The Missing Links: Elucidating Mechanisms of Heterologous GPCR Expression and Trafficking In S. Cerevisiae

Within their native host, G protein-coupled receptors (GPCRs) are ubiquitously expressed, and assist the cell in querying the extracellular environment, initiating cellular responses to diverse sensory and chemical stimuli. Consequently, this large superfamily of membrane proteins that consist of seven transmembrane domains destined for the plasma membrane of eukaryotic cells regulate most physiological processes. In order to reach the plasma membrane, these proteins must navigate the secretory pathway, where they are translocated into the ER, properly fold, and undergo post-translation modifications en route to the cell surface. In recent years, it has been shown that the production of GPCRs in yeast is hindered by host cellular responses, including the unfolded protein response (UPR). Conventionally, the UPR is believed to be triggered when the folding capacity of the endoplasmic reticulum (ER) is exceeded. We hypothesize that the UPR is predominantly induced by an abundance of heterologously expressed GPCRs in the ER, and not due exclusively to improperly folded proteins, which yields an improper balance of protein biosynthesis/maturation or defects in secretory pathway trafficking.

To improve functional production (i.e. ligand-binding yields indicative of active receptors) of GPCRs, we have generated a versatile yeast expression cassette designed to optimize transcription/translation rates; analyze protein structure/function; and incorporate multiple tags for identification and purification. To evaluate sub-cellular localization of GPCRs, we created fluorescent protein variants and codon-optimized fluorophores of organelle targets analyzed by four-color imaging using live-cell confocal microscopy, cryo-TEM, and correlative imaging techniques. Time course analysis, quantitative PCR, co-immunoprecipitation of select proteins, and yeast deletion strains in combination with novel high-resolution imaging techniques have shown differences in GPCR trafficking and quality control initiation, including the UPR, autophagy, and ER associated degradation (ERAD) pathways. We have evaluated GPCR expression profiles; optimized conditions to minimize UPR induction; determined colocalization with organelles and sub-compartments; and confirmed the activity of human adenosine receptors (i.e. hA₂aR, hA₁R, hA₂bR, hA₃R). Furthermore, we engineered domains and identified motifs altering localization and functional production by generating rational chimeric receptors. We continue to analyze the expression, trafficking, and activity of additional GPCR families, including the neurokinin receptors (hNK₁R, hNK₂R, and rNK₂R) in order to determine the generalizability of our observations.