Novel RNAi-mediated approach to G protein-coupled receptor deorphanization: proof of principle and characterization of a planarian 5-HT receptor

PLoS One. 2012;7(7):e40787. doi: 10.1371/journal.pone.0040787. Epub 2012 Jul 18.


G protein-coupled receptors (GPCRs) represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi) was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gα(i/o). The method was then extended to deorphanize a novel Gα(s)-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biogenic Monoamines / metabolism
  • Computational Biology
  • Cyclic AMP / metabolism
  • Gene Knockdown Techniques
  • Ligands
  • Molecular Sequence Data
  • Peptides / metabolism
  • Phylogeny
  • Planarians / metabolism*
  • Polymerase Chain Reaction
  • RNA Interference*
  • Receptors, Serotonin / chemistry
  • Receptors, Serotonin / genetics
  • Receptors, Serotonin / metabolism*
  • Sequence Alignment


  • Biogenic Monoamines
  • Ligands
  • Peptides
  • Receptors, Serotonin
  • Cyclic AMP