title: 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.
authors.affiliation: Departments of Cell Biology and Medicine, Duke University, Durham, NC 27710, USA. l.barak@cellbio.duke.edu
authors.forename: Larry S
authors.initials: LS
authors.lastname: Barak
authors.forename: Robert H
authors.initials: RH
authors.lastname: Oakley
authors.forename: Michael A
authors.initials: MA
authors.lastname: Shetzline
mesh_terms: D019393:Arrestin / Q000378:metabolism*; D019390:Arrestins / Q000378:metabolism; D015374:Biosensing Techniques; D002460:Cell Line; D005755:Gastrins / Q000378:metabolism; D006801:Humans; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010418:Pentagastrin / Q000378:metabolism; D011485:Protein Binding; D043887:Receptor, Cholecystokinin B / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D000071557:beta-Arrestins
chemical_list: D019393:Arrestin; D019390:Arrestins; D005755:Gastrins; D043887:Receptor, Cholecystokinin B; D000071557:beta-Arrestins; D007294:Inositol; C013550:gastrin 17; D010418:Pentagastrin