Agonist-dependent Recruitment of Phosphoinositide 3-Kinase to the Membrane by beta -Adrenergic Receptor Kinase 1

A ROLE IN RECEPTOR SEQUESTRATION*

Sathyamangla V. Naga PrasadDagger , Larry S. Barak§, Antonio RapacciuoloDagger , Marc G. Caron§||, and Howard A. RockmanDagger **

From the Departments of Dagger  Medicine and § Cell Biology and  Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, March 16, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Agonist-dependent desensitization of the beta -adrenergic receptor requires translocation and activation of the beta -adrenergic receptor kinase1 by liberated Gbeta gamma subunits. Subsequent internalization of agonist-occupied receptors occurs as a result of the binding of beta -arrestin to the phosphorylated receptor followed by interaction with the AP2 adaptor and clathrin proteins. Receptor internalization is known to require D-3 phosphoinositides that are generated by the action of phosphoinositide 3-kinase. Phosphoinositide 3-kinases form a family of lipid kinases that couple signals via receptor tyrosine kinases and G-protein-coupled receptors. The molecular mechanism by which phosphoinositide 3-kinase acts to promote beta -adrenergic receptor internalization is not well understood. In the present investigation we demonstrate a novel finding that beta -adrenergic receptor kinase 1 and phosphoinositide 3-kinase form a cytosolic complex, which leads to beta -adrenergic receptor kinase 1-mediated translocation of phosphoinositide 3-kinase to the membrane in an agonist-dependent manner. Furthermore, agonist-induced translocation of phosphoinositide 3-kinase results in rapid interaction with the receptor, which is of functional importance, since inhibition of phosphoinositide 3-kinase activity attenuates beta -adrenergic receptor sequestration. Therefore, agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane is an important step in the process of receptor sequestration and links phosphoinositide 3-kinase to G-protein-coupled receptor activation and sequestration.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphoinositide 3-kinases (PI3Ks)1 are a family of enzymes that can be divided into three classes based on their structure and substrate specificity (1). The class I PI3Ks are heterodimeric enzymes consisting of catalytic and regulatory subunits that are divided into the subgroups IA and IB. The class IA PI3K (consisting of p110 alpha , beta , and delta  catalytic subunits) associates with the p85 regulatory subunit and is essential for coupling signals through receptor tyrosine kinases (1). The class IB PI3K (p110gamma catalytic subunit) associates with the p101 adaptor subunit and is activated by beta gamma subunits of G-proteins (2). Stimulation of a variety of G-protein-coupled receptors (GPCRs) through Gbeta gamma -mediated activation of PI3Kgamma leads to an increase in the level of 3'-phosphorylated phosphatidylinositol (PtdIns), which in turn mediates diverse cellular effects including cell proliferation, cell survival, cytoskeletal rearrangements, and endocytosis (3).

The exposure of a GPCR to agonist produces rapid attenuation of its signaling ability that involves uncoupling of the receptor from its cognate heterotrimeric G-protein. The cellular mechanism mediating agonist-specific or homologous desensitization is a two-step process in which agonist-occupied receptors are phosphorylated by a G-protein-coupled receptor kinase and then bind arrestin proteins (4). Homologous desensitization of agonist-occupied beta -adrenergic receptors (beta ARs) occurs after translocation of G-protein-coupled receptor kinase 2 (GRK2; beta -adrenergic receptor kinase 1 (beta ARK1)) to the plasma membrane. beta ARK1 association with the plasma membrane is facilitated by binding to liberated Gbeta gamma subunits and the interaction of its pleckstrin homology domain to the membrane phospholipids (5). After beta ARK1-mediated beta AR phosphorylation, the phosphorylated receptor becomes desensitized by binding beta -arrestin (4, 6) and is targeted to the clathrin-coated pit for endocytosis (6, 7). In addition to functioning as docking proteins linking GPCRs to components of the endocytic machinery, beta -arrestins also bind other intracellular regulatory proteins such as c-Src (8).

Previous studies have shown that phospholipids are required for receptor internalization (9). In particular, the deletion of PtdIns(3,4,5)P3 binding sites from beta -arrestin results in inhibition of GPCR endocytosis (10), and binding of polyphosphoinositides to AP2 (11) is important for targeting the receptor-arrestin complex to a clathrin-coated pit (12). Since PtdIns(3,4,5)P3 is the main product catalyzed by the lipid kinase activity of P13K (3), and the extended PH domain of beta ARK1 and the helical domain of PI3Kgamma allow for a protein-protein interaction (13, 14), we explored whether beta ARK1 and PI3K might interact to promote beta AR internalization.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Mouse NIH-3T3 and HEK 293 cells were maintained in either Iscove's modified Dulbecco's medium or minimal essential medium supplemented with 10% fetal bovine serum and 1:100 penicillin-streptomycin (10,000 units/ml) at 37 °C. Cells were seeded at a density of ~1-3 × 105 cells/35-mm dish and, at 70-80% confluence, were transiently transfected using the transfection reagent FUGENE6 (Roche Molecular Biochemicals) or calcium phosphate precipitation. Cells were harvested 24 h after transfection, re-plated in triplicate, allowed to grow overnight, and serum-starved for 2-4 h before agonist stimulation.

