G Protein-Coupled Receptors (GPCRs) are proteins consisting of seven transmembrane domains that transmit extracellular signals inside the cell by initiating downstream signaling cascades. GPCRs comprise nearly 4% of all human genes but are targets of approximately 25-40% of all drug discovery efforts. Adenosine receptors (A1, A2a, A2b and A3) are a subfamily of GPCRs, involved in many physiological functions and have been implicated in numerous cardiovascular and neurodegenerative diseases, and therefore are targets for drug development. Here, we utilize yeast as a discovery platform for the adenosine receptor family, because yeast provides a relatively easy and inexpensive platform without the complexities of multiple receptor subtypes, receptor promiscuity and crosstalk that occurs in native mammalian hosts. Yeast share many functionally exchangeable proteins with higher eukaryotes involved in its GPCR signaling pathway. By engineering the yeast genome, we have designed a functional screen that enables us to couple downstream signaling from GPCR-ligand binding to expression of a fluorescent reporter protein for efficiently and rapidly comparing ligand-mediated GPCR signaling.<br/>Previous studies from our laboratory and other groups suggest the role of the C-terminus of the A2a receptor in the observed efficient expression and trafficking to the plasma membrane of the receptor. To better understand the role of C-terminus and to test its efficacy in improving the expression of a subfamily member, we developed A1 receptor chimeras with the A2aR C-terminus using homologous DNA recombination. The role of the C-terminus of the A2a receptor on expression, trafficking and oligomerization state (via fluorescence-resonance energy transfer) of the chimeric fluorescent receptors relative to wildtype receptors will be discussed.<br/>With our engineered yeast strain and chimeric receptors, we could improve expression of other recombinant GPCRs enabling detection of receptor trafficking and oligomerization, and screening ligand library based on receptor binding affinity and signaling efficacy.

Human G protein-coupled receptors expression and signaling in yeast: Design and optimization of the host/protein platform for therapeutic development

251st National Meeting (2016)

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