Pharmacological Characterization of Receptor Redistribution and β-Arrestin Recruitment Assays for the Cannabinoid Receptor 1

2009 ◽  
Vol 14 (7) ◽  
pp. 811-823 ◽  
Author(s):  
Miranda M.C. van der Lee ◽  
Marion Blomenröhr ◽  
Antoon A. van der Doelen ◽  
Jesse W.Y. Wat ◽  
Niels Smits ◽  
...  
Keyword(s):  
G Protein ◽  
Cannabinoid Receptor ◽  
Receptor Activation ◽  
Binding Affinities ◽  
Cannabinoid Receptor 1 ◽  
Pharmacological Characterization ◽  
Cellular Effects ◽  
Partial Agonists ◽  
C Terminus ◽  
G Protein Coupled

Receptor redistribution and β-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular β-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution® assay and 2 nonimaging-based β-arrestin recruitment assays, Tango™ and PathHunter ™, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Gαi coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via β-arrestin recruitment and Gαi-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed. ( Journal of Biomolecular Screening 2009:811-823)

scholarly journals G protein-coupled receptor GPR55 promotes colorectal cancer and has opposing effects to cannabinoid receptor 1

2017 ◽  
Vol 142 (1) ◽  
pp. 121-132 ◽  
Author(s):  
Carina Hasenoehrl ◽  
David Feuersinger ◽  
Eva M Sturm ◽  
Thomas Bärnthaler ◽  
Ellen Heitzer ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
G Protein ◽  
Cannabinoid Receptor ◽  
G Protein Coupled Receptor ◽  
Cannabinoid Receptor 1 ◽  
Opposing Effects ◽  
G Protein Coupled

scholarly journals High-mass MALDI-MS unravels ligand-mediated G protein–coupling selectivity to GPCRs

2021 ◽  
Vol 118 (31) ◽  
pp. e2024146118
Author(s):  
Na Wu ◽  
Agnieszka M. Olechwier ◽  
Cyrill Brunner ◽  
Patricia C. Edwards ◽  
Ching-Ju Tsai ◽  
...  
Keyword(s):  
Complex Formation ◽  
G Protein ◽  
High Sensitivity ◽  
Maldi Ms ◽  
High Mass ◽  
Ionization Mass ◽  
Binding Affinities ◽  
C Terminus ◽  
Protein Complex Formation ◽  
G Protein Coupled

G protein–coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR–G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand–GPCR–partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [β1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using β1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαiβγ and β-arrestin-1 and showed that carvedilol induces an increase in coupling of β-arrestin-1 and Gαiβγ to β1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.

scholarly journals Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hui Li ◽  
Jie Yang ◽  
Cuiping Tian ◽  
Min Diao ◽  
Quan Wang ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescence Imaging ◽  
Cannabinoid Receptor ◽  
Super Resolution ◽  
G Protein Coupled Receptors ◽  
Cellular Functions ◽  
Cannabinoid Receptor 1 ◽  
Intracellular Organization ◽  
Dynamic Movement ◽  
G Protein Coupled

Abstract G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.

scholarly journals CB1 Cannabinoid Receptor Signaling and Biased Signaling

Molecules ◽  
2021 ◽  
Vol 26 (17) ◽  
pp. 5413
Author(s):  
Luciana M. Leo ◽  
Mary E. Abood
Keyword(s):  
Adverse Effects ◽  
G Protein ◽  
Conformational Changes ◽  
Cannabinoid Receptor ◽  
Therapeutic Potential ◽  
Receptor Activation ◽  
Cb1 Receptor ◽  
Cb1 Cannabinoid Receptor ◽  
Biased Signaling ◽  
G Protein Coupled

The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of various disorders, including anxiety, pain, and neurodegeneration. Despite the wide therapeutic potential of CB1, the development of drug candidates is hindered by adverse effects, rapid tolerance development, and abuse potential. Ligands that produce biased signaling—the preferential activation of a signaling transducer in detriment of another—have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. However, biased signaling at the CB1 receptor is poorly understood due to a lack of strongly biased agonists. Here, we review studies that have investigated the biased signaling profile of classical cannabinoid agonists and allosteric ligands, searching for a potential therapeutic advantage of CB1 biased signaling in different pathological states. Agonist and antagonist bound structures of CB1 and proposed mechanisms of action of biased allosteric modulators are used to discuss a putative molecular mechanism for CB1 receptor activation and biased signaling. Current studies suggest that allosteric binding sites on CB1 can be explored to yield biased ligands that favor or hinder conformational changes important for biased signaling.

