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Internalization of β-adrenergic receptors (βARs) occurs by the sequential binding of β-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that βARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from βARK1 and prevents βARK1-mediated translocation of PI3K to activated β2ARs. Furthermore, disruption of the βARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the β2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to βARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.
β-Adrenergic receptors (βARs)* are a member of the large family of G protein–coupled receptors (GPCRs) (Johnson, 1998; Rockman et al., 2002). In the heart, ligand activation of βARs results in the dissociation of the cognate heterotrimeric GTP binding protein (G-protein) into Gα and Gβγ subunits, leading to stimulation of the effector adenylyl cyclase and subsequent physiologic response (Koch et al., 2000). Exposure to agonist also promotes the rapid desensitization of βARs, that not only leads to the attenuation of signaling, but also targets the activated receptor to clathrin-coated pits for internalization (Ferguson et al., 1996; Goodman et al., 1996). An early step in this process involves the rapid phosphorylation of agonist-occupied receptors by a G protein–coupled receptor kinase (GRK, commonly known as the β-adrenergic receptor kinase or βARK) (Lefkowitz, 1998). The phosphorylation of activated βARs by βARK1 requires translocation of the primarily cytosolic βARK to the plasma membrane, a process facilitated by the liberated Gβγ subunits and membrane phospholipids (Pitcher et al., 1998). The second step involves binding of the protein β-arrestin to the phosphorylated receptor resulting in termination of the signal (Lefkowitz, 1998). The binding of β-arrestin serves to target phosphorylated βARs for internalization, through the recruitment of the AP-2 adaptor protein and clathrin to the receptor complex (Laporte et al., 2000). There is increasing evidence that phosphatidylinositol (PtdIns) phospholipids are important molecules in endocytosis of membrane proteins (Czech, 2000). Because a key enzyme for the generation of phospholipids is phosphoinositide 3-kinase (PI3K), it suggests a possible role for PI3K in the internalization of GPCRs. Thus, although the endocytic process is a multistep event involving the coordinate interaction between proteins and as well as the control of lipid modification, the precise molecular mechanisms for this interaction are not well understood.
PI3Ks are a conserved family of lipid kinases that catalyze the addition of phosphate on the third position of the inositol ring (Rameh and Cantley, 1999). Stimulation of a variety of receptor tyrosine kinases and GPCRs results in the activation of PI3K and leads to an increase in the level of D-3 PtdIns, which in turn are potent signaling molecules that modulate a number of diverse cellular effects including: cell proliferation, cell survival, cytoskeletal rearrangements, and receptor endocytosis (Martin, 1998; Sato et al., 2001). In this context, GPCR stimulation leads to the activation of the IB subclass of PI3Ks mediated by the Gβγ subunits of G-proteins (Stoyanov et al., 1995). Studies have suggested a role of phosphoinositides in the process of receptor internalization. For example, deletion of the phosphoinositide binding site from β-arrestin impairs GPCR endocytosis (Gaidarov et al., 1999a), and the binding of PtdIns (3,4,5) P3 and PtdIns (4,5) P2 to AP-2 promotes targeting of the receptor-arrestin complex to clathrin-coated pits (Gaidarov and Keen, 1999).
Recently we have shown a possible involvement of PI3K in the regulation of βAR internalization (Naga Prasad et al., 2001). Considering the potential important role of D-3 phosphoinositides in βAR internalization, we sought to determine: (a) whether there was a direct physical interaction between βARK1 and PI3K, and if so, identify the structural domain responsible for this interaction; (b) whether the interaction of PI3K with βARK1 regulates the translocation PI3K to the agonist-occupied βAR; and (c) whether the lipid products of PI3K modulate the internalization of βARs.
We postulated that the PIK domain of PI3K (Vanhaesebroeck et al., 2001), which is characterized by five pairs of antiparallel helices, might be an important domain to support protein–protein interactions (Walker et al., 1999). To investigate whether there is a direct physical interaction between βARK1 and PI3K, we created PI3K mutants that contained only the PIK domain (FLAG tagged) or had a deletion of the PIK domain PI3KΔPIK (HA tagged) (Fig. 1 a).
βARK1 directly interacts with the PIK domain of PI3K. (a) Schematic representation of full-length PI3Kp110γ and mutants. ABR adaptor binding region, RBD-ras binding domain, C2 similar to PLCδ, which is involved in Ca2+-dependent or -independent phospholipid binding, PIK domain thought to be involved in protein–protein interactions, HA, hemagglutinin tag, FLAG, flag peptide tag. (b) HEK 293 cells cotransfected with plasmids containing βARK1 and FLAG-PIK or PI3KΔPIK cDNAs. FLAG-PIK and PI3KΔPIK were immunoprecipitated from cells, fractionated by SDS-PAGE, and analyzed by immunoblotting with βARK1 monoclonal antibody. βARK1 protein coimmunoprecipitates with FLAG-PIK and not with PI3KΔPIK. (c) GST–PIK fusion protein containing amino acid residues 535–732 of PI3Kγ was produced, which includes the entire PIK domain and flanking upstream 10 amino acids and downstream 7 amino acids. Beads with bound GST alone or GST fusion proteins were incubated with purified βARK1. Beads were washed and bound material was run on SDS-PAGE and immunoblotted with βARK1 monoclonal antibody. Purified βARK1 was found to bind to the GST–PIK fusion protein, whereas no binding was seen with GST alone. (d) PIK domain does not interact with Gβγ subunits of G-protein. Beads with bound GST–PIK fusion protein or βARK1 protein were incubated with purified Gβγ subunits of G-protein. Beads were washed, spun down, and bound material separated by SDS-PAGE electrophoresis followed by immunodetection of Gβ. No interaction between Gβγ and the PIK fusion protein was found, whereas robust interaction between Gβγ and βARK1 purified protein was observed. Purified Gβγ was loaded as a positive control.
HEK 293 cells were transfected with plasmids containing the FLAG-PIK and PI3KΔPIK-HA cDNAs. PIK and PI3KΔPIK proteins were immunoprecipitated from cell extracts using monoclonal FLAG and HA antibodies, respectively. After the addition of purified βARK1 protein to the immune complexes, the presence of βARK1 was assessed by immunoblotting for βARK1. βARK1 was found to associate with the PIK domain protein and not to full-length PI3K lacking this domain (PI3KΔPIK) (Fig. 1 b). Levels of expression of PIK and PI3KΔPIK proteins were similar (Fig. 1 b, bottom). These data suggest that PI3K and βARK1 form a macromolecular complex within the cell.
To investigate whether there was a direct physical interaction between the PIK domain of PI3K and βARK1, GST–PIK fusion protein was immobilized on sepharose beads and incubated with purified βARK1. Purified βARK1 bound specifically to GST–PIK immobilized beads and not to GST alone (Fig. 1 c). No difference in the level of GST and GST–PIK was found (Fig. 1 c, bottom).
Because PI3Kγ interacts with the βγ subunits of G-proteins, we tested whether the PIK domain might directly interact with Gβγ, and thus would compete with βARK1 for these subunits. Purified βARK1 and GST–PIK fusion protein immobilized on Sepharose beads were incubated with purified Gβγ. The beads were washed and recovered proteins were analyzed by SDS-PAGE followed by immunoblotting with a Gβ polyclonal antibody. Whereas a strong association of Gβγ with βARK1 was found, no appreciable association of Gβγ with the PIK domain was detected (Fig. 1 d). Densitometric quantification showed an 11.5 ± 1.1-fold greater binding ability of βARK1 to Gβγ purified proteins compared with the PIK domain.
To test whether overexpression of the PIK domain could displace βARK1 associated PI3K activity in living cells, experiments were performed in HEK 293 cells cotransfected with plasmids containing the βARK1 cDNA (2 μg) and increasing concentrations of the PIK domain cDNA (ranging from 0 to 6 μg). Cell lysates were immunoprecipitated using the βARK1 monoclonal antibody 48 h after transfection and βARK1 associated PI3K activity was assayed. A robust βARK1 associated PI3K activity was found in the absence of PIK domain protein, but this association could be effectively competed away by increasing the concentration of PIK cDNA (Fig. 2 a). The maximal reduction of βARK1 associated PI3K activity occurred when cells were cotransfected with 4 μg of PIK cDNA (Fig. 2 b). Furthermore, we could coimmunoprecipitate FLAG-tagged PIK protein with βARK1 monoclonal antibodies in the cotransfected HEK 293 cells (data not depicted).
Overexpression of the 197 amino acid PIK domain of PI3K competes for endogenous PI3K binding to βARK1. (a) HEK 293 cells were cotransfected with βARK1 and increasing concentrations of PIK domain encoding cDNAs. βARK1 was immunoprecipitated using βARK1 monoclonal antibody and the associated PI3K activity was measured. The lipids were extracted and run on thin layer chromatography plates. Shown is representative autoradiograph of formation of PIP ( PtdIns-monophosphate). Ori, Origin. Mock cells were transfected with vector alone. Increasing the quantity of PIK cDNA used for expression leads to displacement of PI3K activity from βARK1 suggesting PIK protein competes out PI3K from βARK1. Bottom panel shows immunoblotting for βARK1 and PIK in cell lysates. (b) Summary results of n = 5 experiments. *P < 0.0005. The data was normalized to βARK1 associated PI3K activity in cells transfected with βARK1 only. (c) HEK 293 cells were transfected with βARK1 or βARK1 and PIK or βARK1 and PI3KΔPIK encoding cDNAs. βARK1 was immunoprecipitated using βARK1 monoclonal antibody and the associated endogenous PI3K activity was assayed. Extracted lipids were run on TLC plates and a representative autoradiograph showing the formation of PIP is shown here. Mock cells are transfected with vector alone. PIK overexpression lead to displacement of endogenous PI3K activity, whereas PI3KΔPIK overexpression did not alter βARK1 associated PI3K activity. Bottom panels show immunoblotting for βARK1, PI3KΔPIK and PIK in cell lysates. (d) Summary results of n = 4 experiments. *P < 0.001. The data was normalized to βARK1 associated PI3K activity in cells transfected with βARK1 only. (e) HEK 293 cells were transfected with βARK1 or βARK1 and PIK encoding cDNAs. βARK1 was immunoprecipitated using βARK1 monoclonal antibody and the associated endogenous PI3K activity was assayed using PIP2 (PtdIns-4,5-bisphosphate) as a substrate. Extracted lipids were run on TLC plates and the autoradiograph showing the formation of PIP3 (PtdIns-3,4,5-triphosphate) is presented. βARK1 transfected cells were treated with wortmannin (Wort) (50 nM) for 15 min before lysis of cells. Mock cells were transfected with vector alone. Bottom panel shows immunoblotting for βARK1 and PIK from cell lysates. Overexpression of PIK domain or wortmannin treatment led to significant inhibition in the formation of PIP3.
We next tested whether absence of the PIK domain in an otherwise intact PI3K molecule would affect the association of βARK1 with endogenous PI3K in cells. HEK 293 cells were cotransfected with plasmids containing cDNAs for βARK1 (2 μg), βARK1 plus PIK (4 μg), and βARK1 plus PI3KΔPIK (4 μg). Cell lysates were immunoprecipitated with a βARK1 monoclonal antibody and assayed for the associated PI3K activity. Expression of PI3KΔPIK had no effect on the endogenous βARK1/PI3K interaction, whereas overexpression of PIK disrupted this interaction. Treatment with the selective PI3K inhibitor wortmannin (50 nM), abolished this βARK associated PI3K activity (Fig. 2, c and d).
