Article |
Address correspondence to Howard A. Rockman, Duke University Medical Center, DUMC 3104, Durham, NC 27710. Tel.: (919) 668-2521. Fax: (919) 668-2524. E-mail: h.rockman{at}duke.edu
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Abstract |
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Key Words: phosphatidylinositols; phosphoinositide 3-kinase; AP-2; ß-adrenergic receptor; endocytosis
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Introduction |
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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 Gß 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 ß-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 ßAR internalization (Naga Prasad et al., 2001). Considering the potential important role of D-3 phosphoinositides in ßAR internalization, we sought to determine: (a) whether there was a direct physical interaction between ßARK1 and PI3K, and if so, identify the structural domain responsible for this interaction; (b) whether the interaction of PI3K with ßARK1 regulates the translocation PI3K to the agonist-occupied ßAR; and (c) whether the lipid products of PI3K modulate the internalization of ßARs.
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Results |
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To investigate whether there was a direct physical interaction between the PIK domain of PI3K and ßARK1, GSTPIK fusion protein was immobilized on sepharose beads and incubated with purified ßARK1. Purified ßARK1 bound specifically to GSTPIK immobilized beads and not to GST alone (Fig. 1 c). No difference in the level of GST and GSTPIK was found (Fig. 1 c, bottom).
Because PI3K interacts with the ß
subunits of G-proteins, we tested whether the PIK domain might directly interact with Gß
, and thus would compete with ßARK1 for these subunits. Purified ßARK1 and GSTPIK fusion protein immobilized on Sepharose beads were incubated with purified Gß
. The beads were washed and recovered proteins were analyzed by SDS-PAGE followed by immunoblotting with a Gß polyclonal antibody. Whereas a strong association of Gß
with ßARK1 was found, no appreciable association of Gß
with the PIK domain was detected (Fig. 1 d). Densitometric quantification showed an 11.5 ± 1.1-fold greater binding ability of ßARK1 to Gß
purified proteins compared with the PIK domain.
To test whether overexpression of the PIK domain could displace ßARK1 associated PI3K activity in living cells, experiments were performed in HEK 293 cells cotransfected with plasmids containing the ßARK1 cDNA (2 µg) and increasing concentrations of the PIK domain cDNA (ranging from 0 to 6 µg). Cell lysates were immunoprecipitated using the ßARK1 monoclonal antibody 48 h after transfection and ßARK1 associated PI3K activity was assayed. A robust ßARK1 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 ßARK1 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 ßARK1 monoclonal antibodies in the cotransfected HEK 293 cells (data not depicted).
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Because the PIK domain is shared by all members of the PI3K family, we wanted to confirm that the ßARK1-associated endogenous PI3K activity was contributed by Class I PI3K. To test this, HEK 293 cells were transfected with plasmids containing cDNAs for ßARK1 (2 µg) or ßARK1 plus PIK (4 µg). Cell lysates were immunoprecipitated with a ßARK1 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 ßARK1 and the coexpression of PIK completely displaced the ßARK1 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 ßARK1 (Fig. 2 e). Taken together, these data demonstrate that overexpression of the PIK domain can disrupt the interaction between ßARK1 and PI3K, and that the lipid kinase activity belongs to the Class I PI3K family.
Overexpression of PIK blocks ßARK1-mediated translocation of endogenous PI3K
Our results suggest that overexpression of the PIK domain should block the ßARK1-mediated translocation of PI3K to the membrane. In order to test this hypothesis, HEK 293 cells were cotransfected with the ßARK1 (2 µg), ßARK1, and PIK domain (4 µg) containing plasmids, and endogenous ßARs were stimulated with 10 µM isoproterenol for 2 min. Cytosolic and membrane fractions were prepared and analyzed for ßARK1 associated PI3K activity. After isoproterenol stimulation, robust ßARK1-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 ßARK1/PI3K interaction. No change in ßARK1 associated PI3K activity was found in cytosolic fractions after isoproterenol stimulation (data not depicted).