Plasmid Constructs-- The cDNA encoding bovine beta ARK1 and the carboxyl terminus of beta ARK1 (beta ARKct) was described previously (15, 16). Genomic DNA isolated from mouse tail was used for amplification of beta 1AR gene by polymerase chain reaction with Pfu Taq polymerase (Stratagene) using the 5' primer (5'-AATTCgCCgCCATGgACTACAAggACgACgATgATAAgggCgCgggggCgCTCgCCCTg-3') containing an EcoRI site for subcloning followed by Kozak consensus sequence and a FLAG tag and 3' primer (5'-AAGCTTCTACTTGGACTCCGAGGA-3') containing a consensus stop codon with a HindIII site for subcloning. Pfu Taq polymerase-amplified PCR product was subcloned in zero-blunt TOPO vector (Invitrogen) and was cut with EcoRI/HindIII and subcloned into pRK5 mammalian expression vector. The cloned beta 1AR in pRK5 was then sequenced to check for its authenticity. FLAG-tagged beta 2AR was a generous gift from Dr. Robert J. Lefkowitz. HA-tagged pCMV-PI3Kp110gamma wild type (PI3Kgamma ), HA-tagged pCMV-PI3Kp110gamma mutant (Delta PI3Kgamma ) (Delta 942-981, deletion in ATP binding site) were generous gifts from Dr. Charles S. Abrams (17). Myc-PI3Kalpha was a generous gift from Dr. Michael J. Waterfield.

Cytosolic and Membrane Fractionation-- Cell monolayers were scraped in 1 ml of buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml each leupeptin and aprotinin and disrupted further by using Dounce homogenizer. Intact cells and nuclei were removed by centrifugation at 1,000 × g for 5 min. The collected supernatant was further subject to a centrifugation at 38,000 × g for 25 min. The pellet was resuspended in lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 20 mM Tris-Cl (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 2 µg/ml each aprotinin and leupeptin) and used as membrane fraction, and the supernatant was diluted in lysis buffer and used as the cytosolic fraction. Heart samples also underwent similar procedures to obtain membrane and cytosolic fractions. Purity of the membrane fraction preparation was confirmed by measuring enzyme activity of the membrane marker enzyme K+-stimulated p-nitrophenylphosphatase (18) (membrane fraction: 9.4 µmol/mg of protein/min, cytosol: 2.6 µmol/mg of protein/min).

Lipid Kinase Assays-- PI3K assays were carried out as previously described (19). Briefly, cells were lysed in lysis buffer, and cytosolic extract was used for immunoprecipitation with either the C5/1 monoclonal antibody directed against beta ARK1 (20) or the anti-FLAG M2 monoclonal antibody (Sigma). Beads were washed and assayed for PI3K activity. The organic phase was spotted on TLC plates and resolved by chromatography. No associated background PI3K activity was found with beads used for immunoprecipitation (data not shown). TLC plates were subjected to autoradiography, and PIP was quantified by phosphorimaging. PtdIns(4)P (Sigma) was used as a standard. Lipids were prepared as previously described (19).

Immunoblotting and Detection-- Immunoblotting and detection of PI3K, beta ARK1, HA-PI3Kgamma , HA-Delta PI3Kgamma , and Myc-PI3Kalpha were carried out as previously described (19, 20). Immunoprecipitating antibodies were added to 500 µg of cell lysate in lysis buffer followed by the addition of 35 µl of 1:1 protein A- or G-agarose. Samples were rocked overnight at 4 °C then centrifuged at 12,000 × g for 5 min. Immunoprecipitates were washed twice with lysis buffer, twice with 1× phosphate-buffered saline, and resuspended in 1× SDS gel loading buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (Bio-Rad). Blots were incubated with antibodies recognizing PI3K, Myc (Santa-Cruz), and HA (Roche Molecular Biochemicals) at 1:2000 dilution and the beta ARK1 monoclonal antibody at 1:10,000 dilution. Blots were subsequently incubated with appropriate secondary antibody (1:2000 dilution) conjugated to horseradish peroxidase (Amersham Pharmacia Biotech), and detection was carried out using enhanced chemiluminescence.

Determination of beta 2AR Sequestration in HEK 293 Cells by 125I-Cyanopindolol (CYP) Binding-- beta 2AR sequestration was performed as previously described (7). Briefly, HEK 293 cells were plated at a density of 2.5 × 106 cells/dish and transfected the following day with plasmids containing either the beta 2AR (150-250 ng), PI3Kgamma (4.0 µg), or Delta PI3Kgamma (4.0 µg) cDNAs. Twelve hours after transfection, cells were split into six-well Falcon plates at a density of 750,000 cells/well. The following day the media was replaced with minimal essential medium containing 1 µM isoproterenol and 100 µM ascorbate for 0 to 30 min. In separate experiments, cells transfected with beta 2AR were treated with the PI3K inhibitors wortmannin (100 nM) or LY294002 (100 µM) for 15 min before isoproterenol stimulation. To determine the amount of internalized receptor, 100-µl aliquots of whole cells were added to 150 µl of binding buffer (75 mM Tris-HCl, 10 mM MgCl2, 5 mM EDTA (pH 7.5)). Total binding was determined in the presence of 175 pM 125I-CYP alone, the number of internalized receptors was determined by using 175 pM 125I-CYP plus 100 nM CGP12177, and nonspecific binding was determined using 175 pM 125I-CYP plus 1 µM propranolol (7). Sequestration was calculated as the ratio of (specific receptor binding of 125I-CYP in the presence of CGP12177)/(specific receptor binding of 125I-CYP in the absence of CGP12177).

