scholarly journals Reverse optogenetics of G protein signaling by zebrafish non-visual opsin Opn7b for synchronization of neuronal networks

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raziye Karapinar ◽  
Jan Claudius Schwitalla ◽  
Dennis Eickelbeck ◽  
Johanna Pakusch ◽  
Brix Mücher ◽  
...  
Keyword(s):  
G Protein ◽  
Neuronal Networks ◽  
Neural Circuit ◽  
Epileptiform Activity ◽  
Green Light ◽  
Pyramidal Cells ◽  
Protein Signaling ◽  
G Protein Coupled Receptor ◽  
Inwardly Rectifying Potassium Channels ◽  
G Protein Coupled

AbstractOpn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the Gi/o pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light. Consistent with this prediction, illumination of recombinant expressed Opn7b in cortical pyramidal cells results in increased neuronal activity. In awake mice, light stimulation of Opn7b expressed in pyramidal cells of somatosensory cortex reliably induces generalized epileptiform activity within a short (<10 s) delay after onset of stimulation. Our study demonstrates a reversed mechanism for G protein-coupled receptor control and Opn7b as a tool for controlling neural circuit properties.

scholarly journals Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

2011 ◽  
Vol 286 (22) ◽  
pp. 19259-19269 ◽  
Author(s):  
Il-Ha Lee ◽  
Sung-Hee Song ◽  
Craig R. Campbell ◽  
Sharad Kumar ◽  
David I. Cook ◽  
...  
Keyword(s):  
G Protein ◽  
Kinase Activity ◽  
Receptor Activation ◽  
Na Channel ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Α Subunits

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na+ absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant (K220RGRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 (D110AGRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.

scholarly journals Mechanism of β-arrestin recruitment by the μ-opioid G protein-coupled receptor

2020 ◽  
Vol 117 (28) ◽  
pp. 16346-16355 ◽  
Author(s):  
Amirhossein Mafi ◽  
Soo-Kyung Kim ◽  
William A. Goddard
Keyword(s):  
Side Effects ◽  
G Protein ◽  
Three Dimensional ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
G Protein Coupled Receptor ◽  
Intracellular Loop ◽  
Cytoplasmic Region ◽  
Gi Protein ◽  
G Protein Coupled

Agonists to the μ-opioid G protein-coupled receptor (μOR) can alleviate pain through activation of G protein signaling, but they can also induce β-arrestin activation, leading to such side effects as respiratory depression. Biased ligands to μOR that induce G protein signaling without inducing β-arrestin signaling can alleviate pain while reducing side effects. However, the mechanism for stimulating β-arrestin signaling is not known, making it difficult to design optimum biased ligands. We use extensive molecular dynamics simulations to determine three-dimensional (3D) structures of activated β-arrestin2 stabilized by phosphorylated μOR bound to the morphine and D-Ala2,N-MePhe4, Gly-ol]-enkephalin (DAMGO) nonbiased agonists and to the TRV130 biased agonist. For nonbiased agonists, we find that the β-arrestin2 couples to the phosphorylated μOR by forming strong polar interactions with intracellular loop 2 (ICL2) and either the ICL3 or cytoplasmic region of transmembrane (TM6). Strikingly, Gi protein makes identical strong bonds with these same ICLs. Thus, the Gi protein and β-arrestin2 compete for the same binding site even though their recruitment leads to much different outcomes. On the other hand, we find that TRV130 has a greater tendency to bind the extracellular portion of TM2 and TM3, which repositions TM6 in the cytoplasmic region of μOR, hindering β-arrestin2 from making polar anchors to the ICL3 or to the cytosolic end of TM6. This dramatically reduces the affinity between μOR and β-arrestin2.

scholarly journals Activator of G-protein Signaling 1 Blocks GIRK Channel Activation by a G-protein-coupled Receptor

2002 ◽  
Vol 277 (16) ◽  
pp. 13827-13830 ◽  
Author(s):  
Aya Takesono ◽  
Mark W. Nowak ◽  
Mary Cismowski ◽  
Emir Duzic ◽  
Stephen M. Lanier
Keyword(s):  
G Protein ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
G Protein Coupled Receptor ◽  
Channel Activation ◽  
Girk Channel ◽  
G Protein Coupled

scholarly journals In vitro profiling of orphan G protein coupled receptor (GPCR) constitutive activity

