Protein Kinase A and G Protein-coupled Receptor Kinase Phosphorylation Mediates β-1 Adrenergic Receptor Endocytosis through Different Pathways
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m305675200
ISSN1083-351X
AutoresAntonio Rapacciuolo, Shayela Suvarna, Liza Barki‐Harrington, Louis M. Luttrell, Mei Cong, Robert J. Lefkowitz, Howard A. Rockman,
Tópico(s)Caveolin-1 and cellular processes
ResumoAgonist-induced phosphorylation of β-adrenergic receptors (βARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of βARs, particularly the β1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type β1AR (WTβ1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA–β1AR), the putative GRK phosphorylation sites (GRK–β1AR), and both sets of phosphorylation sites (PKA–/GRK–β1AR). Following agonist stimulation, both PKA–β1AR and GRK–β1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WTβ1AR and PKA–β1AR, sequestration of the GRK–β1AR and PKA–/GRK–β1AR was independent of β-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WTβ1AR and PKA–β1AR, whereas caveolae inhibitors prevented internalization only of the GRK–β1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the β1AR and 2) the pathway selected for β1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits. Agonist-induced phosphorylation of β-adrenergic receptors (βARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of βARs, particularly the β1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type β1AR (WTβ1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA–β1AR), the putative GRK phosphorylation sites (GRK–β1AR), and both sets of phosphorylation sites (PKA–/GRK–β1AR). Following agonist stimulation, both PKA–β1AR and GRK–β1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WTβ1AR and PKA–β1AR, sequestration of the GRK–β1AR and PKA–/GRK–β1AR was independent of β-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WTβ1AR and PKA–β1AR, whereas caveolae inhibitors prevented internalization only of the GRK–β1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the β1AR and 2) the pathway selected for β1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits. β-Adrenergic receptors (βARs) 1The abbreviations used are: βAR, β-adrenergic receptor; GPCR, guanine nucleotide-binding regulatory protein-coupled receptor; PKA, protein kinase A; PKC, protein kinase C; GRK, G protein-coupled receptor kinase; HEK, human embryonal kidney; IBMX, 3-isobutyl-1-methylxanthine; ISO, (–)-isoproterenol bitartrate; β-CD, 2-hydroxypropyl-β-cyclodextrin; MDC, monodansylcadaverine; WTβ1AR, wild type β1AR; PKA–β1AR, β1AR lacking putative PKA phosphorylation sites; GRK–β1AR, β1AR lacking putative GRK phosphorylation sites; PKA–/GRK– β1AR, β1AR lacking both sets of phosphorylation sites; GFP, green fluorescent protein; MEM, minimum essential medium; WT, wild type; Gβγ, beta-gamma subunits of G protein; Gαs, stimulatory G protein alpha subunit; GFX, bisindolylmalemide I; PMA, phorbol 12-myristate 13-acetate; VASP, vasodilator- and A kinase-stimulated phosphoprotein; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin; Erk, extracellular signal-regulated kinase; ANOVA, analysis of variance. belong to the large family of G protein-coupled receptors (GPCRs) characterized by a typical structure of seven transmembrane domains (1Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar, 2Rockman H.A. Koch W.J. Lefkowitz R.J. Nature. 2002; 415: 206-212Crossref PubMed Scopus (788) Google Scholar). Three types of βARs, designated β1, β2, and β3ARs, have been cloned from mammalian tissues (1Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar, 3Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1137) Google Scholar). Both β1 and β2ARs contain phosphorylation sites located in the third intracellular loop and the C-terminal tail of the receptor, which serve as targets for cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs) (2Rockman H.A. Koch W.J. Lefkowitz R.J. Nature. 2002; 415: 206-212Crossref PubMed Scopus (788) Google Scholar). Furthermore, site-specific mutagenesis studies of the human β2AR suggest that low concentrations of agonist preferentially induce phosphorylation at PKA sites, whereas higher concentrations of agonist induce phosphorylation at both PKA and GRK sites (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar). Continuous exposure of cells to a stimulus causes βARs to undergo rapid phosphorylation in a process that dampens receptor signaling known as desensitization (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar, 5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 6Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 7Roth N.S. Campbell P.T. Caron M.G. Lefkowitz R.J. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6201-6204Crossref PubMed Scopus (118) Google Scholar, 8Pitcher J.A. Freedman N.J. Lefkowitz R.J. Ann. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar). βARs demonstrate two different mechanisms of desensitization. Agonist-specific or homologous desensitization of βARs consists of a two-step process in which phosphorylation at the C terminus of the βAR is mediated by GRKs followed by binding to an arrestin protein, which sterically interrupts signaling to the G protein (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 8Pitcher J.A. Freedman N.J. Lefkowitz R.J. Ann. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar). Heterologous or non-agonist-specific desensitization is mediated by the second messenger-stimulated protein kinases A and C, which phosphorylate the receptor and effect a change in receptor conformation such that interaction with the G protein is impaired (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). An important consequence of agonist-mediated receptor phosphorylation and desensitization by GRKs is the subsequent internalization of phosphorylated receptors into the cell (9Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (454) Google Scholar). This process is mediated by β-arrestin, which binds to components of the clathrin-mediated endocytic machinery and targets the ligand-bound receptor to clathrin-coated pits for endocytosis (10Lin F. Wang H. Malbon C.C. J. Biol. Chem. 2000; 275: 19025-19034Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 11Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). Interestingly, PKA phosphorylation, although an important mechanism for desensitization (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar, 5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 6Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 7Roth N.S. Campbell P.T. Caron M.G. Lefkowitz R.J. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6201-6204Crossref PubMed Scopus (118) Google Scholar, 8Pitcher J.A. Freedman N.J. Lefkowitz R.J. Ann. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar), appears to play only a small role in β2AR internalization (12Ferguson S.S. 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). Although mechanisms of phosphorylation, desensitization, and internalization by GRKs have been well studied for the β2AR (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar, 5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 6Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 7Roth N.S. Campbell P.T. Caron M.G. Lefkowitz R.J. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6201-6204Crossref PubMed Scopus (118) Google Scholar, 8Pitcher J.A. Freedman N.J. Lefkowitz R.J. Ann. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar), little is known of the role that PKA-mediated phosphorylation plays in the internalization of βARs, particularly the β1AR. GPCRs can internalize via at least two distinct pathways, namely clathrin-coated pits and caveolae. Although very different structurally, clathrin-coated pits and caveolae both serve as microdomains, which, in addition to functioning as transport machinery, also serve as platforms for integrating the cell's signal-transduction pathways (13Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar, 14Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (540) Google Scholar). These membrane domains serve to facilitate cross-talk between different proteins from a particular signaling pathway contained within these distinct regions (15Marx J. Science. 2001; 294: 1862-1865Crossref PubMed Scopus (41) Google Scholar). Proteins to be transported are now thought to have "molecular addresses" located in the amino acid sequences of their cytoplasmic tail regions or to contain a binding site for a particular adaptor protein that directs the molecule to a particular cellular domain (16Anderson R.G. Jacobson K. Science. 