Confocal Microscopy and beta 2AR Phosphorylation-- Confocal microscopy was performed as previously described (21). In brief, HEK 293 cells were transfected with plasmids containing the beta 2AR (2 µg) and beta -arrestin-GFP (2 µg) or the beta 2AR and beta -arrestin-GFP along with either PI3Kgamma (2.5 µg) or Delta PI3Kgamma (2.5 µg). Cells were split into 35-mm dishes with glass bottoms for observation using a Zeiss LSM-510 confocal microscope. The cells were treated with isoproterenol (1 µM) for the indicated times (0 to 5 min), and the fluorescence images were exported as Tiff files by the LSM software to Adobe Photoshop. beta 2AR phosphorylation was performed in intact cells as previously described (7). Cells were transfected with plasmids containing the FLAG beta 2AR (2 µg) and vector DNA (2.5 µg), the FLAG beta 2AR (2 µg) and PI3Kgamma (2.5 µg), or the beta 2AR (2 µg) and Delta PI3Kgamma (2.5 µg). 24 h after transfection, cells were washed and metabolically labeled for 1 h with medium containing 100 µCi of 32P/ml. After stimulation with 10 µM isoproterenol for 5 min, incubations were terminated by adding 2 ml of ice-cold phosphate-buffered saline/well, and then cells were solubilized with the addition of 0.75 ml/well of radioimmune precipitation buffer. After centrifugation at 38000 × g for 20 min at 4 °C, the supernatants were processed for immunoprecipitation of the FLAG-beta 2AR as described above. Phosphorylated receptors were resolved by 10% SDS-polyacrylamide gel electrophoresis, and dried gels were subjected to autoradiography.

In Vivo Pressure Overload Hypertrophy and Isoproterenol Infusion-- Four-month-old adult C57BL/6 wild type mice of either sex were used for this study. Microsurgical procedures and hemodynamic evaluation of pressure overload hypertrophy induced through transverse aortic constriction (T) was performed as previously described (19, 22). After 7 days of aortic constriction, mice were anesthetized, and both carotid arteries were cannulated to measure the trans-stenotic pressure gradient (22). Hearts were then rapidly excised, and individual chambers were separated, weighed, and frozen in liquid N2 for later biochemical analysis. In separate experiments, adult wild type mice underwent intravenous infusion of 10 µM isoproterenol for 3 min at 50 µl/min. Hearts were removed and flash-frozen in liquid N2 for later biochemical analysis. The animals in this study were handled according to approved protocols by the animal welfare regulations of Duke University Medical Center.

Statistical Analysis-- Data are expressed as means ± S.E. Statistical comparisons was performed using an unpaired Student's t test. Results for the beta 2AR sequestration by CYP binding was analyzed using Graphpad Prism.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta ARK1 and PI3K Form a Complex in the Cytoplasm-- Since both beta ARK1 and PI3Kgamma are activated by beta gamma subunits of G-proteins, we explored whether they interact to form a complex in the cytosol. We used a mouse NIH-3T3 cell line since it has a relatively low level of endogenous PI3K activity compared with other cell lines (data not shown). Cells were transfected with beta ARK1 and HA-PI3Kgamma cDNAs, and beta ARK1 was immunoprecipitated from extracts using a monoclonal antibody directed against the beta ARK1 catalytic domain (20). As shown in Fig. 1, A-C, PI3K activity and protein were found associated with beta ARK1. The association was independent of PI3K activity since co-transfection with a catalytically inactive PI3K mutant (HA-Delta PI3Kgamma , deletion in the ATP binding site) did not prevent the association of the PI3K mutant with beta ARK1 (Fig. 1C). Furthermore, the association of PI3K with beta ARK1 occurred with either PI3K isoform (Fig. 1D), and the associated PI3K activity was wortmannin-sensitive (Fig. 1E). Lysates from cell extracts before immunoprecipitation were immunoblotted for beta ARK1, PI3Kgamma , and Delta PI3K to determine levels of expression.


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Fig. 1.   beta ARK1 and PI3K interact to form a cytosolic complex. NIH-3T3 cells were transfected with the cDNAs represented in the boxes on the top of the panel. The control (C) represents transfection with an equivalent quantity of vector DNA. A, 48 h after transfection, 500 µg of cytosolic extract was immunoprecipitated using beta ARK1 monoclonal antibody directed against its catalytic domain, and the associated lipid kinase activity was measured. Shown is a representative autoradiograph of a TLC plate where PIP and phosphatidylinositol bisphosphate (PIP2) are visualized. Ori, origin of resolution. B, beta ARK1-associated PI3K activity quantified by phosphorimaging of the TLC plates from four independent experiments. Results are expressed as fold over control. *, p < 0.05 versus control. IP, immunoprecipitation. C, immunoprecipitations (IP) were performed from cytosolic extracts with an anti-HA or anti-beta ARK1 monoclonal antibody and immunoblotted (IB) with a beta ARK1 monoclonal (upper panel) or anti-HA monoclonal antibody (lower panel). beta ARK1- and HA-PI3Kgamma -transfected cells were used as positive controls (last lane). D, 500 µg of cytosolic extract was immunoprecipitated with a beta ARK1 monoclonal antibody, and the associated lipid kinase activity was measured. Shown is a representative autoradiograph where PIP is visualized. E, 500 µg of cytosolic extract was immunoprecipitated with a beta ARK1 monoclonal antibody, and the associated PI3K activity was measured from cells with and without treatment with 100 nM wortmannin (Wort) for 15 min before lysis. Lysates from cytosolic extracts before immunoprecipitation were used to monitor the levels of beta ARK1 and HA-PI3Kgamma expression.

