Progress toward heterologous expression of active G‐protein‐coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

• Published in: Protein science 2009-11, Vol.18 (11), p.2356-2370
• Main Authors: O'Malley, Michelle A., Mancini, J. Dominic, Young, Carissa L., McCusker, Emily C., Raden, David, Robinson, Anne S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cell Membrane - metabolism; Chromatography, Affinity; Fungal Proteins - metabolism; G‐protein‐coupled receptor; heterologous expression; HSP70 Heat-Shock Proteins - metabolism; Humans; membrane protein; Microscopy, Fluorescence; Molecular Sequence Data; NECA; Protein Sorting Signals; Protein Transport - physiology; Radioligand Assay; Receptor, Adenosine A2A - biosynthesis; Receptor, Adenosine A2A - genetics; Receptor, Adenosine A2A - metabolism; Receptors, G-Protein-Coupled - biosynthesis; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - physiology; Stress, Physiological - physiology; Unfolded Protein Response
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.246

High‐level expression of mammalian G‐protein‐coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high‐resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A2a (hA2aR) receptor is active for ligand‐binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER‐resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA2aR, as expected, and partially cleaved for hA2bR, hCCR5R, and hD2LR. Ligand‐binding assays conducted on the adenosine family (hA1R, hA2aR, hA2bR, and hA3R) of receptors show that hA2aR and hA2bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae.