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.

18 19 20 21 22 23G-protein coupled receptor (GPCR) 68 (GPR68, or OGR1) couples extracellular acidifications and 24 mechanical cues to G protein signaling and plays important roles in vascular physiology, 25 neuroplasticity and cancer progression. The mechanism of mechanosensitivity in GPR68 is 26 currently unkonwn. Here, to study this mechanism, we designed a genetically-encoded fluorescent 27 reporter of GPR68 by fusing a cyclic permuted green fluorescent protein to the third intracellular 28 loop of the receptor. Stimulation with fluid shear stress, extracellular acidifications or the synthetic 29 activator ogerin transiently and robustly increases iGlow's baseline fluorescence up to 4-fold. 30Flow-induced iGlow activation was not suppressed by pharmacological uncoupling of downstream 31 G-protein recruitment, disruption of actin filaments, inhibition of membrane stretch with the 32 peptide toxin GsMTx4, or deletion of a C-terminal amphipathic Helix (Helix 8) proposed to 33 mediate GPCR mechanosensitivity. These results hint that GPR68 uses a hitherto unknown, non-34 canonical mechanism to sense mechanical forces. 35 36 37 38 39 40 G-protein coupled receptors (GPCRs) constitute the largest known family of membrane receptors, 41 comprising at least 831 human homologs organized into 6 functional classes (A to F). They play 42 essential roles in a wide range of biological functions spanning all major physiological systems 43 such as olfaction, energy homeostasis and blood pressure regulation. They also control embryonic 44 development and tissue remodeling in adults. The biological significance of GPCRs is underscored 45 by the fact that ~13% of all known human GPCRs represent the primary targets of ~34% of all 46 pharmaceutical interventions approved by the Food and Drug Administration 1 . 47 GPCRs possess a conserved structure encompassing seven transmembrane helices and 48 switch between resting and active conformations depending on the presence of specific physico-49 chemical stimuli. Besides the vast repertoire of small molecules recognized by GPCRs such as 50 odorants, hormones, cytokines and neurotransmitters, other physico-chemical cues can act as 51 GPCR activators such as photons 2 , ions 3 , membrane depolarizations 4-8 and mechanical forces 9-12 . 52An inherent challenge for the study of GPCR signaling is the fact that GPCRs may often respond 53 to more than one stimulus, therefore acting as complex stimuli integrators. 54 Activated GPCRs physically interact with heterotrimeric G-proteins (Gα, Gβ and Gγ) and 55 promote the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on the 56 Gα subunit upon binding. This process, called G-protein engagement, enables GTP-bound Gα 57 subunits to dissociate from the GPCR:Gβγ complex and activate downstream cellular effectors. 58 Eighteen Gα subunit homologs have been so far identified in mammalian genomes. Gα subunits 59 are clustered into four groups, Gαs, Gαi Gαq and Gα12, each group targeting distinct downstream 60 signaling effector...

Cells generate mechanical force to proliferate, locomote, and perform other cellular functions. Measuring cell forces is vital for understanding their health and behavior, but it has been challenging to find techniques that are compatible with live and fixed samples and do not adversely affect cell function. To