scholarly journals SNAP-tagged nanobodies enable reversible optical control of a G protein-coupled receptor via a remotely tethered photoswitchable ligand

2018 ◽  
Author(s):  
Helen Farrants ◽  
Amanda Acosta Ruiz ◽  
Vanessa A. Gutzeit ◽  
Dirk Trauner ◽  
Kai Johnsson ◽  
...  
Keyword(s):  
G Protein ◽  
Drug Targets ◽  
Metabotropic Glutamate Receptor ◽  
Receptor Activation ◽  
Metabotropic Glutamate ◽  
Extracellular Signals ◽  
Physiological Processes ◽  
Wide Range ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) mediate the transduction of extracellular signals into complex intracellular responses. Despite their ubiquitous roles in physiological processes and as drug targets for a wide range of disorders, the precise mechanisms of GPCR function at the molecular, cellular, and systems levels remain partially understood. In order to dissect the function of individual receptors subtypes with high spatiotemporal precision, various optogenetic and photopharmacological approaches have been reported that use the power of light for receptor activation and deactivation. Here, we introduce a novel and, to date, most remote way of applying photoswitchable orthogonally remotely-tethered ligands (PORTLs) by using a SNAP-tag fused nanobody. Our nanobody-photoswitch conjugates (NPCs) can be used to target a GFP-fused metabotropic glutamate receptor by either gene-free application of purified complexes or co-expression of genetically encoded nanobodies to yield robust, reversible control of agonist binding and subsequent downstream activation. By harboring and combining the selectivity and flexibility of both nanobodies and self-labelling enzymes, we set the stage for targeting endogenous receptors in vivo.

scholarly journals Mechanisms of adhesion G protein–coupled receptor activation

2020 ◽  
Vol 295 (41) ◽  
pp. 14065-14083 ◽  
Author(s):  
Alexander Vizurraga ◽  
Rashmi Adhikari ◽  
Jennifer Yeung ◽  
Maiya Yu ◽  
Gregory G. Tall
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Physiological Processes ◽  
Class B ◽  
Peptide Agonist ◽  
G Protein Coupled ◽  
Existing Structure

Adhesion G protein–coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein–coupling seven-transmembrane–spanning bundle. GAIN domain–mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.

scholarly journals G-Protein Coupled Receptor-Ligand Dissociation Rates and Mechanisms from τRAMD Simulations

2021 ◽  
Author(s):  
Daria B. Kokh ◽  
Rebecca C. Wade
Keyword(s):  
Molecular Dynamics ◽  
G Protein ◽  
Drug Targets ◽  
Dissociation Rate ◽  
Allosteric Modulation ◽  
Ligand Dissociation ◽  
Wide Range ◽  
Dissociation Mechanisms ◽  
G Protein Coupled

There is a growing appreciation of the importance of drug-target binding kinetics for lead optimization. For G protein-coupled receptors (GPCRs), which mediate signaling over a wide range of timescales, the drug dissociation rate is often a better predictor of in vivo efficacy than binding affinity, although it is more challenging to compute. Here, we assess the ability of the τ-Random Acceleration Molecular Dynamics (τRAMD) approach to reproduce relative residence times and reveal dissociation mechanisms and the effects of allosteric modulation for two important membrane-embedded drug targets: the β2-adrenergic receptor and the muscarinic acetylcholine receptor M2. The dissociation mechanisms observed in the relatively short RAMD simulations (in which molecular dynamics (MD) simulations are performed using an additional force with an adaptively assigned random orientation applied to the ligand) are in general agreement with much more computationally intensive conventional MD and metadynamics simulations. Remarkably, although decreasing the magnitude of the random force generally reduces the number of egress routes observed, the ranking of ligands by dissociation rate is hardly affected and agrees well with experiment. The simulations also reproduce changes in residence time due to allosteric modulation and reveal associated changes in ligand dissociation pathways.

Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors

2001 ◽  
Vol 359 (3) ◽  
pp. 465-484 ◽  
Author(s):  
Emmanuel HERMANS ◽  
R. A. John CHALLISS
Keyword(s):  
G Protein ◽  
Drug Targets ◽  
Metabotropic Glutamate Receptors ◽  
Gabab Receptor ◽  
Receptor Activation ◽  
Metabotropic Glutamate ◽  
Calcium Sensing ◽  
Group I ◽  
G Protein Coupled ◽  
The Brain

In 1991 a new type of G-protein-coupled receptor (GPCR) was cloned, the type 1a metabotropic glutamate (mGlu) receptor, which, despite possessing the defining seven-transmembrane topology of the GPCR superfamily, bore little resemblance to the growing number of other cloned GPCRs. Subsequent studies have shown that there are eight mammalian mGlu receptors that, together with the calcium-sensing receptor, the GABAB receptor (where GABA is γ-aminobutyric acid) and a subset of pheromone, olfactory and taste receptors, make up GPCR family C. Currently available data suggest that family C GPCRs share a number of structural, biochemical and regulatory characteristics, which differ markedly from those of the other GPCR families, most notably the rhodopsin/family A GPCRs that have been most widely studied to date. This review will focus on the group I mGlu receptors (mGlu1 and mGlu5). This subgroup of receptors is widely and differentially expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in the control of an array of key signalling events, including roles in the adaptative changes needed for long-term depression or potentiation of neuronal synaptic connectivity. In addition to playing critical physiological roles within the brain, the mGlu receptors are also currently the focus of considerable attention because of their potential as drug targets for the treatment of a variety of neurological and psychiatric disorders.

scholarly journals Structural Characterization of Receptor–Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers

2021 ◽  
Vol 22 (6) ◽  
pp. 3241
Author(s):  
Raudah Lazim ◽  
Donghyuk Suh ◽  
Jai Woo Lee ◽  
Thi Ngoc Lan Vu ◽  
Sanghee Yoon ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Metabotropic Glutamate Receptor ◽  
Three Dimensional ◽  
Allosteric Modulation ◽  
Experimental Investigations ◽  
G Protein Coupled Receptor ◽  
Metabotropic Glutamate ◽  
Gpcr Activation ◽  
G Protein Coupled

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).

G-protein-coupled-receptor activation of the smooth muscle calponin gene

2001 ◽  
Vol 357 (2) ◽  
pp. 587-592 ◽  
Author(s):  
Nickolai O. DULIN ◽  
Sergei N. ORLOV ◽  
Chad M. KITCHEN ◽  
Tatyana A. VOYNO-YASENETSKAYA ◽  
Joseph M. MIANO
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
G Protein ◽  
Transcriptional Activation ◽  
Fold Increase ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Mitogen Activated Protein Kinase ◽  
G Protein Coupled

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

scholarly journals G Protein-coupled Receptor Kinase-mediated Desensitization of Metabotropic Glutamate Receptor 1A Protects against Cell Death

2000 ◽  
Vol 275 (49) ◽  
pp. 38213-38220 ◽  
Author(s):  
Lianne B. Dale ◽  
Moshmi Bhattacharya ◽  
Pieter H. Anborgh ◽  
Barbara Murdoch ◽  
Mickie Bhatia ◽  
...  
Keyword(s):  
Cell Death ◽  
G Protein ◽  
Glutamate Receptor ◽  
Metabotropic Glutamate Receptor ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Metabotropic Glutamate ◽  
G Protein Coupled

scholarly journals Dual Requirement for Rho and Protein Kinase C in Direct Activation of Phospholipase D1 Through G Protein-coupled Receptor Signaling

2000 ◽  
Vol 11 (12) ◽  
pp. 4359-4368 ◽  
Author(s):  
Guangwei Du ◽  
Yelena M. Altshuller ◽  
Yong Kim ◽  
Jung Min Han ◽  
Sung Ho Ryu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
G Protein ◽  
Receptor Activation ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Kinase C ◽  
Phospholipase D1 ◽  
G Protein Coupled

G protein-coupled and tyrosine kinase receptor activation of phospholipase D1 (PLD1) play key roles in agonist-stimulated cellular responses such as regulated exocytosis, actin stress fiber formation, and alterations in cell morphology and motility. Protein Kinase C, ADP-ribosylation factor (ARF), and Rho family members activate PLD1 in vitro; however, the actions of the stimulators on PLD1 in vivo have been proposed to take place through indirect pathways. We have used the yeast split-hybrid system to generate PLD1 alleles that fail to bind to or to be activated by RhoA but that retain wild-type responses to ARF and PKC. These alleles then were employed in combination with alleles unresponsive to PKC or to both stimulators to examine the activation of PLD1 by G protein-coupled receptors. Our results demonstrate that direct stimulation of PLD1 in vivo by RhoA (and by PKC) is critical for significant PLD1 activation but that PLD1 subcellular localization and regulated phosphorylation occur independently of these stimulatory pathways.

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