Background: Activation of GRK2 requires interaction with agonist-occupied GPCRs.Results: Residues on the GRK2 N terminus and kinase domain extension collaborate to create a GPCR docking site.Conclusion: Three GRK subfamilies use similar determinants to create the putative docking site, but subtle differences may dictate selectivity.Significance: Mapping the GRK-GPCR interface is required to understand the mechanism and specificity of GRK activation, and, therefore, the regulation of GPCRs. in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α2A-adrenergic receptors (α2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α2AAR with EC50 values of 15 nm and 8 μm, respectively. We show that mutations in αN  and AST  regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu4, Val7, Leu8, Val11, and Ser12, directly interacts with receptors, whereas residues such as Asp10, Tyr13, Ala16, Met17, Gly475, Val477, and Ile485 are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation  G protein-coupled receptors (GPCRs)1–A).GRKs belong to the AGC kinase family , and thus their kinase domains have many characteristics in common with the catalytic domain of protein kinases A, B (Akt), and C . However6–9–Fig. 1Membrane localization is also key to GRK function, and the various GRKs utilize individual mechanisms to promote membrane localization . For exa14–B and C). Most GRK crystal structures reported thus far display unstructured N-terminal and kinase domain extension regions, and their kinase domains adopt inactive conformations. The sole exception is the GRK6-sangivamycin complex, wherein the small and large lobes assume a conformation similar to that of activated PKA, and the N-terminal region forms an α-helix (αN) that packs against the small lobe and the AST  assay (2A-adrenergic receptor (α2AAR). Our results suggest that, as predicted for GRK1 and GRK6  assay , 25 thatand GRK6 , 21, the2AR cDNA in a mammalian cell expression vector (pcDNA3-FLAG-β2AR) was a gift from Dr. Jeffrey Benovic  (2AR(Y326A) was a gift from Drs. Marc Caron and Larry Barak 355/Ser(P)356, the β2AR carboxyl tail , and GRK2 were obtained from Santa Cruz Biotechnology, Inc. Protein structure figures were generated using the PyMOL Molecular Graphics System.African green monkey kidney cells (COS-7) were from the American Tissue Culture Collection (ATCC). N-terminal FLAG-tagged human βhia, PA) . pcDNA3.ry Barak , 28  (1OMW) (3NYN and 3KRW (The structure of GRK6 in complex with sangivamycin (PDB entry 3NYN)  was usedand 3KRW . The linand 3KRW .6 (pFBD-GRK2-H6)  , 32, 33 GRK2-H6) , 29, butGRK2-H6)  and humaeviously . All con6 constructs. For GRK2-H6 expression, High 5 cells (9 × 106) were plated on 10-cm tissue culture plates in SF900 II medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, and amphotericin B and infected for 48 h with appropriate GRK2 variant baculovirus  before suspension in 0.4 ml lysis buffer , 0.2 mg/ml benzamidine, 10 μg/ml leupeptin, 1 mm dithiothreitol (DTT). Cells were lysed by micro-tip sonication using 90 cycles of alternating 1-s bursts and 2-s rest periods, and then lysates were clarified by centrifugation for 20 min at 40,000 × g. High-speed supernatants were stored in 25% glycerol at −20 °C. GRK2 content in each lysate was determined by Western blot comparison with a GRK2 standard curve.The Bac-to-Bac Expression System (Invitrogen) was used to generate baculoviruses from the various pFBD-GRK2-Hulovirus . Cells wm Tris-HCl, 2 mm EDTA, 7.5 mm MgSO4, pH 7.5). Reactions (12.5 μl) were performed in kinase assay buffer containing 8 μm Rho, 200 μm [γ-32P]ATP (∼1 dpm/fmol ATP), and 2 μl of each lysate. Reactions were equilibrated at 30 °C for 30 s under dim red light illumination before incubation in ambient fluorescent light. Reactions were quenched after 3 min with 12.5 μl of SDS-PAGE sample buffer (containing 50 mm DTT), and then samples were incubated for 30 min at 65 °C with periodic vortex mixing before separating Rho by 10% SDS-PAGE. Gels were stained with Coomassie Blue, and 32P incorporation into Rho bands was determined by liquid scintillation counting. The activity of a High 5 lysate prepared from uninfected cells was subtracted from all samples to account for the activities of endogenous kinases .High 5 cell lysates with overexpressed GRK2 were screened in a rhodopsin (Rho) phosphorylation assay to identify mutants defective in GPCR phosphorylation. Rod outer segments were isolated from frozen dark-adapted bovine retinas , washed with urea as described previously , and res6 variants in High 5 lysates that displayed reduced catalytic activity toward Rho were purified to homogeneity, as described in detail (4 cells/ml) in antibiotic/antimycotic-containing Sf900 II medium were infected with GRK2 baculovirus (volume previously optimized) and incubated at 28 °C for 48–60 h. Cells were collected; washed in STE; resuspended in Ni2+-column buffer (NCB) ; lysed with a Polytron tissue disruptor; and centrifuged at 35,000 × g for 20 min at 4 °C. Supernatants were centrifuged again for 45 min at 100,000 × g, and the resulting supernatants were diluted to 5 mg/ml protein with NCB and adjusted to 20 mm imidazole before loading onto a 5-ml column  containing Ni2+-nitrilotriacetic acid-agarose (Qiagen). Following 15-ml washes with NCB plus 20 mm imidazole and NCB plus 40 mm imidazole, the column was eluted with 40 ml of NCB plus 150 mm imidazole. Fractions (1 ml) were collected and analyzed by Coomassie staining of 8% SDS-polyacrylamide gels. GRK2-H6-containing fractions were pooled, diluted to a NaCl concentration of 50 mm with High S column buffer , and loaded onto tandem 1-ml High Q/High S columns (Bio-Rad). The High Q column was removed, and the washed High S column was eluted with a 20-ml linear gradient of 50–600 mm NaCl in HCB. Triton X-100 was omitted from HCB for the purification of AST mutants, R474A, G475I, N478A, A479S, and A480S. Fractions (0.5 ml) were collected, analyzed by Coomassie staining of 8% SDS-polyacrylamide gels, pooled, and, when necessary, concentrated by centrifugation at 2500 × g using Centricon (Amicon) filtration. Purified proteins were stored in 25% glycerol at −80 °C, and GRK2 content was determined by Coomassie staining of 8% SDS-polyacrylamide gels.GRK2-Hn detail , for furm GRK2, 8 μm Rho, 200 μm [γ-32P]ATP (∼1 dpm/fmol) in GRK2 kinase buffer. Reactions were terminated with 14 μl of SDS-PAGE sample buffer containing 50 mm DTT and processed as described above. The kinase activity observed in the absence of GRK2 (uninfected lysates or a buffer control) represented 2–7% of WT-GRK2 activity and was subtracted from all samples.Phosphorylation of Rho was performed in 10-μl reactions containing 20 nm GRK2 variant, 100 μm [γ-32P]ATP (∼1.5 dpm/fmol), and 1 mm peptide C in kinase assay buffer. Reactions were incubated for 50 min at 30 °C and quenched with 14 μl of SDS-PAGE sample buffer containing 10 mm DTT. Samples were resolved by 18% SDS-PAGE following incubation at 65 °C for 10 min. Peptide C bands were excised and measured for 32P incorporation by scintillation counting.The phosphorylation of peptide C (DDEASTTVSKTETSQVARRR), corresponding to the carboxyl tail of bovine rhodopsin, was measured in 10μl reactions containing 200 n6 variants was measured using endoproteinase Asp-N-treated Rho (329G-Rho), which lacks its carboxyl tail phosphorylation sites , ∼2 μm329G-Rho, and 100 μm peptide C in GRK2 kinase assay buffer were equilibrated at 30 °C for 5 min before exposure to ambient light or continued incubation in the dark for 30 min. SDS-PAGE sample buffer (12 μl) containing 10 mm DTT was used to quench each reaction. Samples were incubated for 10 min at 65 °C and resolved by 18% SDS-PAGE, and the excised peptide C bands were used to measure 32P incorporation as described above. Relative to GRK2 purified in the presence of 0.02% Triton X-100, WT GRK2 purified in its absence displayed a 11-fold decrease in the ability to phosphorylate Rho. Therefore, GRK2 activity presented in Receptor-dependent activation of peptide C phosphorylation by GRK2-Hon sites . ReactiomK and kcat were determined by varying Rho concentration in rod outer segments between 2.5 and 30 μm in 10-μl reactions containing 100 nm GRK2-H6 variant, 100 mm HEPES, pH 7.5, 1 mm EDTA, 10 mm MgCl2, and 1 mm [γ-32P]ATP (∼0.2 dpm/fmol). Phosphorylated Rho was quantified as described above, and initial velocities were fit to the Michaelis-Menten rate equation using GraphPad Prism version 4.0.2AR in COS-7 cells was carried out as described (l-glutamine (4 mm) at 37 °C with 5% CO2. Cells (3.0 × 105 cells/well) were plated in 6-well tissue culture dishes and transfected the following day with 0.8 μg of pcDNA3.1-FLAG-β2AR(WT) or pcDNA3.1-HA-β2AR(Y326A) and 0.4 μg each of pcDNA3.1-Gβ, pcDNA3.1-Gγ, and pcDNA3-GRK2 using 8 μl of FuGENE-HD. After 48 h, cells were serum-starved for 30 min before a 5-min treatment with 10 μm isoproterenol (ISO) or alprenolol, as indicated. Cells were washed twice with cold 20 mm Tris, pH 7.5, 150 mm NaCl and scraped with 200 μl/well receptor solubilization buffer . The resulting lysates were sonicated using a 30-cycle regimen of 1 s on/2 s off, and the receptor was further solubilized by mixing for 30 min on an orbital shaker at 4 °C. We have subsequently determined that sonication is unnecessary. Lysates were clarified by centrifugation at 16,000 × g at 4 °C, and 30 μl of the soluble fractions were treated with 100 units of peptide N-glycosidase F for 2 h at 37 °C. Peptide N-glycosidase F-treated samples were resolved by 10% SDS-PAGE and transferred to nitrocellulose, and immunoblotting was performed sequentially with three primary antibodies: 1) β2AR phosphosite antibody that recognizes agonist-induced phosphorylation (Ser(P)355/Ser(P)356); 2) an antibody that recognizes the β2AR carboxyl tail and reflects total β2AR; and 3) GRK2 polyclonal antibody. Each primary antibody was visualized with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and chemiluminescent substrates, either SuperSignal West Femto for Ser(P) blots or SuperSignal West Pico for β2AR and GRK2 immunoblots. Signals were visualized using the Bio-Rad ChemiDoc XRS System, and band intensities were quantified using Bio-Rad Quantity One software. Stripping of antibodies from nitrocellulose between immunoblotting experiments was achieved by incubation in 25 mm glycine pH 2, 1% SDS for 30 min at room temperature.GRK2-dependent phosphorylation of βescribed . Briefly2. Two days before the experiments, the polyethyleneimine 25-kDa linear transfecting agent  was used with a 3:1 PEI/DNA ratio to transfect either 2.5 μg of total DNA into 106 cells in 6-well plates (for one-point stimulation and titration experiments) or 12 μg of total DNA into 6 × 106 cells in 10-cm dishes (for kinetics and dose-response experiments). α2AAR-GFP2 and RlucII-GRK2 DNA constructs were diluted in 150 mm NaCl in Tris-HCl , and the total amount of DNA transfected in each well or dish was equalized with salmon sperm DNA (Invitrogen). On the day of the BRET experiment (48 h post-transfection), cells were washed, detached, resuspended in PBS, and distributed (105 cells/well) to 96-well microplates . The expression level of α2AAR-GFP2 was measured as total fluorescence using a FlexStationII (Molecular Devices) with excitation and emission filters at 395 and 510 