scholarly journals A Dual GLP-1/GIP Receptor Agonist Does Not Antagonize Glucagon at Its Receptor but May Act as a Biased Agonist at the GLP-1 Receptor

2019 ◽  
Vol 20 (14) ◽  
pp. 3532 ◽  
Author(s):  
Noura Al-Zamel ◽  
Suleiman Al-Sabah ◽  
Yunus Luqmani ◽  
Lobna Adi ◽  
Siby Chacko ◽  
...  
Keyword(s):  
Receptor Agonist ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Glucagon Receptor ◽  
Hek 293 ◽  
293 Cells ◽  
Simultaneous Activation ◽  
Glucagon Like Peptide 1 ◽  
Bret Assay ◽  
Biased Agonist

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of metabolism, making their receptors (GLP-1R and GIPR) attractive targets in the treatment of type 2 diabetes mellitus (T2DM). GLP-1R agonists are used clinically to treat T2DM but the use of GIPR agonists remains controversial. Recent studies suggest that simultaneous activation of GLP-1R and GIPR with a single peptide provides superior glycemic control with fewer adverse effects than activation of GLP-1R alone. We investigated the signaling properties of a recently reported dual-incretin receptor agonist (P18). GLP-1R, GIPR, and the closely related glucagon receptor (GCGR) were expressed in HEK-293 cells. Activation of adenylate cyclase via Gαs was monitored using a luciferase-linked reporter gene (CRE-Luc) assay. Arrestin recruitment was monitored using a bioluminescence resonance energy transfer (BRET) assay. GLP-1, GIP, and glucagon displayed exquisite selectivity for their receptors in the CRE-Luc assay. P18 activated GLP-1R with similar potency to GLP-1 and GIPR with higher potency than GIP. Interestingly, P18 was less effective than GLP-1 at recruiting arrestin to GLP-1R and was inactive at GCGR. These data suggest that P18 can act as both a dual-incretin receptor agonist, and as a G protein-biased agonist at GLP-1R.

GRIF-1–kinesin-1 interactions: a confocal microscopy study

2006 ◽  
Vol 34 (1) ◽  
pp. 48-50 ◽  
Author(s):  
K. Pozo ◽  
F.A. Stephenson
Keyword(s):  
Confocal Microscopy ◽  
Fusion Proteins ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Microscopy Study ◽  
Motor Domain ◽  
African Green Monkey Kidney ◽  
Hek 293 ◽  
293 Cells

GRIF-1 [GABAA (γ-aminobutyric acidA) receptor interacting factor-1] is a member of a coiled-coil family of proteins thought to function as adaptors in the anterograde trafficking of organelles utilizing the kinesin-1 motor proteins to synapses. To study in more detail the molecular interaction between GRIF-1 and the kinesin-1 family member KIF5C, fluorescent yellow- and fluorescent cyan-tagged GRIF-1, KIF5C, the KIF5C MD (motor domain) and the KIF5C NMD (non-motor domain) fusion proteins were generated. Each was characterized with respect to size and ability to co-associate by immunoprecipitation following expression in HEK-293 (human embryonic kidney 293) cells. Further, their distribution in transfected HEK-293 and transformed African green monkey kidney (COS-7) cells was analysed by confocal microscopy. The fluorescent GRIF-1 and KIF5C fusion proteins were all found to behave as wild-type. Double GRIF-1/KIF5C transfectants revealed co-localization. The GRIF-1/KIF5C and GRIF-1/KIF5C NMD double transfectants showed different subcellular distributions compared with single GRIF-1, KIF5C or KIF5C NMD transfections. These studies confirm the association between GRIF-1 and kinesin-1 NMDs. Fluorescence resonance energy transfer studies are ongoing to characterize this interaction in more detail.

