Journal: British journal of pharmacology

Article Title: Uncovering the signalling, structure and function of the 20-HETE-GPR75 pairing: Identifying the chemokine CCL5 as a negative regulator of GPR75

doi: 10.1111/bph.15525

Figure Lengend Snippet: 20-HETE binds to GPR75.(a) Surface plasmon resonance (SPR) analysis of 20-HETE-GPR75 binding, depicting the sensogram (normalized to vehicle (HBSS containing 1% ethanol) (left) of human recombinant GPR75 immobilized to the SPR sensor surface followed by 20-HETE injections (0.0625, 0.125, 0.25, 0.5, 1, 2, 5 and 10 nM) injected at a flow rate of 20 μl min−1 and affinity analysis (right). (b) Structures of arachidonic acid, arachidonoyl ethanolamide, 20-HETE and 12(S)-HETE. (c) SPR sensogram of human recombinant GPR75 immobilized to the SPR sensor surface followed by injections of vehicle (HBSS containing 1% ethanol), arachidonic acid, arachidonoyl ethanolamide, 12(S)-HETE and 20-HETE (1 nM) injected at a flow rate of 20 μl min−1; 20-HETE increases intracellular calcium (iCa2+) via GPR75. (d) FLIPR Calcium 6 assays of mock- or GPR75-transfected HTLA cells treated with vehicle (ethanol), 20-HETE (1 nM) and co-treatment of 20-HETE (1 nM) with AAA (1 nM), a 20-HETE receptor antagonist, showing post-treatment live calcium traces (2 min) or (e) peak treatment response expressed as fold change relative to vehicle-treated cells.Data shown are means ± SEM; n = 6.*P < 0.05, significantly different from vehicle (ethanol), #P < 0.05, significantly different from 20-HETE

Article Snippet: In brief, human full-length recombinant GPR75 (H00010936-G01, Abnova, Taiwan) (isoelectric point [pI] = 8.3) was immobilized onto high sensitivity carboxyl sensors (SEN-HS-8-COOH) using Nicoya’s amine coupling kit (AMINESM-10) (4:1 EDC/NHS) at the desired ~8500–9300 response unit (RU) to detect small molecule binding to GPR75.

Techniques: SPR Assay, Binding Assay, Recombinant, Injection, Transfection

Journal: British journal of pharmacology

Article Title: Uncovering the signalling, structure and function of the 20-HETE-GPR75 pairing: Identifying the chemokine CCL5 as a negative regulator of GPR75

doi: 10.1111/bph.15525

Figure Lengend Snippet: CCL5 increases intracellular calcium (iCa2+) and inositol phosphate-1 (IP-1) via its chemokine receptors and blocks 20-HETE-mediated increases in iCa2+ and IP-1. (a) Surface plasmon resonance (SPR) sensogram of human recombinant GPR75 immobilized to the SPR sensor surface followed by injections of CCL5/RANTES (0.01 nM) injected at a flow rate of 20 μl min−1. (b) FLIPR Calcium 6 assays showing mock- or GPR75-transfected HTLA cells treated with vehicle (PBS), 20-HETE (1 nM) and CCL5 (0.1 nM) in the presence and absence of CCL5 receptor antagonists (CRA) (BX471 targeting CCR1 (25 nM), SB328437 targeting CCR3 (80 nM) and DAPTA targeting CCR5 (20 nM)). (c) FLIPR Calcium 6 assays showing mock or GPR75 transfected HTLA cells treated with vehicle (ethanol), 20-HETE (1 nM), and cells pretreated with CCL5 (0.1 nM) followed by 20-HETE (1 nM) in the presence and absence of CRAs. (d) Peak treatment response expressed as fold change relative to mock-transfected, vehicle (ethanol)-treated cells. Dashed lines represent the maximal response of mock and GPR75-transfected cells to 20-HETE (1 nM). (e) Effect of 20-HETE (1 nM), AAA (1 nM) and CCL5 (0.1 nM) in the presence and absence of CRAs on IP-1 accumulation in HTLA cells transfected with or without WT GPR75-tango overexpression constructs. Data shown are means ± SEM; n = 6. *P < 0.05, significantly different from mock vehicle (PBS), #P < 0.05, significantly different from Mock CCL5, †P < 0.05, significantly different from Mock 20-HETE, **P < 0.05, significantly different from WT GPR75 20-HETE)

Article Snippet: In brief, human full-length recombinant GPR75 (H00010936-G01, Abnova, Taiwan) (isoelectric point [pI] = 8.3) was immobilized onto high sensitivity carboxyl sensors (SEN-HS-8-COOH) using Nicoya’s amine coupling kit (AMINESM-10) (4:1 EDC/NHS) at the desired ~8500–9300 response unit (RU) to detect small molecule binding to GPR75.

Techniques: SPR Assay, Recombinant, Injection, Transfection, Over Expression, Construct

Journal: British journal of pharmacology

Article Title: Uncovering the signalling, structure and function of the 20-HETE-GPR75 pairing: Identifying the chemokine CCL5 as a negative regulator of GPR75

doi: 10.1111/bph.15525

Figure Lengend Snippet: 20-HETE, but not CCL5,increases β-arrestin recruitment to GPR75. (a) PRESTO-tango β-arrestin recruitment assay in HTLA cells overexpressing GPR75-tango constructs treated with increasing concentrations of 20-HETE, arachidonic acid, arachidonoyl ethanolamide and 12(S)-HETE; (b) fold change in β-arrestin recruitment in response to 20-HETE (1 nM), AAA (1 nM) and co-treatment of equimolar concentrations of 20-HETE and AAA (1 nM). (c) CCL5 blocks 20-HETE-mediated β-arrestin recruitment to GPR75. Fold change in β-arrestin recruitment in response to 20-HETE (1 nM), CCL5 (0.1 nM) and the co-treatment of CCL5 (0.1 nM) with 20-HETE (1 nM) in the presence and absence of CCL5 receptor antagonists (CRA) (BX471 targeting CCR1 (25 nM), SB328437 targeting CCR3 (80 nM) and DAPTA targeting CCR5 (20 nM)). (d–f) Molecular modelling of GPR75. GPR75 (grey) alongside the (d) ligand binding site (pink), (e) 20-HETE-GPR75 ligand–receptor binding model (20-HETE in blue) and (f) the CCL5-GPR75 protein–protein interaction model. Data shown are means ± SEM; n = 8–12). *P < 0.05, significantly different from vehicle (ethanol), #P < 0.05, significantly different from 20-HETE)

Article Snippet: In brief, human full-length recombinant GPR75 (H00010936-G01, Abnova, Taiwan) (isoelectric point [pI] = 8.3) was immobilized onto high sensitivity carboxyl sensors (SEN-HS-8-COOH) using Nicoya’s amine coupling kit (AMINESM-10) (4:1 EDC/NHS) at the desired ~8500–9300 response unit (RU) to detect small molecule binding to GPR75.

Techniques: Construct, Ligand Binding Assay, Binding Assay

Journal: British journal of pharmacology

Article Title: Uncovering the signalling, structure and function of the 20-HETE-GPR75 pairing: Identifying the chemokine CCL5 as a negative regulator of GPR75

doi: 10.1111/bph.15525

Figure Lengend Snippet: Mutations across the putative 20-HETE-GPR75 binding site alter the 20-HETE-mediated changes in intracellular calcium (iCa2+), IP-1 and β-arrestin. (a) Molecular modelling depicting the putative 20-HETE-GPR75 binding site (3D left, 2D right). (b) FLIPR Calcium 6 assays showing WT and mutant (S205A, T212N, S219A) GPR75-transfected HTLA cells treated with vehicle 20-HETE (1 nM) or CCL5 (0.1 nM). (c) PRESTO-tango β-arrestin recruitment assays in HTLA cells overexpressing WT and mutant (S205A, T212N, S219A) GPR75-tango constructs treated with increasing concentrations of 20-HETE (n = 8). (d) Effect of 20-HETE (1 nM) on IP-1 accumulation in HTLA cells transfected with mutant (S205A, T212N, S219A) GPR75-tango overexpression constructs. Dotted lines represent the maximal response observed in mock and GPR75-transfected cells to 20-HETE (1 nM). Data shown are means ± SEM; n = 6. *P < 0.05, significantly different from 20-HETE). (e) Web of bias plot illustrating the distinctions between the WT GPR75 and GPR75 mutants (S205A, T212N, S219A) responses to 20-HETE across three signalling pathways (iCa2+, IP-1 and β-arrestin). The signalling responses are normalized to WT (set to 1) and the plot distinguishes each mutant’s degree of deviation from the WT GPR75 transfected cells treated with 20-HETE (1 nM)

Article Snippet: In brief, human full-length recombinant GPR75 (H00010936-G01, Abnova, Taiwan) (isoelectric point [pI] = 8.3) was immobilized onto high sensitivity carboxyl sensors (SEN-HS-8-COOH) using Nicoya’s amine coupling kit (AMINESM-10) (4:1 EDC/NHS) at the desired ~8500–9300 response unit (RU) to detect small molecule binding to GPR75.

