(392b) Engineering Saccharomyces Cerevisiae for the Heterologous Expression of Mammalian G-Protein Coupled Receptors | AIChE

(392b) Engineering Saccharomyces Cerevisiae for the Heterologous Expression of Mammalian G-Protein Coupled Receptors


O'Malley, M. A. - Presenter, Massachusetts Institute of Technology
Mancini, J. D. - Presenter, University of Delaware
Raden, D. - Presenter, University of Delaware
Robinson, A. S. - Presenter, University of Delaware

The G-protein coupled receptors (GPCRs) constitute the largest family of mammalian membrane proteins, and are targets for nearly half of all pharmaceuticals on the market. However, the development of improved, structure-based therapeutics to target these proteins requires milligram amounts of properly folded, purified receptors, which are generally not achievable from native host systems. Thus, over-expression of mammalian G-protein coupled receptors is a necessary first step towards high-resolution structure determination for these important pharmaceutical targets. Though bacterial, yeast, and insect expression systems have been employed to express mammalian GPCRs at high levels, many receptors are improperly processed or otherwise mis-folded in these systems. Even with several host systems available, heterologous expression has not yet systematically allowed for large-scale over-expression of GPCRs, and strategies used to over-express these proteins have typically been empirical.

In this work, we expressed a panel of 12 representative GPCRs from the rhodopsin family of receptors in the yeast Saccharomyces cerevisiae, and systematically compared cellular expression patterns in order to investigate limitations associated with GPCR over-expression in this system. Our findings indicate that only the adenosine A2a receptor can be properly over-expressed in a vacuolar-deficient cell strain, whereas other GPCRs are mainly mis-localized within the peripheral or nuclear endoplasmic reticulum (ER). GPCRs which fail to reach the plasma membrane activate the unfolded protein response pathway within the ER, and many trigger the cellular heat shock response, whereas expression of A2aR escapes both these quality control checkpoints. Furthermore, mis-localized receptors associate with BiP, an ER-resident chaperone, which indicates probable mis-folding of these receptors. N-terminal sequencing has suggested that these problems may stem from translocation failure across the ER membrane. However, expression levels and protein yields for almost all mis-localized receptors are significant (~ 2-4 mg/L of culture), and some are comparable to that of A2aR (~10 mg/L of culture). Overall, our findings suggest that the main limitation associated with heterolgous GPCR expression in S. cerevisiae is associated with a folding problem within the ER.