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

Abstract

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 ligandbinding 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.

The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

“…E. coli expression systems have been employed to produce heterogeneous preparations of neurotensin and olfactory receptors (Grisshammer, 2006(Grisshammer, , 2009White and Grisshammer, 2007;Song et al, 2009). Yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe expression systems have been used to express large quantities of A 2a -adenosine receptor with ligand-binding characteristics near those of wild type (Niebauer et al, 2004;Niebauer and Robinson, 2006;O'Malley et al, 2009). Production through cell-free expression systems has proven useful for structural determinations of both soluble and membrane protein targets (Chen et al, 2007b), but large-scale use of these techniques for GPCR expression have yet to yield a GPCR structure (Klammt et al, 2007a,b;Junge et al, 2008Junge et al, , 2010.…”

Biochemical Characterization of Cardiolipin Synthase Mutations Associated with Daptomycin Resistance in Enterococci

“…Membrane fractions were treated with 0.1 M Na 2 CO 3 at pH 11, 2 M urea, or 1% SDS. SDS (1%) typically releases integrally bound proteins from the membrane (40,41), while milder extraction conditions are more consistent with peripheral proteins. Only treatment with SDS could quantitatively release Cls447a and Cls613a from the membrane (see Fig.…”

Ice breaking in GPCR structural biology

“…Research from different groups raises our concern about the homogeneity of yeast-expressed material [8,[31][32][33] . Their results indicate that the expressed product is a mixture of functional and nonfunctional receptors, because during optimization the expression of the functional GPCR will dramatically increase but the overall expression level is somewhat constant.…”

“…O'MALLEY et al investigated 12 GPCRs expressed in S cerevisiae, and only the A 2A adenosine receptor retained its ligand binding activity. Other receptors, despite being processed and expressed in the same conditions, failed to localize in the plasma membrane and had no detectable ligand binding [31] . A more detailed study using N-terminal sequencing and N-glycosylation detection demonstrated that most of these nonfunctional receptors were not properly processed.…”