(5cb) Heterologous Expression of Membrane Proteins in Saccharomyces Cerevisiae Enables Biophysical Characterization of Pharmaceutical Targets

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.