2002; 296: 1821-1825Crossref PubMed Scopus (1012) Google Scholar, 17Hall R.A. Lefkowitz R.J. Circ. Res. 2002; 91: 672-680Crossref PubMed Scopus (186) Google Scholar). In this regard, although a number of studies have demonstrated a critical role for GRK phosphorylation and β-arrestin binding in the process of clathrin-mediated internalization of the β2AR (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar), the molecular mechanisms that are involved in the internalization of the β1AR are less known. The aim of the present study was to determine the specific role of PKA- and GRK-catalyzed phosphorylation of the β1AR in determining the cellular pathway for agonist-promoted receptor internalization. Materials—All cell culture reagents were procured from Invitrogen. Human embryonal kidney (HEK) 293 cells were obtained from American Type Culture Collection. H-89 was obtained from BIOMOL Research Laboratories. Nonidet P-40 and bisindolylmalemide I (GFX) was obtained from Calbiochem. 3-Isobutyl-1-methylxanthine (IBMX), the anti-flag affinity gels, (–)-isoproterenol bitartrate (ISO), filipin III, 2-hydroxypropyl-β-cyclodextrin (β-CD), water-soluble cholesterol, monodansylcadaverine (MDC), and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma. The β2AR antagonist ICI-118,551·HCl was procured from Research Biochemicals International. [32P]Orthophosphate, and [125I]iodocyanopindolol came from PerkinElmer Life Sciences. Restriction enzymes were obtained from Invitrogen. A cyclic AMP (3H) assay kit and ECL Western blotting detection reagents were obtained from Amersham Biosciences. An alkaline phosphatase substrate kit and protein assay kit were procured from Bio-Rad. Geneticin was obtained from Invitrogen. Plasmid Constructs—We generated mouse wild type β1AR (WTβ1AR) as previously described (18Naga Prasad S.V. Barak L.S. Rapacciuolo A. Caron M.G. Rockman H.A. J. Biol. Chem. 2001; 276: 18953-18959Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Three different mutants lacking, respectively: the putative PKA phosphorylation consensus sites (PKA–β1AR), the putative GRK phosphorylation sites (GRK–β1AR), and both sets of sites (PKA–/GRK–β1AR) (Fig. 1A) were generated using a combination of primers (for details see Supplemental Material). All recombinant DNA-containing plasmids were verified for sequence authenticity and subcloned into mammalian expression vectors. Mammalian Cell Culture and Transfection—HEK 293 cells were maintained as previously described (18Naga Prasad S.V. Barak L.S. Rapacciuolo A. Caron M.G. Rockman H.A. J. Biol. Chem. 2001; 276: 18953-18959Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The evening before transfection, 4 × 106 cells were plated per 75-cm2 flasks. These cells were transfected on day 1 by FuGENE6™ method (Roche Applied Science). For all experiments, each plate received 5 μg of total DNA, comprising 0.25 μg of the mutant β1AR or 0.5 μg of the WTβ1AR, with the balance comprising just pRK5 DNA. For the β-arrestin2 recruitment studies, each plate received 0.5 μg of β1AR along with a 10-fold molar excess GFP-β-arrestin2 plasmid. Cells were split on day 2 into assay dishes as follows: for phosphorylation assays, and cAMP assays, 3 × 106 cells/100-mm dish, for β-arrestin2 recruitment, and confocal studies 1 × 106 cells/9.6-cm2 well. Assays were performed on day 3. HEK 293 cells were transfected with cDNA containing WTβ1AR, PKA–β1AR, GRK–βAR, and PKA–/GRK–1AR to create stable cell lines. 48 h after transfection cells were selected by the addition of Geneticin at a concentration of 0.5 mg/ml. Expression of receptor was determined by radioligand binding assays. WTβ1AR 551 ± 63, PKA–β1AR 896 ± 56, GRK–β1AR 835 ± 11, PKA–/GRK–β1AR 479 ± 15 fmol/mg of protein. These stable cells were used in ELISA assays, confocal experiments, and immunoblotting studies. Intact Cell Phosphorylation—Intact cell phosphorylation was performed as previously described (19Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Briefly, assays were performed at 37 °C, in phosphate-free Dulbecco's modified Eagle's medium, 20 mm HEPES, pH 7.4. Labeling was conducted for 1 h in medium containing 100 μCi of 32Pi/ml (inorganic phosphate, Pi). In PKA inhibition experiments, the labeling medium contained 0.04% Me2SO followed by incubation with ISO (10 μm) for 10 min in the appropriate dishes. The β1AR density (pmol/mg of whole cell protein) of each transfected cell line was determined on a non-radioactive aliquot of each transfected cell population by 