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 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.

scholarly journals C5a-Stimulated Recruitment of β-Arrestin2 to the Nonsignaling 7-Transmembrane Decoy Receptor C5L2

2009 ◽  
Vol 14 (9) ◽  
pp. 1067-1075 ◽  
Author(s):  
Lambertus H.C. Van Lith ◽  
Julia Oosterom ◽  
Andrea Van Elsas ◽  
Guido J.R. Zaman
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Ligand Interaction ◽  
C5a Receptor ◽  
Decoy Receptor ◽  
Complementation Assay ◽  
Fragment Complementation

C5L2 (or GPR77) is a high-affinity receptor for the complement fragment C5a and its desarginated product, C5a-desArg. Unlike the classical C5a receptor CD88, C5L2 does not couple to intracellular G-protein-signaling pathways but is thought to function as a decoy receptor. The authors show that stimulation of C5L2 with C5a and C5a-desArg induces redistribution of green fluorescent protein—labeled β-arrestin2 to cytoplasmic vesicles. C3a and C3a-desArg were inactive in the β-arrestin translocation assay. Direct interaction of ligand-stimulated C5L2 with β-arrestin was confirmed using a novel β-galactosidase fragment complementation assay. In this assay, C5L2 was labeled with a mutationally altered peptide fragment of β-galactosidase, whereas β-arrestin2 was labeled with a corresponding deletion mutant of the enzyme. Stable transfection of the modified C5L2 and subsequent stimulation with C5a or C5a-desArg restored β-galactosidase activity in a dose-dependent manner. The subnanomolar potency of β-arrestin coupling in the β-galactosidase fragment complementation assay is in agreement with the affinity of the receptor-ligand interaction. C5L2 is the first example of a 7-transmembrane decoy receptor that couples to β-arrestin in a ligand-dependent manner. This observation supports the notion that G-protein-signaling and β-arrestin coupling can be 2 separate activities of 7-transmembrane receptors. Furthermore, the β-arrestin assays described in this article provide methods of screening for selective C5L2 modulators. ( Journal of Biomolecular Screening 2009:1067-1075)

scholarly journals Correlation of Autophagosome Formation with Degradation and Endocytosis Arabidopsis Regulator of G-Protein Signaling (RGS1) through ATG8a

2019 ◽  
Vol 20 (17) ◽  
pp. 4190 ◽  
Author(s):  
Yue Jiao ◽  
Miroslav Srba ◽  
Jingchun Wang ◽  
Wenli Chen
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Autophagic Flux ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
In Planta ◽  
Autophagosome Formation ◽  
Root Cells ◽  
Ubiquitin Proteasome

Damaged or unwanted cellular proteins are degraded by either autophagy or the ubiquitin/proteasome pathway. In Arabidopsis thaliana, sensing of D-glucose is achieved by the heterotrimeric G protein complex and regulator of G-protein signaling 1 (AtRGS1). Here, we showed that starvation increases proteasome-independent AtRGS1 degradation, and it is correlated with increased autophagic flux. RGS1 promoted the production of autophagosomes and autophagic flux; RGS1-yellow fluorescent protein (YFP) was surrounded by vacuolar dye FM4-64 (red fluorescence). RGS1 and autophagosomes co-localized in the root cells of Arabidopsis and BY-2 cells. We demonstrated that the autophagosome marker ATG8a interacts with AtRGS1 and its shorter form with truncation of the seven transmembrane and RGS1 domains in planta. Altogether, our data indicated the correlation of autophagosome formation with degradation and endocytosis of AtRGS1 through ATG8a.

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.

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