Novel RNAi-Mediated Approach to G Protein-Coupled Receptor Deorphanization: Proof of Principle and Characterization of a Planarian 5-HT Receptor

Abstract

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 Gαi/o. The method was then extended to deorphanize a novel Gαs-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.

Kinetic profiling an abundantly expressed planarian serotonergic GPCR identifies bromocriptine as a perdurant antagonist

“…Luminescence can therefore be resolved over time in intact cells, enabling kinetic profiling of responses to ligands, an improvement over single (‘endpoint’) measurements from broken cells. The rationale for this approach was based upon the prior demonstration of coupling of the Dugesia tigrina S7.1R homologue (Dt.Ser1, (Zamanian et al., 2012)) and the schistosome S7.1R homologue (Sm.5HTR) to the cAMP signaling pathway (Patocka et al., 2014, Chan et al., 2016b). …”

“…1) and is amenable to miniaturization to facilitate higher throughput drug screening campaigns against flatworm GPCRs (Chan et al., 2016b). Obviously, this approach method is useful only for GPCRs that modulate cellular cAMP levels (G s , G i ), but this reflects a coupling specificity of the majority of flatworm GPCRs examined in heterologous systems to date (Omar et al., 2007, Taman and Ribeiro, 2009, Zamanian et al., 2012, Patocka et al., 2014, MacDonald et al., 2015). Probes for other second messenger pathways (for example genetically encoded Ca 2+ indicators) are available and have also been shown to be useful for deorphanizing lophotrochozoan GPCR activity (Bauknecht and Jekely, 2015).…”

Invertebrate serotonin receptors: a molecular perspective on classification and pharmacology

“…Law et al (2015) used this approach to examine individual 5-HT receptor subtypes from H. contortus and other species in C. elegans made null for all five endogenous 5-HT receptors. In the flatworm Dugesia tigrina, Zamanian et al (2012) developed a loss-of-function technique that could be used to assess the role of specific receptors in drug responses in native tissue. In this approach, responses to drugs were compared in control tissue and following RNAi knockdown (see Glossary) of a 5-HT 7 receptor, allowing identification of drugs dependent on a single receptor (Zamanian et al, 2012).…”

Ca2+ channels and praziquantel: A view from the free world

“…That lateral sidesteps between closely related flatworm groupings can provide fresh insight into the workings of this important clinical therapeutic is not in retrospect surprising as we note PZQ was originally introduced as both an anticestodal and antischistocidal agent [40, 41]. Our work on PZQ is but one evidenced example of a theme of using planarians as a ‘parasite model’ to provide information pertinent to the study of schistosomes [42–44]. …”