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To test whether disruption of the ßARK1/PI3K interaction would prevent the recruitment of PI3K to activated ßARs, HEK 293 cells were transfected with plasmids containing cDNAs encoding FLAG epitopetagged ß2AR (FLAG-ß2AR, 2 µg) or FLAG-ß2AR (2 µg) and PIK domain (4 µg), and assayed for ß2AR associated PI3K activity after stimulation with 10 µM isoproterenol. HEK 293 cells are known to contain adequate levels of ßARK1 to support agonist-induced translocation and receptor phosphorylation (Menard et al., 1997). FLAG-tagged ß2ARs were immunoprecipitated from cell extracts and associated endogenous PI3K activity measured. Significant FLAG-ß2ARassociated PI3K activity was observed as early as 2 min after agonist stimulation, with a decline by 5 min in the cells transfected with ß2AR alone (Fig. 3 c). In contrast, no FLAG-ß2ARassociated 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 ßARK1 complex, thereby preventing its recruitment to the agonist-occupied receptor complex.
Attenuation of ß2AR sequestration by PIK
Previous studies have suggested a role for PI3K in ß2AR internalization (Naga Prasad et al., 2001). We postulated that disruption of the endogenous ßARK1/PI3K interaction by the PIK protein would attenuate ß2AR endocytosis. To test this, agonist-dependent sequestration was studied by [125I]-cyanopindolol binding in HEK 293 cells cotransfected with plasmids containing either the FLAG-ß2AR cDNA, or FLAG-ß2AR and FLAG-PIK cDNAs. A significant (>60%) attenuation in the rate of ß2AR 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 ß2AR sequestration as the initial phase (05 min) was significantly impaired.
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To definitively show that only those cells that contained the PIK domain protein failed to undergo agonist-stimulated ßAR internalization, dual labeling experiments were performed after cotransfection with plasmids encoding HA tagged ß2AR (HA-ß2AR) and PIKGFP. After isoproterenol stimulation, cells were fixed and HA-ß2AR was visualized by Texas red staining and PIK was visualized by GFP fluorescence. Cells that showed restricted distribution of Texas red staining (HA-ß2AR 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 ß2AR internalization (Fig. 4 c, panel 1 and 2, arrowheads) as clearly seen in the overlay (Fig. 4 c, panel 3, arrowheads).
PI3K is not necessary for ß-arrestin recruitment
To test whether PI3K activity was necessary for the recruitment of ß-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 ß2AR-HA and ß-arrestin2GFP 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 ß-arrestin2GFP 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 ß-arrestin2-GFP. With isoproterenol, there was marked redistribution of GFP fluorescence to the membrane, indicating recruitment of ß-arrestin to the membrane (Fig. 5 a, panel 3). Importantly, cells that contained PIK proteins did not affect the membrane recruitment of ß-arrestin (Fig. 5 a, panels 3 and 4, arrowheads), suggesting that D-3 phosphoinositide molecules are not necessary for arrestin recruitment to activated receptors.
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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-ß2AR, 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.
Role of D-3 PtdIns in the recruitment of adaptin
Since overexpression of PIK leads to attenuation of ß2AR endocytosis, we wanted to determine whether the ßARK1-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-ß2AR or FLAG-ß2AR, 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 25 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 ß2AR 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, ß2AR, and PIK were observed in the appropriately transfected cells (Fig. 6 a, bottom).
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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-ß2AR 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-ß2AR 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).
Attenuation of ß2AR endocytosis upon inhibition of PtdIns-3,4,5-P3 production in cells
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 ß2AR-YFP or ß2AR-YFP plus PTEN, and ß2AR endocytosis was evaluated using laser scanning confocal microscopy. ß2AR internalization was followed in the same cell after agonist stimulation. Before agonist, the distribution of ß2AR-YFP was found distinctly at the plasma membrane (Fig. 7 a, panel 1). After agonist stimulation, there was a progressive redistribution of the ß2AR-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 ß2ARs with the PTEN completely prevented the agonist induced redistribution of ß2AR-YFP fluorescence into membrane puncta and intracellular aggregates (Fig. 7, panels 46).
