin receptor with the C-terminal putative phosphorylation sites removed and noted that desensitization was still present. This persistent desensitization suggested that phosphorylation at these sites might not be important in the signal termination of the secretin receptor.
Many questions remain concerning the regulation of the secretin receptor. Is this receptor a substrate for GRK phosphorylation? Are specific GRK-dependent processes involved? Does receptor phosphorylation correlate with desensitization? What is the role of second messenger-dependent phosphorylation? In this paper we demonstrate agonist-dependent secretin receptor phosphorylation by immunoprecipitation of an N-terminal FLAG-tagged secretin receptor, coincident with profound functional receptor desensitization, suggesting a role for receptor phosphorylation in desensitization. Signaling, as determined by cAMP accumulation in human embryonic kidney cells (HEK 293 cells), appears to be unaffected by second messenger-dependent kinases, whereas agonist-activated G protein-coupled receptor kinases play a significant role. GRK-specific phosphorylation of the secretin receptor is shown to correlate with signal attenuation. Understanding the molecular basis for secretin receptor regulation may provide information relevant to an entire class of structurally distinct receptors and should aid in our understanding of the processes they regulate.
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EXPERIMENTAL PROCEDURES |
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Materials--
Basic chemicals and reagents were from Sigma.
Peptides (secretin, glucagon, vasoactive intestinal polypeptide) were
obtained from Peninsula Labs. HEK 293 cells were obtained from the
American Tissue Culture Collection. Tissue culture supplies were
obtained from Life Technologies, Inc. Labeled secretin
(125I) is prepared and purified by high performance liquid
chromatography (9). [2,8-3H]adenine,
[3H]cAMP, [8-14C]cAMP,
[-35S]dATP
S, and [32P]orthophosphate
were obtained from NEN Life Science Products. Restriction enzymes were
from Promega. Sequencing supplies were from U. S. Biochemicals
Corp./Amersham Life Science, Inc. Polymerase chain reaction (PCR)
materials were from Perkin-Elmer (Roche Molecular Systems).
Plasmid Preparation-- Using the known cDNA sequence for the rat secretin receptor (6), oligonucleotides were synthesized, and the full-length nucleotide sequence was amplified from rat heart cDNA by PCR. An epitope-tagged rat secretin receptor was prepared as described (10) by placing the FLAG epitope on the N-terminal region of the mature receptor following a modified influenza hemaglutinin signal sequence to produce a protein that could be recognized with commercially available anti-FLAG antibodies. Fidelity was demonstrated with dideoxy sequencing. The cDNAs were inserted into the pcDNA 1/Amp plasmid (Invitrogen) using HindIII and BamHI. GRK cDNAs were produced as described previously: GRK 2 and GRK 3 (11), GRK 4 (12), GRK 5 (11, 13), and GRK 6 (14). Plasmid purification was performed with Qiagen reagents.
Cell Culture-- HEK 293 cells were grown in modified Eagle's medium (10% fetal bovine serum, 50 mg/liter gentamicin) at 37 °C in 95% air, 5% CO2. One day after transfection, cells were split into appropriate plates after trypsin dissociation. Experiments were performed 24-36 h after transfection.
Transfection-- Transient transfections were performed with calcium phosphate co-precipitation. 1-10 µg of vector DNA was transferred into a 6-ml Falcon tube with 450 µl of sterile water and 50 µl of 2.5 M CaCl2. Then 500 µl of 2× HEPES-buffered saline (0.28 M NaCl, 0.05 M HEPES, 1.5 mM Na3PO4, pH 7.1) was added to the tube and mixed well. This mixture was added dropwise to the 100-mm dish of cells. Cells were plated to a density of approximately 2-3 × 105 cells to each well for cAMP accumulation experiments and 1-1.5 × 106 cells/well for phosphorylation.
