(75c) The Missing Links: Elucidating Mechanisms of Heterologous GPCR Expression and Trafficking In S. Cerevisiae

Within their native host, G
protein-coupled receptors (GPCRs) are ubiquitously expressed, and assist the
cell in querying the extracellular environment, initiating cellular responses to
diverse sensory and chemical stimuli. Consequently, this large superfamily of
membrane proteins that consist of seven transmembrane domains destined for the
plasma membrane of eukaryotic cells regulate most physiological processes. In
order to reach the plasma membrane, these proteins must navigate the secretory
pathway, where they are translocated into the ER, properly fold, and undergo
post-translation modifications en route to the cell surface. In recent years,
it has been shown that the production of GPCRs in yeast is hindered by host
cellular responses, including the unfolded protein response (UPR).
Conventionally, the UPR is believed to be triggered when the folding capacity
of the endoplasmic reticulum (ER) is exceeded. We hypothesize that the UPR is
predominantly induced by an abundance of heterologously expressed GPCRs in the
ER, and not due exclusively to improperly folded proteins, which yields an
improper balance of protein biosynthesis/maturation or defects in secretory
pathway trafficking.

To improve functional production
(i.e. ligand-binding yields indicative of active receptors) of GPCRs, we have
generated a versatile yeast expression cassette designed to optimize
transcription/translation rates; analyze protein structure/function; and
incorporate multiple tags for identification and purification. To evaluate
sub-cellular localization of GPCRs, we created fluorescent protein variants and
codon-optimized fluorophores of organelle targets analyzed by four-color
imaging using live-cell confocal microscopy, cryo-TEM,
and correlative imaging techniques. Time course analysis, quantitative PCR, co-immunoprecipitation of select proteins, and
yeast deletion strains in combination with novel high-resolution imaging
techniques have shown differences in GPCR trafficking and quality control initiation,
including the UPR, autophagy, and ER associated degradation (ERAD) pathways. We
have evaluated GPCR expression profiles; optimized conditions to minimize UPR
induction; determined colocalization with organelles and sub-compartments; and
confirmed the activity of human adenosine receptors (i.e. hA₂aR, hA₁R,
hA₂bR, hA₃R). Furthermore, we engineered domains and
identified motifs altering localization and functional production by generating
rational chimeric receptors. We continue to analyze the expression,
trafficking, and activity of additional GPCR families, including the neurokinin
receptors (hNK₁R, hNK₂R, and rNK₂R) in order
to determine the generalizability of our observations.