Because the PIK domain is shared by all members of the PI3K family, we wanted to confirm that the βARK1-associated endogenous PI3K activity was contributed by Class I PI3K. To test this, HEK 293 cells were transfected with plasmids containing cDNAs for βARK1 (2 μg) or βARK1 plus PIK (4 μg). Cell lysates were immunoprecipitated with a βARK1 monoclonal antibody and assayed for the associated PI3K activity. However, in this experiment PtdIns-4,5-P2 was used as the substrate instead of PtdIns, as in vitro, PtdIns-4,5-P2 can be phosphorylated only by Class I PI3K (Fruman et al., 1998) and not by either the Class II or Class III PI3K enzymes. As shown in Fig. 2 e, robust generation of PtdIns-3,4,5-P3, the product of Class I PI3K catalytic activity, was seen associated with βARK1 and the coexpression of PIK completely displaced the βARK1 associated PI3K activity (Fig. 2 e). Additionally, treatment of cells with wortmannin (50 nM) prior to cell lysis also inhibited the PI3K activity that was coimmunoprecipitated along with βARK1 (Fig. 2 e). Taken together, these data demonstrate that overexpression of the PIK domain can disrupt the interaction between βARK1 and PI3K, and that the lipid kinase activity belongs to the Class I PI3K family.
Our results suggest that overexpression of the PIK domain should block the βARK1-mediated translocation of PI3K to the membrane. In order to test this hypothesis, HEK 293 cells were cotransfected with the βARK1 (2 μg), βARK1, and PIK domain (4 μg) containing plasmids, and endogenous βARs were stimulated with 10 μM isoproterenol for 2 min. Cytosolic and membrane fractions were prepared and analyzed for βARK1 associated PI3K activity. After isoproterenol stimulation, robust βARK1-associated PI3K activity that was wortmannin (50 nM) sensitive could be seen in the membrane fraction (Fig. 3 a). In contrast, overexpression of the PIK domain effectively abolished the agonist-induced translocation of PI3K to the membrane by disrupting the βARK1/PI3K interaction. No change in βARK1 associated PI3K activity was found in cytosolic fractions after isoproterenol stimulation (data not depicted).
Overexpression of PIK domain blocks βARK1 mediated translocation of PI3K to the membrane and to the β2AR. (a) HEK 293 cells were transfected with βARK1 or βARK1 and PIK encoding cDNAs. βARK1 was immunoprecipitated using βARK1 monoclonal antibody from the membrane fraction of unstimulated and stimulated (10 μM isoproterenol for 2 min) cells. βARK1 associated PI3K activity was assayed from these membranes and extracted lipids were separated by thin layer chromatography. The significant increase in βARK1 associated PI3K activity found in the membrane fraction after agonist was completely abolished in presence of PIK domain peptide and with wortmannin (50 nM) treatment of βARK1 transfected cells (Wort). (b) Membrane fraction of cells transfected with βARK1 or βARK1 and PIK encoding cDNAs were immunoprecipitated with a βARK1 monoclonal antibody and immunoblotted for βARK1. Overexpression of PIK did not inhibit βARK1 translocation to the membrane after isoproterenol stimulation. (c) HEK 293 cells were transfected with β2AR or β2AR and PIK encoding cDNAs. The transfected cells were split into separate dishes and individually treated with 10 μM isoproterenol for indicated times. Before isoproterenol treatment, a set of β2AR-transfected cells were treated with wortmannin (Wort) (50 nM). At indicated time points FLAG-β2AR was immunoprecipitated and the associated PI3K activity was measured. Shown is a representative autoradiograph of the thin layer chromatography plate showing the formation of PIP. Lower panel, level of expression of β2AR and PIK domain protein in cell lysates as assessed by immunoblotting.
To investigate whether βARK1 recruitment to the membrane would be inhibited in presence of PIK domain protein, membrane fractions were prepared from the HEK293 cells cotransfected with the βARK1 (2 μg), βARK1, and PIK domain (4 μg) containing plasmids. As shown in Fig. 3 b, overexpression of PIK had no effect on the membrane recruitment of βARK1 after isoproterenol (10 μM) stimulation. These data show that the overexpression of PIK domain protein interrupts the βARK1/PI3K interaction and does not affect agonist-dependent βARK1 translocation to the membrane.
To test whether disruption of the βARK1/PI3K interaction would prevent the recruitment of PI3K to activated βARs, HEK 293 cells were transfected with plasmids containing cDNAs encoding FLAG epitope–tagged β2AR (FLAG-β2AR, 2 μg) or FLAG-β2AR (2 μg) and PIK domain (4 μg), and assayed for β2AR associated PI3K activity after stimulation with 10 μM isoproterenol. HEK 293 cells are known to contain adequate levels of βARK1 to support agonist-induced translocation and receptor phosphorylation (Menard et al., 1997). FLAG-tagged β2ARs were immunoprecipitated from cell extracts and associated endogenous PI3K activity measured. Significant FLAG-β2AR–associated PI3K activity was observed as early as 2 min after agonist stimulation, with a decline by 5 min in the cells transfected with β2AR alone (Fig. 3 c). In contrast, no FLAG-β2AR–associated PI3K activity was found when either the PIK domain was overexpressed or the transfected cells were treated with wortmannin before isoproterenol stimulation (Fig. 3 c). These data indicate that overexpresssion of PIK domain displaces PI3K from βARK1 complex, thereby preventing its recruitment to the agonist-occupied receptor complex.
Previous studies have suggested a role for PI3K in β2AR internalization (Naga Prasad et al., 2001). We postulated that disruption of the endogenous βARK1/PI3K interaction by the PIK protein would attenuate β2AR endocytosis. To test this, agonist-dependent sequestration was studied by [125I]-cyanopindolol binding in HEK 293 cells cotransfected with plasmids containing either the FLAG-β2AR cDNA, or FLAG-β2AR and FLAG-PIK cDNAs. A significant (>60%) attenuation in the rate of β2AR sequestration occurred when the PIK domain protein was overexpressed (Fig. 4 a). Interestingly, overexpression of PIK domain protein was effective in attenuating the early processes of β2AR sequestration as the initial phase (0–5 min) was significantly impaired.
Attenuation of β2AR sequestration upon coexpression of PIK. (a) Agonist-promoted (1 μM isoproterenol) β2AR sequestration by [125I]-CYP binding was performed in HEK 293 cells transfected with β2AR and either the empty vector (▪) or PIK (□) encoding cDNAs over a time course of 0–30 min. Receptor expression (fmol/mg of whole-cell protein) was: β2AR + vector, 330 ± 80; β2AR + PIK, 303 ± 51. n = 5, P < 0.0001 versus β2AR. Inset shows expression of PIK protein. (b) Endocytosis of β2AR-YFP in live cells was monitored for 10 min after isoproterenol (10 μM) stimulation in the absence or presence of PIK domain protein coexpression using confocal microscopy. Panels on the left represent cells transfected with the β2AR-YFP alone (panels 1–3 show the same cell monitored at 0, 5, and 10 min after stimulation. Panel 7 is an example of another cell after 10 min of isoproterenol). Panels on the right represent cell transfected with β2AR-YFP and PIK (panels 4–6 show the same cell monitored as above). Panel 8 is an example of another cell after 10 min of isoproterenol). In the absence of PIK, isoproterenol causes the internalization β2ARs as shown by the formation of distinct cytoplasmic aggregates (arrowheads) and complete loss of membrane fluorescence. In contrast, in the presence of PIK domain protein, there is no redistribution of β2AR-YFP after agonist stimulation, indicating that the process of receptor endocytosis is completely inhibited. (c) Dual staining was performed in cells transfected with plasmids containing HA-β2AR and GFP-PIK cDNAs. After 10 min of 10 μM isoproterenol, cells were fixed with 4% paraformaldehyde, stained for HA-β2AR receptor with Texas red, and PIK was visualized by GFP fluorescence. Panel 1 shows two cells, one with only membrane distribution of HA-β2AR, and another cell with complete redistribution of β2ARs into aggregates (arrowheads). Panel 2, shows that the cell with GFP fluorescence (i.e., PIK domain expression) failed to undergo β2AR internalization, whereas the cell not expressing GFP-PIK showed HA-β2AR aggregates. Panel 3 is an overlay of panels 1 and 2.
We further evaluated β2AR endocytosis using confocal microscopy in transfected cells using β2AR tagged with the YFP (β2AR-YFP) in the presence and absence of PIK. β2AR internalization was followed in the same cell after agonist stimulation. Before agonist, the distribution of β2AR-YFP was found distinctly at the plasma membrane (Fig. 4 b, panel 1). After agonist treatment, there was redistribution of the β2AR-YFP into membrane puncta consistent with entry into clathrin coated pits (Fig. 4 b, panel 2) (Laporte et al., 1999). With time, this was followed by the formation of cytoplasmic aggregates (Fig. 4 b, panel 3), and then with the complete loss of membrane fluorescence (Fig. 4 b, panels 3 and 7, arrowheads). In marked contrast, coexpression with the PIK domain completely prevented redistribution of β2AR-YFP fluorescence into membrane puncta and blocked the formation of intracellular aggregates after isoproterenol stimulation (Fig. 4 b, panels 6 and 8).
To definitively show that only those cells that contained the PIK domain protein failed to undergo agonist-stimulated βAR internalization, dual labeling experiments were performed after cotransfection with plasmids encoding HA tagged β2AR (HA-β2AR) and PIK–GFP. After isoproterenol stimulation, cells were fixed and HA-β2AR was visualized by Texas red staining and PIK was visualized by GFP fluorescence. Cells that showed restricted distribution of Texas red staining (HA-β2AR expression) to the membrane (Fig. 4 c, panel 1), also had GFP fluorescence indicating PIK protein expression (Fig. 4 c, panel 2). In contrast, cells that lacked GFP fluorescence (i.e., PIK protein expression), showed marked agonist-induced β2AR internalization (Fig. 4 c, panel 1 and 2, arrowheads) as clearly seen in the overlay (Fig. 4 c, panel 3, arrowheads).
To test whether PI3K activity was necessary for the recruitment of β-arrestin to the receptor complex, and whether overexpression of the PIK protein alters agonist-induced receptor phosphorylation, we used a HEK 293 cell line with stable expression of both β2AR-HA and β-arrestin2–GFP proteins. Double stably expressing cells were transfected with the plasmid containing FLAG-PIK cDNA and then split in separate dishes. Confocal microscopy was used to visualize fluorescence in cells with 10 μM isoproterenol and subsequently fixed. All cells show β-arrestin2–GFP fluorescence, whereas a smaller percentage shows Texas red staining of the FLAG epitope (Fig. 5 a, panels 1 and 2). In the absence of isoproterenol (Fig. 5 a, panels 1 and 2) cells have a cytosolic distribution of PIK as well as β-arrestin2-GFP. With isoproterenol, there was marked redistribution of GFP fluorescence to the membrane, indicating recruitment of β-arrestin to the membrane (Fig. 5 a, panel 3). Importantly, cells that contained PIK proteins did not affect the membrane recruitment of β-arrestin (Fig. 5 a, panels 3 and 4, arrowheads), suggesting that D-3 phosphoinositide molecules are not necessary for arrestin recruitment to activated receptors.
