TY - JOUR
T1 - Uncovering the signalling, structure and function of the 20-HETE-GPR75 pairing
T2 - Identifying the chemokine CCL5 as a negative regulator of GPR75
AU - Pascale, Jonathan V.
AU - Park, Eon Joo
AU - Adebesin, Adeniyi Michael
AU - Falck, John R.
AU - Schwartzman, Michal Laniado
AU - Garcia, Victor
N1 - Publisher Copyright:
© 2021 The British Pharmacological Society
PY - 2021/9
Y1 - 2021/9
N2 - Background and Purpose: The G-protein-coupled receptor GPR75 (Gq) and its ligand, the cytochrome P450-derived vasoactive eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), are involved in the activation of pro-inflammatory and hypertensive signalling cascades contributing to diabetes, obesity, vascular dysfunction/remodelling, hypertension and cardiovascular disease. Little is known as to how, where and with what affinity 20-HETE interacts with GPR75. Experimental Approach: To better understand the pairing of 20-HETE and its receptor (GPR75), we used surface plasmon resonance (SPR) to determine binding affinity/kinetics. The PRESTO-Tango receptor-ome methodology for GPR75 overexpression was coupled with FLIPR Calcium 6 assays, homogeneous time-resolved fluorescence (HTRF) IP-1 and β-arrestin recruitment assays to determine receptor activation and downstream signalling events. Key Results: SPR confirmed 20-HETE binding to GPR75 with an estimated KD of 1.56 × 10−10 M. In GPR75-transfected HTLA cells, 20-HETE stimulated intracellular Ca2+ levels, IP-1 accumulation and β-arrestin recruitment, all of which were negated by known 20-HETE functional antagonists. Computational modelling of the putative ligand-binding pocket and mutation of Thr212 within the putative 20-HETE binding site abolished 20-HETE's ability to stimulate GPR75 activation. Knockdown of GPR75 in human endothelial cells nullified 20-HETE-stimulated intracellular Ca2+. The chemokine CCL5, a suggested GPR75 ligand, binds to GPR75 (KD of 5.85 × 10−10 M) yet fails to activate GPR75; however, it inhibited 20-HETE's ability to activate GPR75 signalling. Conclusions and Implications: We have identified 20-HETE as a high-affinity ligand for GPR75 and CCL5 as a low-affinity negative regulator of GPR75, providing additional evidence for the deorphanization of GPR75 as a 20-HETE receptor.
AB - Background and Purpose: The G-protein-coupled receptor GPR75 (Gq) and its ligand, the cytochrome P450-derived vasoactive eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), are involved in the activation of pro-inflammatory and hypertensive signalling cascades contributing to diabetes, obesity, vascular dysfunction/remodelling, hypertension and cardiovascular disease. Little is known as to how, where and with what affinity 20-HETE interacts with GPR75. Experimental Approach: To better understand the pairing of 20-HETE and its receptor (GPR75), we used surface plasmon resonance (SPR) to determine binding affinity/kinetics. The PRESTO-Tango receptor-ome methodology for GPR75 overexpression was coupled with FLIPR Calcium 6 assays, homogeneous time-resolved fluorescence (HTRF) IP-1 and β-arrestin recruitment assays to determine receptor activation and downstream signalling events. Key Results: SPR confirmed 20-HETE binding to GPR75 with an estimated KD of 1.56 × 10−10 M. In GPR75-transfected HTLA cells, 20-HETE stimulated intracellular Ca2+ levels, IP-1 accumulation and β-arrestin recruitment, all of which were negated by known 20-HETE functional antagonists. Computational modelling of the putative ligand-binding pocket and mutation of Thr212 within the putative 20-HETE binding site abolished 20-HETE's ability to stimulate GPR75 activation. Knockdown of GPR75 in human endothelial cells nullified 20-HETE-stimulated intracellular Ca2+. The chemokine CCL5, a suggested GPR75 ligand, binds to GPR75 (KD of 5.85 × 10−10 M) yet fails to activate GPR75; however, it inhibited 20-HETE's ability to activate GPR75 signalling. Conclusions and Implications: We have identified 20-HETE as a high-affinity ligand for GPR75 and CCL5 as a low-affinity negative regulator of GPR75, providing additional evidence for the deorphanization of GPR75 as a 20-HETE receptor.
KW - 20-HETE
KW - CCL5
KW - GPR75
KW - cognate pairing
KW - receptor biology
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U2 - 10.1111/bph.15525
DO - 10.1111/bph.15525
M3 - Article
C2 - 33974269
AN - SCOPUS:85107854477
SN - 0007-1188
VL - 178
SP - 3813
EP - 3828
JO - British Journal of Pharmacology
JF - British Journal of Pharmacology
IS - 18
ER -