(52b) The Pivotal Role of Motifs: Elucidating Mechanisms of Heterologous GPCR Expression and Trafficking in Yeast Through Chimeric Receptors

G protein-coupled receptors (GPCRs)
constitute a superfamily of cell surface receptors responsible for the
transduction of extracellular stimuli into intracellular signals. One
sub-family of GPCRs is the adenosine family, identified as A₁R, A₂aR,
A₂bR, and A₃R, which mediates the physiological effects
of extracellular adenosine. Structurally analogous to other GPCRs, each
adenosine receptor consists seven transmembrane-spanning a-helices, an extracellular N-terminal
domain, three extracellular and intracellular loops, and a cytoplasmic
C-terminal domain. Despite this common architecture, adenosine receptor
subtypes are subject to regulation by distinct molecular mechanisms, couple
promiscuously to multiple signaling pathways, and maintain unique ligand
binding properties. Furthermore, the synthesis and export of polytopic membrane
proteins is a complex process involving multiple steps of regulatory control to
ensure proper folding, assembly, selective retention, and transport. As newly
synthesized proteins transverse the secretory pathway from the endoplasmic
reticulum (ER) to their final destination, quality control processes ensure
that the ER-folding capacity is sufficient and adjusted appropriately, in order
to maintain ER homeostasis. Compared to extensive studies of ER quality control
and endogenous trafficking mechanisms in S.
cerevisiae, molecular mechanisms underlying the transport processes of
heterologous membrane proteins from the ER to cell surface are less well
understood. Notably, the regulation of receptor signaling at the plasma
membrane in non-native hosts has remained elusive.

In this study, specific
recognition signals that govern quality control and secretion-dependent
interactions provide a basic
framework for determining trafficking mechanisms of recombinantly expressed
GPCRs. We have investigated processes governing the
heterologous expression of two human adenosine receptors, hA₂aR and hA₃R, and
engineered chimeras of these two receptors in yeast, Saccharomyces cerevisiae. To improve functional production (i.e. ligand-binding
yields indicative of active receptors) of GPCRs, we have assessed protein
structure/function through rational engineered chimeras, determined in vivo receptor activity using
fluorescent ligands, confirmed the activity of purified GPCRs by XAC ligand
chromatography, and quantified total yields of GPCR production using novel
standards. Multiple tags for identification and purification were incorporated
(as reviewed [1]). By
implementing DNA recombination strategies combined with high-resolution imaging
techniques, we have determined intracellular the intracellular localization of
heterologous GPCRs in yeast compared to endogenous organelle markers at the
appropriate spatiotemporal resolution using Structured Illumination Microscopy
(SIM) and evaluated alterations in organelle morphology, such as autophagy due
to prolonged stress, via conventional TEM. In pursuit of a thorough analysis of
protein distribution at the subcellular level, multiple yeast
expression cassettes [2] have been created to test the effects of
codon-optimized fluorescent variants, small epitope tags, polylinker length for
N- and C- terminal tags, and the inclusion of essential retrieval sequences for
ER luminal chaperones and foldases [3]. S.
cerevisiae strains were further engineered to
express fluorescent proteins targeted to various organelles [4]. To investigate
discrete subpopulations of tagged proteins using live-cell imaging methods and
super-resolution techniques (e.g.
Fluorescence-Photoactivation Localization Microscopy, F-PALM), a
photoconvertible GFP variant (i.e. mEos2) and six-residue tetracysteine motif
required for FlAsH (fluorescein arsenical helix binder)-based technology were
implemented.

Utilizing
time course analyses, quantitative PCR, co-immunoprecipitation of select
proteins, and novel tags with state-of-the-art high-resolution imaging
techniques, we 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,
identified select protein interactions with ER folding factors, and confirmed
the activity of human adenosine receptors (i.e. hA₂aR, hA₁R,
hA₂bR, hA₃R) [5,6]. Furthermore, the rational design of
chimeric receptors altered localization and functional production. We continue
to analyze the expression, trafficking, and activity of additional GPCR
families, including the neurokinin receptors (hNK₁R) in order to
determine the generalizability of our observations [7]. Collectively, our analyses provide
mechanistic insight for the successful production of recombinant membrane
proteins in the model eukaryote, S.
cerevisiae.

Publications

1.    
C. L. Young, Z. T. Britton, A. S.
Robinson Recombinant
Protein Expression and Purification: A Comprehensive Review of Affinity Tags
and Microbial Applications, Biotechnology
Journal, 7(4), Jan 10 2012 doi:10.1002/biot.201100155.
[Epub ahead of print]

2.    
C. L. Young, D. Raden, J. Caplan, K. Czymmek, A. S.
Robinson Optimized Cassettes for
Live-Cell Imaging of Proteins and High Resolution Techniques in Yeast,
Yeast, 2012 doi:10.1002/yea.2895. [Epub 2012 Apr 4]

3.    
C. L. Young, D. L. Raden, A. S. Robinson, Analysis of Endoplasmic Reticulum Resident
Proteins in S. cerevisiae: Implementation of H/KDEL Retrieval Sequences,
2012 (submitted).

4.  C.
Young, Z.
Britton, J. Caplan, K. Czymmek, A. Robinson Exploiting
S. cerevisiae: Cellular Systems & Techniques Aimed at Identifying the
Localization of Targeted Proteins, 2012 (in preparation).

5.  C.
Young, E. McCusker, Z.
Britton, S. Modla, J. Caplan, B. Chhun, K. Czymmek, A. Robinson Conserved Trafficking Motifs Regulate
Adenosine Receptors in Yeast: Rational Design of Chimeric hA2a/hA3 Receptors, 2012
(in preparation).

6.  C. Young, E. McCusker, Z. Britton, S. Modla, B.
Chhun, J. Caplan, K. Czymmek, A. Robinson Subcellular
Localization and Trafficking Mechanisms of Functional Adenosine A₁
Receptor Expressed in Saccharomyces cerevisiae: Implications for
Pharmacological Assays, 2012 (in preparation)

7.  C. Young, E. McCusker, B. Chhun, J. Caplan, K.
Czymmek, A. Robinson Regulation
of G protein-coupled receptor, NK₁R: Mechanisms of ER Export and
Plasma Membrane Localization in S. cerevisiae, 2012
(in preparation)