Agonist-dependent beta ARK1-mediated Translocation of PI3Kgamma -- Since beta ARK1 associates with PI3K, we tested whether beta ARK1 could also promote translocation of PI3K to the cell membrane in response to agonist stimulation. Experiments were performed in NIH-3T3 cells co-transfected with beta ARK1 and PI3Kgamma cDNAs, and the endogenous beta AR receptors were stimulated with 10 µM isoproterenol for 2 min. Cytosolic and membrane fractions were prepared and analyzed for beta ARK1-associated PI3K activity. Little change in the beta ARK1-associated PI3K activity was noted in the cytosolic fraction after isoproterenol stimulation (Fig. 2A). In contrast, the membrane fraction showed a significant increase in the beta ARK1-associated PI3K activity (Fig. 2, A and D) at 2 min after agonist stimulation. The beta ARK1-mediated recruitment of PI3Kgamma to the membrane was analyzed by immunoprecipitating beta ARK1 from both fractions and blotting for HA-PI3K. As shown in Fig. 2B, treatment with isoproterenol resulted in a greater level of beta ARK1-associated PI3Kgamma protein in the membrane. Taken together these data show that beta ARK1 and PI3Kgamma interact to form a complex in the cytosol, and beta ARK1 recruits PI3Kgamma to the membrane in an agonist-dependent manner.


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Fig. 2.   beta ARK1 and PI3Kgamma form a complex in the cytosol and translocate to the membrane on agonist stimulation in a Gbeta gamma -dependent manner. A, NIH-3T3 cells were transfected with beta ARK1 and HA-PI3Kgamma and stimulated with isoproterenol (ISO, 10 µM) for a period of 2 min. 250 µg of protein from the membrane and cytosolic fraction was used to immunoprecipitate (IP) beta ARK1 using beta ARK1 monoclonal antibody and assayed for associated PI3K activity. Shown is a representative autoradiograph of a TLC plate where PIP is visualized (samples in duplicate except for control (C), which was transfected with vector DNA and treated with isoproterenol). -ISO, no treatment with isoproterenol; +ISO, treatment with isoproterenol. B, beta ARK1 was immunoprecipitated from the membrane and cytosolic fractions with a beta ARK1 monoclonal antibody and immunoblotted (IB) with an anti-HA monoclonal antibody. Cells transfected with HA-PI3Kgamma cDNA were used as a positive control. MEM, membrane fraction; CYTO, cytosolic fraction. C, cells were co-transfected with beta ARK1 and PI3Kgamma cDNAs along with or without the beta ARKct cDNA (the carboxyl terminus of beta ARK1 that attenuates the Gbeta gamma -dependent signaling). 250 µg of protein from the membrane and cytosolic fraction was used to immunoprecipitate (IP) beta ARK1 with a beta ARK1 monoclonal antibody and assayed for associated PI3K activity after isoproterenol stimulation (2 min). Shown is a representative autoradiograph of the TLC plate where PIP and phosphatidylinositol bis-phosphate (PIP2) are visualized. D, beta ARK1-associated PI3K activity was quantified by phosphorimaging the TLC plates from four independent experiments. Results are expressed as fold over basal (no isoproterenol treatment). *, p < 0.05 versus membrane fraction without isoproterenol treatment. E, NIH-3T3 cells were transfected with the cDNAs represented in the boxes on the top of the panel. Cytosolic extracts were used for immunoprecipitation (IP) with a monoclonal HA-PI3Kgamma or Myc-PI3Kalpha antibody and immunoblotted (IB) with a polyclonal antibody against the carboxyl terminus of beta ARK1 that recognizes both full-length beta ARK1 and the beta ARKct. Control (C), cells transfected with vector DNA. Lysates from cytosolic extracts before immunoprecipitation were used to monitor the level of expression of PI3K, beta ARK1, and beta ARKct. Endogenous beta AR density of NIH-3T3 cells was determined by a 125I-cyanopindolol binding study and found to contain 20.7 ± 3.2 fmols/mg of whole cell protein, which is sufficient to activate and translocate beta ARK1 after agonist stimulation (34, 35).

Agonist-dependent Translocation of beta ARK1-associated PI3K Activity Is Gbeta gamma -dependent-- The agonist-dependent translocation of beta ARK1 to the membrane requires the presence of Gbeta gamma subunits (5). We tested whether Gbeta gamma subunits were required for the translocation of the beta ARK1·PI3Kgamma complex to the membrane by overexpressing the carboxyl-terminal portion of beta ARK1, the beta ARKct, which is known to inhibit the ability of beta ARK1 to bind Gbeta gamma (15). Cells were co-transfected with the beta ARK1 and PI3Kgamma plasmids along with or without the beta ARKct cDNA. Robust PI3K activity was found associated with immunoprecipitated beta ARK1 in the absence of beta ARKct (Fig. 2C). In contrast, the agonist-dependent translocation of beta ARK1-associated PI3K activity was abolished in the presence of the beta ARKct (Fig. 2, C and D). These data suggest that the sequestration of Gbeta gamma by beta ARKct can interrupt the process of beta ARK1-mediated translocation of PI3Kgamma to the membrane (Fig. 2D). Interestingly, in the presence of beta ARKct, there was also loss of beta ARK1-associated PI3K activity in the cytosolic fraction (Fig. 2, C and D). Since the beta ARKct contains the same PH domain as beta ARK1, it appears that beta ARKct competitively inhibits the beta ARK1/PI3K interaction. We tested this using cells expressing beta ARK1, beta ARKct, and either PI3Kgamma or PI3Kalpha followed by immunoprecipitation with antibodies directed against either HA-PI3Kgamma or Myc-PI3Kalpha . As shown in Fig. 2E, both beta ARK1 and beta ARKct were found to interact with either of the PI3K isoforms. To exclude the possibility that overexpression of the beta ARKct altered the level of expression of either beta ARK1 or PI3Kgamma , lysates prepared from the co-transfected cells were immunoblotted for beta ARK1, beta ARKct, and PI3Kgamma . No significant difference in the level of expression levels for either beta ARK1 or PI3Kgamma was seen in presence of the beta ARKct (data not shown).

