Toward an in vivo G Protein-Coupled Receptor (GPCR) expression, interaction, and signaling assay in Saccharomyces cerevisiae
Date
2011
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Publisher
University of Delaware
Abstract
The G Protein-Coupled Receptors (GPCRs) are membrane integral signaling proteins, which play a key role in signal transduction in myriad processes ranging from senses, organ development, chemotaxis, and neurotransmission in humans to cellular mating in yeasts. This diversity has lead to substantial interest in targeting these receptors for drug development. Native tissues typically express very little receptor, while the alternate hydrophilic and hydrophobic nature of the receptors, in conjunction with regulation of GPCRs in all cell types, conspire to present challenges to heterologous receptor expression for characterization. The present work is divided into two main subsections. The first introduces a promoter alteration to the expression of the human adenosine receptors in baker‟s yeast, Saccharomyces cerevisiae, and examines the resulting changes in expression of the receptor for the presence of properly folded and trafficked receptors at the plasma membrane. The second reports the incorporation of an easily monitored assay into the yeast genome for examination of the G protein signaling competence of the heterologous receptors expressed in our lab. Previous assays, particularly in yeast, have focused on ligand-binding competence of the receptor, though most reported crystal structures acknowledge the loss of signaling capacity of the resulting receptor, thus only partially filling the knowledge gap regarding the signaling behavior of these proteins in vivo.