Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Induces Angiogenesis in Progenitor and Mature Endothelial Cells In Vitro: Investigation of the Possible Role of Adenosine

doi: 10.3390/ijms252413343

Figure Lengend Snippet: The effects of the adenosine receptor A 2A antagonist SCH58621, adenosine receptor A 2B inverse agonist, and antagonist MRS1706 on capillary-like tube formation by ( A ) ECFCs and ( B ) HUVECs, and on spheroid sprout formation by ( C ) ECFCs and ( D ) HUVECs, induced by ticagrelor, adenosine, and their combination. The values represent the means ± SDs from at least 6 independent biological replicates each performed in 2 technical replicates. * p < 0.02 and ** p < 0.01 compared to ticagrelor, adenosine, or their combination in the absence of SCH58621 and MRS1706. # p < 0.05 compared to ticagrelor, adenosine, or their combination in the presence of either SCH58621 or MRS1706. HUVECs: human umbilical vein endothelial cells, ECFCs: endothelial colony-forming cells.

Article Snippet: Adenosine, DPCPX (A 1 AR antagonist), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-ac-LDL), and adenosine deaminase were purchased from Sigma-Aldrich (St. Louis, MO, USA), and MRS1220 (A 3 AR antagonist), SCH58621 (A 2A AR antagonist), and MRS1706 (A 2B AR inverse agonist and antagonist) were purchased from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Induces Angiogenesis in Progenitor and Mature Endothelial Cells In Vitro: Investigation of the Possible Role of Adenosine

doi: 10.3390/ijms252413343

Figure Lengend Snippet: Representative images of 2D capillary-like tube formation by ECFCs that were treated with ticagrelor, adenosine, and their combination in the presence of adenosine receptor A 2A antagonist SCH58621, adenosine receptor A 2B inverse agonist, and antagonist MRS1706. Scale bar 100 μΜ.

Article Snippet: Adenosine, DPCPX (A 1 AR antagonist), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-ac-LDL), and adenosine deaminase were purchased from Sigma-Aldrich (St. Louis, MO, USA), and MRS1220 (A 3 AR antagonist), SCH58621 (A 2A AR antagonist), and MRS1706 (A 2B AR inverse agonist and antagonist) were purchased from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Induces Angiogenesis in Progenitor and Mature Endothelial Cells In Vitro: Investigation of the Possible Role of Adenosine

doi: 10.3390/ijms252413343

Figure Lengend Snippet: Representative images of 3D spheroid sprout formation by ECFCs that were treated with ticagrelor, adenosine, and their combination in the presence of adenosine receptor A 2A antagonist SCH58621, adenosine receptor A 2B inverse agonist, and antagonist MRS1706. Scale bar 200 μM.

Article Snippet: Adenosine, DPCPX (A 1 AR antagonist), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-ac-LDL), and adenosine deaminase were purchased from Sigma-Aldrich (St. Louis, MO, USA), and MRS1220 (A 3 AR antagonist), SCH58621 (A 2A AR antagonist), and MRS1706 (A 2B AR inverse agonist and antagonist) were purchased from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Human A 2A AR in different membrane or membrane-mimetic environments and sample preparation workflow. (a) A 2A AR compared in three different environments in this study: (left) detergent micelles, (middle) lipid nanodiscs, and (right) lipid vesicles. (b) Schematic of the sample preparation workflow for preparing lipid vesicles containing 19 F-labeled human A 2A AR for solid-state MAS NMR experiments.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Membrane, Sample Prep, Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Characterization of lipid vesicles containing human A 2A AR and pharmacological validation of A 2A AR in vesicles. ( a and b ) Representative negative stain electron micrographs of unilameller vesicles composed of POPC and POPS (70:30 molar ratio) ( a ) without and ( b ) with reconstituted A 2A AR. ( c ) Dynamic light scattering measurements of the distribution of the sizes of vesicles composed of POPC and POPS (70:30 molar ratio) without (green) and with (purple) A 2A AR. ( d and e ) Pharmacological characterization of A 2A AR in lipid vesicles. ( d ) Saturation binding experiment with 3 H-ZM241385 and A 2A AR in lipid vesicles containing POPC and POPS (70:30 molar ratio). The reported B max value represents the mean and associated error is the s.e.m. from 3 independent trials done in triplicate. ( e ) Radioligand competition experiments. K D and K i values are reported for the antagonist ZM241385 and the agonist NECA, respectively. The reported error represents the s.e.m. from 3 independent trials done in triplicate.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Staining, Binding Assay