nm, respectively. Cells were then incubated in PBS or NOR at room temperature. For one-point stimulation and titration experiments, cells were stimulated with 100 μm NOR for 6 min. For time courses, 100 μm NOR was dispensed for the indicated times. Dose-response curves were performed 6 min after NOR treatment at the indicated concentrations. For all BRET experiments, 2 min before BRET readings, coelenterazine 400A (Biotium) was added to a final concentration of 5 μm, and the expression level of the RlucII-GRK2 variants was measured using a Mithras LB940 multidetector plate reader (Berthold Technologies). BRET measurements were collected using the same plate reader by recording signals detected in the 480 ± 20- and 530 ± 20-nm windows for the Renilla luciferase and GFP2 light emissions, respectively. To determine the specific BRET signal (net BRET), the background signal detected in cells transfected with the luciferase donor alone was subtracted from the BRET values obtained in cells expressing both the energy donor and acceptor. Agonist-promoted BRET reflects the difference between the 530/480 ratios in the presence and absence of NOR. Except for titration experiments, BRET experiments were performed at equivalent acceptor/donor expression ratios for all GRK2 constructs tested. For BRET titrations, a constant amount of the donor was cotransfected with increasing amounts of the acceptor, and for each transfection condition, BRET values were expressed as a function of the total expression level of the acceptor over the total expression level of the donor detected.HEK293T cells were cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin and streptomycin (Wisent Inc.) and incubated at 37 °C in 5% COa posteriori Dunnett's test. For BRET assays, statistical significance was evaluated based on one-way ANOVA with a Tukey's post-test following pooling of all the data.Statistical significance of differences between WT and mutants in phosphorylation assays was assessed with GraphPad Prism version 4.0 software using one-way or two-way analysis of variance (ANOVA) followed by 6 variants were expressed in High 5 insect cells, and cell lysates were screened for their ability to phosphorylate light-activated Rho (A). Most of the purified mutants showed defects comparable with those of their counterparts in cell lysates with the exception of the M15A mutant, which did not exhibit a significant defect when purified. Conversely, M17A showed a more pronounced defect when purified. The reason for this discrepancy is not clear. Of the purified N-terminal mutants, the Y13A mutation continued to show significantly diminished activity compared with WT (90% reduced). Most other mutants lost 50–70% activity (A).The GRK6-sangivamycin crystal structure showed that the extreme N terminus of the enzyme can fold as an extended helix that packs in a cleft formed between the kinase small lobe and the AST. Because point mutations in these interfaces impair function in GRK1, GRK2, GRK5, and GRK6 , 21, 22,ated Rho . The Y13ated Rho A. Most oactivity A.6 variants were assayed for their ability to phosphorylate peptide C, a synthetic substrate derived from the cytoplasmic tail of Rho (B). The D10A, D10R, Y13A, A16V, and M17A mutants lost 47–76% of WT activity. Because these positions are predicted to be oriented at the interface with the small lobe (Ala16 and Met17) or with the AST (Asp10 and Tyr13) (B), their reduced activity is consistent with the N-terminal helix stabilizing the active state of the GRK2 kinase domain. In contrast, the L4A and V7E variants, which were significantly impaired in Rho phosphorylation, retained 176 and 130% of WT activity, respectively. In the homology model, the Leu4 and Val7 side chains point away from the small lobe and AST and are positioned to make intermolecular interactions (B). Our results are consistent with the hypothesis that these GRK residues make direct interactions with GPCRs  B, their ractions B. Our reth GPCRs , 13.6 N-terminal variants  using light-activated Rho as the substrate. The L4A mutation resulted in a 5-fold mK defect with retention of kcat .To better understand the roles of the WT residues in GRK2 function, Michaelis-Menten kinetic constants were measured for selected GRK2-H of kcat . A Km bual helix B.A) and 76–88% reduction in peptide C phosphorylation (B). Thus, these positions are important for both receptor and peptide phosphorylation, consistent with the interactions these residues are predicted to form with the small lobe and/or N-terminal helix. Notably, variants N478A, A479S, A480S, and A482I, whose side chains point toward the kinase large lobe, were not impaired in Rho phosphorylation (B).The GRK2 AST region extends from residue 475 to 485. The G475I, V477D, and I485A mutants displayed a 75–98% reduction in Rho phosphorylation A and 76–rylation B. Thus, rylation B.329G-Rho* stimulated WT activity 6.4-fold, it stimulated D10A, D10R, and Y13A less than 2-fold and did not activate the V477D and I485A mutants  to accelerate phosphorylation of peptide C by our GRK2 variants. Whereas  mutants . The N-tin vitro occur in intact cells, we first assessed the agonist and GRK2 dependence of β2AR phosphorylation in COS-7 cells co-expressing β2AR/GRK2/Gβγ using a GRK phosphosite immunoblotting assay (A). We previously demonstrated that Gβγ enhanced the detection of GRK2-dependent phosphorylation of β2AR  variant is totally impaired in ISO-dependent phosphorylation by endogenous GRKs but that overexpression of GRK2, GRK3, or GRK5 can rescue this defect  variant is capable of full stimulation of adenylyl cyclase activity (2AR(Y326A) variant. Co-transfection of GRK2/Gβγ led to ∼10-fold stimulation of agonist-induced receptor phosphorylation at the Ser(P)355/Ser(P)356 phosphosites (A), consistent with previous results  in intact cells resembled its in vitro activity toward Rho (B). The L4A, V7E, D10R, Y13A, A16V, and M17A variants lost 75–95% of the WT cell-based phosphorylation activity (comparable with the kinase-deficient K220R variant) and were more impaired than in the in vitro Rho phosphorylation assay (A). It is possible that the β2AR(Y326A) mutant exaggerates the GRK2 defects. The M15A mutation again displayed no decrease in activity, mimicking its behavior in the Rho phosphorylation assay. The D3A variant, which showed a 2-fold defect in Rho phosphorylation, also did not exhibit a defect in β2AR phosphorylation. The V477D and I485A AST variants were as deficient as the D10R or Y13A variants in phosphorylation of the β2AR. These data suggest that variants L4A, V7E, D10R, Y13A, M15A, M17A, V477D, and I485 exhibited an overall consistency in their ability to phosphorylate Rho in vitro and the β2AR in intact cells. In contrast, D3A and A16V displayed subtle differences that may be attributed to receptor specificity, the assay milieu, or both.We next surveyed the ability of GRK2 mutants to phosphorylate this receptor. For most mutants, the ability of each GRK2 mutant to phosphorylate the βward Rho B. The L4on assay A. It is in vitro because GRK2 is expected to bind weakly to GPCRs and binds strongly to negatively charged phospholipids, which are required along with Gβγ for GRK2 activity (2AR(Y326A) by GRK2 in intact cells (B), we have thus far been unable to detect a significant ISO-stimulated BRET signal between β2AR-GFP, β2AR-Venus, β2AR-GFP10, and luciferase-GRK2 or between β2AR-luciferase and GRK2-GFP10. The β2AR(Y326A) variant also did not yield a measurable ISO-promoted BRET response. However, we were able to detect agonist-dependent recruitment of GRK2 to the α2AAR, as indicated by the NOR-induced BRET signal observed between α2AAR-GFP2 and luciferase-GRK2 in a time course experiment (A). Co-expression of a constant level of RlucII-GRK2 with increasing concentrations of α2AAR-GFP2 led to an increase in basal and agonist-induced BRET signals in a saturable fashion (B), indicating a specific interaction between the two proteins. For all subsequent BRET experiments, acceptor/donor ratios yielding maximal BRET were used, and the readings were collected 6 min following receptor activation. These conditions provided stable signals and a good dynamic range.One of the primary goals of this work was to distinguish residues on GRK2 that are important for direct interaction with GPCRs. This interaction is difficult to measure activity . Thus, wct cells B, we havperiment A. Co-exp fashion B, indicaA). As a positive control, we anticipated that catalytically inert mutants of GRK2 (such as K220R and D317A) would retain the agonist-induced BRET signal. The K220R and D317A variants are indeed efficiently recruited, with the D317A mutation being recruited 2-fold better than WT . The molecular basis for the increased recruitment of D317A is not clear, but the behavior of the K220R and D317A variants in the recruitment assay is consistent with the reported ability of catalytically inactive GRKs to serve as dominant negative mutants  and β2AR in intact cells (A).We next looked at the GRK2-R195A mutant. Argct cells . We predct cells A.50,H = 15 nm) and a low-affinity component  (A). For WT GRK2, the high- and low-affinity interactions account for ΔBRET values of ∼0.010  and ∼0.027 , respectively. The impairment caused by the R587Q mutation, which prevents the binding of GRK2 to Gβγ, resulted in a nearly complete loss of the BRET signal. On the other hand, the dose-response curve for the R195A mutant exhibited a biphasic curve that retained the high-affinity interaction but a ∼60% reduced low-affinity interaction compared with WT. These results suggest that low agonist concentration promotes the GRK2/Gβγ interaction that accounts for ∼27% of the BRET signal, and higher agonist concentration is necessary to induce GRK2/receptor interaction that generates ∼73% of the agonist-induced BRET signal. Notably, if ∼27 and ∼44%  account for the Gβγ and small lobe/N-terminal/AST-dependent BRET, respectively, ∼30% of the total agonist-induced BRET remains unaccounted for and might be attributed to interactions of the RH domain, the phosphoacceptor peptide, the N terminus, or other regions of GRK2 to the BRET signal. Collectively, these data validate the use of the α2AAR-GFP2:RlucII-GRK2 pair as a BRET biosensor to identify specific mutations in GRK2 that impair its recruitment to an activated receptor.To test whether the residual BRET signal of the R195A mutant was due to Gβγ interaction, which would recruit the mutant to the vicinity of activated cell surface receptors, we measured the BRET signal of WT, R587Q, K220R, and R195A as a function of increasing concentrations of NOR. The WT and K220R variants exhibited a biphasic profile and could be fit by a two-site nonlinear regression analysis that highlighted the presence of a high-affinity component (EC 7.9 μm) A. For WTm NOR (B). In addition to mutants characterized in the in vitro and intact cell assays, we prepared the helix-breaking V7P variant to test the role of the α-helical nature of the N terminus and, because the L8A mutant enhanced Rho phosphorylation (B). Interestingly, the E18A mutant showed a strong potentiation of the recruitment to the activated receptor in line with what was observed for the phosphorylation of Rho in cell lysates (B). The D3A, D10A, D10R, and M15A variants did not show any significant difference in BRET signal