Overexpression of PIK neither inhibits β-arrestin2 recruitment to the receptor complex, transferrin uptake, or downstream PI3K signaling. (a) Confocal microscopy was used to visualize fluorescence in HEK 293 double stably expressing (HA-β2AR-HA and β-arrestin 2-GFP) cells transfected with the FLAG-PIK plasmid. After stimulation with 10 μM isoproterenol for 10 min, cells were fixed and stained with Texas red. All cells show β-arrestin2–GFP (βarr2-GFP) fluorescence whereas a smaller percentage show Texas red staining of the FLAG epitope. Panels 1 and 2 show that in the absence of isoproterenol, both PIK and β-arrestin2–GFP were distributed in the cytoplasm. Panels 3 and 4, after the addition of isoproterenol, the presence of PIK domain protein had no effect on the marked redistribution of β-arrestin–GFP fluorescence to the membrane (arrowheads). (b) HEK 293 cells were transfected with GFP–PIK (4 μg) and plated on to glass-bottom petri dishes. Serum-starved cells were incubated with Transferrin (Tf) conjugated to Texas red for 30 min at 37°C. The cells were thoroughly washed with PBS and fixed in 4% paraformaldehyde. Fixed cells were visualized using laser scanning confocal microscope. Panel 1 shows PIK domain expression in cells as visualized by GFP fluorescence, whereas panel 2 shows the same set of cells that took up Transferrin (Tf) as visualized by Texas red. Cells that overexpressed the PIK domain also had robust Transferrin (Tf) uptake. (c) HEK 293 cells or cells stably expressing PIK were transfected with β2AR encoding cDNA and treated with various agonists. Clarified lysates (50 μg) were immunoblotted for phospho-PKB. MOCK, treated with ascorbic acid; IGF, treated with insulin growth factor (10 nM); Car, treated with carbachol (1 mM); ISO, treated with isoproterenol dissolved in ascorbic acid (10 μM); Cont, positive control for phospho-PKB. (d) Summary results of densitometric analysis of phospho-PKB after stimulation by various agonists (n = 3–5). The data is represented as fold over basal (MOCK).
To determine whether the overexpression of the PIK domain in cells nonspecifically affect other clathrin-mediated processes, we assessed the ability of transferrin to undergo endocytosis. Transferrin receptors are known to constitutively localize and internalize via clathrin-coated vesicles (van Dam and Stoorvogel, 2002). HEK 293 cells were transfected with plasmids encoding PIK–GFP. Transfected cells were incubated with Transferrin–Texas red conjugate at 37°C for 30 min and then fixed. As shown in Fig. 5 b, transferrin uptake was unaffected by the presence of PIK–GFP (Fig. 5 b, panels 1 and 2), indicating that overexpression of PIK domain does not inhibit other clathrin mediated processes.
To investigate whether overexpression of PIK domain protein in cells would alter downstream PI3K signaling, PKB activation was measured in HEK 293 cells stably expressing the PIK domain protein. Cells stably expressing PIK and wild-type HEK 293 cells were transfected with the plasmid containing cDNA for the FLAG-β2AR, and stimulated with various GPCR or growth factor agonists. Agonist stimulation in the absence of PIK resulted in a significant increase of pPKB over mock treatment (Fig. 5, c and d). Importantly, there was robust PKB activation in the cell line overexpressing the PIK domain protein (Fig. 5, c and d). These data show that overexpression of the PIK domain in cells does not interfere with cellular signaling downstream of PI3K.
Since overexpression of PIK leads to attenuation of β2AR endocytosis, we wanted to determine whether the βARK1-mediated localization of PI3K within the activated receptor complex is responsible for the generation of D-3 phosphorylated phosphoinositides that promote recruitment of AP-2 to the agonist-occupied receptor. To test this hypothesis, HEK 293 cells were transfected with FLAG-β2AR or FLAG-β2AR, and PIK plasmids and then stimulated with isoproterenol. The FLAG-epitope was immunoprecipitated from cell extracts and blotted for the associated AP-2 adaptin and clathrin proteins. There was a significant increase in the association of AP-2 adaptin to the agonist-stimulated receptor within 2–5 min, which returned to basal levels by 10 min (Fig. 6 a). In contrast, overexpression of the PIK peptide completely abolished the recruitment of adaptin to the agonist-stimulated β2AR complex (Fig. 6 a). Although the levels of clathrin that coimmunoprecipitated with the receptor showed only modest changes after agonist, this effect appeared to be attenuated in the presence of the PIK peptide (Fig. 6 a). Similar levels of adaptin, clathrin, β2AR, and PIK were observed in the appropriately transfected cells (Fig. 6 a, bottom).
Generation of D-3 PtdIns are necessary for the efficient recruitment of adaptin to the β2AR complex. (a) HEK 293 cells were transfected with FLAG-β2AR, or FLAG-β2AR and PIK encoding cDNAs. Transfected cells were treated with isoproterenol (10 μM) for 0, 2, 5, and 10 min β2AR was immunoprecipitated using the FLAG epitope from the cells lysates and immunoblotted with antibodies for AP-2 adaptin protein and clathrin. Presence of PIK domain protein inhibits AP-2 adaptin association with the β2AR complex. Bottom panels show expression of β2AR and PIK protein in transfected cells and equal quantities of adaptin and clathrin loading. IgG, represents heavy chain of the antibody. C, positive control for clathrin and AP2 adaptin proteins. (b) FLAG-β2AR expressing stable cells were treated with LY294002 (a selective PI3K inhibitor) for 15 min before isoproterenol stimulation. The cells were stimulated for 0, 2, 5, and 10 min and β2AR was immunoprecipitated using a FLAG epitope. Lysates were immunoblotted for AP-2 adaptin protein. Presence of LY294002 inhibited adaptin recruitment to the receptor complex. –LY, untreated cells, +LY cells treated with LY294002. (c) Summary results of densitometric analysis of adaptin recruitment to β2AR in presence and absence of LY294002 (n = 7). The data is represented as fold over basal. (d) FLAG-β2AR expressing stable cells were treated with the permeablizing reagent saponin and the phospholipids as shown, followed by isoproterenol for 5 min. FLAG epitope was immunoprecipitated from the cell lysates and blotted with adaptin antibodies. The upper panel shows cells treated with phosphatidyinositol-4,5-P2, while lower panel were treated with PtdIns-3,4,5-P3. IgG, heavy chain of antibody. (e) Summary results of densitometric analysis of adaptin recruitment to β2AR complex in presence of phosphatidyinositol-4,5-P2 (PIP2) or PtdIns-3,4,5-P3 (PIP3) (n = 7). Data is represented as fold over basal.
To directly demonstrate that the generation of D-3 PtdIns phospholipids are important for the recruitment of AP-2 adaptor proteins to the agonist-occupied receptor complex, the same HEK 293 cells stably expressing FLAG-β2AR were permeabilized with saponin (Jones et al., 1999) and then incubated with increasing concentrations of the phosphorylated lipids, PtdIns-4,5-P2 and PtdIns-3,4,5-P3. After stimulation with isoproterenol, FLAG-β2AR was immunoprecipitated and the immune complexes immunoblotted for the presence of adaptin. The efficiency of recruitment of adaptin to the receptor was significantly enhanced in the presence of PtdIns-3,4,5-P3 compared with a similar concentration of PtdIns-4,5-P2 (Figs. 6, d and e). At high concentrations of PtdIns-4,5-P2, the preferential effect of PtdIns-3,4,5-P3 was lost consistent with previous studies showing that the AP-2 adaptor protein has a higher affinity for PtdIns-3,4,5-P3 compared to other phosphoinositides (Gaidarov et al., 1996).
To directly test whether the local production of PtdIns-3,4,5-P3 is required for receptor internalization, we utilized the lipid phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) (Vanhaesebroeck et al., 2001). PTEN is known to use PtdIns-3,4,5-P3 as its primary substrate converting it to PtdIns-4,5-P2, thus depleting PtdIns-3,4,5-P3 from the membrane (Vanhaesebroeck et al., 2001). HEK 293 cells were transfected with plasmids containing cDNAs with either β2AR-YFP or β2AR-YFP plus PTEN, and β2AR endocytosis was evaluated using laser scanning confocal microscopy. β2AR internalization was followed in the same cell after agonist stimulation. Before agonist, the distribution of β2AR-YFP was found distinctly at the plasma membrane (Fig. 7 a, panel 1). After agonist stimulation, there was a progressive redistribution of the β2AR-YFP into membrane puncta, followed by the formation of cytoplasmic aggregates and complete loss of membrane fluorescence by 10 min (Fig. 4 b, panels 2 and 3). In contrast, coexpression of β2ARs with the PTEN completely prevented the agonist induced redistribution of β2AR-YFP fluorescence into membrane puncta and intracellular aggregates (Fig. 7, panels 4–6).
Depletion of D-3 phosphatidylinositols leads to attenuation in β2AR receptor internalization. (a) Endocytosis of β2AR-YFP in live cells was monitored for 10 min after isoproterenol (10 μM) stimulation in the absence or presence of PTEN protein coexpression using confocal microscopy. Panels on the left represent cells transfected with the β2AR-YFP alone (panels 1–3 show the same cell monitored at 0, 5, and 10 min after stimulation). Panels on the right represent cell transfected with β2AR-YFP and PTEN (panels 4–6 show the same cell monitored as above). In the absence of PTEN, isoproterenol causes the internalization β2ARs as shown by the formation of distinct cytoplasmic aggregates (arrowheads) and complete loss of membrane fluorescence. In contrast, in the presence of PTEN protein, there is no redistribution of β2AR-YFP after agonist stimulation, indicating that the process of receptor endocytosis is completely inhibited. (b) Dual staining was performed in cells transfected with plasmids β2AR-YFP and PTEN cDNAs. After 10 min of 10 μM isoproterenol, cells were fixed with 4% paraformaldehyde, stained for PTEN using Texas red and β2AR was visualized by GFP fluorescence. Panel 1 (a and b) shows the same cell with intact membrane β2AR visualized by GFP fluorescence (panel 1 a) and PTEN Texas red fluorescence (panel 1 b). Panel 2 (a and b) shows the cell with complete redistribution of β2ARs into aggregrates (arrowheads) visualized by GFP fluorescence and very little PTEN expression as visualized by Texas red fluorescence (panel 2 b). Cells from panels 1 and 2 are from the same petri dish but from different fields of view.
To show that only those cells that contained the PTEN failed to undergo agonist-stimulated βAR internalization, dual labeling experiments were performed in cells co-transfected with plasmids containing β2AR-YFP and PTEN. After isoproterenol stimulation cells were fixed and β2AR was visualized by GFP fluorescence and PTEN expression was visualized by Texas red staining. Cells that had the restricted membrane distribution of GFP fluorescence (β2AR) also had the Texas red staining (Fig. 7 b, panel 1, a and b). In contrast, cells that showed β2AR internalization lacked Texas red staining (PTEN expression) (Fig. 7 b, panel 2, a and b, arrowheads).