125I-cyanopindolol. Equivalent amounts of β1AR were immunoprecipitated from each sample and were resolved by SDS-PAGE in 10% gels. Dried gels were subjected to autoradiography and analyzed quantitatively with an Amersham Biosciences PhosphorImager. Radioligand Binding Assays—β1AR expression was evaluated by 125I-cyanopindolol binding studies done at 13 °C for 3 h as previously described (18Naga Prasad S.V. Barak L.S. Rapacciuolo A. Caron M.G. Rockman H.A. J. Biol. Chem. 2001; 276: 18953-18959Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Bound ligand was separated on glass fiber filters (Whatman, GF/C) by vacuum filtration. The filters were washed three times with cold wash buffer (10 mm Tris, 5 mm EDTA (pH 7.4)) and counted in a scintillation counter. Protein concentration was measured with a Bio-Rad DC protein assay kit with bovine serum albumin as the standard. Receptor sequestration after a 30-min exposure to agonist (10 μm ISO) was defined as the loss of 125I-cyanopindolol binding displaced by CGP-12177. Receptor Binding by ELISA—To determine agonist concentration dependence for internalization of the wild type and three mutants, the receptor number was measured by the ELISA method, as described previously (20Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 21Kim J. Ahn S. Guo R. Daaka Y. Biochemistry. 2003; 42: 2887-2894Crossref PubMed Scopus (44) Google Scholar). Briefly, stable cell lines expressing the WTβ1AR or the PKA– or GRK– mutants were plated onto 24-well tissue culture dishes. To improve adhesion of cells to plastic, the wells were treated with 20 μg/ml poly-d-lysine in PBS before plating. 24 h after seeding, cells were incubated with serum-free minimum essential media (MEM) for 10 min at 37 °C. Different concentrations of isoproterenol (0.01–10 μm) were added for 30 min at 37 °C. Reactions were stopped by removing the culture medium followed by fixing cells in 4% formaldehyde/PBS for 5 min at room temperature. Cells were washed three times with PBS followed by blocking with 1% BSA in PBS for 45 min. Mouse monoclonal M2 anti-FLAG IgG was added at a dilution of 1:1000 for 1 h, followed by three subsequent washes with PBS. The samples were then briefly re-blocked, incubated with goat anti-mouse-conjugated alkaline phosphatase at a concentration of 1:1000 in PBS/BSA for 1 h, and washed three times with PBS before the addition of a colorimetric alkaline phosphatase substrate. When adequate color change was reached, 100-μl samples were taken for colorimetric readings at 405 nm using a scanning multiwell spectrophotometer. Non-transfected cells were studied concurrently to determine background signal and all experiments were done in triplicate. GFP-β-arrestin2 Translocation in Live Cells—GFP-β-arrestin2 translocation was visualized in real-time on a 37 °C heated stage Zeiss laser scanning microscope (LSM-510) as previously described (22Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). Cells expressing either the WTβ1AR or one of the mutants and GFP-β-arrestin2 were stimulated with 10 μm ISO in serum-free medium buffered with 10 mm HEPES. Images were collected sequentially every minute for a period of 10 min using a single line excitation filter 488 nm and emission filters at 505–550 nm. For each single experiment, quantitation was performed in the image in which maximal agonist-dependent β-arrestin2 translocation occurred. β-Arrestin2 translocation was calculated and expressed as agonist-promoted percent loss of the green fluorescent color from the cytosol due to GFP-β-arrestin2 expression. Intact Cell β-AR Desensitization: cAMP Assay—HEK 293 cells were incubated for 1 h in serum-free MEM (10 mm HEPES, pH 7.4) prior to the assay. Two identical sets of 100-mm plates were set up; one set was used as controls and the other for desensitization assays. Cells used for desensitization were exposed to 10 μm dobutamine for 5 min at 37 °C and were then washed with serum-free MEM (10 mm HEPES, pH 7.4). Control cells were washed in an identical manner. Both sets of cells were then replaced with assay buffer (serum-free MEM, 10 mm HEPES, 1 mm IBMX, 100 nm ICI 118,551) and exposed to β1-agonist, dobutamine (0.1–10 μm) for 20 min at 37 °C. Reactions were stopped by aspiration of the assay buffer and addition of 200 μl of Tris-EDTA solution (0.05 m Tris, 4 mm EDTA, supplied with cyclic AMP (3H) assay kit) to each plate. Plates