compared with WT. The behavior of N-terminal mutants in the recruitment assay is therefore generally consistent with their activity in β2AR phosphorylation in cells . The P473E substitution had no impact on recruitment to activated α2AAR, whereas G475I and I485A showed 44–50% reduction of the BRET signal. The V477D mutation was the most deleterious of all tested, and its high-agonist concentration BRET signal was inhibited 74% (C). These results paralleled the reduced ability of these AST variants to phosphorylate Rho in vitro (2AR in intact cells (A) or the insignificant decrease in the ability of D481A to phosphorylate Rho in vitro (The effects of AST mutations on receptor recruitment were then investigated C. The P4ited 74% C. These in vitro  and β2ARct cells . Mutantsct cells A or the in vitro  22)..C). The mK but not kcat defects in Michaelis-Menten kinetic studies with Rho as substrate  to assess the roles of specific GRK2 αN and AST residues in forming a potential receptor-docking site and 2) to determine how individual residues contribute to receptor interaction, allosteric activation, or both. To this end, we created a homology model of activated GRK2 based on the GRK6-sangivamycin structure . In thisubstrate , again cA and B) and A and B) and 2AR(Y326A) and peptide C phosphorylation and thus might also fall in this category . Ile485 is predicted to pack against the small lobe but does not directly interact with the αN helix. In general, GRK2 αN and AST variants behave similarly to their GRK1/6 cognates  in COS-7 cells. The results largely replicated the defects for the variants that were observed in vitro. For some variants , the defect in phosphorylating β2AR(Y326A) in intact cells was more pronounced than the defect in phosphorylating Rho in vitro (A and 6B). It is possible that these positions play a more important role in β2AR than Rho phosphorylation or, alternatively, that the β2AR(Y326A) variant exaggerates the GRK2 defects.The use of assays that measure cell-based GRK2 activity was key to extending these studies beyond what has been shown by analogous studies of GRK1 and GRK6. The first of these assays  was a moin vitro A and 6B.2AAR as measured by BRET and proved to be a surprisingly rich source of novel information. The BRET signal in response to increasing concentrations of NOR was biphasic, displaying both high-affinity (EC50 of 15 nm) and low-affinity phases (EC50 ∼8 μm) (in vitro and GRK2-dependent phosphorylation of β2AR(Y326A) and μ-opioid receptor in intact cells  and with receptor (high-NOR concentration phase). The ∼30% of the high-agonist BRET signal that persists when the small lobe/αN/AST interactions are disrupted (i.e. in R195A mutants) could reflect the fact that other regions of GRK2 still retain the ability to bind to receptor, such as the kinase large lobe binding to the third intracellular loop of α2AAR, which is the site of phosphorylation, the RH domain, or some other region of GRK2 binding to the receptor. It could also represent residual binding of the N-terminal region to the receptor despite the fact that these mutants fail to undergo allosteric activation. It will be interesting to see how ligands that are biased toward G protein coupling versus β-arrestin signaling (The second assay developed for this study examined the NOR dependence of GRK2 recruitment to α0 ∼8 μm)  that we ct cells , had a nignaling , 42 affeB). The high NOR requirement to detect this putative GRK2/receptor interaction is consistent with the observation that homologous desensitization requires high receptor occupancy by agonist , the Asp10 side chain makes van der Waals interactions with Val477 and ionic interactions with Arg195. To align these observations, we predict that D10R or D10A variants would retain hydrophobic contacts with the AST residue Val477, but their interactions (or lack thereof) with the critical small lobe residue Arg195 would hinder kinase domain activation. If true, then receptor docking can be uncoupled from kinase domain closure. However, we cannot rule out the possibility that Asp10 plays a more important role in forming interactions with the β2AR and Rho than with the α2AAR.Recruitment of GRK2 mutants to αto α2AAR . In our to α2AAR B, the Aset al.  more efficiently than they phosphorylate Rho . Each of these residues is predicted to be fully or partially solvent-exposed (B and C), and each of the substitutions decreases negative charge and/or increases hydrophobicity at these positions. These results suggest that Ser12, Glu18, Asp481, and Ala482 play a role in direct interactions with receptors. Alternatively, because two of these residues bear negative charge, the mutations may also reduce repulsion with the negatively charged phospholipid bilayer. Regardless, these results demonstrate that it is possible to create GRK2 variants with enhanced function. The variable effects of mutants (e.g. D481A and A482I) on Rho phosphorylation versus recruitment to α2AAR suggest that it is also be possible to create GRK variants with altered GPCR specificity.Some of our variants exhibited gain of function, which gives additional insight into which positions are involved in direct interactions with receptors and, potentially, in mandating receptor selectivity. The V8A and E18A mutants exhibited elevated Rho phosphorylation , and the2AAR , B and C-exposed , B and C13, Met17, Gly475, Val477, and Ile485 (B and C), is necessary for kinase domain activation and that this interaction is required for efficient docking to an activated receptor. Our work suggests that GRK2 Leu4, Val7, Leu8, Val11, and Ser12 directly interact with GPCRs. Finally, our work suggests that Asp10 may be more critical for kinase domain activation than for forming the GPCR docking site. In comparison with previous studies on GRK1 and GRK6, our results with GRK2 highlight differences between GRK subfamilies that may dictate receptor preference. Although BRET- and FRET-based GRK/GPCR recruitment assays have been reported (2AAR seems to tease apart a low-agonist concentration membrane localization event from a high-agonist concentration receptor localization event. This experimental approach should prove very useful in further dissecting the mechanism of GRK2 activation and understanding the regulation of other GPCRs.In summary, our work implies that residues throughout the length of the GRK2 αN helix play a role in receptor interaction and/or substrate phosphorylation. Our results are consistent with the idea that the αN/AST interaction, mediated directly or indirectly by GRK2 Tyrd Ile485 , B and Creported , 53, 54,


Transfer RNAs (tRNAs) are central players in the protein translation machinery and as such are prominent targets for a large number of natural and synthetic antibiotics. This review focuses on the role of tRNAs in bacterial antibiosis. We will discuss examples of antibiotics that target multiple stages in tRNA biology from tRNA biogenesis and modification, mature tRNAs, aminoacylation of tRNA as well as prevention of proper tRNA function by small molecules binding to the ribosome. Finally, the role of deacylated tRNAs in the bacterial “stringent response” mechanism that can lead to bacteria displaying antibiotic persistence phenotypes will be discussed. ThereIn order to fulfill their roles in protein synthesis, tRNA transcripts have to be extensively processed by an assortment of enzymes and ribonucleoprotein complexes . The proMature tRNAs then have to be ligated to the correct amino acid catalyzed by the aminoacyl tRNA synthetase (aaRS) family of enzymes  Figure . AaRSs aMet that is positioned correctly at the first codon. There is then a coordinated binding event where 50S subunit joins the 30S subunit with release of the initiation factors leaving the codon bound fMet-tRNAMet at the P-site. Incoming EF-Tu·GTP·aa-tRNA complexes then interrogate the mRNA codon at the A-site of the 30S subunit-decoding center with their anticodon stem-loops  and then proceed to the ribosome for protein translation . The bacem-loops . Once them-loops . Then, iThus, all these processes where tRNAs are involved can be targets for new drugs. In the sections described below, we aim to summarize the mechanisms of known antibiotics that target tRNAs involved in different processes.E. coli and comprises a catalytic RNA or ribozyme subunit (350 to 450 nucleotides), and a single protein subunit (110 to 150 amino acids) [Bacillus subtilis and E. coli. Importantly, from an antibiotic perspective, there exist striking differences between bacterial and human RNase P (which contains a much higher number of protein subunits). These differences between host and microbe make RNase P an attractive candidate for the design of new antimicrobials.Ribonuclease P is an endoribonuclease that cleaves the 5'-end of pre-tRNA to generate mature tRNA. This is an essential step in the biogenesis of tRNA. A bacterial RNase P holozyme structure has been elucidated from o acids) . A compaE. coli RNase P recognizing the substrate [50) of 60 μM) [50 values ranging from 0.1 to 6 μM [E. coli RNase P inhibitors porphines and porphyrins with inhibition constant (Ki) ranging from 1 to 4 μM work in a similar manner displacing metal ions and/or by altering the tertiary structure of tRNA [E. coli pre-tRNAPhe and prevent RNase P interaction (IC50 of 5–20 μM) [50~3mM); Amicetin and blasticidin S) can also block RNase P activity by binding to pre-tRNA [A number of RNase P targeting antibiotics are known, which use a number of different modes of action. Inhibition mechanisms include: Binding to the pre-tRNA substrate and disrupting RNase P substrate recognition; binding to RNase P itself and altering the P RNA conformation or disrupting the association of RNA and protein in the complex; and finally binding and disruption of the enzyme-substrate complex (RNase P·pre-tRNA). Some examples of antibiotics that bind to pre-tRNA substrates include aminoglycosides, which displace functional metal ions, and have been shown to prevent ubstrate ,18,19,20f 60 μM)   . Interespre-tRNA ,26.E. coli and B. subtilis RNase P with IC50 values obtained of 2 nM to 1 μM, respectively [E. coli RNase P is important in forming interactions with the 3'-end of the pre-tRNA substrate. Gruegelsiepe et al., synthesized a 14 base oligomer that is stable and resistant to nucleases and demonstrated that it binds irreversibly to RNase P. These in vitro studies showed that the antisense oligomer specifically targets the catalytic core (P15 loop) of RNase P and traps it in a partially unfolded state, thereby affecting substrate binding. In addition to this it also affects the coordination of Mg2+ ions that are important for catalysis. The success of this technique is due to the accessibility of the oligomer to this P15 loop region. Further investigations of this oligonucleotide in vivo conjugated it to an invasive peptide via a monoglycine linker (peptide nucleic acid) to facilitate the uptake of the inhibitor into live E. coli cells. Inhibition of growth was observed indicating a successful uptake of the oligomer by the cells and specific targeting of the E. coli RNase P. The formation of hybrid-duplexes with RNase P and subsequent partial unfolding of the ribozyme is thought to make the complex more prone to degradation in the cell.Antisense technology has also been utilized to target