In the present investigation, we provide evidence for a direct protein–protein interaction between PI3K and βARK1, and show that the region of the PI3K molecule that provides the necessary structure for this interaction is the PIK domain. Moreover, we show that the interaction between PI3K and βARK1 is not dependent on Gβγ and that overexpression of PIK domain competitively displaces PI3K from the βARK1/PI3K complex leading to a loss in βARK1 associated PI3K activity. Although overexpression of PIK domain protein disrupts the βARK1/PI3K interaction, it does not inhibit the Gβγ mediated translocation of βARK1 to agonist-occupied receptors or alter other Gβγ-dependent cellular processes. Finally, these experiments demonstrate that overexpression of the PIK domain markedly attenuates β2AR endocytosis in the early phase after agonist stimulation, and suggest that impairing the local production of PtdIns-3,4,5-P3 lipid molecules within the agonist-occupied receptor complex affects the recruitment of critical molecules necessary for efficient receptor endocytosis. Furthermore, we show that overexpression of PTEN, which uses PtdIns-3,4,5-P3 as a substrate, leads to complete inhibition of β2AR internalization, providing strong evidence that the local production D-3 phosphoinositides by PI3K within the agonist-occupied receptor complex is required for endocytosis.
Agonist-occupied βARs are phosphorylated by βARK1 after translocation of βARK1 to the membrane (Lefkowitz, 1998). Subsequently, the phosphorylated receptor can bind with high affinity to β-arrestin (Ferguson et al., 1996), which then recruits AP-2 adaptor molecules and clathrin; two required components in the formation of the endocytic vesicle (Goodman et al., 1996; Laporte et al., 1999, 2000). Previous studies have shown that the association of adaptor proteins with clathrin are critical to the formation of the clathrin lattice complex (Schmid, 1997; Brodsky et al., 2001). Because the recruitment of AP-2 adaptor proteins are at least in part regulated by D-3–phosphorylated phosphoinositides (Gaidarov and Keen, 1999), the generation of these lipids not only play an important role in targeting of the agonist-stimulated receptor to the clathrin coated pit, but are likely important in the initiation/nucleation of the clathrin lattices at sites of endocytosis. This is consistent with data from in vitro studies showing that AP-2 in the assembled coat structure (which is very similar to clathrin-coated pit), binds PtdIns-3,4,5-P3 with greater affinity compared with other phosphoinositides including PtdIns-4,5-P2 (Gaidarov et al., 1996).
Whether the recruitment of AP-2 to agonist-stimulated receptor initiates the nucleation of a new clathrin-coated pit or targets the receptor to a preexisting pit, remains controversial. For example, AP180, a neuronal specific clathrin adaptor protein needs to bind clathrin and phosphoinositides simultaneously to initiate nucleation (Ford et al., 2001), and the assembled receptor/β-arrestin/AP-2 complex may subserve this role and initiate nucleation of the clathrin-coated pit (Laporte et al., 2000). In contrast, studies in living cells have suggested that the targeting of activated GPCRs is to preexisting clathrin-coated pits (Scott et al., 2002). Furthermore, studies using a clathrin–green fluorescent fusion protein suggest that clathrin-coated pits form repeatedly at defined sites and the mobility of these pits is limited by the actin cytoskeleton (Gaidarov et al., 1999b). Studies using receptor tail like synthetic peptides (crosslinked to UV photoreactive molecules) showed enhanced binding to the μ2 subunit of the AP-2 complex in presence of D-3 phosphoinositides, suggesting that phospholipids may provide a mechanism for increasing the specificity in sorting and clathrin coat assembly (Rapoport et al., 1997).
In this study we show that expression of PIK does not block β-arrestin recruitment to the receptor, nor does it impair the ability of βARK to phosphorylate activated receptors, as β-arrestin is only recruited to GRK phosphorylated receptors (Ferguson et al., 1996). Previous studies have shown that deletion of the phosphoinositide binding domain on β-arrestin impairs the formation of clathrin coated pits, but that this process is not altered by wortmannin treatment (Gaidarov et al., 1999a). This suggests that β-arrestin can bind PtdIns-4,5-P2, which is present on the plasma membrane in much greater concentration than PtdIns-3,4,5-P3. Consistent with these findings is our observation showing that even in the absence of receptor-associated PI3K activity, membrane PtdIns-4,5-P2 is sufficient to recruit β-arrestin in an agonist-dependent manner.
Importantly, overexpression of the PIK domain does not block transferrin uptake, whose receptor constitutively localized within clathrin coated vesicles (van Dam and Stoorvogel, 2002). This suggests that recruitment of PI3K by βARK1 to the agonist occupied receptor is a βARK1 specific process. Furthermore, overexpression of the PIK domain did not affect activation of PKB suggesting a specific role in displacement of PI3K from βARK1. Thus, agonist dependent phosphorylation of PKB in PIK expressing cells may enable signaling either by direct receptor stimulation or through transactivation of receptor tyrosine kinases, as shown for other GPCRs (Kowalski-Chauvel et al., 1996; Saward and Zahradka, 1997).
To test the hypothesis that the local generation of PtdIns-3,4,5-P3 is a necessary step for β2AR endocytosis, we performed experiments in cells that had overexpression of PTEN, a PtdIns-3,4,5-P3 lipid phosphatase. Because PtdIns-3,4,5-P3 generated by PI3K within the receptor complex would be immediately hydrolyzed by PTEN we could determine the importance for PtdIns-3,4,5-P3 in supporting the internalization process. The presence of PTEN resulted in significant inhibition of β2AR endocytosis showing the requirement for PI3K activity within the receptor complex for effective agonist-induced endocytosis. We postulate that inhibition of PtdIns-3,4,5-P3 production within the receptor complex prevents the effective interaction of various components needed for receptor endocytosis particularly, adaptin leading to an impairment in receptor endocytosis.
The crystal structure of PI3Kγ shows the PIK domain to be centrally positioned with a solvent exposed surface suitable for protein-protein interactions (Walker et al., 1999). Therefore, it is possible that other molecules containing a PIK domain, such as enzymes belonging to the family of PI3K (all the classes of PI3K), can potentially interact with βARK1 depending up the tissue and the abundance of the various isoforms. This is consistent with our previous data where we have shown in HEK 293 cells that βARK1 could also interact with the PI3Kα isoform when overexpressed (Naga Prasad et al., 2001). Interestingly, it has recently been shown that another PI3K, the class II PI3K C2α, interacts with clathrin and regulates clathrin-mediated membrane trafficking particularly in the process of vesicle uncoating (Gaidarov et al., 2001). This suggests possible redundancy for the production of phosphoinositides within the receptor complex, a finding not surprising considering that receptor sequestration is a multistep process, highly regulated by many molecules at different stages (Brodsky et al., 2001).
Taken together, these data show that overexpression of the PIK domain displaces endogenous PI3K from βARK1 leading to impairment of PI3K translocation to the receptor after agonist stimulation. The loss in receptor associated PI3K activity impairs the ability of the agonist-occupied receptor/PI3K complex to generate D-3 phospholipid molecules. We propose that the products of PI3K play a critical role in determining the dynamics of receptor endocytosis. Agonist-induced recruitment of class I PI3Ks by βARK1 to the receptor complex functions to increase the production of D-3 phospholipid molecules, that in turn regulates the recruitment of AP-2 and cargo (i.e., receptor/β-arrestin complex) to clathrin-coated pits on the membrane. The generation of PtdIns-3,4,5-P3 by PI3K within the activated receptor complex promotes more efficient recruitment of AP-2 and receptor sequestration. The rise in the local concentration of PtdIns-3,4,5-P3 within the receptor complex, which enhances the recruitment of AP-2 to the complex, likely plays a significant role in the initiation/nucleation of new clathrin-coated pits. The efficiency of clathrin coated pit formation will depend on the association of the various critical components that, in part, are regulated by their affinity to bind newly generated D-3 phospholipids.
HEK 293 cells were maintained and transfected as previously described (Naga Prasad et al., 2001). Cells were harvested 24 h after transfection, replated in triplicate, allowed to grow overnight, and serum starved either overnight or 2–4 h before agonist stimulation. FLAG-PIK domain expressing permanent HEK 293 cell line was prepared by selecting cells against Geneticin (Life Technolgies) as described earlier (Laporte et al., 2000). All cells with stable FLAG-PIK expression showed both diffuse cytoplasmic distribution of PIK and inhibition of β2AR-YFP internalization as visualized by confocal microscopy (unpublished data). HA-β2AR and β-arrestin double stable cell lines were prepared by selecting the cells against two different antibiotics (Menard et al., 1997). Cell lines with stable expression of FLAG-β2AR, a gift from Dr. Robert Lefkowitz (Duke University Medical Center, Durham, NC) and HA-β2AR were used for the confocal experiments.
PIK and PI3KΔPIK mutants of PI3K were prepared by PCR amplification using the full-length p110γ cDNA as template (Fig. 1 a). The PIK domain was amplified using Pfu platinum turbo Taq high fidelity enzyme (STRATAGENE) with the 5′ primer (5′-TCTCGAGGATCCGCCGCCATGGACTACA AGGACGACGATGATAAGCACCCGATAGCCCTGCCT-3′) containing XhoI and BamHI sites for subcloning, followed by Kozak consensus sequence and a FLAG epitope tag and the 3′ primer (5′-GTCGACCTAGTCGTGCAGCATGGC-3′) containing consensus stop codon with a SalI site for subcloning. The PCR product was subcloned in zero-blunt TOPO vector (Invitrogen) and sequence verified for authenticity. After digestion with the restriction enzymes BamHI and SalI, the PIK domain fragment was subcloned into the following expression plasmids: the pRK5 mammalian expression vector, the pGEX-4T1 bacterial expression vector to generate GST fusion proteins, and the EGFP vector pEGFP-C1.
PI3KΔPIK was constructed using two PCR reactions that selectively amplified upstream and downstream from the PIK domain (Fig. 1 a). PI3K upstream from PIK was amplified using the forward primer (5′-TGCGGATCCGCCACCATGGAGCTGGAGAACTATAAACAG-3′) containing a BamHI site and Kozak consensus sequence, and the reverse primer (5′-TTCTGCTCGACCGCGGTCCCCTTCCGG-3′) containing a SacI site. The region of PI3K downstream of the PIK domain was amplified using the forward primer (5′-CTGAGGGGCCGCGGCACAGCCATG-3′) containing a SacI site and the reverse primer (5′-ACCCGGGATCCTTAAGCGTAGTCTGGTACGT-3′) containing a BamHI site and a consensus stop codon. The upstream and downstream regions of PI3K were separately subcloned in the zero-blunt TOPO vector and sequence was verified by dideoxy sequencing. After digestion with the restriction enzymes BamHI/SacI, a three-fragment ligation was carried with the mammalian expression vector pRK5, to generate the plasmid containing the PI3KΔPIK cDNA (Fig. 1 a). The catalytic activity of PI3KΔPIK was indistinguishable from wild-type PI3K (unpublished data). The plasmids containing cDNAs encoding βARK1, FLAG-β2AR, HA-β2AR, and p110γ have been described previously (Naga Prasad et al., 2001). The PTEN plasmid was gift from Dr. Christopher Kontos (Duke University Medical Center).
Plasmid DNAs were transformed in Escherichia coli BL21 cells. Overnight cultures were grown in LB medium supplemented with ampicillin (100 μg/ml), diluted to an A600 of 0.2 in the same medium, and grown for another 1 h at 37°C. Cultured cells were then induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside for 2 h. Cells were then pelleted, washed once with PBS, and resuspended in PBS containing 1 mM PMSF, 2 mg/ml lysozyme, and incubated for 15 min on ice. Cells were lysed by adding Triton X-100 1%. Solublized cells were incubated with DNase (300 units) for 15 min on ice and centrifuged at 13,000 rpm for 10 min. Glutathione-Sepharose beads were added to the supernatant and gently agitated at 4°C for 2 h. Beads were washed three times with ice-cold PBS containing 1% Triton X-100 followed by three washes with cold PBS without detergent. Protein concentration was determined using a DC protein assay kit (Bio-Rad Laboratories), and the integrity of the fusion protein was analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie staining.