were frozen at –80 °C for subsequent processing of cells. Cells were scraped and collected, boiled for 10 min and placed on ice. Samples were then centrifuged at 15,000 × g for 15 min, and supernatant was transferred to a tube for cAMP measurement. cAMP was measured using the procedure outlined in the assay kit. The amount of protein per fraction was determined using a dye-binding protein assay kit. Confocal Microscopy—Confocal microscopy was carried out as previously described (18Naga Prasad S.V. Barak L.S. Rapacciuolo A. Caron M.G. Rockman H.A. J. Biol. Chem. 2001; 276: 18953-18959Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). HEK 293 cells were transfected with the plasmids containing cDNAs encoding either the FLAG-WTβ1AR, FLAG-β2AR, or one of the mutant FLAG-β1ARs as well as pRK5. Live cells were incubated in the absence or presence of filipin, β-CD, cholesterol, MDC along with sucrose, H-89, and GFX for the indicated times and stimulated with ISO (10 μm) for 30 min. All incubations were carried out at 37 °C. Staining of FLAG-tagged receptor was carried out as previously described (23Naga Prasad S.V. Laporte S.A. Chamberlain D. Caron M.G. Barak L. Rockman H.A. J. Cell Biol. 2002; 158: 563-575Crossref PubMed Scopus (154) Google Scholar). Transferrin uptake was carried out as described previously (24van Dam E.M. Stoorvogel W. Mol. Biol. Cell. 2002; 13: 169-182Crossref PubMed Scopus (178) Google Scholar). All samples were visualized under the Olympus 1X70 laser scanning confocal microscope, using single sequential line excitation filters of 568 nm and emission filters of 585 nm for Texas Red detection. Images were viewed using Fluoview software and processed using Adobe Illustrator 9.0.1 and Adobe Photoshop 6.0.1. Immunoblotting—Pretreatment of cells with inhibitors and stimulation with agonist were carried out at 37 °C in serum starvation medium as described in the figure legends. After stimulation, cells were lysed directly with 100 μl/well Laemmli sample buffer and proteins (30 μg/lane) were resolved by SDS-PAGE. Phosphorylation of Erk1/2 was detected by protein immunoblotting using a 1:1000 dilution of rabbit polyclonal phospho-specific mitogen-activated protein kinase IgG (New England BioLabs) with horseradish-peroxidase conjugated donkey anti-rabbit IgG as secondary antibody. Vasodilator- and A kinase-stimulated phosphoprotein (VASP) was detected by using 1:1000 anti-FLAG M2 antibody with horseradish-peroxidase conjugated anti-mouse IgG as secondary antibody. Blots were developed in ECL reagents for 1 min. Agonist-induced Phosphorylation of the β 1AR in Intact 293 Cells—To study the role of PKA- and GRK-mediated phosphorylation of the β1AR in an agonist-dependent manner, we used HEK 293 cells transiently transfected with plasmids containing the WTβ1AR, PKA–β1AR, GRK–β1AR, or PKA–/GRK–β1AR cDNAs. In unstimulated cells, the WTβ1AR exists as a phosphoprotein migrating with a molecular mass of ∼70 kDa (Fig. 1B). Upon stimulation, phosphorylation of the WTβ1AR as well as PKA–β1AR and GRK–β1AR mutants increased ∼2-fold above basal levels (Fig. 1B). However, when both the PKA and GRK sites were mutated (PKA–/GRK–β1AR), no agonist-dependent phosphorylation of the receptor was observed. Agonist-induced Desensitization of the β 1AR in Intact 293 Cells—To determine the role of PKA and GRK phosphorylation in the desensitization of the β1AR we measured cAMP accumulation in HEK 293 cells transiently transfected with the various β1AR mutants. The PKA and GRK phosphorylation mutants caused a 6- to 7-fold increase in cAMP, similar to the wild type receptor, indicating that they were fully coupled to Gs (Fig. 2). In contrast, stimulation of the PKA–/GRK–β1AR mutant produced only a 3-fold increase in cAMP, suggesting that removal of all of the sites resulted in a general impairment of receptor function (Fig. 2). Furthermore, cells overexpressing the WTβ1AR, PKA–β1AR, or GRK–β1AR showed ∼70% desensitization measured as a reduction in catecholamine-induced cAMP generation on repeated exposure to the β1-selective agonist dobutamine. In contrast to the wild type receptor, which fully desensitized even at agonist concentrations that produced a less than maximal increase in intracellular cAMP, cells transfected with the PKA–/GRK–β1AR were not significantly desensitized by pretreatment with agonist. Consistent with the β2AR model of