A Role for Receptor Kinases in the Regulation of Class II G Protein-coupled Receptors
1998; Elsevier BV; Volume: 273; Issue: 12 Linguagem: Inglês
10.1074/jbc.273.12.6756
ISSN1083-351X
AutoresMichael A. Shetzline, Richard T. Premont, Julia K. L. Walker, Steven R. Vigna, Marc G. Caron,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe 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. 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. 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) 1The abbreviations used are: GPCR, G protein-coupled receptors; GRK, GPCR kinase; HEK, human embryonic kidney; dATPαS, deoxyadenosine 5′-(α-thiol)triphosphate; PCR, polymerase chain reaction; PKA and PKC, protein kinase A and C, respectively. into subfamilies: I) rhodopsin/β-adrenergic, II) secretin/glucagon, and III) the metabotropic glutamate receptor families (1Birnbaumer M. J. Recept. Signal Transduct. Res. 1995; 15: 131-160Crossref PubMed Scopus (77) Google Scholar, 2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar). 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/β-adrenergic receptor family of GPCR (Class I) (1Birnbaumer M. J. Recept. Signal Transduct. Res. 1995; 15: 131-160Crossref PubMed Scopus (77) Google Scholar). 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 β-adrenergic receptor family, the mechanisms regulating Class II GPCR signal transduction are largely unknown. Typically, signal termination occurs via two distinct pathways (2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar). 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 (3Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar, 4Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-353PubMed Google Scholar). Other modes of signal attenuation that involve second messengerdependent protein kinases acting on both active and unstimulated receptors are termed heterologous desensitization (2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar, 5Lohse M.J. Andexinger S. Pitcher J. Trukawinski S. Codina S. Faure J.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 8558-8564Abstract Full Text PDF PubMed Google Scholar). Both protein kinase A and protein kinase C have been shown to play a role in this mode of desensitization (2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar). With the cloning of the secretin receptor (6Ishihara T. Nakamura S. Kaziro T. Takahashi T. Takahashi K. Nagata S. EMBO J. 1991; 10: 1635-1641Crossref PubMed Scopus (369) Google Scholar), 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. (7Ozcelebi F. Holtmann M.H. Rentsch R.U. Rao R. Miller L.J. Mol. Pharmacol. 1995; 48: 818-824PubMed Google Scholar) 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.(8Holtmann M.H. Roettger B.F. Pinon D.I. Miller L.J. J. Biol. Chem. 1996; 271: 23566-23571Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) 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. 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 (9Farouk M. Vigna S.R. McVey D.C. Meyers W.C. Gastroenterology. 1992; 102: 963-968Abstract Full Text PDF PubMed Google Scholar). [2,8-3H]adenine, [3H]cAMP, [8-14C]cAMP, [α-35S]dATPαS, 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). Using the known cDNA sequence for the rat secretin receptor (6Ishihara T. Nakamura S. Kaziro T. Takahashi T. Takahashi K. Nagata S. EMBO J. 1991; 10: 1635-1641Crossref PubMed Scopus (369) Google Scholar), 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 (10Guan X-M. Kobilka T.S. Kobilka B.K. J. Biol. Chem. 1992; 267: 21995-21998Abstract Full Text PDF PubMed Google Scholar) 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 (11Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), GRK 4 (12Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), GRK 5 (11Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Tiberi M. Nash S.R. Bertrand L. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1996; 271: 3771-3778Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), and GRK 6 (14Stoffel R.H. Randall R.R. Premont R.T. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 27791-27794Abstract Full Text PDF PubMed Google Scholar). Plasmid purification was performed with Qiagen reagents. 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. 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 mHEPES, 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. 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 mmphenylmethylsulfonyl 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. Membrane binding was performed as published (9Farouk M. Vigna S.R. McVey D.C. Meyers W.C. Gastroenterology. 1992; 102: 963-968Abstract Full Text PDF PubMed Google Scholar). Briefly, using a constant amount of HEK 293 membrane protein, cold displacement (porcine secretin; Peninsula Labs) of 125I-porcine secretin binding was performed in duplicate tubes. Nonspecific binding was defined in the presence of 1 μm unlabeled porcine secretin. Data was analyzed using Graph Pad-Prism and LIGAND software as described (9Farouk M. Vigna S.R. McVey D.C. Meyers W.C. Gastroenterology. 1992; 102: 963-968Abstract Full Text PDF PubMed Google Scholar). The accumulation of cAMP in intact cells was quantitated chromatographically by the method Salomon (15Salomon Y. Methods Enzymol. 1991; 150: 22-29Crossref Scopus (130) Google Scholar). 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 (15Salomon Y. Methods Enzymol. 