283769 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 ). 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  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 . S. cerevisiae strains were further engineered to express fluorescent proteins targeted to various organelles . 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 . Collectively, our analyses provide mechanistic insight for the successful production of recombinant membrane proteins in the model eukaryote, S. cerevisiae.
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)
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