Membrane proteins have become an important focus of recent biochemical research, since they play a vital role in cellular communication as well as most biological functions.  The G-Protein Coupled Receptors (GPCRs) represent the largest class of membrane proteins, which mediate cellular responses in the presence of extracellular stimuli.  GPCRs serve as the primary detection mechanism for sight and smell, and also play direct roles in cancer, diabetes, and HIV infection among other debilitating diseases.  In fact, about 50% of all pharmaceuticals on the market target GPCRs, earning annual revenues of over $30 billion.  Although there are over 1000 suspected GPCRs in humans, they have proven difficult to study as they are expressed in extremely low levels in native tissues.  Biophysical characterization would help elucidate GPCR structure and function, but usually requires micrograms to milligrams of functional protein, which is extremely difficult to achieve due to low natural abundance.  Even though GPCRs have been the focus of therapeutic targets for some time, relatively little is known about their expression, folding, interactions, and detailed structure.          

The ultimate goal of this work is to elucidate protein folding and explore conformational changes for individual GPCRs by using a non-native, yeast expression system to over-express them.  Specifically, the work presented here focuses on (1) understanding heterologous expression of mammalian G-protein coupled receptors in the yeast S. cerevisiae and (2) using biophysical techniques to characterize functional GPCRs that have been expressed, purified, and reconstituted from this system. 

Engineering Saccharomyces cerevisiae for the Expression of Mammalian GPCRs

Given its overall low expense, ease of genetic manipulation, and eukaryotic secretory pathway, yeast are an attractive host organism for the over-expression of complex membrane proteins.  In an effort to understand potential limitations to the use of a yeast system for the expression of mammalian GPCRs, recombinant DNA techniques were used to sub-clone 12 GPCRs into the genome of S. cerevisiae.  Trafficking of these GPCRs through the secretory pathway was monitored using confocal microscopy to follow the expression of green fluorescent protein (GFP) tagged receptors.  Overall, our results indicate that GPCRs which fail to localize to the plasma membrane activate the stress response pathway within the endoplasmic reticulum (ER) and preferentially associate with an ER-resident chaperone BiP, as determined through affinity precipitation studies.  Additionally, many mis-localized GPCRs trigger the heat shock response pathway within the cytosol.  However, by optimizing cell culture conditions, we have managed to express milligram amounts of the human adenosine A₂a receptor (hA₂aR) in this system, which is membrane localized and properly folded.  Currently, purified yields obtained for the A₂a receptor are the highest achieved from any heterologous or native expression system.  Collectively, these studies have shown that the main bottleneck in yeast-based GPCR expression is located within the ER of the cell.  Current efforts are underway to manipulate folding in this organelle to promote proper folding and trafficking of mammalian GPCRs in S. cerevisiae.      

Characterization of GPCRs using Biophysical Techniques

Expression and purification of milligram amounts of the human adenosine A₂a receptor from S. cerevisiae has allowed for extensive biophysical characterization of this G-protein coupled receptor through biophysical studies.  GPCRs, as with all membrane proteins, require surfactants for their stabilization outside of their native plasma membrane environment.  Therefore, numerous surfactants and lipid additives were screened for their ability to stabilize the active conformation of hA₂aR in a protein detergent complex (PDC), as assessed through ligand binding and neutron scattering techniques. Circular dichroism and intrinsic fluorescence spectroscopy were used to study purified protein actively reconstituted in a protein-detergent complex in order to elucidate general protein stability and explore conformational changes under a variety of conditions.  Binding of agonist caused a small blue shift in the emission peak of hA₂aR's intrinsic fluorescence spectra, suggesting a rearrangement of hA₂aR tryptophan residues upon agonist binding. In contrast, no detectable changes in CD spectra of hA₂aR are seen upon binding agonist or antagonist ligands, implying that hA₂aR's alpha helices may rearrange but otherwise remain unaffected during ligand binding.  Thermal denaturation of purified receptors shows that hA₂aR is a highly thermostable protein, yet undergoes irreversible aggregation at higher temperatures.  The secondary and tertiary structures of hA₂aR are also somewhat more resistant to thermal unfolding in a ligand-bound state.  Unfolding with chemical denaturants and hydrostatic pressure leads to an apparent red-shift of the fluorescence emission maxima, which suggests that hA₂aR's tryptophan residues are solvent exposed in an unfolded state.  We have also applied these methods towards the characterization of a disulfide bond deficient mutant of hA₂aR, which has suggested that disulfide bonding is critical to maintenance of overall protein stability, yet its absence preserves functionality of the receptor. These results have provided the first experimental insights into conformational changes and protein folding for the hA₂a receptor, and may further direct future folding and re-folding studies for other GPCRs.