1991; 150: 22-29Crossref Scopus (130) Google Scholar). 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 (15Salomon Y. Methods Enzymol. 1991; 150: 22-29Crossref Scopus (130) Google Scholar). Cellular proteins were resolved by SDS-polyacrylamide gel electrophoresis. Protein was transferred to nitrocellulose and then subjected to immunoblotting with appropriate GRK antisera (12Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 16Arriza J.L. Dawson T.M. Simerly R.B. Martin L.J. Caron M.G. Snyder S.H. Lefkowitz R.J. J. Neurosci. 1992; 12: 4045-4055Crossref PubMed Google Scholar, 17Premont R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Abstract Full Text PDF PubMed Google Scholar). 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 mmNaCl, 50 mm Tris-HCl, pH 8, 5 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mmNaF, 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. 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.1 A). The rat secretin receptor demonstrated rapid and complete desensitization in response to agonist occupancy (Fig. 1 B). The rate of cAMP accumulation decreased with a half-time of 7 min, and no further cAMP accumulation occurred after 20 min (Fig. 1 B). 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. Studies of desensitization of the rhodopsin and adrenergic receptors have demonstrated the importance of receptor phosphorylation (2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar, 3Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar, 4Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-353PubMed Google Scholar). 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. 2 B). There is a small component of basal (agonist-independent) phosphorylation; however, agonist stimulation increased this signal 4–10-fold (Fig.2 A). The functional N-terminal FLAG-tagged receptor represents a suitable tool for studying specific secretin receptor protein phosphorylation. Phosphorylation of Class I GPCRs can occur by second messenger-dependent mechanisms or by G protein-coupled receptor kinase activity (2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar, 4Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-353PubMed Google Scholar). 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 μmstaurosporine, 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 (18Menard L. Ferguson S.S.G. Zhang J. Lin F.-T. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: 800-808Crossref PubMed Scopus (214) Google Scholar).Figure 4Secretin receptor phosphorylation by immunoprecipitation. HEK 293 cells overexpressing secretin receptor with preincubation of 30 μm H89 or 1 μm staurosporine (Stauro) or media (vehicle) receptor phosphorylation was determined as under "Experimental Procedures." Preincubation was for 18 min, and stimulation with secretin (0.1 μm) was performed for 2 min. A, representative gel of receptor protein demonstrating basal (−) and agonist-stimulated (+) receptor phosphorylation. One band of precipitable radioreactivity is apparent in each treatment group corresponding to the size of the phosphorylated secretin receptor (50–60 kDa, Bio-Rad low range markers). B, phosphorImager analysis of two independent experiments. Data are mean ± S.E. and normalized to basal receptor phosphorylation in the absense of agonist and preincubation with vehicle (vehicle). Similar data were generated with 1 nm agonist stimulation (not shown).View Large Image Figure ViewerDownload (PPT) 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) (19Lohse M. Benovic J.L. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1990; 265: 3202-3209Abstract Full Text PDF PubMed Google Scholar). 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. 5 B). 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 TableI). These GRKs also caused a shift in the secretin EC50 to the right (Fig. 6). The EC50shifted from 0.48 nm for the native receptor to 0.69 and 1.28 nm with cotransfection of GRK 2 and 3, respectively (Fig. 6 A, 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. 6 B, TableI).Figure 6cAMP accumulation in HEK 293 cells cotransfected with empty vector or with GRKs. cAMP accumulation was measured as under "Experimental Procedures." Whole cell stimulation with secretin was performed at doses indicated for 2 min. All curves represent three independent experiments, with each experimental point performed in triplicate. Data are mean ± S.E. and normalized to maximal stimulation, determined with secretin receptor cotransfected with empty vector and stimulated with 1 μm secretin. A, secretin receptor-only transfected cells compared with cells cotransfected with 5 μg of GRK 2 or GRK 3. B, secretin receptor-only transfected cells compared with cells cotransfected with 5 μg of GRK 4, GRK 5, or GRK 6. Experimental values for mean cAMP accumulation for basal and maximal stimulation for the three independent experiments (determined with 0.1 pm and 1 μm secretin, respectively) for secretin, GRK 2, GRK 3, GRK 4, GRK 5, and GRK 6 were 0.05, 2.82; 0.03, 1.86; 0.04, 2.19; 0.07, 2.83; 0.04, 1.70; 0.05, 2.97.View Large Image Figure ViewerDownload (PPT)Table IDesensitization of secretin-stimulated cAMP accumulation by GRKs 2, 3, 4, 5, or 6TransfectionEC50 (95% CI)Vmax(±S.E.)nm+Empty vector0.48 (0.31–0.73)0.92 (± 0.03)+GRK 20.70 (0.42–1.13)0.61 (±0.02)+GRK 31.28 (0.87–1.88)0.74 (±0.02)+GRK 40.78 (0.42–1.47)
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