Techniques: Binding Assay, Mutagenesis, Transfection, Construct, Over Expression

Journal: British journal of pharmacology

Article Title: Uncovering the signalling, structure and function of the 20-HETE-GPR75 pairing: Identifying the chemokine CCL5 as a negative regulator of GPR75

doi: 10.1111/bph.15525

Figure Lengend Snippet: 20-HETE, but not CCL5, increases intracellular calcium (iCa2+) via GPR75 in human umbilical vein endothelial cells (HUVECs). FLIPR Calcium 6 assays of HUVECs treated with; (a) vehicle (ethanol), 20-HETE (1 nM) and co-treatment of 20-HETE (1 nM) with AAA (1 nM), a 20-HETE receptor antagonist; (b) GPR75 knockdown in HUVECs abolishes 20-HETE-mediated increases in iCa2+. Effect of 20-HETE (1 nM) on intracellular calcium in control (Ctrl) and GPR75 siRNA-treated HUVECs. (c) CCL5 increases iCa2+ via its chemokine receptors. Effects of vehicle (PBS) and CCL5 (0.1 nM) in the presence and absence of CCL5 receptor antagonists (CRA) (BX471 targeting CCR1 [25 nM], SB328437 targeting CCR3 [80 nM] and DAPTA targeting CCR5 [20 nM]); (d) CCL5 blocks 20-HETE-stimulated increases in iCa2+. Effects of pretreatment of CCL5 (0.1 nM) ± CRAs followed by 20-HETE (1 nM). (e) Gpr75 knockdown intensifies CCL5 (RANTES)-mediated changes in iCa2+ in HUVECs. Effect of CCL5 (0.1 nM) on intracellular calcium in control (Ctrl) or GPR75 siRNA-treated HUVECs. Data shown are means ± SEM; n = 6

Article Snippet: In brief, human full-length recombinant GPR75 (H00010936-G01, Abnova, Taiwan) (isoelectric point [pI] = 8.3) was immobilized onto high sensitivity carboxyl sensors (SEN-HS-8-COOH) using Nicoya’s amine coupling kit (AMINESM-10) (4:1 EDC/NHS) at the desired ~8500–9300 response unit (RU) to detect small molecule binding to GPR75.

Techniques: Knockdown, Control

Journal: British journal of pharmacology

Article Title: Uncovering the signalling, structure and function of the 20-HETE-GPR75 pairing: Identifying the chemokine CCL5 as a negative regulator of GPR75

doi: 10.1111/bph.15525

Figure Lengend Snippet: 20-HETE promotes Ca2+ signalling, IP-1 accumulation and β-arrestin recruitment through its receptor GPR75. (1) 20-HETE binds to and activates GPR75, setting in motion a signalling program that promotes Gq-coupled changes to calcium, the accumulation of IP-1 and the recruitment of β-arrestin to GPR75. These events precede 20-HETE-mediated changes to pro-inflammatory signals such as the activation of NF-kB, uncoupling of endothelial nitric oxide synthase (eNOS) and the upregulation of angiotensin converting enzyme (ACE). These effects are prevented by 20-HETE receptor antagonists (e.g., AAA, 2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyeicosa-6(Z),15(Z)-dienoate (20-SOLA) and 19(R)-HETE). (2) The chemokine CCL5 predominantly interacts with the chemokine receptors CCR1, CCR3 and CCR5 to elicit Gi-coupled changes to cAMP and secondary changes to intracellular calcium. (3) CCL5, through its interaction with GPR75, can prevent and block the 20-HETE-GPR75 pairing and the ensuing changes in both calcium and PLC-mediated signalling cascades

Article Snippet: In brief, human full-length recombinant GPR75 (H00010936-G01, Abnova, Taiwan) (isoelectric point [pI] = 8.3) was immobilized onto high sensitivity carboxyl sensors (SEN-HS-8-COOH) using Nicoya’s amine coupling kit (AMINESM-10) (4:1 EDC/NHS) at the desired ~8500–9300 response unit (RU) to detect small molecule binding to GPR75.

Techniques: Activation Assay, Blocking Assay

Journal: British journal of pharmacology

Article Title: Uncovering the signalling, structure and function of the 20-HETE-GPR75 pairing: Identifying the chemokine CCL5 as a negative regulator of GPR75

doi: 10.1111/bph.15525

Figure Lengend Snippet:

Article Snippet: In brief, human full-length recombinant GPR75 (H00010936-G01, Abnova, Taiwan) (isoelectric point [pI] = 8.3) was immobilized onto high sensitivity carboxyl sensors (SEN-HS-8-COOH) using Nicoya’s amine coupling kit (AMINESM-10) (4:1 EDC/NHS) at the desired ~8500–9300 response unit (RU) to detect small molecule binding to GPR75.

Techniques: Sequencing