Membrane Preparation/Binding--
All steps were performed at
4 °C. Plates were placed on ice, media was aspirated, and the cells
were washed with 10 ml of ice-cold phosphate-buffered saline. 5-10 ml
of lysis buffer (10 mM Tris, 5 mM EDTA with
protease inhibitors: 10 µg/ml aprotinin, 5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 10 µg/ml benzamidine, 0.2 mM
phenylmethylsulfonyl fluoride) were added to each plate. With a cell
lifter, cells were scraped off the plate and placed in 15-ml conical
tubes on ice. Cell fragments were homogenized with a Polytron PT 3000 for 20-30 s at 14,000-16,000 cps. Material was centrifuged at
300-400 × g for 10 min to remove unlysed cells and
nuclei. Supernatant was transferred to 13 × 100-mm tubes on ice
and centrifuged at 18,000 rpm (40,000 × g) (Sorval
SM24 rotor) for 30 min at 4 °C. Supernatant was discarded, and the
membrane pellet was resuspended in binding buffer for immediate assay
or lysis buffer and stored at 80 °C.
Adenylyl Cyclase Assays-- The accumulation of cAMP in intact cells was quantitated chromatographically by the method Salomon (15). Cells were labeled with [3H]adenine (1 µCi/ml) in modified Eagle's medium, 5% fetal bovine serum, 50 mg/liter gentamicin (1 ml/well) 12-16 h before experimentation. To assay the accumulation of cAMP, labeling media was aspirated, and cells were washed with 1 ml of phosphate-buffered saline and preincubated in 1 ml of media/well (modified Eagle's medium, 0% fetal bovine serum, 10 mM HEPES, 1 mM isobutylmethylxanthine; assay medium) for 15-30 min. Cells were stimulated with appropriate agonist, and at the end of the experimental duration, media was aspirated, and 1 ml of ice-cold stop solution (0.1 mM cAMP, 4 nCi/ml [14C]cAMP, 2.5% perchloric acid) was placed in each well. Plates remained on ice at 4 °C for 20-30 min, after which solution was transferred to 12 × 75 tubes containing 100 µl of 4.2 M KOH. Tubes were vortexed and stored at 4 °C for cAMP determination by column chromatography (15). For experiments requiring pretreatment with protein kinase inhibitors, transfected cells were incubated in 30 µM H89 or staurosporine for 20 min and then stimulated with secretin in the presence of H89 or staurosporine at doses noted for 10 min. Control experiments were performed in parallel to ensure activity of the inhibitors. Data is normalized for total cellular uptake using [14C]cAMP as described previously (15).
Western Blotting-- Cellular proteins were resolved by SDS-polyacrylamide gel electrophoresis. Protein was transferred to nitrocellulose and then subjected to immunoblotting with appropriate GRK antisera (12, 16, 17).
Cell Phosphorylation-- Cells were labeled with [32P]orthophosphate (66 µCi/well) for 1 h in phosphate-free modified Eagle's medium, 20 mM HEPES, pH 7.4, at 37 °C. Agonist was applied as indicated in figure legends. Treatment was stopped by placing the cells at 4 °C and washing with ice-cold phosphate-buffered saline (3 ml/well) twice and then adding 400 µl/well of radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM disodium pyrophosphate, 5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 10 µg/ml benzamidine). Lysed cells from 2 wells (800 µl) were transferred to 1.5-ml tubes on ice and rotated on an inversion wheel for 1 h. Solubilized material was transferred to Beckman TLA 100.2 tubes for centrifugation at 200,000 × g for 15 min at 4 °C. The supernatant was transferred to 1.5-ml tubes on ice with 100-µl protein A-Sepharose beads (Pharmacia Biotech Inc.) in 3% bovine serum albumin and radioimmune precipitation buffer. An aliquot of supernatant was removed for protein determination (Bio-Rad DC protein assay kit). After a 1-h preclearing, beads were pelleted, and the supernatant was transferred to 1.5-ml tubes with 100 µl of protein A-Sepharose beads and 16 µg of monoclonal IgG-M2-FLAG (Eastman Kodak Co.). Samples were placed on an inversion wheel at 4 °C. After 2 h, beads were pelleted, and supernatant was discarded. Beads were washed three times with ice-cold radioimmune precipitation buffer. SDS-polyacrylamide gel electrophoresis sample buffer was added to each sample to provide the same membrane protein/volume of sample for gel loading. Immune complexes were dissociated by heating to 65 °C for 10 min and resolved on a 1-mm thick, 10% SDS-polyacrylamide gel electrophoresis gel. Dried gels were analyzed quantitatively with a Molecular Dynamics PhosphorImager.