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Discussion |
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Agonist-occupied ßARs are phosphorylated by ßARK1 after translocation of ßARK1 to the membrane (Lefkowitz, 1998). Subsequently, the phosphorylated receptor can bind with high affinity to ß-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-3phosphorylated 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/ß-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 clathringreen 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 ß-arrestin recruitment to the receptor, nor does it impair the ability of ßARK to phosphorylate activated receptors, as ß-arrestin is only recruited to GRK phosphorylated receptors (Ferguson et al., 1996). Previous studies have shown that deletion of the phosphoinositide binding domain on ß-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 ß-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 ß-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 ßARK1 to the agonist occupied receptor is a ßARK1 specific process. Furthermore, overexpression of the PIK domain did not affect activation of PKB suggesting a specific role in displacement of PI3K from ßARK1. 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 ß2AR 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 ß2AR 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 PI3K 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 ßARK1 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 ßARK1 could also interact with the PI3K
isoform when overexpressed (Naga Prasad et al., 2001). Interestingly, it has recently been shown that another PI3K, the class II PI3K C2
, 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 ßARK1 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 ßARK1 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/ß-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.
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Materials and methods |
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Plasmid constructs
PIK and PI3KPIK mutants of PI3K were prepared by PCR amplification using the full-length p110
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.
PI3KPIK 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 PI3K
PIK cDNA (Fig. 1 a). The catalytic activity of PI3K
PIK was indistinguishable from wild-type PI3K (unpublished data). The plasmids containing cDNAs encoding ßARK1, FLAG-ß2AR, HA-ß2AR, and p110
have been described previously (Naga Prasad et al., 2001). The PTEN plasmid was gift from Dr. Christopher Kontos (Duke University Medical Center).
GST fusion protein expression and pulldown experiments
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-ß-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 (11.5 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 ßARK1 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 ßARK1 protein was prepared by incubating ßARK1 monoclonal antibodies with protein G agarose beads for 1 h at 4°C, followed by the addition of purified ßARK1 protein. Subsequently, 10 µg of purified Gß was added to either ßARK1 immobilized beads (5 µg), or the GSTPIK fusion protein beads (5 µg), and gently rocked for 45 min at room temperature. Beads were spun down, washed in binding buffer x2, and resolved by gel electrophoresis. The presence of Gß
was detected by immunoblotting with an antibody directed against the Gß subunit. Purified Gß
and ßARK1 were gifts from Dr. Robert Lefkowitz.
Membrane fractionation
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.
Lipid kinase assay
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 ßARK1 (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 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 510 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
Immunoblotting and detection of ßARK1, FLAG-PIK, PI3KPIK-HA, FLAG-ß2AR, HA-ß2AR, ß-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 ß-adaptin, clathrin heavy chain (BD Transduction Laboratories), HA (Roche Molecular Biochemicals), and FLAG (Sigma-Aldrich) at a 1:2,000 dilution and the ßARK1 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.
Determination of ß2AR sequestration in HEK 293 cells by [125I]-cyanopindolol binding
ß2AR sequestration was performed as previously described (Naga Prasad et al., 2001). HEK 293 cells were transfected with plasmids containing ß2AR (250 ng) or ß2AR (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 in living and fixed cells
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 ß2AR-YFP (2 µg) or ß2AR-YFP (2 µg) and PIK domain (4 µg) or ß2AR-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 010 min. For dual staining of ß2AR-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 antimouse 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 505550 nm for GFP detection and 585 for Texas red detection.
Treatment of cells with phospholipids
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
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. TransferrinTexas 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.
Statistical analysis
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 ß2AR sequestration by CYP binding was analyzed using Graph-pad prism.
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Footnotes |
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* Abbreviations used in this paper: ßAR, ß-adrenergic receptor; CYP, cyanopindolol; GPCR, G proteincoupled receptor; PI3K, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol(s).
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Acknowledgments |
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Submitted: 22 February 2002
Revised: 12 June 2002
Accepted: 12 June 2002
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References |
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