GST fusion proteins (1–1.5 μg) on beads were incubated in 0.5 ml of binding buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.2% Triton X-100) for 2 h at 25°C together with purified βARK1 protein (5 μg). The beads were spun and washed three times with binding buffer followed by three washes with binding buffer without detergent. The beads were resuspended in SDS gel loading buffer and resolved by gel electrophoresis, immunoblotting and detection was carried out as described later.
Immobilized purified βARK1 protein was prepared by incubating βARK1 monoclonal antibodies with protein G agarose beads for 1 h at 4°C, followed by the addition of purified βARK1 protein. Subsequently, 10 μg of purified Gβγ was added to either βARK1 immobilized beads (5 μg), or the GST–PIK fusion protein beads (5 μg), and gently rocked for 45 min at room temperature. Beads were spun down, washed in binding buffer ×2, and resolved by gel electrophoresis. The presence of Gβγ was detected by immunoblotting with an antibody directed against the Gβ subunit. Purified Gβγ and βARK1 were gifts from Dr. Robert Lefkowitz.
Membrane fractions were prepared as previously described (Naga Prasad et al., 2001). Briefly, cells were scraped in 1 ml of buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 2 μg/ml each leupeptin and aprotinin, and disrupted by using Dounce homogenizer. Intact cells and nuclei were removed by centrifugation at 1,000 g for 5 min. The supernatant was subjected to 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-HCl, pH 7.4, 1 mM sodium orthvanadate, and 2 μg/ml each leupeptin and aprotinin) and used as membrane fraction.
PI3K assays were carried out as previously described (Naga Prasad et al., 2001). Briefly, cells were lysed in lysis buffer in presence of protease inhibitors and membrane and cytosolic fraction was prepared as described above. 500 μg of membrane or cytosolic fraction was used for immunoprecipitation with either the C5/1 monoclonal antibody directed against βARK1 (Choi et al., 1997) or the anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) in presence of 35 μl of protein G-agarose (Life technologies). The samples were centrifuged at 10,000 rpm for 1 min and sedimented beads were washed once with lysis buffer, thrice with PBS containing 1% NP40 and 100 μM sodium-orthovanadate, three times with 100 mM Tris.Cl, pH 7.4, containing 5 mM LiCl and 100 μM sodium-orthovanadate, twice with TNE (10 mM Tris.Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 100 μM sodium-orthovandate). The last traces of buffer were completely removed using the insulin syringe and the pelleted beads were resuspended in 50 μl fresh TNE. To the resuspended pellet 10 μl of 100 mM MgCl2 and 10 μl of 2 mg/ml PtdIns (20 μg) sonicated in TE (10 mM Tris.Cl, pH 7.4, and 1 mM EDTA) were added. The reactions were started by adding 10 μl of 440 μM ATP, 10 μCi 32p γ ATP, and were incubated at 23°C for 10 min with continuous agitation. The reactions were stopped with 20 μl 6N HCl. Extraction of the lipids were done by adding 160 μl of chloroform:methanol (1:1) and the samples were vortexed and centrifuged at room temperature to separate the phases. 30 μl of the lower organic phase was spotted on to the 200-μ silica-coated flexi-TLC plates (Selecto-flexible; Fischer Scientific) precoated with 1% potassium oxalate. The spots were allowed to dry and resolved chromatographically with 2N glacial acetic acid:1-propanol (1:1.87). The plates were dried after resolution, exposed, and the autoradiographic signals were quantitated using Bio-Rad PhosphoImager. Lipid Preparation: PtdIns (Sigma-Aldrich or Avanti) or PtdIns-4,5-P2 (Echelon) was dissolved in chloroform at a concentration of 10 mg/ml. 50 μl of this stock was dried down in a stream of air in a 1.5-ml Eppendorf tube. 250 μl of TE was added to the Eppendorf to bring the concentration to 2 mg/ml. The lipids were suspended by sonicating them in an ice bath for 5–10 min. Sonicated lipids were then added to each reaction. PtdIns(4)P (Sigma-Aldrich) or PtdIns-3,4,5-P3 (Echelon) was used as a standard. The lipid standards were run as a separate lane on the TLC plate to identify the migration of PIP or PIP3 (Renkonen and Luukkonen, 1976). TLC plates were stained with iodine to identify the formation lipids products (Renkonen and Luukkonen, 1976).
Immunoblotting and detection of βARK1, FLAG-PIK, PI3KΔPIK-HA, FLAG-β2AR, HA-β2AR, β-adaptin, and clathrin were blotted as described previously (Laporte et al., 1999, 2000). Immunoprecipitating antibodies were added to 500 μg of cell lysate and the immune complexes were washed and resuspended in gel-loading buffer. Blots were incubated with antibodies recognizing β-adaptin, clathrin heavy chain (BD Transduction Laboratories), HA (Roche Molecular Biochemicals), and FLAG (Sigma-Aldrich) at a 1:2,000 dilution and the βARK1 monoclonal antibody at a 1:10,000 dilution. 50 μg of cell lysates were resolved by a 10% SDS-PAGE gel and transferred to PVDF membrane, phospho-PKB primary antibody was used at 1:1,000 dilution. Detection was carried out using enhanced chemiluminescence (Amersham Pharmacia Biotech). Densitometric analysis was carried out using Bio-Rad Flouro-S Multiimage software.
β2AR sequestration was performed as previously described (Naga Prasad et al., 2001). HEK 293 cells were transfected with plasmids containing β2AR (250 ng) or β2AR (250 ng) and PIK domain (4 μg) cDNAs. Total binding was determined in the presence of 175 pM [125I]-cyanopindolol (CYP) alone, 175 pM [125I]-CYP plus 100 nM CGP12177 was used to determine internalized receptors and nonspecific binding was determined using 175 pM [125I]-CYP plus 1 μM propranolol (Menard et al., 1997). Sequestration was calculated as the ratio of (specific receptor binding of [125I]-CYP in the presence of CGP12177 and/or specific receptor binding of [125I]-CYP in the absence of CGP12177).
Confocal microscopy was carried out as previously described (Naga Prasad et al., 2001). HEK 293 cells were transfected with the plasmids containing cDNAs encoding either the β2AR-YFP (2 μg) or β2AR-YFP (2 μg) and PIK domain (4 μg) or β2AR-YFP (2 μg) and PTEN (2 μg). Cells were plated onto glass-bottom dishes for observation in the confocal microscope. Live cells were treated with isoproterenol (10 μM) and images were collected sequentially over a time course of 0–10 min. For dual staining of β2AR-HA or FLAG-PIK, cells were fixed in 4% paraformaldehyde in PBS for 30 min after 10 min of 10 μM isoproterenol stimulation. Cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min, incubated in 1% BSA in PBS for 1 h. Cells were washed with PBS and incubated with anti-HA or anti-FLAG monoclonal antibody (1:250) with 1% BSA in PBS for 1 h. Cells were washed and incubated with goat anti–mouse IgG conjugated with Texas red (1:500; Molecular Probes) for 1 h. Samples were visualized using single sequential line excitation filters at 488 and 568 nm and emission filter sets at 505–550 nm for GFP detection and 585 for Texas red detection.
Modification of a previously described method was used to vary the concentration of D-3 phospholipids in living cells (Jones et al., 1999). Briefly, one of the synthetic phospholipids DiC16 PtdIns-3-P, DiC16 PtdIns-4,5-P2, or DiCPtdIns-3,4,5-P3 (AVANTI), was mixed with phosphatidylcholine and phosphoinositol (Sigma-Aldrich) at a 1:100:100 ratio and dried under N2. Phospholipids were then re-suspended in 10 mM Hepes, pH 7.4, containing 1 mM EDTA and sonicated. Cells were treated with Saponin (0.04 mg/ml) in serum-free medium along with the vesicles containing the phospholipids at given concentration of PtdIns-3-P or PtdIns-4,5-P2 or PtdIns-3,4,5-P3 for 10 min at 25°C. Cells were then treated with isoproterenol (10 μM) for 5 min at 37°C, then lysed for immunoprecipitation experiments with a buffer containing 0.8% Triton X-100, 20 mM tris-HCl, pH 7.4, 300 mM NaCl, 1 mM EDTA, 20% glycerol, 0.1 PMSF, 10 μg/ml leupeptin and aprotinin.
Transferrin uptake was carried out as described previously (van Dam and Stoorvogel, 2002). Briefly, HEK 293 cells were transfected with GFP-PIK (4 μg). 24 h after transfection, the cells were split into six glass-bottom petri dishes (Mat Tek Corporation). The following day the cells were serum starved for 1 h before transferrin treatment. Transferrin–Texas red conjugate was added to the cells at a final concentration of 33 μg/ml and incubated at 37°C for 30 min. After 30 min the cells were washed with PBS and fixed in 4% paraformaldehyde. Confocal microscopy was carried out on these cells as described earlier above.
Data are expressed as mean ± SEM. Statistical comparisons were performed using an unpaired Student's t test and analysis of variance where appropriate. Results for the β2AR sequestration by CYP binding was analyzed using Graph-pad prism.
This work was supported in part by National Institutes of Health grants HL56687 and HL61558 (H.A. Rockman), the Burroughs Wellcome Fund (H.A. Rockman), and an AHA Postdoctoral fellowship form the American Heart Association, Mid-Atlantic Affiliate (S.V. Naga Prasad). H.A. Rockman is a recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. M.G. Caron is an Investigator of the Howard Hughes Medical Institute.
↵* Abbreviations used in this paper: βAR, β-adrenergic receptor; CYP, cyanopindolol; GPCR, G protein–coupled receptor; PI3K, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol(s).
Stéphane A. Laporte's present address is Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada, H3A 1A1.
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Rapoport, I., M. Miyazaki, W. Boll, B. Duckworth, L.C. Cantley, S. Shoelson, and T. Kirchhausen. 1997. Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes. EMBO J. 16:2240–2250.
Renkonen, O., and A. Luukkonen. 1976. Thin-Layer Chromatography of Phospholipids and Glycolipids. Marcel Dekker, Inc., New York. 2–54.
Rockman, H.A., W.J. Koch, and R.J. Lefkowitz. 2002. Seven-transmembrane-spanning receptors and heart function. Nature. 415:206–212.
Sato, T.K., M. Overduin, and S.D. Emr. 2001. Location, location, location: membrane targeting directed by PX domains. Science. 294:1881–1885.
Saward, L., and P. Zahradka. 1997. Angiotensin II activates phosphatidylinositol 3-kinase in vascular smooth muscle cells. Circ. Res. 81:249–257.
Schmid, S.L. 1997. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu. Rev. Biochem. 66:511–548.
Scott, M.G.H., A. Benmerah, O. Muntaner, and S. Marullo. 2002. Recruitment of activated G protein-coupled receptors to pre-existing clathrin-coated pits in living cells. J. Biol. Chem. 277:3552–3559.
Stoyanov, B., S. Volinia, T. Hanck, I. Rubio, M. Loubtchenkov, D. Malek, S. Stoyanova, B. Vanhaesebroeck, R. Dhand, B. Nurnberg, et al. 1995. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science. 269:690–693.
Vanhaesebroeck, B., S.J. Leevers, K. Ahmadi, J. Timms, R. Katso, P.C. Driscoll, R. Woscholski, P.J. Parker, and M.D. Waterfield. 2001. Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev. Biochem. 70:535–602.
Walker, E.H., O. Perisic, C. Ried, L. Stephens, and R.L. Williams. 1999. Structural insights into phosphoinositide 3-kinase catalysis and signalling. Nature. 402:313–320.


text: One of the more popular sites in the overclocking community, HardOCP has its finger on the pulse of tweaker culture and related technology. The site provides tests and tips on the latest hardware, especially the rarified memory and motherboards that overclockers prefer. Links to related sites are included, and the site monitors issues crucial to stable hardware design, like cooling and chip steppings.

industry_corpus2: Computing & Telecommunications


text:
Jordan Brand is keeping the ball rolling with the second installment in Russell Westbrook’s signature line. As you all know, each and every colorway of the Jordan “Why Not?” Zer0.2 has a special meaning behind it. The lastest colorway of the Zer0.2 is releasing soon and is dubbed The ‘Family’ colorway. Not much to guess about regarding the story behind this colorway, Brodie is a family man and it comes as to no surprise he’s dedicating a colorway to them.
The colorway is really simple and this has got to be the colorway that I cop. The upper of the shoe is all black with some gold on the tongue logo, heel of the shoe, and medial tab that has ‘The Family’ written on it. The midsole and outsole are both a solid white color to finish off the second release colorway of the “Why Not?” Zer0.2.

industry_corpus2: Sports


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Only 12% of financial institutions are digitally mature. Can you afford not to be one of them?
Visits to retail bank branches are set to drop 36% by 2022. At the same time, mobile transactions will increase by 121%.
The lesson: sectors spanning the financial services specturm including; Banking, Wealth Management and Insurance Institutions must prepare for a digital-first future. Your online experiences need to be as social as Instagram, convenient as Amazon, personalized as Netflix, and intuitive as Apple - even if you are 150-years old.
Episerver’s Digital Experience Cloud™ Platform makes it easy for financial service providers to deliver better experiences. You can quickly create mobile experiences, combine your services with personalized content, and use intelligent marketing automation to move the customer journey forward.
Use Episerver’s Intelligent Campaigns to send triggered emails, texts and messages at exactly the right time. Our marketing automation can deliver always-on campaigns to welcome or re-engage your customers, as well as personalized content to segments or individuals.
Episerver’s solution combines artificial intelligence and rule-based personalization to automatically deliver the right content for each customer. This helps them find what they want faster and improves your results. All secured by our cloud service running on Microsoft Azure.
Easily deliver mobile experiences with optimized layouts and simplified navigation. Use Episerver’s headless content management to add content to your online banking sites and apps. Customers can also log in to online banking directly from your digital marketing campaigns.
Replicate the service, expertise and experience from your branches online. Episerver helps you easily provide searchable FAQs, knowledge bases, and live chat from LivePerson. You can also increase account originations and inquiries with intuitive, personalized forms.
Mesirow had to make the brand come alive digitally. Episerver’s solution was chosen for its ease of use and ability to deliver an authentic web and brand experience.
Metro Bank took action by revamping their digital estate to better meet the needs of their more than one million customers.
Secura Insurance had several clear goals for the upgrade. The first was to move from the existing site with minimal disruption to business. Next, the new platform needed to drive organic traffic to increase Secura’s reach.
Eastern Alliance sought a platform that would allow secure access to resources and tools easily. This innovation would help agents and clients view policy & claims data as well manage billing and payments.
Watch how the Episerver Digital Experience Cloud helps you create modern digital experiences. The platform automatically personalizes experiences with artificial intelligence, integrates with live chat functionality, and delivers “headless” content anywhere.
In the financial services industry, transformation strategies are no longer an option. To improve operations, drive growth and become a successful organization, you must leverage data analytics, improve CX and eliminate the silos.

industry_corpus2: Finance & Economics


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I used to think I liked Sushi and Sashimi.
Then I thought I really liked it.
Now that I've started dreaming about it and having it sometimes twice a day, I think addiciton is probably a more fitting term!
Kevin (my room mate) and I just came back from a Sushi Bar in Northgate (shopping centre in the northern suburbs of Adelaide, as the name implies) for my fourth serve of the stuff in one and a half days. My favourites by far are the salmon sashimi and sushi with tuna. The sushi with avocado atop the seaweed is also tantalising, but can be a tad filling after you've had a few!
I think I'm training myself to take a bit more heat too, the Rubenerd Wasabi/SoySauce Ratio is narrowing every time, though I make sure to only dab a minute amount on as not to smother the flavour of the sashimic goodness.
The girls at the sushi bars here are very friendly and nice too, haha!
Newer post ← Is WordPress.org Down?

industry_corpus2: Hospitality & Catering


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After opening of new airport, traffic will be able to travel from the airport to Islamabad and Rawalpindi through Thalian Interchange. Metro Bus Service project work is under speedy development, and the project is planned to be ready by December 2017.
Boarding bridges have been installed for 15 passenger planes and test trials of latest machines of passenger luggage are near completion. Computerized electronic boards of arrival and departure information have been tested successfully. Construction of Rainwater dam for water supply has been completed.

industry_corpus2: Transportation


text: After graduating from the Prague Conservatoy, the tenor Josef Moravec studied at the Academy of Performing Arts in Prague and at the Sibelius Academy in Helsinki. He won thid prize in the Junior category and the Vilém Zítek Award at the Antonín Dvořák International Vocal Competition in Karlovy Vary. He has frequently appeared as a guest singer at the J. K. Tyl Theatre in Plzeň (Salome, Adriana Lecouvreur, Die lustigen Weiber von Windsor, Die Zauberflöte, Turandot, Polish Blood, Le contes d’Hoffmann, The Bartered Bride), at the South Bohemia Theatre in České Budějovice (Der Vögelhändler, Otello, Don Giovanni, La bohème, Pagliacci, Rigoletto). Since the 2010/11 season, he has been a permanent guest at the National Moravian-Silesian Theatre in Ostrava (La traviata, Fedora, Cardillac, Orphée aux enfers, Polish blood, Eine Nacht in Venedig, Die Csárdásfürstin, Roméo et Juliette, The Bartered Bride, Armida, Nabucco, Anna Bolena, Mirandolina). He potrayed numerous roles at the Prague State Opera, including Pong (Turandot), Junkman and Herman Augustus (Bernstein: Candide), the First Priest and the Armed Man (Die Zauberflöte), Daland’s Steersman (Der fliegende Holländer), the Boy, First American, Strolling Player (Britten: Death in Venice), Rodriguez (Massenet: Don Quichotte), the Young Sailor and Shepherd (Tristan und Isolde). His roles at the Prague National Theatre include the hockey player Petr Svoboda (Smolka: Nagano), Avenant (Glass: La Belle et la Bête) and Joe (La fanciulla del West), Remendado (Carmen), Alfredo (La traviata) and Vašek (The Bartered Bride). Since the 2014/15 season, he has been a soloist of the National Theatre Opera, where he his repertoire has encompassed Janáček’s Skuratov (From the House of the Dead), the Schoolmaster/Mosquito (The Cunning Little Wixen), the Yurodivy (Boris Godunov), the Fisherman (Stravinsky: The Nightingale), Alméric (Tchaikovsky: Iolanta), the Abbe/the Incredible (Andrea Chénier), and other roles. He has also appeared as a guest at the Silesian Theatre in Opava, the F. X. Šalda Theatre in Liberec and the National Theatre in Brno. He has performed abroad too (Japan, Scandinavia, Algeria, Austria, Germany, Switzerland, Spain, France and Slovakia).

industry_corpus2: Film & Entertainment


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10 Basic Habits Every Stylish Woman Tends To Follow !!
There’s no universal code or to-do list that should be followed to be and look stylish everyday. We see many women looking stylish and classy everyday. If you don’t see them panicking about wrinkled blouses and hobbling in painful heels. You may think that how are they making it possible so easily. There is no magic behind that, they are well prepared. And the truth is, it’s not about filling your wardrobe with expensive clothes and following all the latest trends. Real style starts at your own home the moment you wake up. And some very basic habits every stylish woman tends to follow which leads to a great outfit every single day.
Stylish women are always aware with the perfect sizes according to their body. You certainly don’t want to look baggy or squeezed in any of your outfits. Wearing loose blouses will make look baggy and a too tight jeans will never do you good.
Staying messy is not the right choice for you if you want to look fashionable everyday. Because being messy will hide many clothes that you can pair together to create a great outfit. So stay organised which will help you select clothes for your outfit of the day. And a big benefit is that it will save a little time.
Mornings can be really hectic. So if you have already decided your outfit in the night, you will be ready to rock the world. No hassle in the pretty mornings.
It is certainly not necessary to buy expensive clothes all the time. You can look as much as stylish in low end fashion too. Just pair a expensive piece of accessory, top wear or bag to your outfit and you are good for the day.
Wearing wrong undergarments can make you look like a fashion disaster. So always keep you undergarment collection updated according to your clothes.
Following your favorite style icons and recreating their looks is not at all a bad idea. You liked what they wore? Go on, recreate it. And you can buy them from online fashion stores and make use of offers such as Debenhams Fashion offers.
If you shop according to the latest trends, Don’t. Trends come and go, they are not certain. Buy what you like and you will always have something to wear.
Backup outfit is a necessity. You thought of pairing a outfit and the result was not what you wanted. So your backup will save the day for you.
Never rush while getting ready if you want to look stylish. Give enough time to your outfit, makeup and other essential things.
Always be updated with the weather. You obviously don’t want to wear white on a rainy day.
8 Craziest And Embarrassing Things Everyone Has Done Once In Their Life!
Pierced Nose? 9 Things Only You Girls Can Understand Instantly!
5 super easy DIY tips to distress your Denim to your heart’s content.
8 Fierce Coco Chanel Quotes That'll Define The Power Of Women And Fashion!!

industry_corpus2: Others


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With our leading technology also as our spirit of innovation,mutual cooperation, benefits and advancement, we'll build a prosperous future together with your esteemed organization for Art Paint Paste , art paint paste , art paint date , and continuously make improvements to service high-quality.
We have been ready to share our knowledge of internet marketing worldwide and recommend you suitable items at most aggressive selling prices. So Profi Tools give you ideal benefit of money and we have been ready to create with each other with Art Paint Paste , art paint paste , art paint date , We'll continue to devote ourselves to market and product development and build a well-knit service to our customer to create a more prosperous future. Please contact us today to find out how we can work together.


text: The Blossom collection is designed with our Signature Stretch fabric. It is soft yet crisp to the touch along with a hint of spandex allowing lasting comfort and durability. This breathable and flexible Signature fabric holds its shape and is sure to keep up with you all day long. Try it today!

industry_corpus2: Manufacturing


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A honeymoon is the perfect excuse to purchase some beautiful pyjamas and nightwear. Not always cheap, but definitely a long term investment buy and most of the brands mentioned below offer a monogramming service...the perfect way to personalise your pj's! You will be pleased to know ivory and white aren't the only options either.
Poplin Bardot classic pyjamas £100.
Olivia Von Halle Lila silk pyjamas £350.
Yolke Scallop stretch silk pyjamas £290.
Olivia Von Halle Ava Ivory silk pyjamas £395.
Olivia Von Halle Lila silk striped pyjamas £350.
Olivia Von Halle Audrey silk robe £550.
Three Graces Drums in the night robe £800.
Yolke Fern silk robe. £200.
Bodas Shadow stripe cotton robe £72.
Hermione de Paula Coral floral robe £199.
Liberty Blue poppy cotton robe. £145.
Yolke Bluebell silk robe. £160.
Three Graces Alice maxi cotton dress £350.
Eberjey Love letters jersey nightdress £80.
Zara embroidered flowing gown £49.99.
Eres silk-satin night slip £370.
One of my favourite new nightwear brands is Three Graces London which was founded in 2015 by Catherine Johnson. Everything is completely dreamy and made from the best quality handmade silk and lace and Swiss cotton. I have been eyeing up their Victorian inspired Alice Fell Cotton Maxi Dress for weeks now which would be wonderful as a nightdress and equally perfect worn floating around on a warm Summers evening.


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Prepares students to design using three-dimensional digital graphics for games, visual effects, movies, virtual worlds, simulations, and many other applications. The curriculum integrates traditional art and design skills, the utilization of commercial 3D software, and design principles related to time, motion, and lighting. Students also study research methods and a range of problem-solving principles, and develop critical thinking and creative capacities. Most important, as a graduate of the 3DDD major, you will have the ability to adapt to the constantly-changing needs of the industry in order to create 3D models, renders, and simulations for a wide variety ofindustries.
Cooperative Education is an option for students in the 3D Digital Design program. Students are available during the summer and the curriculum is flexible enough that they can opt to co-op other terms as well.
Extensive laboratory facilities (15 labs with over $40M in up-to-date equipment) are housed within the College of Imaging Arts and Sciences.
Labs are state-of-the-art, featuring the latest software packages used in design, art, printing, publishing, photography, and film for traditional and digital production.
Most artists and designers need at least a bachelor's degree and a strong portfolio or reel of work.
We appreciate your interest in your career and we will make every effort to help you succeed. Feel free to contact James Bondi, the career services coordinator who works with the 3D Digital Design program. You can access information about services through our web site at www.rit.edu/careerservices.

industry_corpus2: Subject Education


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It is vital to recognize that the lifestyles tactics of human cells are basically one of a kind from the lifestyles techniques of bacterial cells. Antibiotics, in assessment to hydrogen peroxide and ethyl alcohol have a selective effect on existence techniques of bacteria, suppressing them, and do no longer affect the strategies happening inside the cells of the human frame. consequently, presently recognised antibiotics are classified based totally on their mechanism of action and chemical shape. some antibiotics inhibit the synthesis of outer shell (membrane) of the bacterial cellular – shape, is absolutely absent in human cells. most critical amongst these tablets are antibiotics of penicillin group, cephalosporins and a few other preparations. other antibiotics inhibit exclusive tiers of protein synthesis bacterial cells are tablets belonging to the group of tetracyclines, macrolides, aminoglycosides. The antibiotics differ considerably in their basic property – antibacterial activity. in the instructions for every antibacterial drug is a list of bacteria that the drug has a spectrum of its activity; some antibiotics paintings on many sorts of micro organism, others only for certain types of microbes. Sorry, nonetheless not detected antibiotics, which would have inhibited the hobby simultaneously micro organism and viruses, as the variations within the structure and peculiarities of metabolism of these microorganisms are fundamental. capsules which could act on viruses, notwithstanding enormous advances of recent years, continues to be absolutely insufficient, and their performance is rather low.
All dwelling things, such as micro organism, quick adapt to damaging environmental situations. The development of resistance to antibiotics is one of the most hanging examples of this kind of tool. it can be argued that subsequently, any type of micro organism can increase resistance to any antimicrobial drug. improvement of resistance occurs quicker, at large scale is used the substance. as the bacteria increase resistance to antibiotics, mankind has been forced to invent new tablets. therefore, we can assume that if today we had been to prescribe antibiotics to all children, grandchildren day after today we should deal with is honestly not anything. within the course of this race in society there are conflicts of interest. Society, in general, is interested in lowering the fee of antibiotic therapy and keeping the balance between cost and effectiveness of remedy. To achieve this aim it's miles necessary to restriction the use of antibiotics strict situations to keep away from immoderate charges for improvement and manufacturing of new tablets. manufacturers of the equal antibiotics on the contrary are interested by increasing income (due to the growth of symptoms), so that it will inevitably lead to more rapid unfold of resistance of microorganisms to pills and, as a consequence, the need to broaden new capsules. unluckily, the big and out of control use of antibiotics has caused significant resistance of microorganisms to them. moreover, in Russia the uncontrolled use of antibiotics combined with the shortage of finances for health care. these days in our us of a maximum of the causative agents of most not unusual infections resistant to tablets of the tetracycline organization. So the selection of drug for the treatment the physician wishes to realize no longer best what form of pathogen brought about the infection, however to what this pathogen is drug touchy. it might appear that this trouble is without problems solved in laboratory research. however, unfortunately, inside the software of present day methods of research the answer may be received only after 2 – three days. for this reason, in actual existence, antibiotics are prescribed empirically, i.e. on the basis of to be had sensible enjoy. but even the most amazing doctor can not independently gather experience inside the utility of all feasible antibiotics and is secure to say that the drug A higher than drug B. further, you should recollect how a good deal widely in concrete geographical region amongst bacteria is vast resistance to a selected drug. health practitioner will unavoidably should depend on the consequences of unique researches, their essential analysis of global and national revel in, as well as on pointers for standards of care advanced by means of professionals.
Having said that, it's far obvious that antibiotics should be used simplest for infections resulting from bacteria. health facility with severe and lifestyles-threatening infectious diseases (e.g., meningitis – inflammation of the meninges, pneumonia – irritation of lungs, and many others.) the duty for the correctness of the choice lies absolutely with the health practitioner, which is primarily based on information tracking of the patient and on consequences of special research. In slight infections going on within the outpatient putting, the state of affairs is essentially distinct. The doctor examines the child and prescribes a medication, sometimes accompanied via factors and answers to questions, on occasion now not. frequently a doctor is asked to prescribe an antibiotic. In such situations, the doctor is once in a while psychologically simpler to write down a prescription, and not to compromise their reputation and waste time explaining inexpediency of such appointment. So never ask the health practitioner to prescribe your infant antibiotics, specifically after the departure of the doctor is generally happens domestic recommendation, calls to household and friends and only then decide to provide the child antibiotics or not.

industry_corpus2: Biopharmaceuticals


text: Three-Bedroom Holiday Home in Dannemare is situated in Dannemare. Guests staying at this holiday home have access to a fully equipped kitchen. The holiday home comes with a TV and 3 bedrooms.

industry_corpus2: Travel & Geography


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If you missed out on the Vespa’s 1950s incarnation as a sleek functional objet that came to symbolize postwar Modernism (think Gregory Peck and Audrey Hepburn in Roman Holiday) or its 1960s and ’70s version as a fetish of British youth culture (think mohair suits, bennies, and gang wars between Mods and Rockers), here’s your chance to personify the scooter boy of the twenty-first century. Picture yourself cruising the East Village, organic coffee in the glove box, Beat Pod blaring 50 Cent, your dachshund behind you in a Vespet carrier.
The iconic Vespa returned to the American market in 2000, nearly 15 years since Piaggio had stopped selling the scooters here. A new age naturally demanded new accessories, and after the reintroduction of the classic PX model last year, creative director Evelyn Kim asked the Parsons Design Lab—a program that links students with companies for real-world experience—to conceive a line of Vespa accessories for the iPod generation.
Working in multidisciplinary groups of five, the students researched Vespa’s cultural history, observed current trends, and developed concepts for products that would easily complement the bikes and appeal to American consumers. Three are expected to go into production by the end of next year. Product-design student Eddie Chiu learned from a dealer that most New York Vespa buyers are now 28 and older, which inspired him to digitally model a reclining seat that converts the bikes into lounge chairs. Soner Onen, a design-technology student, came up with the concept for Vespet, a carrier for four-legged creatures with a grille in front, a window in back, and padding inside in case of bumps.