PKA-mediated heterologous desensitization and GRK-mediated homologous desensitization, these data suggest that β1ARs could become fully desensitized by either phosphorylation of PKA and/or GRK sites and only when all the phosphorylation sites were removed did a dramatic reduction in agonist promoted desensitization occur. Both PKA and GRKs Mediate Agonist-induced β 1AR Internalization—As opposed to desensitization, agonist-induced internalization of β2ARs is thought to be mediated predominantly through GRK phosphorylation and β-arrestin binding. To determine the contribution of PKA and GRK phosphorylation to agonist promoted β1AR internalization, we measured the loss of β1ARs from the cell surface in response to isoproterenol by radioligand binding. As shown in Fig. 3A, agonist stimulation resulted in a marked loss of WTβ1ARs from the cell surface. In contrast, at saturating agonist concentrations the extent of agonist-induced sequestration of both the PKA–β1AR and GRK–β1AR was about half that of the WTβ1AR. The double mutant exhibited minimal agonist-induced internalization. These results contrast with the desensitization data (Fig. 2), which indicate that either PKA or GRK sites alone were sufficient for full receptor desensitization, and suggest that both PKA and GRK phosphorylation are required for full internalization of the β1AR. To further examine the role of PKA and GRK phosphorylation in β1AR receptor endocytosis, we determined the dose dependence of isoproterenol-stimulated sequestration of the wild type and mutant receptors (Fig. 3B). Whereas WTβ1AR and PKA–β1AR exhibited half-maximal internalization at similar agonist concentrations, the EC50 for GRK–β1AR internalization was ∼10-fold higher (EC50 values for WTβ1AR = 56 nm, PKA–β1AR = 46 nm, GRK–β1AR = 632 nm, and PKA–/GRK–β1AR = 534 nm). Thus, two distinct mechanisms appear to contribute additively to β1AR sequestration, one a GRK site-dependent mechanism that predominates at lower agonist concentrations, and the other a PKA site-dependent mechanism that accounts for approximately half of the agonist-dependent β1AR sequestration at higher agonist concentrations. Importantly, the reduced efficiency of sequestration for the β1AR mutants was observed over a broad range of agonist concentrations (Fig. 3B). PKA-mediated β 1AR Internalization Does Not Involve β-Arrestin Recruitment to the Membrane—Previous studies have shown that GRK-mediated desensitization involves recruitment of β-arrestin to the phosphorylated β2AR (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). We therefore studied the ability of the wild type and mutant receptors to recruit GFP-β-arrestin2 to the membrane. As shown in Fig. 4A, agonist stimulation of the WTβ1AR resulted in marked translocation of β-arrestin from the cytosol to the plasma membrane. In the absence of PKA sites (PKA–β1AR), β-arrestin recruitment was significantly increased. In contrast, cells expressing the GRK–β1AR mutant showed marked impairment of β-arrestin recruitment to the membrane (Fig. 4, A and B). Removal of all phosphorylation sites in the double mutant resulted in very low β-arrestin recruitment comparable to that of the GRK–β1AR (Fig. 4, A and B). Importantly, the ability of the GRK–β1AR (PKA sites intact) to internalize (Fig. 3), despite the marked reduction of β-arrestin recruitment (Fig. 4), suggests that internalization of the β1AR can proceed via multiple mechanisms. Phosphorylation of the β 1AR Directs Internalization via Two Separate Pathways—β-Arrestin recruitment has been shown to be important for targeting the β2AR to clathrin-coated pits for internalization (25Miller W.E. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (281) Google Scholar). Because the GRK–β1AR mutant showed marked impairment of β-arrestin recruitment compared with the PKA–β1AR mutant, we sought to determine the pathway(s) of internalization following PKA- and GRK-mediated phosphorylation of the β1AR. In these experiments cells expressing FLAG epitope-tagged WTβ1AR, PKA–β1AR, GRK–β1AR, or β2AR (used as a control) were used for the internalization studies assessed by laser scanning confocal microscopy. Because caveolae have been reported to play a significant role in both the signaling and internalization of several GPCRs, cells were pretreated with two different caveolae pathway inhi
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