Premium
Progress toward heterologous expression of active G‐protein‐coupled receptors in Saccharomyces cerevisiae : Linking cellular stress response with translocation and trafficking
Author(s) -
O'Malley Michelle A.,
Mancini J. Dominic,
Young Carissa L.,
McCusker Emily C.,
Raden David,
Robinson Anne S.
Publication year - 2009
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.246
Subject(s) - g protein coupled receptor , receptor , saccharomyces cerevisiae , heterologous expression , biology , rhodopsin like receptors , microbiology and biotechnology , adenosine receptor , biochemistry , signal transduction , yeast , gene , recombinant dna , glutamate receptor , metabotropic receptor , agonist
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 A 2 a (hA 2 aR) 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 hA 2 aR, as expected, and partially cleaved for hA 2 bR, hCCR5R, and hD 2L R. Ligand‐binding assays conducted on the adenosine family (hA 1 R, hA 2 aR, hA 2 bR, and hA 3 R) of receptors show that hA 2 aR and hA 2 bR, 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 .