G Protein-Coupled Receptor Desensitization as a Measure of Signaling: Modeling of Arrestin Recruitment to Activated CCK-B Receptors

Abstract

Gastrin is one of the principle hormonal mediators of gastric acid secretion, and its cognate receptor (CCK-B) is a member of the superfamily of GPCRs. Patients with hypergastrinemia may present with a variety of symptoms, including gastric ulcers or malignant tumors. Thus, the molecular mechanisms that terminate CCK-B receptor signaling, as well as an ability to measure gastrin bioactivity in a timely manner, have important clinical implications. In order to assess CCK-B receptor regulation, we have constructed a single cell biosensor containing the CCK-B receptor and an arrestin/GFP chimera. The gastrin biosensor responded to both immunologically detectable gastrin-17 and undetectable pentagastrin, and was able to determine the gastrin bioactivity of serum from a patient with clinical hypergastrinemia. We determined that the CCK-B receptor binds arrestin with a pharmacology mirroring CCK-B receptor signaling through inositol phosphate, and that the rate of arrestin dissociation from internalized receptor mirrors receptor recycling to the plasma membrane. Moreover, the CCK-B recycling rate is intermediate between that of Class A GPCRs such as the beta2-adrenergic receptor and Class B GPCRs such as the vasopressin type 2 receptor. Mathematical modeling of these results indicates that a common receptor conformation may underlie both CCK-B signaling and desensitization. In addition to its use in drug screening, this methodology should generalize to other receptors for use in diagnosis and monitoring of bioactive ligands involved in GPCR-based disease.

Targeting of the Orphan Receptor GPR35 by Pamoic Acid: A Potent Activator of Extracellular Signal-Regulated Kinase and β-Arrestin2 with Antinociceptive Activity

“…Upon agonist-mediated GPCR activation, ␤-arrestins rapidly redistribute en masse to ligand-activated plasma membrane receptors, and then the activated GPCR/arrestin complexes concentrate in clathrin-coated pits and/or internal vesicles. The agonistmediated redistribution of ␤-arrestin fits a sigmoid dose-response model in which the ligand affinities approximate the measured EC 50 values of the active compounds (Barak et al, 2003;Ozawa et al, 2005).…”

Identification of Drosophila Neuropeptide Receptors by G Protein-coupled Receptors-β-Arrestin2 Interactions

“…The ␤arr2-GFP translocation response following exposure to cognate ligands is known to be dependent on the high level of receptor expression that is typically found in transfected cell lines (29). The translocation assay produced robust responses in the case of the Drosophila proctolin receptor CG6986.…”

“…When used in a qualitative "all-or-none" fashion as we have done in this work with saturating ligand concentrations, ␤arr2-GFP translocation is of limited value to evaluate systematically the rank order potencies of "related" ligands, such as the case of the six related DTK peptides. We note that the assay can also be used quantitatively (with available confocal microscopy platforms) to establish potency orders as effectively as other signaling or receptor binding assays (29,32). Quantitative assays should now be employed to rank the potential interactions of multiple and related DTK peptides with the CG6515 and CG7887 receptors.…”

Relative Opioid Efficacy Is Determined by the Complements of the G Protein-Coupled Receptor Desensitization Machinery

“…The ␤-arrestin-GFP translocation experiments have been designed such that the endogenous arrestin complement is comparable with the ␤-arrestin-GFP complement and the receptor complement is larger than both of them. Because the complement of endogenous arrestins remains fixed throughout all the different drug treatments the behavior of the receptor reflects the efficacy of the drug in inducing ␤-arrestin-GFP translocation to the plasma membrane in the presence of a particular complement of GRKs in a qualitative manner (Oakley et al, 2000;Barak et al, 2003).…”