beta ARK1 Translocates PI3K to beta AR-- The ability of beta ARK1 to translocate PI3K to the cell membrane suggests a mechanism for co-localization of PI3K with beta ARs. To test this, we used HEK 293 cells transfected with either FLAG epitope-tagged beta 1AR or beta 2AR plasmids and monitored the association of PI3K to the receptor after agonist stimulation. HEK 293 cells are known to contain adequate levels of beta ARK1 to support agonist-induced receptor phosphorylation (8). Transfected cells were split into separate dishes and stimulated with 10 µM isoproterenol from 0 to 10 min. The FLAG epitope was immunoprecipitated from cell extracts, and PI3K activity was measured. As shown in Fig. 3, A-D, FLAG beta 1AR- and FLAG beta 2AR-associated PI3K activity was observed by 2 min after agonist stimulation, with gradual decline by 10 min. Moreover, the PI3K activity associated with the FLAG beta 1AR and beta 2AR was wortmannin-sensitive (Fig. 3E). In addition, we could also detect the agonist-dependent association of endogenous PI3Kgamma protein with the transfected FLAG beta 2AR at 2-10 min after isoproterenol treatment (data not shown).


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Fig. 3.   beta ARK1 translocates PI3Kgamma to the beta -adrenergic receptor. A, HEK 293 cells were transfected with FLAG beta 1AR and split into separate dishes that were then individually treated with 10 µM isoproterenol for the indicated times. Control (C), transfected with vector DNA. At indicated times points, cell monolayers were scraped, and 500 µg of protein from the cytosolic extract was used for immunoprecipitation (IP) with an anti-FLAG monoclonal antibody, and the associated PI3K activity was measured. Shown is a representative autoradiograph of the TLC plate where PIP is visualized. B, FLAG beta 1AR-associated PI3K activity was quantified by phosphorimaging of the TLC plates from five independent experiments. Results are expressed as fold over basal (no isoproterenol treatment). *, p < 0.05 versus 0 min. C, as for panel A, except cells were transfected with FLAG beta 2AR cDNA. D, as for panel B, except the levels of FLAG beta 2AR-associated PI3K activity was quantified. *, p < 0.05 versus 0 min. E, HEK 293 cells were transfected with FLAG beta 1AR (upper panel) and FLAG beta 2AR (lower panel). A set of transfected cells was treated with 100 nM wortmannin 15 min before agonist stimulation. Both wortmannin-treated and untreated cells were then stimulated with 10 µM isoproterenol for the indicated times. Cell extracts were used to immunoprecipitate beta 1AR and beta 2AR using the anti-FLAG antibody, and PI3K activity was measured in the immunoprecipitates. Shown are the autoradiograph of TLC plates where PIP was visualized.

PI3K Activity Is Required for the Sequestration of the Receptor-- Because beta 2AR endocytosis is regulated by beta ARK1, and beta ARK1-mediated PI3K translocation provides a mechanism for the interaction of PI3K with receptor, we determined whether the PI3K inhibitors, wortmannin and LY294002, could attenuate beta AR internalization. Agonist-dependent sequestration was studied in HEK 293 cells transfected with the FLAG beta 2AR plasmid. As shown in Fig. 4A, a significant attenuation in the rate of beta 2AR sequestration was observed in wortmannin-treated cells for up to 20-30 min. Similarly, a 50% reduction in sequestration was also observed with LY294002-treated cells (data not shown). To directly address whether PI3K is required for the process of beta AR sequestration, a time course of agonist-dependent sequestration was studied in HEK 293 cells transfected either with the FLAG beta 2AR, FLAG beta 2AR and PI3Kgamma , or FLAG beta 2AR and Delta PI3Kgamma plasmids. Similar to the pattern of inhibition by wortmannin, Delta PI3Kgamma -transfected cells showed a significant attenuation in the rate of receptor sequestration (Fig. 4B). These data demonstrate a role for PI3K in the process of beta AR internalization possibly due to the local production of PtdIns(3,4,5)P3.


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Fig. 4.   PI3K activity is required for beta AR sequestration. A, agonist promoted (1 µM isoproterenol) beta 2AR sequestration in transfected HEK 293 cells studied by 125I-cyanopindolol binding over a time course of 0-30 min in untreated (black-square) and wortmannin (100 nM)-treated () cells. Receptor sequestration at each time point has been normalized to the value of internalized beta 2AR at 30 min in the absence of wortmannin. The maximal absolute level of sequestration for the beta 2AR in the untreated cells at 30 min was 22.3 ± 3.9%, n = 5. Receptor expression (fmol/mg of whole-cell protein) was 378 ± 123; *, p < 0.05 versus untreated. Inset, upper panel, inhibition of endogenous lipid kinase activity with wortmannin. beta 2AR and + represents beta 2AR-transfected wortmannin-treated cells; beta 2AR and - represents beta 2AR-transfected untreated cells. The first lane represents endogenous PI3K activity in the untransfected cells. Lower panel, the level of endogenous PI3Kgamma expression. B, agonist-promoted beta 2AR sequestration in cells co-transfected with beta 2AR along with either empty vector (black-square) or PI3Kgamma () or Delta PI3Kgamma (open circle ) cDNAs in HEK 293 cells. Receptor expression (fmol/mg of whole-cell protein) was: beta 2AR + vector, 316 ± 75; beta 2AR + PI3Kgamma , 367 ± 81; beta 2AR + Delta PI3Kgamma , 267 ± 157. n = 5, *, p < 0.05 versus beta 2AR. Inset upper panel, immunoblot showing the levels of expression of HA-PI3Kgamma , HA-Delta PI3Kgamma , and PI3Kgamma in the transfected cells. Cells were transfected with beta 2AR (-), beta 2AR and wild type PI3Kgamma (WT) or beta 2AR and Delta PI3Kgamma (Delta ) cDNAs. Inset lower panel, lipid kinase activity in the HEK 293 cells transfected with the PI3Kgamma (WT) and Delta PI3Kgamma (Delta ) cDNAs. The receptor sequestration at each time point has been normalized to the value of internalized beta 2AR at 30 min. The maximal absolute level for the beta 2AR in the untreated cells at 30 min was 25.0 ± 2.1%, n = 5. C, beta -arrestin 2-GFP recruitment to the membrane on isoproterenol stimulation (1 µM) was monitored by confocal microscopy in HEK 293 cells transfected with beta 2AR and beta -arrestin 2-GFP cDNAs along with either empty vector pRK5, PI3Kgamma , or Delta PI3Kgamma at the indicated times. Marked redistribution of beta -arrestin 2-GFP to the membrane occurs within 2.5 min. Arrows on the 5-min panel highlight regions of translocated beta -arrestin 2-GFP. The upper panel is an immunoblot for HA from extracts of the same cells showing equal levels of HA-PI3Kgamma and HA-Delta PI3Kgamma expression. D, agonist (isoproterenol (ISO), 5 min) promoted beta 2AR phosphorylation in transfected cells after 32Pi metabolic labeling for 1 h before stimulation (upper panel). Lower panel, immunoblot for HA showing equal levels of expression of HA-PI3Kgamma and HA-Delta PI3Kgamma . + represents HA-PI3Kgamma control. IB, immunoblot; IP, immunoprecipitate.

Alteration of receptor endocytosis with overexpression of the Delta PI3Kgamma mutant could result if Delta PI3Kgamma inhibited either recruitment of beta -arrestin to the phosphorylated receptor or directly inhibited receptor phosphorylation. To exclude these possibilities, cells were co-transfected with plasmids containing FLAG beta 2AR, beta -arrestin 2-GFP, and either PI3Kgamma or Delta PI3Kgamma cDNAs and were monitored for the recruitment of beta -arrestin 2-GFP using confocal microscopy. In cells transfected with Delta PI3Kgamma , there was prompt recruitment of beta -arrestin 2-GFP to the receptor after exposure to agonist (Fig. 4C). To directly test the effect of Delta PI3Kgamma expression on receptor phosphorylation, 32Pi metabolic labeling was performed in HEK 293 cells transfected with plasmids containing FLAG beta 2AR and either the PI3Kgamma or Delta PI3Kgamma cDNAs. As shown in Fig. 4D, the level of agonist-induced receptor phosphorylation was not affected by the expression of Delta PI3Kgamma . Thus, the effect of overexpression of Delta PI3Kgamma on beta 2AR sequestration is not related to processes that are involved with beta 2AR phosphorylation or beta -arrestin 2 recruitment.

beta ARK1 and PI3K Form a Complex in the Heart-- Since normal beta AR function is critical for maintenance of cardiac function, particularly during periods of increased workload (23), we wanted to determine whether PI3K and beta ARK1 interact in an organ of in vivo relevance such as the heart. The monoclonal beta ARK1 antibody was used to immunoprecipitate beta ARK1 from myocardial extracts prepared from normal mouse hearts. The immunoprecipitate was tested for the presence of associated PI3K protein by immunoblotting. As a positive control, the PI3K polyclonal antibody was used to immunoprecipitate total PI3K from separate extracts prepared from the same heart. As shown in Fig. 5A, PI3K was co-immunoprecipitated along with beta ARK1 from the myocardial extract. To determine whether the association of beta ARK1 with PI3K in the heart would also result in the beta ARK1-mediated translocation of PI3K to the membrane, anesthetized mice were stimulated with isoproterenol (10 µM) by infusion through a cannulated jugular vein for 3 min. Membrane and cytosolic fractions were prepared from the treated hearts, and PI3K activity was measured after immunoprecipitation with the beta ARK1 monoclonal antibody. As show in Fig. 5B, a significant increase in beta ARK1-associated PI3K activity was found in the membrane fraction after isoproterenol treatment (fold induction over untreated, 5.60 ± 1.10, p < 0.05, n = 3). No difference in beta ARK1-associated PI3K activity was found in the cytosolic fraction after isoproterenol treatment (fold induction over untreated, 1.19 ± 0.47, n = 3). We previously documented an induction of both beta ARK1 and PI3Kgamma in the pressure overloaded heart (19, 20) and, therefore, tested whether the interaction of beta ARK1 with PI3K would also be enhanced in the hypertrophied heart. As shown in Fig. 5, C and D, greater wortmannin-sensitive PI3K activity and protein were found complexed with beta ARK1 in hypertrophied hearts compared with sham-treated hearts.