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Determination of the orientation of A 2A AR in lipid vesicles. ( a ) Schematic of the fluorescence-quenching assay used to quantify receptor orientation within vesicles. Green circles represent position C289 labeled with Cy3. Receptors with Cy3 covalently attached to position C289 facing toward the vesicle interior are labeled “A 2A AR inside”, while receptors with Cy3 facing away from the vesicle interior are labeled “A 2A AR outside”. Grey circles represent Cy3-labels that have been chemically quenched. ( b ) Quantitative comparison of the orientation of A 2A AR in vesicles made from POPC or POPC and POPS (70:30 molar ratio). The orientations “A 2A AR inside” and “A 2A AR outside” are as defined in ( a ). Error bars indicate the s.e.m. calculated from n≥3 independent experiments. Statistically significant values are illustrated as ***p<0.005 using a 2-tailed unpaired t-test.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Fluorescence, Labeling, Comparison

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Fluorescence thermal melting profiles of the A 2A AR complex with the antagonist in three different membrane mimetics. ( a ) Schematic of the fluorescence thermal shift assay as applied to A 2A AR in lipid vesicles. The inactive fluorescent dye (grey stars) shows increased emission upon covalent attachment with cysteines that become solvent accessible upon protein unfolding (orange stars). ( b ) Representative thermal melting profiles for A 2A AR in DDM/CHS detergent micelles, lipid nanodics containing POPC and POPS (70:30 molar ratio), and lipid vesicles containing POPC and POPS (70:30 molar ratio). ( c ) The melting temperature for A 2A AR in each membrane mimetic is reported as the mean of three independent experiments ± s.e.m.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Fluorescence, Membrane, Thermal Shift Assay, Solvent

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F MAS NMR-observed conformational equilibria of human A 2A AR in complex with an antagonist in lipid vesicles measured with different experimental parameters. ( a ) 1-dimensional 19 F-MAS spectra of A 2A AR[A289C TET ] in complex with the antagonist ZM241385 reconstituted into POPC/POPS lipid vesicles recorded with 105 kHz 1 H TPPM decoupling at three different MAS frequencies. ( b ) NMR spectra from (a) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 and P3. ( c ) 1-dimensional 19 F-MAS spectra of the same sample used to measure the data in (a) recorded with 1 H TPPM decoupling power set to one-quarter of the applied MAS frequency. ( d ) NMR spectra from (c) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 and P3. ( e and f ) ( e ) 1-dimensional 19 F-MAS spectra of the same sample used to measure the data in (a) recorded with no 1 H decoupling and ( f ) NMR spectra from ( e ) are shown superimposed with Lorentzian deconvolutions. ( g-j ) Linewidths measured for populations P1 and P3 for A 2A AR in ( g-i ) lipid vesicles or ( j ) lipid nanodiscs or detergent micelles. Components colored green are from free TET, consistent with earlier NMR studies (see text).

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F MAS NMR-observed conformational equilibria of human A 2A AR in complex with the agonist NECA in lipid vesicles measured with low power TPPM 1 H decoupling at several MAS frequencies. ( a ) 1-dimensional 19 F-MAS spectra of A 2A AR[A289C TET ] in complex with the agonist NECA reconstituted into POPC/POPS lipid vesicles recorded with three different MAS frequencies and 1 H TPPM decoupling at an applied 1 H power of one-quarter of the MAS frequency. ( b ) NMR spectra from (a) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 through P5. Components colored green are from free TET, consistent with earlier NMR studies (see text).

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F-NMR systematic comparison of the conformational equilibria of antagonist-bound and agonist-bound human A 2A AR[A289C TET ] across three membrane or membrane-mimetic systems by solution NMR in (a) detergent micelles and (b) lipid nanodiscs and by MAS solid-state NMR in (c) lipid vesicles. Same color scheme as in and .

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Comparison, Membrane