G-protein coupled receptor (GPCR) 68 (GPR68, or OGR1) couples extracellular acidifications and mechanical stimuli to G-protein signaling and plays important roles in vascular physiology, neuroplasticity, and cancer progression. Inspired by previous GPCR-based reporters, here, we inserted a cyclic permuted fluorescent protein into the third intracellular loop of GPR68 to create a genetically-encoded fluorescent reporter of GPR68 activation we call "iGlow". iGlow responds to known physiological GPR68 activators such as fluid shear stress and extracellular acidifications. In addition, iGlow responds to Ogerin, a synthetic GPR68-selective agonist, but not to a non-active Ogerin analog, showing the specificity of iGlow-mediated fluorescence signals. Flow-induced iGlow activation is not eliminated by pharmacological modulation of downstream G-protein signaling, disruption of actin filaments, or application of GsMTx4, an inhibitor of certain mechanosensitive ion channels activated by membrane stretch. Deletion of the conserved Helix 8, proposed to mediate mechanosensitivity in certain GPCRs, does not eliminate flow-induced iGlow activation. iGlow could be useful to investigate the contribution of GPR68-dependent signaling in health and disease.
Mechanical and chemical activation of GPR68 (OGR1) probed with a genetically-encoded fluorescent reporter
- Award Group:
- Funder(s): Foundation for the National Institutes of Health
- Award Id(s): GM130834
- Funder(s):
Currently Viewing Accepted Manuscript - Newer Version Available
Alper D. Ozkan, Tina Gettas, Audrey Sogata, Wynn Phaychanpheng, Miou Zhou, Jérôme J. Lacroix; Mechanical and chemical activation of GPR68 (OGR1) probed with a genetically-encoded fluorescent reporter. J Cell Sci 2021; jcs.255455. doi: https://doi.org/10.1242/jcs.255455
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