13 research outputs found

    Surface to intracellular fluorescence ratio for cell expressing WT/mutant AVPR2 with or without SCTR co-expression.

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    <p>The presence of SCTR increased the amount of fluorescence on the cell surface in cell expressing R137H AVPR2 tagged with YFP, indicating a rescue of the mutant receptor. The data were mean±SEM from three independent experiments from 5–6 ROIs per sample.</p

    Surface expression of mAVPR1a, mAVPR1b, mAVPR2 and mSCTR are similar.

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    <p>Shown are representative images of CHOK1 cells expressing mAVPR1a/mAVPR1b/mAVPR2 or mSCTR constructs. Surface to intracellular fluorescence ratios were similar for these four types of cells. The data were mean±SEM from three independent experiments with 5–6 ROIs per sample. Scale bar, 10μM.</p

    mSCTR specifically oligomerizes with mAVPR2, and mAVPR1a, but not mAVPR1b.

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    <p>Shown are the net BRET ratios for CHO-K1 cells expressing a combination of mSCTR-Rlu donor and mAVPR-YFP acceptor constructs. Saturable curves from BRET assays were obtained for mAVPR2 and mAVPR1a, but not for mAVPR1b. The data were mean±SEM from three to five independent experiments in triplicate. ***, P<0.001. **, P<0.01. *, P<0.05.</p

    Rescue of R137H mutant by SCTR as reflected by reduced affinity to β-arrestin.

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    <p>The affinity between WT or mutant AVPR2 with β-arrestin were determined by BRET, using AVRP2 tagged with Rlu and β-arrestin tagged with YFP. A) In the native state, the R137H mutant shows significantly higher affinity to β-arrestin than the WT AVPR2. While the two other mutants, A89P and Q174R, showed slightly higher BRET than the WT receptor, but the increase was not significant. Upon co-expression of SCTR, β-arrestin affinity of R137H was significantly reduced compared to the scenario when no SCTR was present. B) BRET was measured at 10 min after addition of 1μM Vp to stimulate receptor internalization. WT AVPR2 showed increase affinity to β-arrestin, but not the R137H mutant without SCTR co-expression. With SCTR, R137H demonstrated increased β-arrestin binding, suggesting functional rescue of the receptor. Data are presented as means±SEM from three independent experiments in duplicate. ***, P<0.001, **, P<0.01.</p

    Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells

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    A new bioorthogonal reactant pair, spiro[2.3]­hex-1-ene (Sph) and 3,6-di­(2-pyridyl)-<i>s</i>-tetrazine (DpTz), for the strain-promoted inverse electron-demand Diels–Alder cycloaddition, that is, tetrazine ligation, is reported. As compared to the previously reported strained alkenes such as <i>trans</i>-cyclooctene (TCO) and 1,3-disubstituted cyclopropene, Sph exhibits balanced reactivity and stability in tetrazine ligation with the protein substrates. A lysine derivative of Sph, SphK, was site-selectively incorporated into the extracellular loop regions (ECLs) of GCGR and GLP-1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells with the incorporation efficiency dependent on the location. Subsequent bioorthogonal reactions with the fluorophore-conjugated DpTz reagents afforded the fluorescently labeled GCGR and GLP-1R ECL mutants with labeling yield as high as 68%. A multitude of functional assays were performed with these GPCR mutants, including ligand binding, ligand-induced receptor internalization, and ligand-stimulated intracellular cAMP accumulation. Several positions in the ECL3s of GCGR and GLP-1R were identified that tolerate SphK mutagenesis and subsequent bioorthogonal labeling. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP-1R should allow biophysical studies of conformation dynamics of this important class of GPCRs in their native environment in live cells

    Lrp5 coexpression enhances glucagon and GLP1-induced β-catenin signaling.

