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.
Footnotes
This work was supported by the Intramural Research program of the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- GRK
- G protein-coupled receptor kinases
- CNO
- clozapine-N-oxide
- DREADD
- designer receptor exclusively activated by designer drug
- MIN6
- mouse insulinoma
- ACh
- acetylcholine
- OXO-M
- oxotremorine-M
- [3H]NMS
- [3H]N-methylscopolamine
- HA
- hemagglutinin
- BRET
- bioluminescence resonance energy transfer
- Luc
- R. reniformis luciferase 8
- V-arr2
- Venus-tagged version of arrestin-2
- V-arr3
- Venus-tagged version of arrestin-3
- siRNA
- small interfering RNA
- pERK1/2
- phosphorylated ERK1/2.
- Received May 31, 2012.
- Accepted July 20, 2012.
- U.S. Government work not protected by U.S. copyright
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|