industry_corpus2: Automobiles


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Inspired by all the top Pop artists from around the world, this pack will give your Pop tracks the edge and take you to the next level in your production, you have everything here to create your next Vocal Pop hit with 24-Bit WAV Stems with wet & dry versions for the Main Vocal Parts/Vocal Chops, Drums, Percussion, Main Melody Parts, Bass, Guitars, Pianos, Plucks, FX, Leads, MIDI Files & Presets For Each Kit, listen to the demo and be blown away by this top quality pack.
* Please Use Avenger 1.2.4 Or Higher.
7 x FULL Top Future Pop Construction Kits.
209 x 24-Bit Wav Files.
All Kits Named, BPM & Key Labelled. (All Kits BPM 110).
1.96GB Zipped | 2.49GB Unzipped.
* 50 x Jaw Dropping Colossal EDM Drop Midi Files.
* 30 x Spire Named Presets (Individual & Bank Files).
* 10 x Room Shaking Kicks In Key.
* 9 x Folders With Spire Presets, Kicks & Midi Used For The Showcase Demo.


text:
Stay connected with what's happening at MIP UK with the latest news and updates from our Leighton Buzzard HQ!
Stay connected with the latest news from MIP UK!
Watch engaging content, ranging from "How-to" product Videos, to new Corporate initiatives, to specific industry related solutions and information.
Discover industry specific content and relevant MIP information, as well as Job openings and other corporate initiatives.
Be inspired and informed by images and content relevant to the healthcare industry, as well as to our products and solutions!

industry_corpus2: Health & Medicine


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Cyprus President Nikos Anastasiades and Turkish – Cypriot leader Mustafa Akinci Turkish will have their first meeting for 2017 on Wednesday morning.
Another meeting between the two leaders has been scheduled to take place on 7 January; before the Geneva talks on 9, 10 and 11 January and the Conference on Cyprus on the 12th of the month.
Contacts between negotiators Andreas Mavroyiannis and Ozdil Nami who met Tuesday are ongoing, and today Greek Foreign Nikos Kotzias minister will meet with the special adviser of Secretary General UN Cyprus, Espen Barth Εide and tomorrow he will meet with the Foreign Minister for European issues of Great Britain Sir Alan Duncan before departing for New York.
On Thursday the Nicos Anastasiades will inform the National Council on developments in view of the upcoming Geneva Conference, while today late afternoon he will brief the Holy Synod in the Archdiocese of Cyprus on the Cyprus Issue.
On Monday, January 9, the greek Prime Minister will inform political leaders about developments on Cyprus, while on Monday Alexis Tsipras will meet with Greek President Prokopis Pavlopoulos. The Cyprus Issue will be high on the meeting’s agenda with a focus on information and positions of political leaders and the positions of the Greek and Greek Cypriot side in the Geneva Conference.
Turkish Foreign Minister Mevlut Cavusoglu will be in New York on 5 January, according to a statement by the Turkish Foreign Ministry, where he will meet with UN Secretary General Antonio Guterres, a day before the meeting between Guterres and Kotzias on January 6.

industry_corpus2: Politics & Administration


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With PRIV™, DTEK60™, and DTEK50™, the power of BlackBerry is now on Android™. That means you get access to over a million apps on Google Play™ and great experiences that come with this OS. Combine that with the best integrated messaging experience on Android, and the added security of BlackBerry, and you truly have the best of both worlds.
On top of all the great experiences that come with the Android 6.0, Marshmallow, BlackBerry adds incredible new features that make sharing ideas and connecting with people faster and easier.
Android Marshmallow now on PRIV and DTEK series.
Whether it’s your financial reports or your selfies, your data is encrypted on the PRIV and DTEK series smartphones. We could get really technical and tell you about all the things we do to ensure our devices are ultra secure, but just know that BlackBerry lives and breathes security and has taken extra steps to help protect you from malware, data breaches and any attempts to hack and tamper with your OS.
BlackBerry smartphones powered by Android are packed with lots of other security features like Password Keeper to store all your passwords, log in information and important records, Picture Password (on PRIV) to make it virtually impossible for someone to break into your phone and encrypted messaging with BBM ®. Take control and be confident knowing your personal data is being kept private.
Compose email messages and social posts1, or respond directly to messages and calendar invites.
Snooze items and come back to them later, based on time/date, your location or your network connection.
Get the best out-of-box calendar experience on Android. It seamlessly syncs with work accounts, as well as personal, for a truly integrated experience.
When it’s time to join a call, the BlackBerry® Calendar will present you with a simple Join Now button that automatically dials you in. When receiving or creating meeting invites, you are presented with adjacent or conflicting appointments and time zone previews. And with Google Now™ voice commands, booking meetings has never been easier.
Late for a meeting? Send an auto-populated “I’m running late” message to attendees with just a few gestures.
Widgets allow you to see app content quickly on Android devices, displaying information from an app directly on your home screen. They can take up a lot of space there though, and potentially include sensitive information. BlackBerry Pop-up Widgets are a pre-loaded feature on BlackBerry smartphones powered by Android, which allow you to view any application widget by simply swiping up or down across the icon on your home screen, giving you fast access to the information you need.
Gesture controls have been improving the way information is accessed on BlackBerry smartphones for years. On your PRIV or DTEK series smartphone, swiping up from the bottom edge of the screen will allow you to quickly open BlackBerry Hub, Device Search, or Google Now, so your most important information is always one swipe away.
With access to Google Play on your PRIV, you automatically have access to over one million apps. Whether it’s Uber, Spotify®, Instagram or BBM®, you’ll find it there.
Connect to your TV, car, thermostat, wearables, fitness trackers, and so much more on your PRIV.
With Miracast built-in, you can display what is on the screen of your PRIV on your TV, game console, and other smart devices. Or easily stream music from apps like Spotify.
Learn about the all-touch DTEK50 and DTEK60, powered by Android.
Get help setting up and using your BlackBerry smartphone powered by Android.
1. Supported social apps only.


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Charming and private Hamlet made up by an historic farmhouse and 3 cottages situated in a beautiful wooded valley in the Lucca hills, at the start of the Garfagnana National Park.
Despite the not very high spot among the high hills, the house is very sunny even in winter season.
The houses date from the mid 1700s and have many original features, including exposed stone walls and original ceilings with chestnut beams and cotto tiles. The property is set in three and a half hectares of pine and chestnut wood, with over 100 abundant olive trees and other fruit trees, with beautiful views over the valley. The small swimming pool and its sun terrace seem to hover over the trees – ideal for lounging in the Tuscan sunshine.
The original, three storey farmhouse. Currently laid out with two double bedrooms, three large sitting rooms, large dining room, massive kitchen, master bathroom and utility / shower room. The house could easily rearranged with four bedrooms.
The converted barn. Two double bedrooms, double sitting room with fireplace, eat-in kitchen with fireplace, family bathroom with tub and separate large shower room.
The converted stable. Pretty sitting room, small kitchen, double bedroom and shower room.
The converted bakehouse. Pretty sitting room with original working, full-size pizza / bread oven, small kitchen, double bedroom and shower room.
The property is accessed via a long concrete drive. The property is completely private. At the end of the drive is a wall with large wrought iron gates. Next to the gates is shrine dating from the 1740s, which is also owned by the property. This has an original, 18th century marble bas-relief of Santa Zita, patron saint of Lucca and born in Pescaglia in the 11th century.
The pool measures 8x4m and seems to hang above the trees, giving wonderful views across the valley. The whole area was remodelled in 2004, with the addition of a brick path with stone steps, a large brick patio area and a wooden deck suspended over the valley.

industry_corpus2: Agriculture & Fisheries


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Valuable Content » Arts-and-entertainment » Best Television set holders.
No matter whether you might have a LCD or maybe Plasma TV SET you are going to would like to don it something. There is a fantastic collection of TELEVISION stands for smooth monitors available on the market which will enable you to select one who accommodates your personal seems and complements your own home's design. Many people may request exactly why can easily they will certainly not location their own TELEVISION SET on the regular piece of furniture. Properly, there is nothing to stop these people other than the truth that a new TELEVISION endure cost not nearly as expensive a good expensively developed piece of furniture made to store an appartment screen TELEVISION SET.
Precisely what other considerations are you looking to check? Nicely, most people never only have a very TELEVISION, they will often employ a DVD participant, some sort of gaming system, what about a TIVO hence the tv set remain you buy will be able to cater to your audio-visual gear plus the real ripped display screen TELEVISION alone. Several holders may also appear complete with a new cable television more tidy which can be utilized to risk-free all your cables firmly in addition to efficiently rather than acquiring strands connected with spaghetti major out of your TELEVISION along with tools.
Dimension must also consider. That you do not have to get some sort of have that is below sort of along with your TELEVISION seems like it'll slide away from with the merest impression definitely not would you like to obtain a TV SET remain which is outsized and swallows up important living space inside your space. The opposite matter to check for would be the true style and design in the stay. Some individuals enjoy stainless and consider it appears ideal for their house. Some others choose a far more healthy appear with what about a timber complete. As well as you may decide a new matte african american end together with frosted glass to present a complex glimpse. Merely you possibly can determine exactly what appears very best to your house.
A brand new ripped tv screen merely signifies that your property will have this activity centre you've got imagined during the last 12 months. The thing you need to select this can be a TV remain generated for the item. Having fresh TELEVISION SET are a symbol of level projection screens you will get something that ended up being created exclusively for your brand-new TELEVISION. Along with a wide variety designs and finishes to decide on among you're secured to uncover the one which matches the present interior decoration. Realizing that almost everything look like it has been acquired while doing so can help you keep house hunting clean and pointed creating the best amusement region.
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Welcome to another edition of our newsletter, in this week’s mail we have a host of results including the Invitation Day and Ashridge Cup, Kian wins the Spooner Stableford, and time is running out to take advantage of our Complete Equipment Solution. Enjoy the read!
Ashridge won 3 – 2.
We have three sets of Lynx irons in stock, as well as a driver, wood and hybrid that I am looking to clear. Please pop into the shop and see what excellent offers we have available!!!
I need to clear my own Callaway Big Bertha Staff bag (which retailed for around £300) and am happy to accept £120 for it, or nearest offer. This is a superb bag that has a ton of space for anything and everything you could ever want to put in your golf bag.
Come in before it gets snapped up!