In plasmid co-transfection experiments, receptor expression was determined by flow cytometry analysis of a sample from each transfection group. The fluorescence was determined by incubation for 1 h at 37 °C with monoclonal IgG-M2-FLAG (1:500 dilution, Kodak), three washes with phosphate-buffered saline, and detection with Fc-specific, fluorescein-labeled goat anti-mouse (1:200 dilution, Sigma). Cells were then washed, removed from the plate with 10 mM Tris, pH 7.4, 5 mM EDTA, and fixed with 3.6% formaldehyde. Samples were analyzed within 1 h on a Becton-Dickinson flow cytometer. Base-line fluorescence was determined from a sample of HEK 293 cells untransfected and/or a sample of HEK 293 cells transfected with the secretin receptor not exposed to primary antibody (IgG-M2-FLAG). Base-line fluorescence was subtracted from each sample. Receptor fluorescence was normalized to total cellular protein determined from an aliquot of each transfection sample before immunoprecipitation. Gel lanes were loaded with the same amount of receptor protein. ![]() |
RESULTS |
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Signaling and Desensitization of the Rat Secretin Receptor-- Binding studies with cell membranes prepared from HEK 293 cells transiently transfected with the native rat secretin receptor and the N-terminal FLAG secretin receptor cDNA demonstrated an identical KD for secretin binding (2.3 nM) with a Vmax of 0.5-1.0 pmol/mg of protein (data not shown). Ligand binding was specific and not significantly competed for by either excess glucagon or vasoactive intestinal polypeptide (data not shown). Secretin elicited dose-dependent whole cell cAMP accumulation, with an EC50 of 0.4 nM and 0.07 nM for the native and N-terminal FLAG-tagged rat secretin receptor, respectively, with identical Vmax values (Fig. 1A). The rat secretin receptor demonstrated rapid and complete desensitization in response to agonist occupancy (Fig. 1B). The rate of cAMP accumulation decreased with a half-time of 7 min, and no further cAMP accumulation occurred after 20 min (Fig. 1B). Cross-desensitization experiments using either vasoactive intestinal polypeptide or glucagon as the stimulus in cells coexpressing these receptors indicate that this desensitization is homologous (data not shown). Given the rapid nature of secretin receptor desensitization, receptor phosphorylation was examined as a likely mechanism for signal attenuation.
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Secretin Receptor Phosphorylation-- Studies of desensitization of the rhodopsin and adrenergic receptors have demonstrated the importance of receptor phosphorylation (2-4). The addition of the FLAG epitope to the N terminus of the rat secretin receptor provides the opportunity to demonstrate unequivocal secretin receptor protein phosphorylation by immunoprecipitation (Fig. 2, A and B). The major phosphorylated protein in receptor-expressing cells runs as a broad band of 55-65 kDa and is not present in immunoprecipitates from cells not expressing FLAG-secretin receptor. Agonist-dependent phosphorylation occurs with an EC50 of 14 nM (Fig. 2B). There is a small component of basal (agonist-independent) phosphorylation; however, agonist stimulation increased this signal 4-10-fold (Fig. 2A). The functional N-terminal FLAG-tagged receptor represents a suitable tool for studying specific secretin receptor protein phosphorylation.
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The Role of Second Messenger-dependent Protein Kinases in Secretin Receptor Desensitization/Phosphorylation in HEK 293 Cells-- Phosphorylation of Class I GPCRs can occur by second messenger-dependent mechanisms or by G protein-coupled receptor kinase activity (2, 4). In HEK 293 cells transiently transfected with the secretin receptor and pretreated with 30 µM H89, a PKA inhibitor, no significant enhancement in cAMP generation was observed. Similarly, 1 µM staurosporine, a PKC inhibitor, had no significant effect on cAMP accumulation (Fig. 3). Dose-response curves were generated to investigate the potential effects of these kinases over the full range of agonist concentrations. The EC50 for cAMP accumulation was 0.3 nM for secretin alone, or in the presence of H89 or staurosporine and only a minor increase in the maximal cAMP, accumulation was evident at high agonist concentrations (Fig. 3). Under these conditions, H89 and staurosporine can effectively inhibit protein kinase A and C. Despite the lack of effect of PKA and PKC inhibition on secretin receptor signaling, the effect of the kinase inhibitors on receptor phosphorylation was examined. Interestingly, preincubation with 30 µM H89 and 1 µM staurosporine produced a 50% decrease in secretin receptor phosphorylation (Fig. 4, A and B). This effect was present whether cells were stimulated with high (0.1 µM) or low (1 nM) secretin concentrations for 2 min. These results suggest that in HEK cells, phosphorylation of the secretin receptor by PKA or PKC occurs but does not appreciably modulate its signaling efficiency. However, in COS 7 cells similarly transfected with the rat secretin receptor, preincubation with these protein kinase inhibitors significantly augmented secretin-stimulated cAMP accumulation, indicating that the second messenger-regulated protein kinases may be involved in receptor regulation in other cell types (data not shown). The ability of PKA and PKC to regulate secretin receptor signaling in COS 7, but not in HEK 293 cells, is likely due to the relatively low content of GRKs in COS 7 cells as compared with HEK 293 cells (18).