Abstract 022: Evidence for AT2-receptor-MAS Dimerisation from Fluorescence Resonance Energy Transfer (FRET) Imaging and Fluorescence Cross Correlation Spectroscopy (FCCS)

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Daniel C Villela ◽  
Anke Teichmann ◽  
Sebastian Kirsch ◽  
Maibritt Mardahl ◽  
Lisa M Münter ◽  
...  
Keyword(s):  
Cross Correlation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Expression Vectors ◽  
Physical Interaction ◽  
At2 Receptor ◽  
Hek 293 ◽  
Fret Efficiency ◽  
In Cells

The angiotensin AT2-receptor (AT2R) and the receptor MAS share a strinkingly similar spectrum of signaling mechanisms and protective, physiological actions. Furthermore, cross-inhibition by the respective receptor antagonists has been observed. Therefore we hypothesised that a physical interaction between these two receptors may exist. HEK-293 cells were transfected with vectors encoding MAS or AT2R fused in the C-terminus with the fluorophores CFP or YFP for FRET and GFP or mCherry for FCCS. FRET with photobleaching was used to detect, whether MAS and AT2R are localised in very close proximity (1-10nm) in cell membranes thus indicating dimerisation. FCCS was used to follow simultaneously occurring fluctuations in fluorescence intensity of both labeled molecules. Several controls were applied such as co-transfection of equal amounts of fused and non-fused MAS/AT2R expression vectors for competition, co-tranfection of coding and uncoding pcDNA vectors or co-transfection with an unrelated transmembrane receptor. Experiments were conducted under baseline conditions and in cells treated with AT2R/MAS agonists and antagonists Significant FRET efficiency of 10.8±0.8% was measured for AT2-YFP/MAS-CFP strongly indicating heterodimerisation. FRET efficiency was not altered by AT2R or MAS agonists or antagonists. Non-fluorescent MAS and AT2R competed with fluorescent receptors as indicated by a 50% reduction in FRET efficiency (6.0±0.6%), while empty vectors did not compete (9.6±0.6%). No FRET efficiency was observed with an unrelated transmembrane receptor (0.44±1.44%) indicating specificity of receptor interactions. Both, MAS and AT2R also formed homodimers (7.4±0.8% for MAS, 9.2±0.8% for AT2R). Hetero- and homodimerisations were absent if amino acid C35 of the AT2R was mutated (3,9 ± 1,2%). FCCS corroborated the FRET results and revealed a significantly enhanced cross correlation in cells tranfected with fluorophore-tagged MAS/AT2R when compared to vectors only expressing the fluorophores (8.5±3% vs 11.1±4%; p<0.0001). Our data strongly suggest that MAS and the AT2R form homo- and heterodimers. Studies to investigate the physiological relevance of MAS/AT2R dimerisation are currently being conducted.

scholarly journals Dual-acting peptide with prolonged glucagon-like peptide-1 receptor agonist and glucagon receptor antagonist activity for the treatment of type 2 diabetes

2007 ◽  
Vol 192 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Thomas H Claus ◽  
Clark Q Pan ◽  
Joanne M Buxton ◽  
Ling Yang ◽  
Jennifer C Reynolds ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Insulin Secretion ◽  
Receptor Agonist ◽  
Antagonist Activity ◽  
Glucagon Receptor ◽  
Glucagon Receptor Antagonist ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Type 2 diabetes is characterized by reduced insulin secretion from the pancreas and overproduction of glucose by the liver. Glucagon-like peptide-1 (GLP-1) promotes glucose-dependent insulin secretion from the pancreas, while glucagon promotes glucose output from the liver. Taking advantage of the homology between GLP-1 and glucagon, a GLP-1/glucagon hybrid peptide, dual-acting peptide for diabetes (DAPD), was identified with combined GLP-1 receptor agonist and glucagon receptor antagonist activity. To overcome its short plasma half-life DAPD was PEGylated, resulting in dramatically prolonged activity in vivo. PEGylated DAPD (PEG-DAPD) increases insulin and decreases glucose in a glucose tolerance test, evidence of GLP-1 receptor agonism. It also reduces blood glucose following a glucagon challenge and elevates fasting glucagon levels in mice, evidence of glucagon receptor antagonism. The PEG-DAPD effects on glucose tolerance are also observed in the presence of the GLP-1 antagonist peptide, exendin(9–39). An antidiabetic effect of PEG-DAPD is observed in db/db mice. Furthermore, PEGylation of DAPD eliminates the inhibition of gastrointestinal motility observed with GLP-1 and its analogues. Thus, PEG-DAPD has the potential to be developed as a novel dual-acting peptide to treat type 2 diabetes, with prolonged in vivo activity, and without the GI side-effects.

