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In vivo assembly and large-scale purification of a GPCR - Gα fusion with Gβγ, and characterization of the active complex


Kumar, Abhinav; Plückthun, Andreas (2019). In vivo assembly and large-scale purification of a GPCR - Gα fusion with Gβγ, and characterization of the active complex. PLoS ONE, 14(1):e0210131.

Abstract

G protein coupled receptors (GPCRs) are central players in recognizing a variety of stimuli to mediate diverse cellular responses. This myriad of functions is accomplished by their modular interactions with downstream intracellular transducers, such as heterotrimeric G proteins and arrestins. Assembling a specific GPCR-G protein pair as a purified complex for their structural and functional investigations remains a challenging task, however, because of the low affinity of the interaction. Here, we optimized fusion constructs of the Gα subunit of the heterotrimeric G protein and engineered versions of rat Neurotensin receptor 1 (NTR1), coexpressed and assembled in vivo with Gβ and Gγ. This was achieved by using the baculovirus-based MultiBac system. We thus generated a functional receptor-G protein fusion complex, which can be efficiently purified using ligand-based affinity chromatography on large scales. Additionally, we utilized a purification method based on a designed ankyrin repeat protein tightly binding to Green Fluorescent Protein (GFP-DARPin) that may be used as a generic approach for a large-scale purification of GPCR-G protein fusion complexes for which no ligands column can be generated. The purification methods described herein will support future studies that aim to understand the structural and functional framework of GPCR activation and signaling.

Abstract

G protein coupled receptors (GPCRs) are central players in recognizing a variety of stimuli to mediate diverse cellular responses. This myriad of functions is accomplished by their modular interactions with downstream intracellular transducers, such as heterotrimeric G proteins and arrestins. Assembling a specific GPCR-G protein pair as a purified complex for their structural and functional investigations remains a challenging task, however, because of the low affinity of the interaction. Here, we optimized fusion constructs of the Gα subunit of the heterotrimeric G protein and engineered versions of rat Neurotensin receptor 1 (NTR1), coexpressed and assembled in vivo with Gβ and Gγ. This was achieved by using the baculovirus-based MultiBac system. We thus generated a functional receptor-G protein fusion complex, which can be efficiently purified using ligand-based affinity chromatography on large scales. Additionally, we utilized a purification method based on a designed ankyrin repeat protein tightly binding to Green Fluorescent Protein (GFP-DARPin) that may be used as a generic approach for a large-scale purification of GPCR-G protein fusion complexes for which no ligands column can be generated. The purification methods described herein will support future studies that aim to understand the structural and functional framework of GPCR activation and signaling.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2019
Deposited On:05 Sep 2019 15:24
Last Modified:01 Oct 2019 12:31
Publisher:Public Library of Science (PLoS)
ISSN:1932-6203
OA Status:Gold
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1371/journal.pone.0210131
PubMed ID:30620756

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