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Role of G Protein-coupled Receptor Kinases in Secretin Receptor Desensitization and Phosphorylation in HEK 293 Cells-- The lack of effect of PKA and PKC inhibitors on cAMP signaling in HEK 293 cells transfected with the secretin receptor suggests that GRKs might play the predominant role in receptor phosphorylation in these cells. This is supported by the short time course of desensitization and the fact that receptor phosphorylation occurred within 1 min of agonist stimulation (data not shown) (19). Therefore, we examined the potential involvement of G protein-coupled receptor kinases on both phosphorylation and signaling by co-transfecting HEK 293 cells with each of five different GRKs (GRK 2-6). Since GRK-mediated phosphorylation has been shown to occur within seconds to minutes, phosphorylation was examined at 2 min after agonist exposure. As shown in Fig. 5, secretin receptor phosphorylation varied by GRK subtype. GRKs 2, 3, and 5 significantly enhanced agonist-stimulated receptor protein phosphorylation. Expression of GRKs 3 and 5 increased phosphorylation up to -15 fold compared with the level apparent with endogenous cellular GRKs. However, overexpression of GRKs 4 and 6 did not significantly increase basal or agonist-stimulated secretin receptor phosphorylation (Fig. 5, A and B). GRK 5 was the only GRK to have an effect on basal phosphorylation (Fig. 5B). In the absense of agonist, overexpressed GRK 5 doubled basal secretin receptor phosphorylation. Coexpression of members of the GRK 2 subfamily (GRK 2 and 3) with the secretin receptor caused significant signal attenuation, reducing maximal cAMP accumulation by 39 and 26%, respectively (Fig. 6 and Table I). These GRKs also caused a shift in the secretin EC50 to the right (Fig. 6). The EC50 shifted from 0.48 nM for the native receptor to 0.69 and 1.28 nM with cotransfection of GRK 2 and 3, respectively (Fig. 6A, Table I). This is in contrast to GRKs 4 and 6, which had no significant effect on signal generation. The Vmax was 89 and 92% with EC50 0.78 and 0.85 nM for GRK 4 and 6, respectively (Table I). GRK 5 was the most potent of the GRKs evaluated, in reducing both Vmax (40% reduction) and shifting the EC50 (to 1.37 nM) (Fig. 6B, Table I).
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DISCUSSION |
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In this paper we show that homologous desensitization of the
secretin receptor, a Class II G protein-coupled receptor, is mediated
by phosphorylation via G protein-coupled receptor kinases in HEK 293 cells. In these cells, PKA and PKC inhibitors did not significantly
alter secretin receptor signaling. Direct assessment of phosphorylation
of the secretin receptor was performed with a N-terminal FLAG-tagged
rat secretin receptor, which retains the properties of the native
receptor. Receptor phosphorylation correlates with desensitization of
cAMP signaling for all GRKs investigated. Agonist-dependent
receptor phosphorylation occurs in the presence of PKA and PKC
inhibition; moreover, these inhibitors do not appear to alter signal
attenuation. These data suggest that receptors of the Class II family
of G protein-coupled receptors can be regulated by mechanisms similar
to those of the rhodopsin/ adrenergic (Class I) receptor family.
