Expression, purification, and biophysical characterization of G-protein coupled receptors expressed from Saccharomyces cerevisiae

We have achieved high-level expression and membrane localization of one type of G-protein coupled receptor (GPCR), the human adenosine A2a receptor (hA2aR), in the yeast Saccharomyces cerevisiae at levels of approximately 10 milligrams per liter of culture, which has facilitated its purification and structural characterization. The development of a suitable purification scheme via immobilized metal affinity chromatography and ligand affinity has led to the purification of greater than 6 milligrams per liter of culture ligand-binding hA2aR. Throughout purification, the presence of a mammalian cholesterol analog, cholesterol hemisuccinate (CHS), to the protein detergent complex (PDC) was critical to the maintenance of hA2aR activity in vitro. A variety of biophysical characterization techniques, including circular dichroism and fluorescence spectroscopy, were applied to receptors reconstituted in surfactant micelles to characterize receptor stability.;We sought to understand the critical parameters governing stability and structure of PDCs to enable downstream characterization of GPCRs, using hA 2aR as a model protein. The ability of different micellar compositions to stabilize the active conformation of hA2aR was measured through radio-ligand binding, providing a sensitive probe of protein structure, and micelle structure was analyzed via small angle neutron scattering (SANS). We found that most micelles used in this study were welldescribed by an oblate ellipsoidal shape, and that morphology varied widely upon lipid content increase. Activity of hA2aR depended significantly on the alkyl chain length of CibetaG2 and CiSbetaG2 surfactants used in this study as well as the surfactant/lipid ratio. 7-8 CHS monomers per micelle were necessary to maintain hA2aR activity among different systems, but CHS content alone within the micelle was not sufficient to preserve activity. Therefore, activity changes among different systems are likely the result of both structural changes within the micelle environment as well as specific protein-lipid interactions.;Although high-level expression of hA2aR was accomplished in Saccharomyces cerevisiae, proper folding and membrane localization of these proteins proved GPCR-dependent. Localization, stress response activation, and activity for several GPCRs were systematically analyzed in S. cerevisiae in order to understand limitations associated with their over-expression in this system. Although all receptors were expressed at the mg/L scale, only the human adenosine A2a receptor was plasma membrane localized within a vacuolar-deficient cell strain, whereas other GPCRs were largely retained within the cell. Receptors which failed to reach the plasma membrane activated the unfolded protein response pathway within the ER, and many triggered the cellular heat shock response, yet expression of hA 2aR escaped both these quality control checkpoints.Through radio-ligand binding studies conducted on the adenosine family of receptors, we have demonstrated that only receptors that have been properly processed exhibit ligand-binding activity.;In order to investigate the membrane insertion and leader sequence processing for individual transmembrane domains, a set of transmembrane domain truncation mutants for the hA2aR and hA3R were constructed. Processing of the pre-pro leader sequence fused to the N-terminus of each truncated receptor was evaluated through a gel mobility shift assay, where expressed receptor fragments were compared to fragments sub-cloned without the leader sequence. In our system, expression of full-length hA2aR is characterized by complete leader sequence processing and ultimate plasma membrane localization, while full-length hA3R is predominantly intracellular, with the N-terminal leader sequence left intact. Interestingly, we observe that truncations which include only the N-terminus, first transmembrane domain, and first intracellular loop for both hA2aR and hA3R display a population of mature receptors and a population of pre-Kex2 proteins, indicating that downstream transmembrane domains within each protein influence translocation and leader sequence processing for the full-length protein. Therefore, it is likely that the interaction of transmembrane domains, either with the leader sequence or with each other while within the translocon, influences the orientation and insertion of domains as they are translocated into the ER membrane. (Abstract shortened by UMI.)...