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Fig. 5.   beta ARK1 and PI3K form a complex in heart. A, 4 mg of myocardial extract from a mouse heart was used to immunoprecipitate (IP) beta ARK1 (left lane), and 2 mg of the extract was used to immunoprecipitate PI3K as positive control (right lane). IB, immunoblot. B, myocardial membrane fractions were prepared, and beta ARK1 was immunoprecipitated using beta ARK1 monoclonal antibody, assayed for the associated PI3K activity. -ISO, hearts without isoproterenol treatment; +ISO, hearts treated with isoproterenol. C, beta ARK1-associated PI3K activity was measured in the myocardial extracts from the sham (S) and transverse aortic constricted (T) hearts (19, 20). 4 mg of the myocardial extracts was used for immunoprecipitation with beta ARK1 monoclonal antibody and then assayed for the PI3K activity (fold induction in T over S 2.1 ± 0.13; p, < 0.05, n = 3). -Wort and +Wort, reactions performed in the absence or presence of wortmannin. D, myocardial extracts from sham (S) and T hearts were used to immunoprecipitate beta ARK1 and PI3Kgamma with a monoclonal antibody directed against beta ARK1 (4 mg of extract) and a polyclonal antibody for PI3Kgamma (2 mg of extract), respectively, as a positive control (C). The protein bands were visualized using ECL chemiluminescence.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is increasing evidence for a requirement of D-3 phosphoinositides in the process of membrane trafficking and receptor internalization (9-12). Since, PtdIns(3,4,5)P3 is the main product catalyzed by the lipid kinase activity of P13K (3), we investigated whether there is a mechanism that links the agonist-dependent recruitment of PI3K to GPCR endocytosis. The data presented here provide evidence for an interaction between beta ARK1 and PI3K in the cytosol. Then, in response to agonist, PI3K undergoes beta ARK1-mediated translocation to the membrane. Furthermore, the beta ARK1-dependent translocation of PI3K allows for colocalization of PI3K with the receptor, which is of functional importance since catalytically inactive PI3K or inhibitors of PI3K activity attenuate beta AR sequestration. These studies thus demonstrate that the agonist-dependent recruitment of PI3K to the membrane is an important step in the process of receptor sequestration and links class I PI3Ks to GPCR signaling and endocytosis.

Recent structural studies of PI3Kgamma show that it contains many modular regions including a helical domain (also called a HEAT sequence motif) (14) that could be involved in protein-protein interactions (24, 25). Since the HEAT sequence is common to PI3Kalpha , -beta , and -gamma isoforms, it is possible that this is the common interacting domain for all PI3Ks. Indeed, in this study we show that beta ARK1 can interact with both PI3Kgamma and -alpha isoforms but cannot rule out the possibility of beta ARK1 interacting with PI3Kbeta , since it was not tested. In this regard there is increasing interest in the role of the PI3Kbeta isoform in cell signaling as shown by a recent study demonstrating activation of downstream signal molecules like v-Akt (protein kinase B) and protein kinase Cepsilon by PI3Kbeta after GPCR stimulation (26). Thus, the HEAT domain of PI3K could provide the necessary structure for the interaction of PI3Ks with beta ARK1. Depending on the cellular signaling pathway that is activated (i.e. growth factor stimulation versus GPCR stimulation), the PI3K isoform that interacts with beta ARK1 might change.

Our data are consistent with a mechanism that allows beta ARK1 to mediate agonist-dependent translocation of PI3Kgamma to the membrane. Several studies have shown activation of PI3Kgamma in the presence of Gbeta gamma but have not documented PI3Kgamma recruitment to the membrane after agonist stimulation. Our data suggest that a PI3K molecule interacting with beta ARK1 is recruited to the plasma membrane by beta ARK1, and this recruitment is Gbeta gamma -dependent, since expression of the beta ARKct (a Gbeta gamma -sequestering peptide) interrupted the beta ARK1-mediated translocation of PI3K to the membrane. It is interesting that there was a loss of beta ARK1-associated PI3K activity in cytosolic fractions with overexpression of beta ARKct despite the equal expression of beta ARK1 or PI3Kgamma . These data indicate that the beta ARKct can compete with beta ARK1 for interaction with PI3Kgamma and further points to the carboxyl-terminal PH domain of beta ARK1 as the interacting domain, since it is common within both beta ARKct and beta ARK1. This conclusion is also supported by our data showing that both the beta ARKct and beta ARK1 can be co-immunoprecipitated along with either of the PI3K isoforms. Based on these experiments, it appears that overexpression of the beta ARKct acts to inhibit the recruitment of beta ARK1 to the membrane by sequestering Gbeta gamma subunits (16) and may also directly interrupt the beta ARK1/PI3Kgamma interaction.

A previous study shows that the PI3K yeast homologue, Vps34p, plays an important role in endocytosis (27). Furthermore, a recent study shows that treatment with wortmannin blocks agonist-induced beta 2AR endocytosis (28). Our data clearly show that in transfected HEK 293 cells, the presence of agonist promotes the association of wortmannin-sensitive PI3Kgamma activity with both beta 1- and beta 2-adrenergic receptors. Moreover, treatment with PI3K inhibitors or overexpression of catalytically inactive PI3K attenuates beta 2AR sequestration.