scholarly journals Ackr3-Venus knock-in mouse lights up brain vasculature

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Aliza T. Ehrlich ◽  
Meriem Semache ◽  
Pierre Couvineau ◽  
Stefan Wojcik ◽  
Hiroyuki Kobayashi ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adult Brain ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Membrane Expression ◽  
Hek 293 ◽  
Brain Vasculature ◽  
Intact Gene ◽  
The Brain

AbstractThe atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits βarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

scholarly journals Bioluminescence Resonance Energy Transfer assay (BRET assay)

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (24) ◽  
Author(s):  
Yan Yan ◽  
Ting-Hai Xu ◽  
Kaleeckal G. Harikumar ◽  
Laurence J. Miller
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bret Assay

scholarly journals AlphaScreen HTS and Live-Cell Bioluminescence Resonance Energy Transfer (BRET) Assays for Identification of Tau–Fyn SH3 Interaction Inhibitors for Alzheimer Disease

2014 ◽  
Vol 19 (10) ◽  
pp. 1338-1349 ◽  
Author(s):  
J. Nicholas Cochran ◽  
Pauleatha V. Diggs ◽  
N. Miranda Nebane ◽  
Lynn Rasmussen ◽  
E. Lucile White ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Alzheimer Disease ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Amyloid Β ◽  
Resonance Energy ◽  
Live Cell ◽  
Dominant Negative ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bret Assay

Alzheimer disease (AD) is the most common neurodegenerative disease, and with Americans’ increasing longevity, it is becoming an epidemic. There are currently no effective treatments for this disorder. Abnormalities of Tau track more closely with cognitive decline than the most studied therapeutic target in AD, amyloid-β, but the optimal strategy for targeting Tau has not yet been identified. On the basis of considerable preclinical data from AD models, we hypothesize that interactions between Tau and the Src-family tyrosine kinase, Fyn, are pathogenic in AD. Genetically reducing either Tau or Fyn is protective in AD mouse models, and a dominant negative fragment of Tau that alters Fyn localization is also protective. Here, we describe a new AlphaScreen assay and a live-cell bioluminescence resonance energy transfer (BRET) assay using a novel BRET pair for quantifying the Tau–Fyn interaction. We used these assays to map the binding site on Tau for Fyn to the fifth and sixth PXXP motifs to show that AD-associated phosphorylation at microtubule affinity regulating kinase sites increases the affinity of the Tau–Fyn interaction and to identify Tau–Fyn interaction inhibitors by high-throughput screening. This screen has identified a variety of chemically tractable hits, suggesting that the Tau–Fyn interaction may represent a good drug target for AD.

scholarly journals Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

2021 ◽  
Author(s):  
Sunday Okutachi ◽  
Ganesh babu Manoharan ◽  
Daniel Abankwa
Keyword(s):  
Cancer Cells ◽  
Mapk Pathway ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Monoamine Neurotransmitter ◽  
Braf Mutations ◽  
Anti Cancer ◽  
Phosphatase 2A ◽  
Bret Assay

Phenothiazines (PTZ) are well known as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they are used for decades as antipsychotic drugs. In addition, significant anti-cancer properties have been ascribed to them. Several attempts for their repurposing were made, however, their incompletely understood polypharmacology is challenging. Here we examined the potential of PTZ to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras-MAPK pathway activity. Consistently, combinations of a CaM inhibitor and a PP2A activator synergistically inhibited cancer cells with KRAS or BRAF mutations. We identified the covalently reactive PTZ derivative fluphenazine mustard as an inhibitor of Ras driven proliferation and Ras membrane organization. We confirmed its anti-CaM activity in vitro and through a cellular CaM target engagement bioluminescence resonance energy transfer (BRET) assay. Our results suggest that improved PTZ derivatives retaining their synergistic CaM inhibitory and PP2A activating properties, but without neurological side-effects, may be interesting to pursue further as anti-cancer agents.

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