Interactions between G-protein-coupled receptors and periplakin: a selective means to regulate G-protein activation

2004 ◽  
Vol 32 (5) ◽  
pp. 878-880 ◽  
Author(s):  
G. Milligan ◽  
H. Murdoch ◽  
E. Kellett ◽  
J.H. White ◽  
G.-J. Feng
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Protein Activation ◽  
G Protein Activation ◽  
Melanin Concentrating Hormone ◽  
Receptor Interacting Proteins ◽  
G Protein Coupled ◽  
Two Hybrid

A substantial number of G-protein-coupled receptor-interacting proteins have been identified initially by the use of yeast two-hybrid screens. Using the C-terminal tail of both opioid receptors and the melanin concentrating hormone receptor-1 as bait, the actin and intermediate filament-binding protein periplakin was isolated. In each case, the site of interaction is within helix VIII of the receptor and periplakin limits agonist-mediated G-protein activation potentially by competing with G-protein for this region of the receptor.

scholarly journals Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist

2015 ◽  
Vol 112 (19) ◽  
pp. 6194-6199 ◽  
Author(s):  
Hannah M. Stoveken ◽  
Alexander G. Hajduczok ◽  
Lei Xu ◽  
Gregory G. Tall
Keyword(s):  
G Protein ◽  
Clinical Utility ◽  
G Protein Coupled Receptors ◽  
Activation Mechanism ◽  
Inhibitory Influence ◽  
Neighboring Cell ◽  
Protein Activation ◽  
G Protein Activation ◽  
G Protein Coupled

The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease.

scholarly journals Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system

Parasitology ◽  
2019 ◽  
Vol 147 (8) ◽  
pp. 865-872 ◽  
Author(s):  
Oliver Weth ◽  
Simone Haeberlein ◽  
Martin Haimann ◽  
Yinjie Zhang ◽  
Christoph G. Grevelding
Keyword(s):  
Schistosoma Mansoni ◽  
G Protein ◽  
Hybrid System ◽  
G Protein Coupled Receptors ◽  
Dependent Manner ◽  
Yeast Two Hybrid ◽  
Ligand Interaction ◽  
Yeast Two Hybrid System ◽  
G Protein Coupled ◽  
Two Hybrid

AbstractSchistosomiasis is an acute and chronic disease caused by parasitic worms of the genus Schistosoma. Treatment is solely dependent on praziquantel. In the face of the worldwide dimension, projects have been initiated to develop new chemotherapies. Due to their proven druggability, G protein-coupled receptors (GPCRs) are promising targets for anthelmintics. However, to identify candidate receptors, a deeper understanding of GPCR signalling in schistosome biology is essential. Comparative transcriptomics of paired and unpaired worms and their gonads revealed 59 differentially regulated GPCR-coding genes putatively involved in neuronal processes. In general, the diversity among GPCRs and their integral membrane topology make it difficult to characterize and deorphanize these receptors. To overcome existing limitations, we performed a pilot approach and utilized the innovative Membrane-Anchored Ligand And Receptor yeast two-hybrid system (MALAR-Y2H) to associate potential neuropeptide ligands with their cognate receptors. Here, we demonstrated the ability to express full-length GPCRs of Schistosoma mansoni in a heterologous yeast-based system. Additionally, we localized GPCRs and chimeras of neuropeptides fused to the WBP1 transmembrane domain of yeast to the plasma membrane of yeast cells. Reporter gene assays indicated ligand-receptor binding, which allowed us to identify certain neuropeptides as potential ligands for two GPCRs, which had been found before to be differentially expressed in schistosomes in a pairing-dependent manner. Thus, the MALAR-Y2H system appears suitable to unravel schistosome GPCR–ligand interactions. Besides its relevance for understanding schistosome biology, identifying and characterizing GPCR–ligand interaction will also contribute to applied research aspects.

scholarly journals GPR11, a Putative Seven-Transmembrane G Protein-Coupled Receptor, Controls Zoospore Development and Virulence of Phytophthora sojae

2009 ◽  
Vol 9 (2) ◽  
pp. 242-250 ◽  
Author(s):  
Yonglin Wang ◽  
Aining Li ◽  
Xiaoli Wang ◽  
Xin Zhang ◽  
Wei Zhao ◽  
...  
Keyword(s):  
G Protein ◽  
Hyphal Growth ◽  
Phytophthora Sojae ◽  
G Protein Coupled Receptors ◽  
Yeast Two Hybrid ◽  
Content Type ◽  
Signaling Mechanisms ◽  
G Protein Coupled ◽  
Two Hybrid ◽  
Receptor Family

