G16 as a universal G protein adapter: implications for agonist screening strategies

doi:10.1016/0165-6147(96)10026-2

Trends in Pharmacological Sciences

Volume 17, Issue 7, July 1996, Pages 235-237

https://doi.org/10.1016/0165-6147(96)10026-2 Get rights and content

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Cited by (110)

Using in vitro receptor activity studies of synthetic cannabinoids to support the risk assessment of new psychoactive substances – A Swedish strategy to protect public health from harm
2023, Forensic Science International
In the past 15 years, close to 1000 of new psychoactive substances (NPS) have been reported in Europe and globally. At the time of identification, data on safety, toxicity and carcinogenic potential of many NPS are not available or very limited. To work more efficiently, a strategy and collaboration between the Public Health Agency of Sweden (PHAS) and the National Board of Forensic Medicine was established involving in vitro receptor activity assays to demonstrate neurological activity of NPS. This report summarizes the first results on the synthetic cannabinoid receptor agonists (SCRAs), and subsequent actions taken by PHAS. A total of 18 potential SCRAs were selected by PHAS for in vitro pharmacological characterization. 17 compounds could be acquired and investigated for their activity on the human cannabinoid-1 (CB1) receptors expressed together with the AequoScreen system in CHO-K1 cells. Dose-response curves were established using eight different concentrations in triplicates at three occasions with JWH-018 as reference. For the MDMB-4en-PINACA, MMB-022, ACHMINACA, ADB-BUTINACA, 5F-CUMYL-PeGACLONE, 5C-AKB48, NM-2201, 5F-CUMYL-PINACA, JWH-022, 5Cl-AB-PINACA, MPhP-2201, 5F-AKB57 the half maximal effective concentration values ranged from 2.2 nM (5F-CUMYL-PINACA) to 171 nM (MMB-022). EG-018 and 3,5-AB-CHMFUPPYCA were none-active. The results contributed to 14 of these compounds being scheduled as narcotics in Sweden. In conclusion, many of the emerging SCRAs are potent activators of the CB1 receptor in vitro, although some lack activity or are partial agonists. The new strategy proved useful when data on psychoactive effects of the SCRAs under investigation were not available or limited.
Neuropeptide B/W receptor 1 peptidomimetic agonists: Structure-activity relationships and plasma stability
2022, European Journal of Medicinal Chemistry
Citation Excerpt :
Biological activities were assessed in a calcium mobilization-based assay utilizing CHO-RD-HGA16 cells (Molecular Devices, CA, US) stably expressing the human NPBWR1. The CHO-RD-HGA16 cells express the promiscuous Gα16 protein, which allows coupling of receptors to the calcium mobilization-based signal transduction pathway [25]. In our assay, the control NPW23 peptide has an EC50 (±S.E.M.) of 8 ± 0.8 nM (Fig. 3), which matches literature results (NPW30 EC50 = 10.9 nM [3] and NPW23 EC80 = 20 nM [18]) and has no response in parental CHO-RD-HGA16 cells.
Neuropeptides B and W (NPB and NPW) are endogenous ligands of the Neuropeptide B/W Receptor 1 (NPBWR1) which has been implicated in a wide range of functions including regulation of pain and energy homeostasis. There is currently little information on the structure-activity relationships (SAR) of these two neuropeptides. In a quest to develop stable and potent NPBWR1 peptidomimetic agonists, we performed systematic SAR by truncation, Alanine/Glycine and d-amino acid scans, and replacement with unnatural amino acids. Evaluation in the NPBWR1 calcium assay revealed that the C-terminal GRAAGLL and N-terminal WYK regions constitute the two-epitope pharmacophore for NPBWR1 agonism. Replacement of the N-terminal Trp with its desaminoTrp residue resulted in compound 30 which exhibited nanomolar potency comparable to the endogenous NPB at NPBWR1 (Calcium assay: EC₅₀ = 8 nM vs. 13 nM, cAMP assay: 2.7 nM vs 3.5 nM) and enhanced metabolic stability against rat plasma (39.1 min vs. 11.9 min).
Genome Editing Provides New Insights into Receptor-Controlled Signalling Pathways
2018, Trends in Pharmacological Sciences
Citation Excerpt :
Ligand-screening strategies for novel activators of GPCRs have often centred to trying to develop assays that can be utilised broadly, irrespective of the G protein-coupling preference and profile of the receptor. Approaches to this have included transfection alongside a receptor of interest of the ‘universal’ G protein Gα16 [37], and cotransfection of a cocktail of chimeric G protein α subunits [38]. Recently a transforming growth factor α (TGF-α) shedding assay was introduced [39] and was shown to be able to report activation of a broad range of heterologously expressed GPCRs.
Rapid developments in genome editing, based largely on CRISPR/Cas9 technologies, are offering unprecedented opportunities to eliminate the expression of single or multiple gene products in intact organisms and in model cell systems. Elimination of individual G protein-coupled receptors (GPCRs), both single and multiple G protein subunits, and arrestin adaptor proteins is providing new and sometimes unanticipated insights into molecular details of the regulation of cell signalling pathways and the behaviour of receptor ligands. Genome editing is certain to become a central component of therapeutic target validation, and will provide pharmacologists with new understanding of the complexities of action of novel and previously studied ligands, as well as of the transmission of signals from individual cell-surface receptors to intracellular signalling cascades.