The native secretin receptor and the N-terminal FLAG-tagged secretin receptor bound ligand identically, consistent with the KD determined for the secretin receptor by others (6, 7). The N-terminal portion of the native secretin receptor has been shown to be important for ligand binding and signaling (20). It is therefore interesting that addition of the FLAG epitope does not alter ligand binding but does enhance receptor signaling. The additional FLAG residues caused a shift in the EC50 to the left, indicating an increased ability to stimulate cAMP accumulation. The mechanism for this enhanced signaling is unclear. The presence of this epitope may alter the conformation of the native receptor or the agonist-bound receptor to promote signaling and adenylyl cyclase activation. Alterations in receptor sequence have been shown to result in both increased and decreased signaling (8, 20). Despite these subtle differences, the N-terminal FLAG-tagged receptor is an excellent tool for the study of secretin receptor phosphorylation, desensitization, and cellular trafficking.
Termination of G protein-coupled receptor-mediated signaling is
facilitated by different mechanisms (3, 4, 19, 21). Evidence that
phosphorylation plays a role in GPCR desensitization has been obtained
from several members of the large Class I receptor family (11, 13, 19,
22). Phosphorylation of GPCRs has been shown to involve two types of
serine/threonine protein kinases: second messenger-activated protein
kinases (PKA and PKC) and the GRKs that phosphorylate agonist-occupied
GPCRs (2-4). A family of these GRKs exist that have been shown to
phosphorylate GPCRs but differ significantly in their mechanisms of
membrane association and/or activation (3, 4). Here we show that in HEK
293 cells transfected with the secretin receptor, desensitization of
the secretin-mediated signal is rapid and accompanied by significant phosphorylation of the receptor protein. The time course for the attenuation of the signal is consistent with that which has been shown
for the 2-adrenergic receptor in such cells as the
BEAS-2B cells (human airway epithelial cells) but is slower than that found in Chinese hamster ovary cells permanently expressing the secretin receptor (8, 23). We have also noted that kinetic differences
exist between HEK 293 cells and COS 7 cells transiently expressing the
rat secretin receptor.2 These
slower rates in Cos 7 cells are consistent with recent findings from
our laboratory that demonstrated that the complement of GRKs in COS 7 cells is significantly lower than that of many other cell lines
(18).
That secretin receptor signaling in HEK 293 cells is modulated by
GRK-mediated phosphorylation is supported by two lines of evidence.
First, inhibitors of PKA and PKC had essentially no effect on the
secretin-mediated accumulation of cAMP, suggesting these kinases do not
play a dominant role in regulating the receptor signaling function in
HEK 293 cells. PKA has been shown to be effective in the
desensitization of the D1A dopamine and the -adrenergic receptor (22). The effectiveness of PKA and PKC in 293 cells is evident
in the reduction in receptor phosphorylation by inhibitors (Fig. 4).
The role of second messenger-regulated phosphorylation of the receptor
in 293 cells is unknown. Second, overexpression of specific GRKs
enhanced the blunting of the receptor response as well as increased
receptor phosphorylation. Among the GRKs tested, GRK 2, 3, and 5 produced the largest enhancement of desensitization and the greatest
enhancement of phosphorylation. On the other hand, GRKs 4 and 6 were
without effect on signaling and did not cause any significant increase
in receptor phosphorylation over endogenous kinases. These data are
reminiscent of data obtained with adrenergic, dopaminergic, and
angiotensin receptors (13, 22, 24) in which preferential action of the
various GRKs has been demonstrated. GRK mediation of the
secretin-dependent attenuation of the receptor response and
phosphorylation in HEK 293 cells is consistent with the rapid time
course of receptor desensitization. The half-time for the GRK-mediated
desensitization of the
2-adrenergic receptor is 15 s, whereas that for the PKA-mediated response is severalfold slower
(t1/2 > 3 min) (19, 22, 25). The role of
receptor sequestration in desensitization has been reviewed recently
(21). Sequestration is enhanced by phosphorylation in the adrenergic
receptor family (21). Previous work on the
1-adrenergic
and the secretin receptors demonstrate only 10-15% surface receptor
loss by 2-3 min of agonist exposure (23).2 Also, using a
fluorescent secretin agonist, Holtmann et al. (8) demonstrated that label remained at the cell surface after 1 min of
agonist stimulation. Our desensitization and phosphorylation experiments were performed at 2 min, and sequestration is likely not a
predominant feature of this signal attenuation. These data support a
role for GRKs in the desensitization process. The use of specific GRK
phosphorylation inhibitors was attempted (K220R and a C-terminal GRK 2 construct); however, the effect of agonist-induced phosphorylation is
so dramatic (up to 15-fold stimulation over basal), we were unable to
block the effect of endogenous kinase activity. Mutation of the
phosphorylation sites may provide further information; however, the
secretin receptor contains no less than 22 putative phosphorylation
residues in its intracellular domains. Pertubation of all these sites
would require characterization of receptor binding, signaling, and
desensitization properties of all these mutants. Additionally,
site-directed mutagenesis is known to alter the conformation of
receptor proteins, and this may alter phosphorylation and/or
desensitization.
