(185g) Understanding Translocation Bottlenecks for Mammalian G-Protein Coupled Receptors Heterologously Expressed in Saccharomyces Cerevisiae

G-protein coupled receptors are 7-transmembrane domain integral
membrane proteins that are targets for nearly half of all pharmaceuticals on
the market.  Defining the structure of these proteins on an individual basis is
of utmost importance for rational drug design, yet structural characterization
is generally hindered by low-level expression in native tissues.  In order to
facilitate high-resolution structure determination for these proteins, we have
achieved high-level expression (~mg/L of culture) for several human GPCRs by
expressing them in the yeast Saccharomyces cerevisiae.  However, we have
observed that improper translocation into the ER membrane, as evidenced by an
intact N-terminal leader sequence fused to expressed receptors, is correlated
with intracellular retention and probable misfolding of yeast-expressed human
GPCRs.  Examining the mechanism by which translocation fails is critical to
understanding and overcoming this bottleneck in yeast, and could lead to
re-engineering this host for the robust over-expression of mammalian membrane
proteins.      

In this work, we seek to elucidate the limitations for
membrane insertion of GPCRs within the yeast ER membrane by creating systematic
TM domain truncation mutants for the human adenosine A₂a and A₃
receptors, each with a C-terminal GFP tag.  Trafficking, leader sequence
processing, and transmembrane domain topology within the membrane for each
receptor truncation mutant were monitored in order to model membrane insertion
for the full-length, wild-type protein.  In our system, expression of
full-length A₂aR is characterized by complete leader sequence
processing and ultimate plasma membrane localization, while full-length A₃R
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 A₂aR
and A₃R 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.  In the case of A₂aR, the addition of subsequent
transmembrane domains leading up to the full-length protein was characterized
by an increase in mature protein relative to the pre-Kex2 population.  Current
studies are focused on determining the orientation of GPCR transmembrane domain
segments within the ER membrane through the use of a fluorescence protease
protection (FPP) assay, which is applied to yeast cells in vivo.