Related Experiment Video

Updated: Dec 9, 2025

Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery
08:21

Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Published on: June 28, 2019

7.2K

The Effect of Cell Surface Expression and Linker Sequence on the Recruitment of Arrestin to the GIP Receptor

Suleiman Al-Sabah1, Lobna Adi1, Moritz Bünemann2

  • 1Department of Pharmacology and Toxicology, Faculty of Medicine, Kuwait University, Kuwait City, Kuwait.

Frontiers in Pharmacology
|September 9, 2020
PubMed

Insights

Arrestin recruitment to the glucose-dependent insulinotropic polypeptide receptor (GIPR) is sensitive to assay configuration. Modifications like linker removal and specific amino acid substitutions significantly alter GIPR-arrestin interactions, impacting drug discovery for obesity and type 2 diabetes.

Area of Science:

  • Pharmacology and Molecular Biology
  • G protein-coupled receptor (GPCR) signaling
  • Diabetes and Obesity Therapeutics

Background:

  • Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors are key targets for treating type 2 diabetes mellitus (T2DM) and obesity.
  • Arrestins, initially known for receptor desensitization, also mediate G protein-independent signaling pathways.
  • While GLP-1 receptor (GLP-1R) arrestin interaction is established, GIP receptor (GIPR) interaction remains debated.

Purpose of the Study:

  • To investigate the controversial arrestin recruitment to the GIP receptor.
  • To determine the impact of assay configuration, including receptor modifications and epitope tags, on GIPR-arrestin interactions.
  • To understand how these findings influence the discovery of biased agonists for GIPR.

Main Methods:

  • Utilized Förster Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) assays to measure arrestin recruitment.
  • Employed site-directed mutagenesis to substitute specific amino acids (SR to GG) at the GIPR C-terminus.
  • Assessed the role of N-terminal FLAG epitope and influenza hemagglutinin signal peptide in GIPR cell surface expression and arrestin recruitment.

Main Results:

  • A modified GIPR construct (GIPR-SYFP2) lacking a 10 amino acid linker showed arrestin recruitment, unlike the original GIPR-YFP construct.
  • Substitution of the serine/arginine (SR) motif with glycine/glycine (GG) abolished arrestin recruitment in BRET assays and significantly reduced it in FRET assays.
  • N-terminal FLAG and HA signal peptide tags were necessary for detectable arrestin recruitment, likely by enhancing receptor expression.

Conclusions:

  • Assay configuration, including receptor linker sequences, specific amino acid residues, and epitope tags, critically influences the detection of GIPR-arrestin interactions.
  • These findings highlight the complexity of studying arrestin recruitment to GIPR and the potential for artifactual results.
  • Careful consideration of assay design is crucial for drug discovery programs seeking biased agonists targeting GPCRs like GIPR.
Keywords:
G protein-coupled receptorarrestinbioluminescence resonance energy transferfluorescence resonance energy transferglucagon-like peptide-1glucose-dependent insulinotropic polypeptide

More Related Videos

Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay09:03

Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

Published on: March 10, 2020

13.4K
Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking11:57

Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Published on: March 28, 2014

16.2K

Related Concept Videos

Assembly of Signaling Complexes01:30

Assembly of Signaling Complexes

Multiprotein signaling complexes are formed in a dynamic process involving protein-protein interactions at the cytoplasmic domain of transmembrane receptors or enzymatic and non-enzymatic proteins associated with the receptor. These complexes ensure the activation and propagation of intracellular signals that regulate cell functions.
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...
6.3K
Intracellular Signaling Affects Focal Adhesions01:17

Intracellular Signaling Affects Focal Adhesions

Integrins act both as extracellular input receivers and as intracellular processing activators. As their name suggests, integrins are entirely integrated into the membrane structure. Their hydrophobic membrane-spanning regions interact with the phospholipid bilayer's hydrophobic region. These membrane receptors provide extracellular attachment sites for effectors like hormones and growth factors. They activate intracellular response cascades when their effectors are bound and active.
Some...
3.3K
IP3/DAG Signaling Pathway01:11

IP3/DAG Signaling Pathway

Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and...
13.8K
GPCR Desensitization01:12

GPCR Desensitization

G protein-coupled receptor (GPCR) signaling plays a crucial role in cell functioning. GPCR desensitization is an equally essential process. It allows cells to respond to changing environments and regain sensitivity to new stimuli while preventing unnecessary stimulation when no longer needed. Prolonged exposure to stimuli leads to GPCR desensitization. It involves blocking the receptors from binding and activating additional G proteins. This inhibits activation of downstream effectors, thereby...
7.6K
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze...
16.4K
GPI Anchoring of Proteins in the ER Membrane01:29

GPI Anchoring of Proteins in the ER Membrane

GPI-anchoring is a post-translational, reversible protein modification that is ubiquitous in eukaryotes. Such proteins are primarily present on the exoplasmic leaflet of the plasma membrane.
GPI-anchor structure
A sequence of 11 enzymatic reactions results in the synthesis of the complete GPI anchor consisting of a hydrophobic and a hydrophilic portion. The hydrophobic portion comprises phosphatidylinositol, while the hydrophilic part comprises polar groups like phosphoethanolamine,...
5.0K