the RNA moiety of RNase P itself  with inhectively ,28. The Since bacterial RNase P differs markedly from its eukaryotic counterpart, it is possible to specifically target only the bacterial RNase P. In addition, RNase P is present in relatively lower concentrations as compared to ribosomes . Therefo50 = 820 nM) has also been identified. Ir6Ac is a semisynthetic derivative of irigenol that is produced by plants of the Leguminosae family. This compound binds to RNase P with a unique mechanism, which is unlike that of aminoglycosides. This inhibitor not only binds to the RNase P alone (Ki = 130 nM), but also binds to the RNase P·pre-tRNAAsp complex (Kis = 480 nM). Contrary to aminoaglycosides, such as neomycin B, Ir6Ac cannot bind pre-tRNAAsp or tRNAAsp alone, but requires the presence of RNase P for its action [Recently a potent inhibitor, Ir6Ac , which is essential and universally conserved in all three domains of life and found on the anticodon loop on tRNAs that decode the ANN codon [6A’s biosynthesis [tRNAs contain a large number of modified bases that are involved in a variety of roles from structural stability, recognition elements required for discrimination by aaRSs, to hyper modified bases on the anticodon loop of the tRNA allowing a single tRNA isoacceptor to read multiple codons and prevent frameshifting on the mRNA . A numbeeumoniae , Bacillusubtilis  and E. c[E. coli . TrmD meribosome . This esribosome . AnotherNN codon ,41. Receynthesis ,43. Althynthesis . Deletio2+, and positively charged polyamines, such as spermidine, which bind non-specifically to nucleic acids. Aminoglycoside antibiotics mimic polyamines due to the high number of positive charges and this allows them to bind to the negatively charged backbone of RNA [Asp and fluorescence anisotropy experiments demonstrated this binding destabilizes the native conformation of the tRNA. Tobramycin binding also leads to a substantial 30-fold decrease in the catalytic affinity (or Km) of aspartyl-tRNA synthetase (AspRS) for its tRNAAsp substrate resulting in inhibition of the aminoacylation reaction [E. coli tRNAPhe at positions G20, A44 and G45 [tRNA structures can be stabilized by metal ions, such as Mge of RNA ,46. In ae of RNA . Importareaction . Detailereaction ,49. Anot and G45 . It obstLeu [Another compound, pentamidine   of 0.06 mg/L; Candida albicans with MIC of 1 mg/L and protozoa, such as Trichomonas sp. A number of synthetic derivatives of purpuromycin have been synthesized to increase activity [Purpuromycin  is an anino acid ,54,55. Tactivity ,57,58 anactivity .E. coli (about 30% of E. coli contain them). They are produced as a tool to decrease competition from other bacteria living side by side in the same environment [Colicins are antibacterial toxins secreted out into the extracellular medium by members of the enterobacteriaceae family, such as ironment . Several in vivo as well as in vitro. In addition to the nuclease activity by colicin E5 and D, there exists another type of nuclease called PrrC [E. coli in response to a T4 phage infection. PrrC is a suicidal nuclease since it cleaves its own lysine tRNA as a result of the phage infection [Lys [Colicin E5 RNase targets tRNAs specific for tyrosine, histidine, asparagine and aspartic acid by cleaving anticodon QUN that contains the hyper-modified queuosine nucleotide (Q) at the wobble position 34 . On the led PrrC . These anfection  and it dion [Lys . Thus thion [Lys . This iswww.tranadiscovery.com) have used modern bioinformatics tools to conduct a detailed analysis of the tRNA of certain organisms with a particular focus on the ASL region of tRNAs. From this analysis, probes that mimic the ALS region have been designed. Using this probe, high throughput sequencing (HTS) analysis has been performed to screen for compounds that can bind with high affinity and specificity to the ASL region of the particular tRNA. Some examples using this novel patented technology include the HTS assay for methicillin resistant Staphylococcus aureus that identified compounds that bind specifically to tRNAArg. By utilizing a fluorescein-labeled oligonucleotide mimic of the ASL loop of tRNAArg on a ribosome scaffold, biochemically active compounds could then be isolated and then tested for dose response [Lys3 of HIV [The anticodon stem loop region (ASL) of microbial tRNAs from bacteria and fungi are diverse in RNA sequence as well as containing a range of modifications . Scientiresponse ,65,66. T3 of HIV ,68,69 anS. aureus. This antibiotic is a naturally occurring isoleucyl-tRNA synthetase inhibitor produced by Pseudomonas fluorescens strains and works by docking onto the enzyme catalytic active site and competing with the isoleucine and ATP substrates for binding. This effectively prevents formation of the isoleucyl-adenylate reaction intermediate and therefore aminoacylation of tRNAIle. A number of natural aaRS inhibitors employ similar mechanisms to inhibit their respective aaRSs. Rationally designed stable mimics of aminoacyl-adenylate reaction intermediates have also been studied extensively and prove to be potent inhibitors of their respective aaRS in vitro. However, these inhibitors have issues with bioavailability and bacterial uptake and often do not sufficiently discriminate between bacterial and eukaryotic aaRSs for use as an effective antibiotic.The aminoacylation of tRNAs by aaRSs has been found to be targeted by a large number of natural occurring antibiotics. This is perhaps not surprising considering the essential role aaRSs play in translation and with typically 18–20 different amino acid specific enzymes per bacterial cell. What is surprising is that very few aaRS inhibitors are actually used in a clinical setting. Pseudomonic acid or mupirocin is one example, yet is one of the most effective topically applied antibiotics used to combat methicillin resistant Other aaRS inhibition mechanisms that prevent tRNA aminoacylation include amino acid mimics, non-competitive inhibitors that bind outside of the active site but modulate enzyme activity and antibiotics that directly bind to the substrate tRNA (mentioned earlier in text). We have recently reviewed these aaRS inhibitor mechanisms in detail  as have Leu. Recently, a pharmaceutical company (Anacor) used combinatorial screening of a library of organoboron compounds and discovered a novel synthetic compound, AN2690  was found to show broad-spectrum activity with minimum inhibitory concentration (MIC) in the range of 0.5 to 1 μg/mL. Importantly, when S. cerevisiae colonies resistant to AN2690 were generated, the mutations were all tracked to the CDC60 gene, which encodes the cytoplasmic LeuRS [Leu-AN2690 adduct [A number of aaRSs, which catalyze the aminoacylation of branched chain amino acids, such as LeuRS, valyl-tRNA synthetase  and IleRS, possess an additional proofreading domain called the CP1 domain ,78,79. T, AN2690  that inhic LeuRS . Most inic LeuRS . This obic LeuRS  in a non0 adduct . This ad50 values in the range of 5 to 400 nM. AN2690 was found to be most potent of all the compounds and very effective against fungi, yeast, molds or dermatophytes causing skin infections. AN2690 is specifically effective against Trichophyton rubrum and Trichophyton mentagrophytes the causative agent of onychomychosis, a disease of the human nail bed  This was also confirmed by the X-ray crystal structure of the complex of E. coli LeuRS·tRNALeu·TM84, which demonstrated that TM84 does indeed bind to the catalytic active site; But the CCA 3'-end of tRNALeu is positioned into the enzyme active site, trapped into an “aminoacylation-like” conformation. Specifically, the terminal adenosine of the CCA 3'-end directly interacts with the TM84 toxin. Additionally, TM84 forms interactions with the highly conserved, mobile KMSKS loop of LeuRS, thereby allowing the loop to adopt a closed-conformation encapsulating TM84 and explaining the high affinity of the toxin in the presence of tRNALeu [Agrocin 84 acts as a molecular Trojan horse by mimicking a tumor-derived substrate called agrocinopine ,104 and the cell ,106,107.the cell ,109 . EF-Tu is a GTPase that only binds to the aminoacyl-tRNAs in a GTP bound form and then transfers the aminoacyl-tRNAs to the A-site of the ribosome and is therefore critical to the protein translation machinery. Upon codon-anticodon recognition in the ribosome, EF-Tu undergoes a conformational change leading to the hydrolysis of the active EF-Tu·GTP complex to the inactive EF-Tu·GDP complex ,112,113.N-methyl derivative of kirromycin and is produced by Streptomyces goldiniensis [Thermus thermophilus EF-Tu with GDP and aurodox in a 1:1:1 ratio at 2 Å resolution indicates that binding of aurodox resembles the EF-Tu·GTP bound conformation [Frateuria sp. W-315 and is active against both Gram-positive and Gram-negative organisms [Kirromycin is an antibiotic that binds to the ribosome·aa-tRNA·EF-Tu·GDP complex. This results in inhibition of the release of the EF-Tu·GDP complex from the ribosome. Failure of the release of EF-Tu from the ribosome does not affect the binding of aminoacyl-tRNA to the A-site of the ribosome, but blocks the subsequent peptide bond formation step ,116. Thuiniensis , and cryormation . Additiorganisms ,123,124.rganisms ,126.E. coli EF-Tu·GDP·GE2270 complex has confirmed that this compound directly competes with aminoacyl-tRNA for the same binding site on EF-Tu. It also blocks the GTP to GDP conformational change in EF-Tu. A comparison of binding modes indicates that GE2270 therefore has a different binding mode compared to kirromycin and enacyloxin IIa but is limited as an antibiotic due to its low solubility [GE2270A is a thiazolyl peptide antibiotic that is active against Gram-positive bacteria. Crystal structure of lubility ,128,129.lubility ,118,119.There exist three tRNA binding sites on the ribosome: an A-site for the incoming aminoacylated tRNAs, a P-site for the peptidyl-tRNAs and an E site where the deacylated tRNAs exit. The tRNA species bound to these sites adopt a large number of different conformations during the three-stage translation process of initiation, elongation, and termination. As a consequence of this highly complex and essential process, there exists a vast array of natural and synthetic drugs that target different stages of the process binding to the ribosome. Many of these toxins directly interact or interfere with tRNA species in mechanisms ranging from prevention of tRNA anticodon loop binding to mRNA in the decoding center to prevention of aminoacyl-tRNA movement into the P-site the peptidyl transferase center (PTC). The space available in the article limits the detail in which we discuss the large number of antibiotics targeting the ribosome but we direct the reader to a number of excellent reviews recently written on the topic ,132,133.Initiation of protein synthesis involves, the formation the 70S subunit of the ribosome along with the initiator fMet-tRNA and start codon of mRNA positioned at the P-site. Drugs that inhibit this step are broadly classified either as 50S or 30S inhibitors. Examples of antibiotics binding to the 50S subunit of the ribosome include macrolide  , lincosaSimilarly, antibiotics inhibiting the 30S subunit include tetracyclines and aminocyclitols (made up of aminoglycosides and spectinomycin). Tetracyclines bind to the 30S subunit