ABSTRACT G protein-coupled receptors (GPCRs) represent a large receptor family involved in a broad spectrum of cell signaling. To understand signaling mechanisms mediated by GPCRs in Phytophthora sojae, we identified and characterized the PsGPR11 gene, which encodes a putative seven-transmembrane GPCR. An expression analysis revealed that PsGPR11 was differentially expressed during asexual development. The highest expression level occurred in zoospores and was upregulated during early infection. PsGPR11-deficienct transformants were obtained by gene silencing strategies. Silenced transformants exhibited no differences in hyphal growth or morphology, sporangium production or size, or mating behavior. However, the release of zoospores from sporangia was severely impaired in the silenced transformants, and about 50% of the sporangia did not completely release their zoospores. Zoospore encystment and germination were also impaired, and zoospores of the transformants lost their pathogenicity to soybean. In addition, no interaction was observed between PsGPR11 and PsGPA1 with a conventional yeast two-hybrid assay, and the transcriptional levels of some genes which were identified as being negatively regulated by PsGPA1 were not clearly altered in PsGPR11-silenced mutants. These results suggest that PsGPR11-mediated signaling controls P. sojae zoospore development and virulence through the pathways independent of G protein.

scholarly journals Interaction of class A G protein-coupled receptors with G proteins.

2004 ◽  
Vol 51 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Rafał Slusarz ◽  
Jerzy Ciarkowski
Keyword(s):  
G Protein ◽  
G Proteins ◽  
G Protein Coupled Receptors ◽  
Alpha Subunit ◽  
Protein Activation ◽  
Class A ◽  
G Protein Activation ◽  
G Protein Coupled ◽  
Receptor Dimer

A model for interaction of class A G protein-coupled receptor with the G protein G(alpha) subunit is proposed using the rhodopsin-transducin (RD/Gt) prototype. The model combines the resolved interactions/distances, essential in the active RD*/Gt system, with the structure of Gt(alpha) C-terminal peptide bound to RD* while stabilizing it. Assuming the interactions involve conserved parts of the partners, the model specifies the conserved Helix 2 non-polar X- - -X, Helix 3 DRY and Helix 7/8 NP- -Y- - F RD* motifs interacting with the Gt(alpha) C-terminal peptide, in compliance with the structure of the latter. A concomitant role of Gt(alpha) and Gt(gamma) C-termini in stabilizing RD* could possibly be resolved assuming a receptor dimer as requisite for G protein activation.

scholarly journals Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations

2020 ◽  
Vol 295 (51) ◽  
pp. 17486-17496
Author(s):  
Christopher T. Schafer ◽  
Anthony Shumate ◽  
David L. Farrens
Keyword(s):  
G Protein ◽  
Protein Activation ◽  
Canonical Class ◽  
Equilibrium Binding ◽  
C Terminus ◽  
G Protein Activation ◽  
Ligand Discovery ◽  
Pharmaceutical Agents ◽  
G Protein Coupled ◽  
Covalently Bound

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

scholarly journals Bioluminescence Resonance Energy Transfer Based G Protein-Activation Assay to Probe Duration of Antagonism at the Histamine H3 Receptor

2019 ◽  
Vol 20 (15) ◽  
pp. 3724 ◽  
Author(s):  
Tamara A. M. Mocking ◽  
Maurice C. M. L. Buzink ◽  
Rob Leurs ◽  
Henry F. Vischer
Keyword(s):  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Short Residence Time ◽  
Protein Activation ◽  
G Protein Activation ◽  
Activation Assay ◽  
G Protein Coupled ◽  
Histamine H3

Duration of receptor antagonism, measured as the recovery of agonist responsiveness, is gaining attention as a method to evaluate the ‘effective’ target-residence for antagonists. These functional assays might be a good alternative for kinetic binding assays in competition with radiolabeled or fluorescent ligands, as they are performed on intact cells and better reflect consequences of dynamic cellular processes on duration of receptor antagonism. Here, we used a bioluminescence resonance energy transfer (BRET)-based assay that monitors heterotrimeric G protein activation via scavenging of released Venus-Gβ1γ2 by NanoLuc (Nluc)-tagged membrane-associated-C-terminal fragment of G protein-coupled receptor kinase 3 (masGRK3ct-Nluc) as a tool to probe duration of G protein-coupled receptor (GPCR) antagonism. The Gαi-coupled histamine H3 receptor (H3R) was used in this study as prolonged antagonism is associated with adverse events (e.g., insomnia) and consequently, short-residence time ligands might be preferred. Due to its fast and prolonged response, this assay can be used to determine the duration of functional antagonism by measuring the recovery of agonist responsiveness upon washout of pre-bound antagonist, and to assess antagonist re-equilibration time via Schild-plot analysis. Re-equilibration of pre-incubated antagonist with agonist and receptor could be followed in time to monitor the transition from insurmountable to surmountable antagonism. The BRET-based G protein activation assay can detect differences in the recovery of H3R responsiveness and re-equilibration of pre-bound antagonists between the tested H3R antagonists. Fast dissociation kinetics were observed for marketed drug pitolisant (Wakix®) in this assay, which suggests that short residence time might be beneficial for therapeutic targeting of the H3R.

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