2021 ◽  
Author(s):  
Lyndsay R. Watkins ◽  
Cesare Orlandi
Keyword(s):  
G Protein ◽  
Luciferase Reporter ◽  
List Type ◽  
Constitutive Activity ◽  
Protein Signaling ◽  
G Protein Coupled Receptor ◽  
Orphan Receptors ◽  
Gpcr Family ◽  
Orphan Gpcrs ◽  
G Protein Coupled

AbstractBackground and PurposeMembers of the G protein coupled receptor (GPCR) family are targeted by a significant fraction of the available FDA-approved drugs. However, the physiological role and pharmacological properties of many GPCRs remain unknown, representing untapped potential in drug design. Of particular interest are ~100 less-studied GPCRs known as orphans because their endogenous ligands are unknown. Intriguingly, disease-causing mutations identified in patients, together with animal studies, have demonstrated that many orphan receptors play crucial physiological roles, and thus, represent attractive drug targets.Experimental ApproachThe majority of deorphanized GPCRs demonstrate coupling to Gi/o, however a limited number of techniques allow the detection of intrinsically small constitutive activity associated with Gi/o protein activation which represents a significant barrier in our ability to study orphan GPCR signaling. Using luciferase reporter assays, we effectively detected constitutive Gs, Gq, and G12/13 protein signaling by unliganded receptors, and introducing various G protein chimeras, we provide a novel, highly-sensitive tool capable of identifying Gi/o coupling in unliganded orphan GPCRs.Key ResultsUsing this approach, we measured the constitutive activity of the entire class C GPCR family that includes 8 orphan receptors, and a subset of 20 prototypical class A GPCR members, including 11 orphans. Excitingly, this approach illuminated the G protein coupling profile of 8 orphan GPCRs (GPR22, GPR137b, GPR88, GPR156, GPR158, GPR179, GPRC5D, and GPRC6A) previously linked to pathophysiological processes.Conclusion and ImplicationsWe provide a new platform that could be utilized in ongoing studies in orphan receptor signaling and deorphanization efforts.What is already knownA large group of understudied orphan GPCRs controls a variety of physiological process.What this study addsA new strategy to identify G protein signaling associated with orphan GPCRs.Identification of Gi/o coupling for 8 orphan GPCRs.What is the clinical significanceMany orphan GPCRs are associated with pathological conditions and represent promising druggable targets.

scholarly journals Genetic analysis of the role of G protein–coupled receptor signaling in electrotaxis

2002 ◽  
Vol 157 (6) ◽  
pp. 921-928 ◽  
Author(s):  
Min Zhao ◽  
Tian Jin ◽  
Colin D. McCaig ◽  
John V. Forrester ◽  
Peter N. Devreotes
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
Electrical Potential ◽  
Leading Edge ◽  
Receptor Signaling ◽  
Protein Signaling ◽  
G Protein Coupled Receptor ◽  
Camp Receptor ◽  
Green Fluorescent ◽  
G Protein Coupled

Cells display chemotaxis and electrotaxis by migrating directionally in gradients of specific chemicals or electrical potential. Chemotaxis in Dictyostelium discoideum is mediated by G protein–coupled receptors. The unique Gβ is essential for all chemotactic responses, although different chemoattractants use different receptors and Gα subunits. Dictyostelium amoebae show striking electrotaxis in an applied direct current electric field. Perhaps electrotaxis and chemotaxis share similar signaling mechanisms? Null mutation of Gβ and cAMP receptor 1 and Gα2 did not abolish electrotaxis, although Gβ-null mutations showed suppressed electrotaxis. By contrast, G protein signaling plays an essential role in chemotaxis. G protein–coupled receptor signaling was monitored with PHcrac–green fluorescent protein, which translocates to inositol phospholipids at the leading edge of cells during chemotaxis. There was no intracellular gradient of this protein during electrotaxis. However, F-actin was polymerized at the leading edge of cells during electrotaxis. We conclude that reception and transduction of the electrotaxis signal are largely independent of G protein–coupled receptor signaling and that the pathways driving chemotaxis and electrotaxis intersect downstream of heterotrimeric G proteins to invoke cytoskeletal elements.

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