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    <p>A). HEK293 cells cultured in 12-well plate were transfected with a combination of indicated plasmids (GCGR 1000 ng, Lrp5 500 ng) for 24 h and then treated with or without 50 nM GCG1-29 for 1 h. The cells were harvested and lysed, and samples were used for western blot analysis. The blot was first probed with anti-β-catenin antibody and then stripped and reprobed for anti-β-actin antibody as a loading control. B). 293STF cells cultured in 24-well plate were transfected with 100 ng of empty vector (pcDNA3.1) or GCGR plasmid along with the indicated amount of Lrp5 and 5 ng TKRlu plasmids on day 1, and then treated with or without 50 nM GCG1-29 on day 2. Cells were harvested for luciferase activity measurement on day 3. Triplicate samples were used for each treatment. *p<0.005 compared with non-treated group. 4C). 293STF cells cultured in 24-well plate were transfected with 100 ng GLP1R, 100 ng Lrp5 and 5 ng TKRlu plasmids on day 1 and then treated with the GLP1 agonist GLP1(7–36) (50 nM) or the antagonist Exendin(9–39) (50 nM) on day 2. Cells were harvested on day 3 to measure luciferase activity. Duplicate samples were used for each treatment. *p<0.05 compared with the non-treated (NT) group.</p

    Blocking Lrp5/6 inhibited glucagon-induced β-catenin signaling.

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    <p>A). 293STF cells cultured in 24-well plate were transfected with a combination of GCGR (100 ng), Lrp5 (100 ng), Lrp5ECD (200 ng) and TKRlu (5 ng) plasmids as indicated on day 1, and then were treated with or without 50 nM GCG1-29 on day 2. Cells were harvested on day 3 to measure the TCF-mediated luciferase activity. Triplicate samples were used for each treatment. *p<0.005 compared with the non-treated (NT) group. <sup>#</sup>p<0.005 compared with the group without the Lrp5ECD transfection. B). DKK1 protein inhibits glucagon-induced β-catenin signaling. 293STF cells cultured in 24-well plate were transfected with pcDNA3.1 and GCGR plasmids (100 ng each) or GCGR and Lrp5 plasmids (100 ng each) along with 5 ng TKRlu plasmid on day 1, and then were treated with 50 nM GCG1-29±2 µg/ml DKK1 on day 2. Cells were harvested on day 3 to measure the luciferase activity. Duplicate samples were used for each treatment. *p<0.02 compared with the GCG1-29-treated group.</p

    Lrp5 physically interacts with GCGR.

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    <p>A). HEK293 cells cultured in 6-well plate were transfected with 2000 ng of a control vector (GFP), v5-tagged Lrp5, HA-tagged GCGR, or both for 24 h and then were treated with or without 50 nM GCG1-29 for 1 h. Cells were harvested and lysed, and equal amounts of protein were used for western blot analysis. The blot was first probed with v5 antibody and then stripped and reprobed with HA antibody. The β-actin blot was used as a loading control. B). HEK293 cells were transfected and treated same as in A. The cells were harvested and lysed, and equal amounts of lysate were immunoprecipitated with the indicated antibody. For HA antibody, the antibody complex was pulled down by protein G beads. For v5 antibody, it was a single-step pull-down because the antibody was directly conjugated to the agarose beads. After pull-down, the beads were washed three times with 1× TBST and then incubated in 1× SDS sample buffer to release the bound proteins. The lysates were used for western blot analysis and probed with the indicated antibody. C–E). BRET data for Rlu-tagged Lrp5 and YFP-tagged GCGR expressed on COS-1 cells. Shown are the static BRET signals (C), saturation BRET analysis (D), and effect of natural agonist ligand binding on the BRET signals (E). Shaded area represents the background signal of 0.12 determined using Rlu-tagged Lrp5 with soluble YFP as noted. Coexpression of untagged GCGR or Lrp5 competitively reduced the BRET signals obtained between Lrp5-Rlu and GCGR-YFP (black bars). Saturation BRET analysis supported the specific interaction of Lrp5 and GCGR. The non-specific bystander type BRET signal (linear) was observed when the non-structurally-related CCK1 receptors were co-expressed with Lrp5. Incubation with the natural agonist peptide ligand, glucagon (up to 1 µM), did not significantly change the Lrp5 and GCGR BRET signal. Data are represented as the means ± S.E.M. from four to six independent experiments performed in triplicate. Data marked with ** were significantly different from background signals at the level of p<0.01.</p
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