scholarly journals Mechanical and chemical activation of GPR68 (OGR1) probed with a genetically-encoded fluorescent reporter

2020 ◽  
Author(s):  
Alper D Ozkan ◽  
Tina Gettas ◽  
Audrey Sogata ◽  
Wynn Phaychanpheng ◽  
Jérôme J Lacroix
Keyword(s):  
G Protein ◽  
Cancer Progression ◽  
Primary Cilia ◽  
Fluid Shear Stress ◽  
Chemical Activation ◽  
Specific Inhibitor ◽  
G Protein Signaling ◽  
Mechanical Stimuli ◽  
Protein Signaling ◽  
Fluorescent Reporter

AbstractG-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. Here, we designed a genetically-encoded fluorescent reporter of GPR68 activation called “iGlow”. iGlow responds to known GPR68 activators including fluid shear stress, extracellular pH and the synthetic agonist ogerin. Remarkably, iGlow activation occurred from both primary cilia-like structures and from intracellular vesicles, showing iGlow senses extracellular flow from within the cell. Flow-induced iGlow activation is not eliminated by pharmacological modulation of G protein signaling, disruption of actin filaments, or the presence of GsMTx4, a non-specific inhibitor of mechanosensitive ion channels. Genetic deletion of the conserved Helix 8, proposed to mediate GPCR mechanosensitivity, did not eliminate flow-induced iGlow activation, suggesting GPR68 uses a hitherto unkonwn, Helix8-independent mechanism to sense mechanical stimuli. iGlow will be useful to investigate the contribution of GPR68-mediated mechanotransduction in health and diseases.

Mechanical and chemical activation of GPR68 (OGR1) probed with a genetically-encoded fluorescent reporter

2021 ◽  
Author(s):  
Alper D. Ozkan ◽  
Tina Gettas ◽  
Audrey Sogata ◽  
Wynn Phaychanpheng ◽  
Miou Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
Cancer Progression ◽  
Fluid Shear Stress ◽  
Fluorescent Protein ◽  
Chemical Activation ◽  
G Protein Signaling ◽  
Mechanical Stimuli ◽  
Protein Signaling ◽  
Fluorescent Reporter ◽  
Membrane Stretch

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.

scholarly journals Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region

2017 ◽  
Vol 114 (38) ◽  
pp. 10095-10100 ◽  
Author(s):  
Gabriel S. Salzman ◽  
Shu Zhang ◽  
Ankit Gupta ◽  
Akiko Koide ◽  
Shohei Koide ◽  
...  
Keyword(s):  
G Protein ◽  
Cancer Progression ◽  
G Protein Coupled Receptors ◽  
G Protein Signaling ◽  
Biological Processes ◽  
Protein Signaling ◽  
Extracellular Region ◽  
Mechanistic Analysis ◽  
Signaling Domains ◽  
G Protein Coupled

Adhesion G protein-coupled receptors (aGPCRs) play critical roles in diverse biological processes, including neurodevelopment and cancer progression. aGPCRs are characterized by large and diverse extracellular regions (ECRs) that are autoproteolytically cleaved from their membrane-embedded signaling domains. Although ECRs regulate receptor function, it is not clear whether ECRs play a direct regulatory role in G-protein signaling or simply serve as a protective cap for the activating “Stachel” sequence. Here, we present a mechanistic analysis of ECR-mediated regulation of GPR56/ADGRG1, an aGPCR with two domains [pentraxin and laminin/neurexin/sex hormonebinding globulin-like (PLL) and G protein-coupled receptor autoproteolysis-inducing (GAIN)] in its ECR. We generated a panel of high-affinity monobodies directed to each of these domains, from which we identified activators and inhibitors of GPR56-mediated signaling. Surprisingly, these synthetic ligands modulated signaling of a GPR56 mutant defective in autoproteolysis and hence, inStachelpeptide exposure. These results provide compelling support for a ligand-induced and ECR-mediated mechanism that regulates aGPCR signaling in a transient and reversible manner, which occurs in addition to theStachel-mediated activation.

Metabolic Acidosis Regulates RGS16 and G-protein Signaling in Osteoblasts

2021 ◽  
Author(s):  
Nancy S. Krieger ◽  
David A. Bushinsky
Keyword(s):  
Metabolic Acidosis ◽  
Bone Resorption ◽  
G Protein ◽  
Specific Inhibitor ◽  
Osteoclastic Bone Resorption ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Mrna Levels ◽  
Protein Signaling ◽  
Stimulation Of

Chronic metabolic acidosis stimulates cell-mediated net calcium efflux from bone mediated by increased osteoblastic cyclooxygenase 2 (COX2), leading to prostaglandin E2-induced stimulation of RANKL-induced osteoclastic bone resorption. The osteoblastic H+-sensing G-protein coupled receptor (GPCR), OGR1, is activated by acidosis and leads to increased bne resorption. As regulators of G protein signaling (RGS) proteins limit GPCR signaling, we tested whether RGS proteins themselves are regulated by metabolic acidosis. Primary osteoblasts were isolated from neonatal mouse calvariae and incubated in physiological neutral (NTL) or acidic (MET) medium. Cells were collected and RNA extracted for real time PCR analysis with mRNA levels normalized to RPL13a. RGS1, RGS2, RGS3, RGS4, RGS10, RGS11 or RGS18mRNA did not differ between MET and NTL; however by 30' MET decreased RGS16 which persisted for 60' and 3h. Incubation of osteoblasts with the OGR1 inhibitor CuCl2 inhibited the MET induced increase in RGS16 mRNA. Gallein, a specific inhibitor of Gβγ signaling, was used to determine if downstream signaling by the βγ subunit was critical for the response to acidosis. Gallein decreased net Ca efflux from calvariae and COX2 and RANKL gene expression from isolated osteoblasts. These results indicate that regulation of RGS16 plays an important role in modulating the response of the osteoblastic GPCR, OGR1, to metabolic acidosis and subsequent stimulation of osteoclastic bone resorption.

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