The direct demonstration of secretin receptor phosphorylation by specific immunoprecipitation of an N-terminal FLAG-tagged receptor correlates well with previous data by Ozcelebi et al. (7) on agonist-dependent phosphorylation of a protein, with the size expected for the transfected secretin receptor in COS 7 cells and Chinese hamster ovary cells. The effect of overexpressed GRK 2, 3 and 5 on the agonist-mediated response and consequent phosphorylation, implies a role for phosphorylation by these kinases in signal termination of the secretin receptor. Holtmann et al. (8) have proposed internalization of the secretin receptor as the main mode of desensitization rather than receptor phosphorylation. This contention was based on the observation that in a stimulation/restimulation protocol, a C-terminal-truncated mutant secretin receptor was capable of desensitizing and internalizing a fluorescently labeled agonist similar to the wild-type receptor. However, interpretation of these results may have been confounded by the incomplete removal of bound agonist before restimulation (8). Whereas sequestration/internalization is an important phenomenon in the desensitization process, evidence suggests that it plays a more important role in the GPCR resensitization process (21). Indeed, phosphorylation of GPCRs facilitates the interaction of the phosphorylated receptors with arrestin proteins, which uncouple the receptor/G-protein interaction. At the same time though, phosphorylation and arrestin protein binding provide a trigger for phosphorylated receptor internalization and resensitization (21). Thus, phosphorylation and arrestin binding play a dual role in that not only do they uncouple the response but also act as a trigger or adaptor to mediate the resensitization process (21).
G protein-coupled receptors are ubiquitous. Presently GPCRs are known to be activated by peptides and protein hormones, monoamine and amino neurotransmitters, calcium ions, light photons, tastants, and odorants. To ensure the stability of the cellular environment, cells must be able to terminate signals in a timely manner to prevent overstimulation and to be prepared for the acquisition of new information. The secretin receptor has an important role in many physiologic processes of the gastrointestinal tract, pancreas, and biliary epithelium (26, 27). Understanding the role of GRKs in Class II receptor signal transduction should provide insight into the regulation of pancreatic fluid secretion, bile flow, and other gastrointestinal neuroendocrine responses.
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ACKNOWLEDGEMENT |
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We thank Linda Czyzyk, Anne Marie Colapietro, and Lucie Bertrand for expert technical support. We are grateful to Drs. Neil Freedman and Luc Menard for helpful suggestions and comments and to Dr. Robert J. Lefkowitz for GRK specific antisera.
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FOOTNOTES |
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* This work supported in part by a National Institutes of Health Grant NS19576 and grants from Bristol-Myers Squibb and Zeneca Pharmaceuticals.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A fellow supported by an National Institutes of Health Training
Grant 5T32DK07568.
§ To whom correspondence should be addressed: Duke University Medical Center, Box 3287, Durham, NC 27710. Tel.: 919-684-5433; Fax: 919-681-8641.
1
The abbreviations used are: GPCR, G
protein-coupled receptors; GRK, GPCR kinase; HEK, human embryonic
kidney; dATPS, deoxyadenosine 5'-(
-thiol)triphosphate; PCR,
polymerase chain reaction; PKA and PKC, protein kinase A and C,
respectively.
2 M. A. Shetzline, R. T. Premont, J. K. L. Walker, S. R. Vigna, and M. G. Caron, unpublished observation.
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REFERENCES |
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