期刊
JOURNAL OF CELL SCIENCE
卷 134, 期 16, 页码 -出版社
COMPANY BIOLOGISTS LTD
DOI: 10.1242/jcs.255455
关键词
Mechanobiology; GPCR; Fluid shear stress; Genetically encoded fluorescent reporter
类别
资金
- Western University of Health Sciences
- Federal Work-Study
- National Institutes of Health [GM130834, NS101384]
The study developed a genetically encoded fluorescent reporter, iGlow, by inserting a fluorescent protein into the third intracellular loop of GPR68, which responds to physiological GPR68 activators and synthetic agonists with specificity. iGlow activation is unaffected by pharmacological modulation of downstream G-protein signaling or disruption of actin filaments, providing a new tool for investigating GPR68-dependent signaling in health and disease.
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.
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