scholarly journals Biased Coupling to β-Arrestin of Two Common Variants of the CB2 Cannabinoid Receptor

2021 ◽  
Vol 12 ◽  
Author(s):  
Gábor Turu ◽  
Eszter Soltész-Katona ◽  
András Dávid Tóth ◽  
Cintia Juhász ◽  
Miklós Cserző ◽  
...  
Keyword(s):  
G Protein ◽  
Intracellular Trafficking ◽  
Cannabinoid Receptor ◽  
Therapeutic Potential ◽  
Receptor Activation ◽  
Missense Mutations ◽  
Common Variants ◽  
Biased Signaling ◽  
The Central Nervous System ◽  
G Protein Coupled

β-arrestins are partners of the G protein-coupled receptors (GPCRs), regulating their intracellular trafficking and signaling. Development of biased GPCR agonists, selectively targeting either G protein or β-arrestin pathways, are in the focus of interest due to their therapeutic potential in different pathological conditions. The CB2 cannabinoid receptor (CB2R) is a GPCR involved in various functions in the periphery and the central nervous system. Two common occurring variants of CB2R, harboring Q63R or L133I missense mutations, have been implicated in the development of a diverse set of disorders. To evaluate the effect of these mutations, we characterized the binding profile of these mutant CB2 receptors to G proteins and β-arrestin2. Although their ability to inhibit cAMP signaling was similar, the Q63R mutant had increased, whereas the L133I mutant receptor had decreased β-arrestin2 binding. In line with these observations, the variants also had altered intracellular trafficking. Our results show that two common variants of the CB2 receptor have biased signaling properties, which may contribute to the pathogenesis of the associated disorders and may offer CB2R as a target for further development of biased receptor activation strategies.

scholarly journals Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging

2020 ◽  
Author(s):  
Hui Li ◽  
Jie Yang ◽  
Tian Cuiping ◽  
Min Diao ◽  
Quan Wang ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescence Imaging ◽  
Cannabinoid Receptor ◽  
Super Resolution ◽  
G Protein Coupled Receptors ◽  
Cellular Functions ◽  
Cannabinoid Receptor 1 ◽  
Intracellular Organization ◽  
Dynamic Movement ◽  
G Protein Coupled

AbstractG-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy when being activated.

CSIG-07. ACTIVATION OF THE ADHESION G PROTEIN-COUPLED RECEPTOR GPR133 BY ANTIBODIES AGAINST THE N-TERMINUS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi34-vi34
Author(s):  
Gabriele Stephan ◽  
Joshua Frenster ◽  
Niklas Ravn-Boess ◽  
Devin Bready ◽  
Jordan Wilcox ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Receptor Activation ◽  
Cell Culture Supernatant ◽  
Dependent Manner ◽  
G Protein Coupled Receptor ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus ◽  
G Protein Coupled

Abstract We recently demonstrated that GPR133 (ADGRD1), a member of the adhesion G protein-coupled receptor (aGPCR) family, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We therefore postulate that GPR133 represents a novel target in GBM, which merits development of therapeutics. Like most aGPCRs, GPR133 is characterized by an intracellular C-terminus, 7 plasma membrane-spanning α-helices and a large extracellular N-terminus. The N-terminus possesses a conserved GPCR autoproteolysis-inducing (GAIN) domain that catalyzes cleavage at a GPCR proteolysis site (GPS), resulting in a C-terminal fragment (CTF) and an N-terminal fragment (NTF). We showed that dissociation of the cleaved NTF and CTF at the plasma membrane increases canonical signaling of GPR133, which is mediated by coupling to Gs and increase in cytosolic cAMP. Toward characterizing the effect of biologics on GPR133 function, we overexpressed wild-type or mutant forms of GPR133 in HEK293T cells and patient-derived GBM cells lines. Treatment of these cells with antibodies specifically targeting the NTF of GPR133 increased receptor activation in a dose-dependent manner. No effects were elicited with an antibody against the receptor’s intracellular C-terminus. Interestingly, cells overexpressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. Following antibody treatment, co-purification of the GPR133 NTF and the N-terminal antibody from the cell culture supernatant indicated the formation of antibody-NTF complexes. Analysis of these complexes suggested that antibody binding stimulated the dissociation of the NTF from the CTF. However, the increased flexibility of the GAIN domain and NTF after cleavage, independently of dissociation, may also endow the receptor with responsiveness to the effects of the antibodies. These data constitute a proof-of-concept paradigm of modulation of GPR133 function with antibodies. This work provides rationale for pursuing development of biologics targeting GPR133 in GBM.

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