and block the binding of aminoacylated tRNAs to the A-site of the ribosome ,144. SpeMycobacterium tuberculosis infections including the multi-drug resistant types [Other examples of antibiotics binding to the 30S subunit include tuberactinomycins, thermorubins  and blasnt types ,153,154.nt types .Streptomyces griseochromogenes [E. coli cells was 50 μg/mL and towards mammalian cells was 2 to 10 μg/mL. Unlike other blasticidins, BlaS possesses a unique mechanism of inhibition of the protein translation machinery. Analysis of the crystal structure of BlaS bound the 70S ribosome complex revealed that BlaS binds to the 50S subunit of the ribosome at the P-site and not at the A-site like other Bla antibiotics [Blasticidin S is an antibiotic produced by omogenes . BlaS haibiotics ,158. Upoibiotics .Gln and Asn-tRNAAsn. The pathway first involves production of Glu-tRNAGln and/or Asp-tRNAAsn by a non-discriminating synthetase (ND) followed by transamidation to Gln-tRNAGln and Asn-tRNAAsn by glutamine amidotransferase [gatC, gatA and gatB (Genomic studies have shown that certain microorganisms lack glutaminyl-tRNA synthetase (GlnRS) and asparaginyl-tRNA synthetase (AsnRS) . These onsferase . This enand gatB .Helicobacter pylori (H. pylori) GatCAB with respect to Asp-tRNAAsn [Streptomyces alboniger). A number of puromycin derivatives have been synthesized to test their effect on H. pylori GatCAB complex [Recently synthetic inhibitors of the amidotransferase (adT) enzyme have been synthesized. These inhibitors, aspartycin and glutamycin mimic the 3'-end of the mis-aminoacylated tRNA. Both aspartycin and glutamycin are competitive inhibitors of -tRNAAsn ,163. Glu complex . TherefoN-acetyl glucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) connected by a β-1, 4 linkage. The GlcNAc and MurNAc subunits are linked to short peptides of variable sizes [Streptococcus pneumoniae and S. aureus, these peptide chains contain additional Ala-Ala and Ser-Ala inter-subunit crosslinks [S. aureus using Gly-tRNAGly [S. pneumoniae using Ala-tRNAAla and Ser-tRNASer, respectively. Thus, these enzymes and their aminoacyl-tRNA substrates are important targets for antibiotic design against multi-drug resistant bacteria [Ala. Using this approach, an analogue of aminoacyl-tRNA linked to 1, 2, 4 oxazolidine ring has been synthesized that inhibits Fem transferases in Weissellaviridescens (FemXWv (IC50 = 1.4 μM) [S. pneumoniae was inhibited by a 2'-deoxyadenosine 3'-phosphonate analogue [Peptidoglycan is the major component of the Gram-positive bacterial cell wall and is composed of alternating le sizes . In someosslinks . Interesosslinks . FemXAB -tRNAGly . MurMN bbacteria . Attempt 1.4 μM) . Similaranalogue .Mycobacterium tuberculosis and Mycobacterium smegmatis form biofilms under conditions of stress and can tolerate about 50 times the MIC of antituberculosis drugs, such as isoniazid and rifampicin [Pseudomonas aeruginosa, quorum sensing gets elevated upon expression of the relA gene [Pseudomonas sp., Samonella sp., Vibrio cholera and Legionella sp. [Streptomyces coelicolor [Myxococcus sp. due to increased levels of (p)ppGpp [Nutritional stress or inhibition of the aminoacylation reaction of aaRSs results in the accumulation of deacylated tRNA. These uncharged tRNAs then bind to the A-site on the ribosome and are recognized by RelA and/or SpoT, which then trigger the stringent response mechanism ,175,176.fampicin ,182,183,elA gene . Elevateella sp. ,187,188.elicolor . Some ot(p)ppGpp ,191 and (p)ppGpp ,193. Tar(p)ppGpp . tuf B gene), which leads to kirromycin resistance. Of course, spontaneous resistance mutations can appear in aaRSs genes in organisms exposed to aaRS targeting antibiotics, which we recently reviewed elsewhere [C. albicans strains appeared as a result of a single point mutation in the LeuRS CP1 editing domain [Agrobacterium radiobacter strain K84 to the plant pathogen A. tumefaciens [Resistance to antibiotics that interfere with tRNA biology can be mediated by a variety of different mechanisms. Some of these involve alterations in membrane permeability to impair the influx of the drug or to have an active efflux of the drug from the cell. Examples of these include chloramphenicol, erythromycin, neomycin, streptomycin and spectinomycin ,194,195.lsewhere . Of the g domain . While ag domain , from thefaciens .Modification of the binding site of the antibiotic is another mechanism to decrease the affinity of the drug. For example, methylation of rRNA by rRNA methyltransferases confer resistance to aminoglycosides, chloramphenicols, oxazolidinones and some macrolides ,202. ThiIn order to lower the effective concentration of the drug, some microorganisms increase the expression of target or a mimic of the target so as to lower the effective concentration of the drug and thus leave some target molecules uninhibited. For example, overexpression of the rRNA fragment that resembles h34 of the 16S rRNA is involved in conferring resistance to spectinomycin . SimilarThe central role of the numerous tRNA species in the microbial protein synthesis apparatus makes these ribosome associated adaptor molecules prominent targets for antibiotics. In this review, we have highlighted that antibiotics can also target a significant number of key processing and tailoring steps that are also essential for tRNAs to carry out their role in translation. With the ever-looming specter of microbial antibiotic resistance threatening the usefulness of standard medical treatments against human pathogens, there is a growing need for the development of new anti-infectives. This search has to confront a decreasing number of new essential targets to explore and also find antibiotics less prone to imparting resistance in the microbes on which they are trained. In this respect, the novel tRNA-targeting antibiotics discussed in this review hold out some hope. From the design of inhibitors targeting ASL region of tRNA  and esse


Release of pro-inflammatory mediators by mast cells is a key feature of allergic disease. The ‘dogma’ is that IgE molecules merely sensitise mast cells by binding FcεRI prior to cross-linking by multivalent allergen, receptor aggregation and mast cell activation. However, certain monoclonal IgE antibodies have been shown to elicit mast cell activation in an antigen-independent cytokinergic manner, and DNP-specific murine SPE-7 IgE is the most highly cytokinergic antibody known. We show that both monovalent hapten and recombinant SPE-7 IgE Fab inhibit its cytokinergic activity as measured by mast cell degranulation and TNF-α release. Using SPE-7 IgE, a non-cytokinergic human IgE and a poorly cytokinergic murine IgE, we reveal that interaction of the Fab region of ‘free’ SPE-7 IgE with the Fab of FcεRI-bound SPE-7 IgE is the basis of its cytokinergic activity. We rule out involvement of IgE Fc, Cε1 and Cλ/κ domains, and propose that ‘free’ SPE-7 IgE binds to FcεRI-bound SPE-7 IgE by an Fv-Fv interaction. Initial formation of a tri-molecular complex (one ‘free’ IgE molecule cross-linking two receptor-bound IgE molecules) leads to capture of further ‘free’ and receptor-bound IgEs to form larger clusters that trigger mast cell activation. IgE plays a critical role in mast cell mediated type I hypersensitivity in allergic disease. The ‘dogma’ of mast cell activation is that IgE bound to its high-affinity receptor, FcεRI, must be cross-linked by multivalent antigen  to cause receptor aggregation, signal transduction and the release of pro-inflammatory mediators that initiate the allergic response1245678910111213141516171819202122The DNP-specific murine IgE, SPE-7, is the most highly cytokinergic antibody known, inducing mast cell survival, migration, fibronectin adhesion, FcεRI upregulation, cytokine release and degranulation in the absence of antigen810152022et al. reported that murine IgEs differing only in their VH and/or VL domain sequences elicit a wide range of cytokinergic activities10Kitaura and co-workers ranked a number of murine IgEs, from the most poorly to the most highly cytokinergic IgEs, on the basis of their ability to perform an increasing number cytokinergic activities and also the strength of these activities87In the present study we focus on the mechanism of cytokinergic SPE-7 IgE activity in a reproducible human system using the LAD-2 human mast cell line. We demonstrate that some of the important features observed in the rodent system hold true in this human system, including the critical role of the Fv region of SPE-7 IgE. We also demonstrate for the first time that the cytokinergic activity of SPE-7 IgE requires both the saturation of FcεRI on the LAD-2 cells by SPE-7 IgE and the presence of ‘free’ SPE-7 IgE in solution. We therefore propose a mechanism by which the Fv regions of ‘free’ SPE-7 IgE molecules cross-link the Fv regions of adjacent receptor-bound SPE-7 IgE molecules to induce mast cell activation.in vitro when incubated with cord blood or human lung primary mast cells9181921Human IgE does not bind to murine FcεRI, but paradoxically both human and murine IgE bind to human FcεRI5 rat FcεRI molecules per cell, similar to the level of receptor expressed by naïve LAD-2 cells (mean ± SEM of 0.7 ± 0.3 × 105 human FcεRI per cell), which increased to 1.7 ± 0.2 × 105 upon addition of 6 ng/ml IL-4 to the cell culture for 5 days prior to receptor quantification , were not activated upon addition of SPE-7 IgE, even at 30 nM (5 μg/ml). The non-cytokinergic and poorly cytokinergic nature of 102.1F10 and 27–74 IgE antibodies, respectively, were demonstrated in non-sensitised LAD-2 human mast cells, primed with IL-4 and stem cell factor alone, and in all sensitisation conditions  or poorly-cytokinergic murine IgE 27–74 (with murine Fc) did not activate cells sensitised with SPE-7 IgE . FurtherIn the present work we have demonstrated that human mast cells of the LAD-2 cell line can be activated by highly cytokinergic murine SPE-7 IgE  and C. T7810The inhibition of SPE-7 IgE-dependent mast cell degranulation and cytokine release by recombinant SPE-7 IgE Fab in the present study  further 293031et al. showed that calcium flux in RBL-2H3 cells was significantly reduced upon removal of ‘free’ SPE-7 IgE by washing7In the light of these results, the necessity for cytokinergic IgE to be bound via its Fc to FcεRI and/or to be ‘free’ in solution was then studied. Two previous studies in rodent systems have shown that the presence of ‘free’ IgE is required for cytokinergic activity. Asai and co-workers demonstrated that increased survival of murine BMMCs was immediately ablated on removal of ‘free’ IgE, and Pandey −10 M), followed by the capture of ‘free’ IgE by a Fab-Fab interaction to form a tri-molecular complex (It was not clear from this earlier work whether FcεRI-bound IgE and ‘free’ IgE must both be cytokinergic for activation of the mast cells. We have now shown that mast cells must be sensitised with SPE-7 IgE and stimulated with high concentrations of ‘free’ SPE-7 IgE to trigger degranulation in the absence of antigen . We sugg complex . The capIn this view of the mechanism we have been able to rule out interactions between the Fab region of receptor-bound IgE and Fc of ‘free’ IgE  or conve1 (PDB ID: 1OAQ) was observed in 80% of SPE-7 molecules2 conformer (PDB ID: 1OCW) was seen in around 20% of SPE-7 