Previous studies show that both the clathrin adaptor molecule AP2 (11) and beta -arrestins (10) bind PtdIns(3,4,5)P3 with high affinity and PtdIns (4,5)P2 at a 1000-fold lower affinity. Therefore, in our experiments with overexpression of Delta PI3K, generation of PtdIns(3,4,5)P3 would be inhibited, resulting in an increase in the concentration of PtdIns(4,5)P2, which would be much less efficient in promoting receptor endocytosis. Studies in macrophages have shown inhibition of phagocytosis in the presence of the PI3K inhibitor wortmannin, and this inhibition prevented recruitment of dynamin to endocytic vesicles (29). This suggests the possibility that generation of phosphoinositides by PI3K within the receptor complex may regulate internalization of beta 2ARs through recruitment of dynamin (30). Importantly, whereas phosphoinositides have been implicated in receptor internalization after platelet-derived growth factor (31) and insulin receptor (9) stimulation, we provide evidence here for the involvement of phosphoinositides after beta AR stimulation. Taken together these data demonstrate an important role of PI3Kgamma in the process of beta AR internalization, possibly due to the local production of PtdIns(3,4,5)P3.

Although the observed attenuation of beta AR sequestration by Delta PI3K suggests a critical role for generation of phosphoinositides in beta AR sequestration, it is also possible that Delta PI3K interrupts either beta -arrestin recruitment to the phosphorylated receptor or directly inhibits beta ARK1-mediated beta AR phosphorylation by sequestering Gbeta gamma . To exclude these possibilities we showed that overexpression of Delta PI3K inhibits neither the beta -arrestin 2-GFP recruitment to the receptor nor beta ARK1-mediated beta 2AR phosphorylation. These data suggest that inhibition of beta AR internalization is not linked to events that are involved with phosphorylation and desensitization of the receptor.

To determine the association of PI3K with beta ARK1 in a tissue of in vivo relevance, we studied the heart under several conditions. In the unstimulated heart, beta ARK1 and PI3K form a complex in myocardial extracts. Importantly, after isoproterenol stimulation there was increased beta ARK1-associated PI3K activity in myocardial membranes corroborating the cell culture studies. We have shown in earlier experiments an increase in both beta ARK1 and PI3Kgamma activity in the pathophysiological state of in vivo pressure overload hypertrophy (19, 20). Our present results show that in the hypertrophied heart there is an increase in beta ARK1-associated PI3K activity and protein that may be important for the regulation of adrenergic receptors during this pathologic state. For instance, under conditions of in vivo pressure overload hypertrophy, there would be a higher level of beta ARK1-mediated PI3K recruitment to the membrane. This would lead to both diminished receptor number (due to more efficient receptor sequestration) and impaired receptor function (enhanced phosphorylation from increased beta ARK1 activity). Consistent with this hypothesis are the characteristic findings of diminished beta AR number and reduced beta AR coupling to G-proteins in failing human hearts and in experimental models of heart failure (23, 32). Our studies suggest that in pathophysiological states such as hypertrophy and heart failure, alterations in PI3K activation and recruitment may contribute to abnormalities in beta AR function.

A recent study using rat cardiomyocytes links the v-Akt (protein kinase B)-glycogen synthase kinase 3beta pathway to activation of atrial natriuretic factor (ANF) transcription (26), which is widely considered a marker for cardiac hypertrophy and heart failure. Furthermore, studies using adenoviral-mediated expression system in neonatal rat cardiomyocytes show that activation of glycogen synthase kinase 3beta , a negative regulator of cardiomyocyte hypertrophy, is mediated by a PI3K pathway (33). These studies are consistent with our earlier findings of activation of PI3K in hearts with pressure overload hypertrophy (19) and, combined with our present study, support our view for an important role of phosphoinositides and PI3K in cardiac hypertrophy and failure.

Based on our study we propose the idea that agonist-stimulated recruitment of PI3K is linked to beta AR internalization. PI3K that colocalizes with the receptor may serve to increase the local membrane concentration of PtdIns(3,4,5)P3, thus enhancing the functional interaction of determinants of the endocytic process such as beta -arrestin, AP-2, clathrin, and dynamin. Whether the process leads to a more efficient recruitment of GPCRs to preformed clathrin-coated pits or the newly formed pits are interesting possibilities to explore further.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL56687 (to H. A. R.), NS19576 (to M. G. C.), and HL61365 (to L. S. B.) and the Burroughs Wellcome Fund (to H. A. R.).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.

|| An Investigator of the Howard Hughes Medical Institute.

** A recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. To whom correspondence should be addressed: Dept. of Medicine and Cell Biology, Duke University Medical Center, CARL Bldg., Rm. 226, DUMC 3104, Durham, NC 27710. Tel.: 919-668-2520; Fax: 919-668-2524; E-mail: h.rockman@duke.edu.

Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M102376200

    ABBREVIATIONS

The abbreviations used are: PI3K, phosphoinositide 3-kinase; GPCR, G-protein-coupled receptor; beta AR, beta -adrenergic receptor; beta ARK1, beta -adrenergic receptor kinase 1; beta ARKct, carboxyl-terminal peptide of beta -adrenergic receptor kinase; PtdIns, phosphatidylinositol; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; PIP, phosphatidylinositol monophosphate; T, transverse aortic constriction; GFP, green fluorescent protein; HEK cells, human embryonic kidney cells; PCR, polymerase chain reaction; HA, hemagglutinin; CYP, cyanopindolol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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