Pharmacological and signalling properties of a D2-like dopamine receptor (Dop3) in Tribolium castaneum
2015, Insect Biochemistry and Molecular Biology
Dopamine is an important neurotransmitter in the central nervous system of vertebrates and invertebrates. Despite their evolutionary distance, striking parallels exist between deuterostomian and protostomian dopaminergic systems. In both, signalling is achieved via a complement of functionally distinct dopamine receptors. In this study, we investigated the sequence, pharmacology and tissue distribution of a D2-like dopamine receptor from the red flour beetle Tribolium castaneum (TricaDop3) and compared it with related G protein-coupled receptors in other invertebrate species.
The TricaDop3 receptor-encoding cDNA shows considerable sequence similarity with members of the Dop3 receptor class. Real time qRT-PCR showed high expression in both the central brain and the optic lobes, consistent with the role of dopamine as neurotransmitter. Activation of TricaDop3 expressed in mammalian cells increased intracellular Ca²⁺ signalling and decreased NKH-477 (a forskolin analogue)-stimulated cyclic AMP levels in a dose-dependent manner. We studied the pharmacological profile of the TricaDop3 receptor and demonstrated that the synthetic vertebrate dopamine receptor agonists, 2 – amino- 6,7 – dihydroxy – 1,2,3,4 – tetrahydronaphthalene hydrobromide (6,7-ADTN) and bromocriptine acted as agonists. Methysergide was the most potent of the antagonists tested and showed competitive inhibition in the presence of dopamine. This study offers important information on the Dop3 receptor from Tribolium castaneum that will facilitate functional analyses of dopamine receptors in insects and other invertebrates.
Regulation of cyclic adenosine monophosphate release by selective β<inf>2</inf>-adrenergic receptor stimulation in human terminal failing myocardium before and after ventricular assist device support
2012, Journal of Heart and Lung Transplantation
Response to catecholamines is blunted in terminal heart failure due to β-receptor downregulation and uncoupling from adenylyl cyclase (AC). Improved myocardial responsiveness to catecholamines after ventricular assist device (VAD) support is associated with upregulation of β₁-adrenergic receptors (β₁-ARs). Little is known about the regulation of AC and β₂-AR coupling after VAD; moreover β₂-AR stimulation during VAD was claimed to induce myocardial recovery.
We analyzed in VAD-supported human myocardium the regulation of AC activity upon β₁-AR and selective β₂-AR stimulation in 8 non-failing hearts (NF) and 17 paired samples of VAD patients. AC messenger RNA was measured by TaqMan. AC was stimulated via β₂-AR using clenbuterol (β₂-AR agonist) and bisoprolol (β₁-AR blocker). Organ bath experiments were done with trabeculae from both ventricles. Samples were stratified according to chronic or acute heart failure history.
Isoprenaline-induced AC activity was downregulated (p < 0.001) pre-VAD and increased significantly (p < 0.05) after unloading (mean ± standard deviation pmole/mg/min) in NF (47.9 ± 14.9), pre-VAD (24.35 ± 13.3), and post-VAD (50.04 ± 50.25). Forskolin stimulation revealed significant (p < 0.05) upregulation of AC activity during VAD, especially in acutely failing hearts (NF, 192.1 ± 68.7; pre-VAD, 191.1 ± 60.4; post-VAD, 281.5 ± 133). However, forskolin stimulation relative to isoprenaline-induced inotropy remained reduced before and after VAD compared with NF. The selective stimulation of β₂-AR did not reveal influence of VAD support on β₂-AR–AC coupling. Stimulation of ventricular trabeculae by > 100 μmole/liter clenbuterol revealed negative inotropic responses.
VAD does not influence β₂-AR coupling to AC stimulation. Elevated response to catecholamines after VAD support is influenced by β₁-AR upregulation and modulation of AC activity. Restoration of β-adrenergic responsiveness was restricted to acutely failing hearts.
Experimental Challenges to Targeting Poorly Characterized GPCRs. Uncovering the Therapeutic Potential for Free Fatty Acid Receptors
2011, Advances in Pharmacology
The G protein-coupled receptors (GPCRs) are extremely successful drug targets, with recent estimates suggesting that approximately 30% of all currently available therapeutics act at these receptors. Despite this success, only a small number of the over 400 known nonodorant GPCRs are currently targeted, suggesting there is still untapped therapeutic potential. However, as most GPCRs were identified based on their sequence homology to other members of the superfamily, many still remain “orphan” receptors without known ligands. Indeed, even once a GPCR has been deorphanized, the receptor typically is still poorly characterized in terms of its pharmacology and biological functions, presenting a unique set of experimental challenges in order to define its therapeutic potential. We discuss some of these challenges and how they have been addressed in order to uncover the therapeutic potential of five recently deorphanized receptors that are activated by short- and long-chain free fatty acids.

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Trends in Pharmacological Sciences

Current awarenesG16 as a universal G protein adapter: implications for agonist screening strategies

First page preview

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

Cell Calcium

J. Biol. Chem.

Curr. Biol.

J. Biol. Chem.

Current awarenes
G₁₆ as a universal G protein adapter: implications for agonist screening strategies