title: Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor.
abstract: Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M₃ muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types.
authors: Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Ken-ichiro
authors: K
authors: Nakajima
authors: Jürgen
authors: J
authors: Wess
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019393:Arrestin / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D003024:Clozapine / Q000031:analogs & derivatives / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D015202:Protein Engineering; D055503:Protein Multimerization; D011869:Radioligand Assay; D043587:Receptor, Muscarinic M3 / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D019393:Arrestin; D007328:Insulin; D043587:Receptor, Muscarinic M3; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D003024:Clozapine; C079149:clozapine N-oxide; D002118:Calcium