molecules, and has a deep pocket, which binds a wide range of haptens, such as alizarin red and furazolidone , with a range of affinities (20 nM to 100 μM). Furthermore, upon binding of the higher affinity hapten DNP-lysine (KD = 2 × 10−8 M), the SPE-7 IgE Fab domain undergoes induced fit rearrangement to conformer Ab3 (PDB: ID 1OAU)2930The ability of a cytokinergic IgE to form Fv-Fv interactions may also be due to inherent conformational lability and polyreactivity of SPE-7 IgE. James and colleagues revealed the conformational isomerism of SPE-7 IgE by structural studies, where the predominant conformer Abet al. showed interactions between VH and VL domains of two single chain Fvs (scFv) of MFE-23, and Calarese and colleagues demonstrated VH domain swapping between neighbouring anti-HIV 2G12 Fabs3134There are examples of Fv-mediated self-association in other immunoglobulin classes, and crystallographic studies have shown a number of potential modes of self-association. Boehm 5 when compared to the same reactants in solution5 FcεRI molecules per cell, which is close to the mean ± SEM of 1.7 ± 0.2 × 105 FcεRI molecules per LAD-2 human mast cell or RBL-2H3 cell measured in our work . DeLisi demonstrated this by calculating a reduction in KD by a factor of 10our work , there wWe attempted to detect Fv-Fv mediated interactions between SPE-7 IgE molecules and/or recombinant SPE-7 IgE Fab by ELISA, surface plasmon resonance (SPR) and flow cytometry (data not shown). Despite a high density of molecules on the SPR surface and the same high concentrations of SPE-7 IgE and SPE-7 IgE Fab as in the activation assays, we were unable to detect interactions between the molecules. This probably reflects the low affinity of an individual Fv-Fv mediated interaction between receptor-bound and ‘free’ cytokinergic IgE molecules, and explains why extremely high concentrations of IgE are required to manifest the avidity of the interaction between ‘free’ IgE and receptor-bound IgE on the mast cell membrane.We have demonstrated an Fv-Fv mediated mechanism for the cytokinergic activity of SPE-7 IgE. Our study has made use of purified recombinant Fabs, and a similar approach could be extended to locate the interaction sites in the ‘free’ and bound IgEs more precisely. Exchange of heavy and light chains between recombinant IgEs would establish whether only one or both chains in SPE-7 IgE are required to form these sites, and mutagenesis or structural studies such as X-ray crystallography or NMR could be applied to determine the epitopes involved. Currently we do not know whether the mechanisms or locations of the binding sites are the same for cytokinergic IgEs other than SPE-7 IgE.A continuing effort to understand the cytokinergic activity of IgE is important because this activity may well be involved in the pathogenesis of chronic diseases associated with IgE, including eczema, allergic rhinitis, asthma, and food allergies. Cytokinergic IgE may be involved in both atopic and non-atopic  forms of these diseases. However, it is not yet known what effect cytokinergic IgE may have on other FcεRI- and CD23-expressing cellsOur conclusion that Fv-Fv interaction is the basis for cytokinergic activity does not contradict the ‘dogma’ that cross-linking is required for receptor aggregation and mast cell activation12341SPE-7 IgE hybridoma cells . Light-chain and heavy chain forward and reverse primers see  were des33Polymerase incomplete primer extension (PIPE) cloningThis purified pVITRO1_SPE-7 IgE light-chain plasmid was linearised at MCS2 by AgeI and NheI restriction enzyme digest (New England Biolabs). SPE-7 IgE Fab heavy-chain (comprising the SPE-7 IgE VH-region coupled to the Cε1 constant domain) was amplified and a 6×-histidine tag added at the N-terminus using round 1 PCR primers, followed by a second round of PCR to add regions homologous to linearised pVITRO1_SPE-7 IgE light-chain plasmid. Both rounds of PCR were set up and carried out as for the light-chain  were transfected with purified plasmid DNA using PEI (Sigma-Aldrich) and cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% FCS, 2 mmol/L L-glutamine, 10 U penicillin/streptomycin  at 37°C and 5% CO2. Following selection with 50 ng/ml hygromycin B (Invitrogen), cells were transferred into Freestyle™ 293 expression media (Invitrogen), supplemented with hygromycin B, to spinner culture flasks. Culture supernatants were harvested and filtered.Human embryonic kidney cells  coated overnight with 5 μg/ml DNP-HSA (Sigma-Aldrich), blocked with PBS-1% BSA and incubated with 50 μl cell-free culture supernatants for 2 hours, followed by HRP-conjugated anti-His-tag detection antibody diluted 1/1000 (Novagen). His-tagged Fab was detected with TMB (R&D Systems) by measuring absorbance at 450 nm .His-tagged Fab protein was purified by affinity chromatography using a HisTrap FF Crude column and äKTA Prime system . Captured His-tagged protein was eluted with 500 mM imidazole phosphate buffer. Size-exclusion purification was carried out using an HPLC system (Gilson), with Superdex™ 200 column  and fractions were collected between 10 and 30 minutes . Purity 2 . Simultaneously, Qifikit® set-up bead cocktail, a mixture of blank beads and beads bearing a high number of murine IgG monoclonal antibodies, and calibration bead cocktail, a mixture of five populations of beads bearing different, but well-defined numbers of murine IgG monoclonal antibodies, were also incubated with the secondary F(ab')2. All incubations were carried out in darkness, at 4°C for 45 minutes. FITC fluorescence was analysed by flow cytometry .LAD-2 human mast cells (NIH) were isolated from continuous culture in StemPro-34 media with 100 ng/ml stem cell factor (both Invitrogen) or following 5 days priming with 100 ng/ml stem cell factor and 6 ng/ml IL-4 (R&D Systems). RBL-2H3 cells were removed from adherent culture in RPMI supplemented with 10% FCS, 2 mmol/L L-glutamine, 10 U penicillin/streptomycin . Cells were incubated with 1 μg primary anti-human or anti-rat FcεRI mouse IgG antibody or isotype control (eBioscience), washed again, and incubated with secondary FITC-conjugated anti-mouse IgG F(ab')The set-up bead cocktail was first used to adjust the flow cytometer voltage settings to detect the required range of FITC fluorescence. The median fluorescence intensity (MFI) for each of the five calibration bead populations was determined and plotted against the known number of IgG molecules on the beads to produce a calibration curve. The number of human FcεRI molecules expressed per LAD-2 mast cell was interpolated from the MFI of the stained cell peak using the constructed calibration curve. Flow cytometry data was analysed by FlowJo software (Treestar).2 overnight. Following washing with HBSS, cells were stimulated with 0.5–5 μg/ml SPE-7 IgE (Sigma-Aldrich).RBL-2H3 cells were seeded at 20,000 cells/well in RPMI supplemented with 10% FCS, 2 mmol/L L-glutamine, 10 U penicillin/streptomycin  and incubated at 37°C, 5% CO2−4 (0.2 μg/ml)LAD-2 human mast cells (NIH) were primed for 5 days in StemPro-34 media with 100 ng/ml stem cell factor (Invitrogen) and 6 ng/ml IL-4 (R&D Systems) to up-regulate FcεRI expression. In some cases, cells were also sensitised with 3 nM human IgE FcεAfter each incubation, supernatants were harvested . Cells were finally lysed with 0.5% Triton X-100 (Sigma-Aldrich) for 30 minutes and the lysates collected. β-hexosaminidase release was measured using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide substrate (Sigma-Aldrich) as previously described50 values were performed by GraphPad Prism .Statistically significant difference between experimental conditions was determined by one-way ANOVA with Dunnett's or Tukey's post-test. All statistical analyses and calculation of ICH.J.B. and H.J.G. carried out experimental design, H.J.B. performed functional experiments, H.J.B. and H.B. produced the IgE Fab with help from T.S.D., and the manuscript was written by H.J.B., B.J.S. and H.J.G. All authors reviewed the manuscript.Supplementary Information


Technical developments for improving the safety and accuracy of pedicle screw placement play an increasingly important role in spine surgery. In addition to the standard techniques of free-hand placement and fluoroscopic navigation, the rate of complications is reduced by 3D fluoroscopy, cone-beam CT, intraoperative CT/MRI, and various other navigation techniques. Another important aspect that should be emphasized is the reduction of intraoperative radiation exposure for personnel and patient.The aim of this study was to investigate the accuracy of a new navigation system for the spine based on an electromagnetic field.Twenty pedicle screws were placed in the lumbar spine of human cadavers using EMF navigation. Navigation was based on data from a preoperative thin-slice CT scan. The cadavers were positioned on a special field generator and the system was matched using a patient tracker on the spinous process. Navigation was conducted using especially developed instruments that can be tracked in the electromagnetic field. Another thin-slice CT scan was made postoperatively to assess the result. The evaluation included the position of the screws in the direction of trajectory and any injury to the surrounding cortical bone. The results were classified in 5 groups: grade 1: ideal screw position in the center of the pedicle with no cortical bone injury; grade 2: acceptable screw position, cortical bone injury with cortical penetration ≤ 2 mm; grade 3: cortical bone injury with cortical penetration 2,1-4 mm, grad 4: cortical bone injury with cortical penetration 4,1-6 mm, grade 5: cortical bone injury with cortical penetration >6 mm.The initial evaluation of the system showed good accuracy for the lumbar spine . A comparison of the initial results with other navigation techniques in literature  shows that the accuracy of this system is comparable.EMF navigation offers a high accuracy in Pedicle screw placement with additional advantages compared to other techniques. The short set-up time and easy handling of EMF navigation should be emphasized. Additional advantages are the absence of intraoperative radiation exposure for the operator and surgical team in the current set-up and the operator’s free mobility without interfering with navigation. Further studies with navigation at higher levels of the spine, larger numbers of cases and studies with control group are planned. Pedicle screw instrumentation is one of the standard techniques in spine surgery. The correct position of the screws ensures good pullout strength in the bone and good control of rotation and possibility of repositioning the respective vertebral body . The tecTo achieve an optimal central position of the pedicle screw in the pedicle with the standard technique without injuring the medial, lateral, cranial, or caudal wall of the pedicle, it is necessary to select the correct point of insertion of the pedicle screw into the vertebra, correct spatial placement of the screw through the pedicle, and perfect fit of the diameter of the screw to the diameter of the pedicle. The optimal screw diameter can be estimated individually based on the pre-operative CT or MRI images. The correct point of insertion of the screw is determined intra-operatively as in the conventional technique using