Date of Award
Doctor of Philosophy
Theses & dissertations (Interdisciplinary)
Timothy A. Day
G protein-coupled receptors (GPCRs) constitute the largest known superfamily of integral membrane proteins, and represent a particularly lucrative set of chemotherapeutic targets. These seven transmembrane receptors play a central role in eukaryotic signal transduction and physiology, mediating cellular responses to a diverse range of extracellular stimuli. The phylum Platyhelminthes is of considerable medical and biological importance, housing prominent human pathogens as well as established model organisms in the realm of developmental and stem cell biology. There exists ample motivation to elucidate the structural and functional properties of GPCRs in this phylum.
The availability of whole genome sequence data for the human blood fluke Schistosoma mansoni and the model planarian Schmidtea mediterranea paves the way for the first genome-wide analyses of platyhelminth GPCRs. Extensive efforts were made to delineate the receptor complements of these organisms. Further work primarily focuses on validation of a novel method for elucidating receptor function in the native cell membrane environment. Together, these genomic and functional data improve our understanding of basic platyhelminth receptor biology and shed light on a promising set of anthelmintic drug targets.
Application of a transmembrane-focused it in silico protocol led to the discovery of 116 S. mansoni and 333 S. mediterranea GPCRs, followed by extensive curation of underlying gene models. Phylogenetic analysis of the resulting dataset confirmed the presence of the primary metazoan GRAFS families and revealed novel lineage-specific receptor groupings, including a large platyhelminth-specific Rhodopsin-like subfamily (PROF1) and a planarian-specific Adhesion-like family (PARF1). Support vector machines (SVMs) were trained and used for ligand-based classification of full-length Rhodopsin GPCRs, complementing phylogenetic and homology-based classification. PROF1 receptors were further revealed as neuronally-expressed endoGPCRs via whole mount in situ hybridization.
In light of the unreliable nature of heterologous approaches to GPCR deorphanization, a novel loss-of-function assay was developed for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. RNA interference (RNAi) was used in conjunction with a cAMP radioimmunoassay (RIA) to monitor second messenger modulation in response to the translational suppression of individual receptors. This strategy was applied to the deorphanization of both neuropeptide and aminergic GPCRs, allowing for the determination of Gαs and Gαi/o-mediated signaling. Loss-of-function phenotypic assays were performed in parallel. While these results establish the potential of this approach, future work can lead to further optimizations and the eventual adaptation of this protocol to higher-throughput platforms.
Zamanian, Mostafa, "Genomic and functional characterization of G protein-coupled receptors in the human pathogen Schistosoma mansoni and the model planarian Schmidtea mediterranea" (2011). Graduate Theses and Dissertations. 12153.