anatomical landmarks. Two different approaches to the pedicle are available: the Roy-Camille or the Weinstein approach. Precise positioning of the screws in the pedicle is essential for the stability of the pedicle systems.From a biomechanical standpoint, the pedicle screws should ideally be placed along the pedicle axis. This makes it possible to utilize the greatest transversal and sagittal pedicle diameter and insert thicker pedicle screws. The stability of the screw in the bone is determined to a large extent by the thickness of the screw and its length inside the vertebra. Stability is increased by a greater screw diameter and insertion depth. In addition to the biomechanical aspect of stability, the ideal position also ensures optimal protection of adjacent structuresTechnical advances have made several different techniques for placing pedicle screws available . In addiCommercially available navigation systems can be classified into active and passive systems, i.e. they either transmit or receive signals , 18. AmoElectromagnetic navigation uses electromagnetic fields that penetrate the body , 22, 23.This study was conducted in compliance with the strict ethical guidelines for human cadaver studies. All body donors were fully legally competent and had a will in which they agreed to the use of their body or body parts for research, study, or teaching purposes . The ethics committee of the medical Association of Westfalen-Lippe gave its approval for this study (214-037-f-S).The study was conducted on two human bodies  with intact spines that were preserved using the Thiel method . The bodies were examined in advance by a CT scan to check for previous operations on the spine, pathological changes, tumors, and severe anomalies.A new EMF navigation system with clinical approval for ENT and neurosurgery  and adapted for spine surgery was tested. Special instruments were developed  to make navigation in the EM field possible.The principle of the new navigation system based on an electromagnetic measuring system is the continuous tracking of the instruments and the patient’s anatomical structures during the surgical procedure. The navigation system thus supports the operative procedure by providing additional information without changing or hindering the operator’s conventional workflow. The calibration and correction process can correct static errors that occur when measuring the position. The method takes errors into account that are attributable to the position and direction of the navigated instrument. This improves the accuracy of the position in a space adapted to the application. Navigation takes place virtually in real time in a 3D data set. Theoretically, precise intraoperative evaluation is thus possible in all planes.A special field generator was used to generate the electromagnetic field (EMF) . The fieAll required and especially developed instruments were free of ferromagnetic substances to prevent measuring and instrument errors. The coils required for navigation are firmly anchored in the instruments using a Luer-Lock system. This makes a standardized sterilization process possible by removing the coil system. Special instruments with hollow cavities had to be developed for this.4 Spinal System, BBraun, Melsungen, Germany).The instruments used were a “CenterPointer”, “AwlPointer”, “SpinePointer” and a navigable screwdriver. All instruments were adapted from standard instruments for spine surgery. The CenterPointer is used for surface matching and also to center punch and open the pedicle. The AwlPointer is used to open the vertebral body in the specified direction of trajectory and to determine the screw length required. The SpinePointer makes it possible to palpate the pedicle to detect faulty positions and injuries to the surrounding cortical bone. All instruments are connected with the navigation system by a wire. All pedicle screws used were polyaxial screws with a diameter of 6 mm . These data were used to determine the diameter of the pedicles and the direction of trajectory. The CT data set was stored in DICOM format for further processing and instrumentation in the navigation system. A 3D data set and high-resolution VRT model were calculated by the navigation system .A total of 20 pedicle screws were inserted in the lumbar spine  using EMF navigation.The torsos of the cadavers were placed in prone position on a special, non-metallic, carbon fibre OR table  to prevent interference. The field generator was placed below the patient so that the frame encompassed the entire surgical field. Access was via a standard midline incision followed by dissection of the soft tissues up to the screw insertion sites. After preparation of the operation site in the region of the affected segment of the spine, the patient tracker was firmly attached to the spinous process. This step forms the reference in the coordinate space of the navigation unit. The tracker was used to navigate the respective vertebral body.First, surface matching (10 points) on the respective vertebral body was conducted for the purpose of comparing the CT image data set with the electromagnetic coordinate system. After error calculation of the system and release for navigation, the operator performed an optical check using anatomical landmarks. Navigation was conducted only after this optical check for errors. This was followed by the virtual determination and center punch of the standard insertion point into the pedicle and subsequent opening of the pedicle and the vertebral body using the AwlPointer. Particular attention was paid to positioning the AwlPointer precisely in the center of the pedicle in all planes. A virtual tool was used to calculate the length and diameter of the pedicle screws . As an aAfter the study, the cadavers were again examined by CT scan and evaluated. The results were classified in 5 groups: grade 1 –ideal screw position in the center of the pedicle with no cortical bone injury; grade 2 –acceptable screw position, cortical bone injury with cortical penetration ≤ 2 mm; grade 3: cortical bone injury with cortical penetration 2,1–4 mm, grad 4: cortical bone injury with cortical penetration 4,1–6 mm, grade 5: cortical bone injury with cortical penetration >6 mm . A statiA total of 20 pedicle screws and the technical components of the system were evaluated. The evaluation of the screws was performed by 3 evaluators.The following average pedicle diameters were calculated: L1–6.2 mm, L2–7.4 mm, L3–8.9 mm, L4–11.2 mm, and L5–14.3 mm. The mean pedicle diameter was 8.6 mm. The minimum lumbar pedicle width was 6.1 mm and maximum 12.9 mm; the range was therefore 6.8 mm. The pedicle screws used all had a diameter of 6 mm; all screws were polyaxial screws.An excellent position (grade 1) was found in 13 pedicles, a minor penetration (grade 2) in 4 pedicles, a moderate deviation (grade 3) in 3 pedicles, and there was no malposition >4,1mm (grade 4 and 5).Maximum pedicle perforation of no more than 2 mm was thus found in 85% of all screw positions; 17 of 20 screws  were classified as group 1 or 2.Instrumentation using pedicle screws is a standard procedure in spine surgery . Many diSome disadvantages of the existing methods are that they are prone to errors regarding screw position. In addition, the intraoperative exposure to radiation presents an increased risk to operators, surgery team, and patients due to the intraoperative radiation needed  , 33. AnoThe disadvantage of navigation systems that work on the basis of detecting optical sensors is that the line of sight can never be interrupted . This liThe aim of this study was the experimental evaluation of a new EMF navigation system for placing pedicle screws. Navigation was carried out using a pre-interventional CT-based data set with an EMF navigation system modified especially for spine surgery.EMF navigation of pedicle screws using a CT-based data set has not yet been described in literature. The method has several advantages over existing techniques. For one, the intraoperative multiplanar visualization in real time provides a considerable advantage over 2D visualization with a fluoroscopic data set. Furthermore, the system used features easy handling, because nearly unchanged standard instruments can be used for the procedure. It is not necessary to use cannulated screws or K-Wires. In addition, due to the positioning of the patient on the coil generating the EM field and matching using a minimized patient tracker, there are no parts of the navigation system that interfere in the surgical site. The operator’s standard workflow is not impaired. When EMF navigation is used alone, in the current workflow only slight radiation exposure of the operator and surgical team occurs during anatomical positioning of the patient tracker. The slight radiation described here refers to the necessary use of fluoroscopy to determine the correct level, especially for higher regions of the spine. The preoperative CT scan required for navigation can result in an increased radiation dose for the patient compared with other techniques; additional studies are required to compare exposure in different techniques. Another advantage of EMF navigation in comparison with optical methods is the position of the reference coil on the instruments. It is placed near the tip of the instrument, which can result in lower torsion errors.The evaluation of the pedicle screw position showed a lower rate of faulty positioning . However, it should be emphasized that due to the large mean size of the lumbar pedicles and a screw size of 6 mm, the allowable error tolerance is greater here than in higher segments of the spine .One limitation of this study is the small sample size and the non-comparative study. Therefore, the results have to be evaluated in future studies with larger sample sizes and the control group.The relative mobility of the polyaxial screws in the navigated screwdriver must be emphasized. This also results in a slight angle deviation and greater probability of error. The rigidity of the instruments must also be established in larger patient groups. High bending forces can cause problems, especially on the awl, as here the navigation coil is currently not directly attached to the tip of the instrument. This can also cause errors in virtual visualization and navigation.In summary, the results indicate a good placement technique for pedicle screws using a new EMF navigation system for the lumbar spine. Additional studies with larger populations and smaller pedicle diameters in higher segments of the spine should be conducted. It should be emphasized that the system is easy to handle and does not limit the operator’s workflow or use of standardized instruments. The electromagnetic navigation system has the technical advantage with respect to handling, as no permanent optical connection (line of sight) of the components is required and the operator’s workflow is not impaired. In addition, navigation can take place without intraoperative X-ray guidance, resulting in lower radiation dose for the operator and surgical team. It should be emphasized that special instruments and techniques are employed for EMF navigation that are associated with higher costs than those used in the conventional technique. However, a special operating table (e.g. carbon fiber table) is not required.


AMPK is a sensor of cellular energy status and a promising target for drugs aimed at metabolic disorders. We have studied the selectivity and mechanism of a recently described activator, C2, and its cell-permeable prodrug, C13. C2 was a potent allosteric activator of α1-complexes that, like AMP, also protected against Thr172 dephosphorylation. Compared with AMP, C2 caused only partial allosteric activation of α2-complexes and failed to protect them against dephosphorylation. We show that both effects could be fully restored by exchanging part of the linker between the autoinhibitory and C-terminal domains in α2, containing the equivalent region from α1 thought to interact with AMP bound in site 3 of the γ subunit. Consistent with our results in cell-free assays, C13 potently inhibited lipid synthesis in hepatocytes from wild-type and was largely ineffective in AMPK-knockout hepatocytes; its effects were more severely affected by knockout of α1 than of α2, β1, or β2. •C2 activates AMPK via direct activation and protection against dephosphorylation•C2 and its cell-permeable prodrug C13 selectively activate AMPKα1•A sequence located in the C-terminal alpha-linker of α1 confers specificity of C2•C13 inhibits hepatic lipid synthesis via an AMPK-dependent and α1-selective mechanism This study reports the selectivity and mechanism of a new AMPK activator. It inhibits hepatic lipid synthesis via an AMPKα1-isoform-selective mechanism and a sequence located in the C-terminal region of α1 that confers specificity. AMP-activated protein kinase (AMPK) is a central energy sensor and regulator of energy homeostasis . AMPK is2+-mediated process catalyzed by Ca2+/calmodulin-dependent protein kinase kinases  as well as transcriptional variants exist for each of the subunits, generating at least 12 distinct heterotrimeric complexes . There a kinases .AMPK is considered a major target for drugs to combat the growing epidemic of metabolic disorders  because Recently, 50) at 6.3 nM, but the exact isoform combination used and whether it was purified from bacteria, insect, or mammalian cells were not specified . We also tested the effects of C2 and AMP on recombinant human α1β1γ1 and α2β1γ1 complexes expressed in E. coli and obtained similar results (data not shown). The small, partial activation of the α2β1γ1 complex was also potent . Characterization of the subunit composition of the recombinant complexes used in this study is shown in We initially compared the ability of C2 and AMP to activate various recombinant human AMPK complexes, expressed in insect cells, in cell-free assays B. As rep50 of 33 μM and inhibited FBP1 with a half-maximal inhibitory concentration (IC50) of 5 μM (AMP (and ZMP) are known to allosterically modulate several enzymes other than AMPK; for example, AMP activates 6-phosphofructo-1 kinase (PFK1), and AMP and ZMP inhibit the gluconeogenic enzyme fructose-1,6-bisphosphatase-1 (FBP1) . As expe of 5 μM C. By con of 5 μM C, nor diTo determine whether C2 affects the activity of any other protein kinases, we screened it in cell-free assays against a panel of 138 protein kinases. The majority were not affected by 10 μM C2 , includiSpeculating that C2 exerts its effects by exploiting the same binding site on the γ subunits as AMP, we found the modest allosteric activation of α2 complexes by C2 compared to AMP unexpected. One explanation is the presence of a second, inhibitory site unique to α2. Indeed, at the low concentrations of ATP typically used in radiometric kinase assays, activation by AMP itself is biphasic, with a pronounced inhibitory effect at high concentrations due to competition with ATP at the catalytic site . HoweverIn addition to allosterically activating AMPK  than α2 complexes, whose activation was evident only at concentrations above 30 μM B. Note t2O2 increased these ratios  and He ratios D, but th not C13 E. We nex/− cells F.14C]acetate incorporation into saponifiable lipid (fatty acids) and nonsaponifiable lipids  was assessed. There was a concentration-dependent inhibition of lipid synthesis in response to C13 (IC50 of 1.7 μM for saponifiable lipids and 1.5 μM for nonsaponifiable lipids), with a maximal effect at 30 μM  mice or WT controls. C13 dose-dependently inhibited the synthesis of saponifiable and nonsaponifiable lipids in WT hepatocytes, correlating with increases in the phosphorylation of AMPK and ACC , which is >20-fold more potent than A769662  and inhibited de novo lipid synthesis and fatty acid esterification, effects (at least lipid synthesis) that were abolished in AMPK-KO hepatocytes. C13 was more effective than A769662, which was evident from the inability of A769662 to stimulate robust phosphorylation of Raptor, which in our experience requires a higher threshold of AMPK activity than ACC phosphorylation. We found no change in adenine nucleotide levels during treatment with C13 at concentrations up to 100 μM in primary hepatocytes, showing that the compound does not act indirectly via that mechanism. Furthermore, the preference for α1 was confirmed in intact cells by the poor activation of α2 complexes revealed by isoform-specific immunoprecipitation. This was also demonstrated by the marked increase in ICAlthough we have not identified the precise binding sites used by C2, it is structurally analogous to AMP and is also equally effective with AMP in displacing a GST-γ2 fusion protein from ATP-Sepharose. Moreover, C2 failed to activate AMPK containing a mutant γ2 subunit (R531G), which renders AMPK complexes insensitive to AMP. The side chain of Arg531 interacts with the phosphate group of AMP bound in site 3, so this suggests that C2 binds in that site. These results suggest that C2 uses the same binding sites on the AMPKγ subunit as AMP and ATP, most likely sites 1 and 3 . This is2Y94) with a revised version (4CFH). This model suggests that the region from 343 to 353 in human α1 (Q13131), termed α-regulatory subunit-interacting motif-1 (α-RIM1), associates with the unoccupied site 2, whereas the region from 369 to 379 (α-RIM2) associates with AMP bound in site 3  is unused, leaving three  where adenine nucleotides can bind. In the structure for an active α1β2γ1 complex , the α ln site 3 . Using oIn summary, we report the detailed characterization and potential mechanism of action of an AMP-mimetic but α1-selective AMPK activator that, unlike AICAR or ZMP, is completely selective for AMPK compared to other AMP-regulated or -metabolizing enzymes. Although the preference for α1-containing complexes may limit its use for some indications, this shows that it is possible to develop α isoform-specific activators, along with the β isoform-selective activators typified by A769662. Recently, AMP-activated protein kinase (AMPK) is a central energy sensor and regulator of metabolic homeostasis. The activation of AMPK provides desirable therapeutic effects in metabolic disorders such as type 2 diabetes. However, there is currently no direct AMPK activator available for the treatment of metabolic disorders. Only a handful of small molecules have been reported to directly stimulate AMPK with no defined mechanism of action mode elaborated except for A769662, which stimulates β1-containing complexes. We performed an extensive characterization of a recently identified AMPK activator, a nucleotide mimetic, termed Compound 2 (C2) and its prodrug C13. We observed that C2 stimulates AMPK at least 20-fold more potently than A769662 in cell-free assays, with absolute specificity over other AMP-regulated or -metabolizing enzymes. We also found that C2 stimulates AMPK by mimicking both effects of AMP, allosteric activation and inhibition of dephosphorylation via protein phosphatase (PP2C). Strikingly, we identified that C2 preferentially stimulates α1-containing complexes and identified a sequence located in the C-terminal region of α1, outside the catalytic domain, which confers this specificity. The selectivity of the compound for α1 complexes in cell-free assays was consistent with the ability of the cell-permeable prodrug C13 to potently inhibit hepatic lipogenesis in primary mouse hepatocytes, which was reversed in AMPKα1-deficient hepatocytes. This demonstrates that it is possible to develop isoform-selective compounds outside the β subunit carbohydrate-binding-module-dependent compounds, typified by A769662. A more complete understanding of the mechanism by which small molecules activate AMPK may also facilitate the design of novel AMPK activators that could be used to treat patients with metabolic disorders.−/−, AMPKα2−/− and liver AMPK-null (AMPKα1−/− and liver-specific AMPKα2−/−) mice were generated and bred, as previously described  and 200 μM AMARA (AMARAASAAALARRR). Reactions were started by the addition of AMPK (5 mU), incubated for 20 min at 30°C and quenched by spotting onto P81 and immersion in 75 mM H3PO4. Washed filters were dried and [32P] incorporation determined by Cherenkov counting. AMPK activity in cell extracts was determined by immunoprecipitation with AMPKα1 or AMPKα2 antibodies from 50 μg material, as previously described  containing 50 mM HEPES, pH 7.4, 10 mM MgClescribed . Results2, 0.1 mM EGTA, 0.03% BRIJ-35, 1 mM DTT, and the indicated compounds for 15 min at 30°C. Mg2+ was omitted from the negative control. Reactions were terminated by 20-fold dilution and storage on ice. AMPK activity was assayed under standard conditions in the presence of saturating AMP (200 μM). The results are expressed as percentage activity of the negative control. In control experiments, PP2Cα activity was determined in reactions containing 50 mM TES pH 7.4, 0.1 mM EGTA, 25 mM MgCl2, 0.01% BRIJ-35, 0.02% (v/v) 2-mercaptoethanol, and 100 μM EFLR(pT)SCGS (168–176 AMPKα2). Liberated phosphate was determined using malachite green  were washed with warm PBS and allowed to rest for 3 hr in fresh M199. Cells were treated with vehicle (0.1% DMSO) or the indicated compounds and labeled with 1 mCi⋅mmol−1 [1-14C]acetate for 3 hr. Cells were washed with ice-cold PBS, gently scraped into 0.5 ml PBS and saponified in methanolic KOH at 80°C for 1 hr. Nonsaponifiable and saponifiable lipids were extracted with petroleum ether before and after acidification with HCl. Lipid fractions were washed with 5% HAc, dried using N2, and dissolved in scintillant for the determination of [14C] incorporation. Lipogenesis in hepatocytes from AMPKβ1/β2-KO mice was determined in media containing [3H]acetate (0.2 mCi⋅mmol−1). After 4 hr labeling, total lipids were extracted using the method of 3H] was determined by scintillation counting. The results are expressed as micromoles acetate incorporated per gram-hour.Lipid synthesis was determined by labeling adherent cultures of primary hepatocytes with [1-3H(N)]palmitate. After overnight culture, hepatocytes (5 × 105) were washed with warm PBS and allowed to rest for 3 hr in fresh M199 containing 0.5 mM carnitine. Cells were treated with vehicle (0.1% DMSO) or the indicated compounds for 30 min and labeled with 1 mCi⋅mmol−1 palmitic acid  for an additional 1 hr. Cells were washed with ice-cold PBS, lipids extracted using the method of Bligh and Dyer, and neutral lipids were resolved on TLC plates (Partisil K6) in 70:30:1 petroleum ether:diethyl ether:acetic acid. Lipids were stained with iodine vapor and triglyceride eluted from TLC scrapings with 1:1 ethanol:Triton X-100, and the incorporation of [3H] was determined by scintillation counting.Fatty acid esterification was determined by labeling primary hepatocytes with [9,10-