Heterologous expression of G-protein-coupled receptors

doi:10.1016/0167-7799(96)10059-7

Trends in Biotechnology

Volume 14, Issue 11, November 1996, Pages 426-430

https://doi.org/10.1016/0167-7799(96)10059-7 Get rights and content

G-protein-coupled receptors (GPCRs) are integral membrane proteins of great pharmacological importance owing to their central role in the regulation of cellular responses to external stimuli. Heterologous expression systems have been used to explore ligand binding, G protein and effector coupling, and structural aspects of the receptors. GPCRs can be expressed in a functional form in all expression systems, but with varying degrees of success because of differences in receptor and host cell characteristics. This article will discuss aspects related to the choice and suitability of expression systems for the intended analysis of GPCR properties.

References (49)

MarjamäkiA.
Eur. J. Pharmacol.
(1994)
KarnikS.S. et al.
J. Biol. Chem.
(1990)
VuT-K.H. et al.
Cell
(1991)
O'DowdB.
J. Biol. Chem.
(1989)
EidneK.A.
Mol. Cell. Endocrinol.
(1992)
MouillacB.
J. Biol. Chem.
(1992)
NgG.Y.K.
Eur. J. Pharmacol.
(1994)
ParkerE.M. et al.
J. Biol. Chem.
(1991)
BertinB.
J. Biol. Chem.
(1992)
SanderP. et al.
FEBS Lett.
(1994)

ArkinstallS. et al.

FEBS Lett.

(1995)

HarfstE.

Mol. Cell. Endocrinol.

(1992)

ButkeraitP.

J. Biol. Chem.

(1995)

GetherU. et al.

J. Biol. Chem.

(1995)

SchwartzT.W.

Curr. Opin. Biotechnol.

(1994)

KenakinT.

Life Sci.

(1988)

EasonM.G.

J. Biol. Chem.

(1992)

MigeonJ. et al.

J. Biol. Chem.

(1994)

WalkerP.

Mol. Cell. Endocrinol.

(1993)

NeedhamM.

Protein Expression Purific.

(1995)

ChenW. et al.

Anal. Biochem.

(1995)

StratowaC. et al.

Curr. Opin. Biotechnol.

(1995)

LernerM.R.

Trends Neurosci.

(1994)

McClintockT.S.

Anal. Biochem.

(1993)

Cited by (109)

Heterologous expression of melanopsin: Present, problems and prospects
2016, Progress in Retinal and Eye Research
Melanopsin, the photosensory pigment of specialized mammalian retinal ganglion cells, is involved in various non-image forming tasks such as pupillary light reflex, circadian entrainment and irradiance detection. Melanopsin genes have been detected in all vertebrate classes and are resolved in two lineages, Opn4m and Opn4x. In addition, two splice variants have been found in several species leading to a short (OPN4-S) and a long (OPN4-L) isoform, mainly differing in the length of the C terminus. Since its discovery in Xenopus laevis in 1998, this novel photopigment has received tremendous interest, but has been very refractory to the many attempts to unravel its photochemical and structural properties. Largely, some insight has been collected in its downstream signaling. Due to its low natural abundance most molecular data have been gathered via recombinant expression in heterologous hosts. A variety of expression hosts has been utilized, but to date only a restricted set of to some extent conflicting data has become available, which we here aim to put into perspective. We first briefly recall the most popular hosts and solubilization and purification approaches reported for GPCRs. Subsequently, a critical overview is presented of the outcome of the various host systems employed for recombinant expression of melanopsins, categorized by host type. These data finally are compiled in a general conclusion, and followed by a critical assessment and potential future directions.
Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system
2015, Protein Expression and Purification
Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas.
Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform.
In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln₁₅–Cys₁₃₀) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody–antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90⁺ cells in a flow cytometry experiment.
A crystal clear solution for determining G-protein-coupled receptor structures
2012, Trends in Biochemical Sciences
G-protein-coupled receptors (GPCRs) are medically important membrane proteins that are targeted by over 30% of small molecule drugs. At the time of writing, 15 unique GPCR structures have been determined, with 77 structures deposited in the PDB database, which offers new opportunities for drug development and for understanding the molecular mechanisms of GPCR activation. Many different factors have contributed to this success, but if there is one single factor that can be singled out as the foundation for producing well-diffracting GPCR crystals, it is the stabilisation of the detergent-solubilised receptor–ligand complex. This review will focus predominantly on one of the successful strategies for the stabilisation of GPCRs, namely the thermostabilisation of GPCRs using systematic mutagenesis coupled with thermostability assays. Structures of thermostabilised GPCRs bound to a wide variety of ligands have been determined, which has led to an understanding of ligand specificity; why some ligands act as agonists as opposed to partial or inverse agonists; and the structural basis for receptor activation.
Investigation of the potential involvement of eicosanoid metabolites in anti-diuretic hormone signaling in Rhodnius prolixus
2012, Peptides
Citation Excerpt :
Importantly, we must note that the heterologous system utilized for this receptor assay is dependent on calcium mobilization, and thus, this would appear to contradict the above-mentioned in vitro experiments demonstrating the lack of calcium involvement. However, studies have shown that differences in post-translational processing or receptor promiscuity often occurs when utilizing heterologous systems [20,47], which in some cases can be advantageous in determining receptor–ligand pairing when downstream signaling partners are unknown. This is also supported by the sizeable disparity in RhoprCAPA-2 potency (∼100-fold) observed between physiological assays involving inhibition of tubule fluid secretion (IC50 = 4 nM) [36] and heterologous receptor assays (EC50 = 385 nM) [35].
The use of naturally occurring plant-derived compounds for controlling insect pests remains an attractive alternative to potentially dangerous synthetic chemical compounds. One prospective plant-based compound, isoforms of the so-called jack bean urease (JBU) from the jack bean, Canavalia ensiformis, as well a derived peptide, Jaburetox-2Ec, have insecticidal effects on an array of insect species. In the Chagas’ disease vector, Rhodnius prolixus, some of the physiological effects attributed to these urease isoforms include inhibition of serotonin (5-HT)-stimulated fluid secretion by the Malpighian tubules (MTs). Here, we investigated whether the effects of these exogenous urease isoforms were targeting the neuroendocrine network involved in the anti-diuretic hormone (RhoprCAPA-2) signaling cascade. We show that pharmacological agents known to interfere with eicosanoid metabolite biosynthesis do not affect RhoprCAPA-2 inhibition of 5-HT-stimulated fluid secretion by MTs. In addition, we demonstrate that RhoprCAPA-2 inhibition of MTs is independent of extracellular or intracellular calcium. Using a heterologous system for analysis of receptor activation, we show that neither JBU nor Jaburetox-2Ec are agonists of the anti-diuretic hormone receptor, RhoprCAPAr1. Finally, activation of the receptor using sub-maximal doses of the natural ligand, RhoprCAPA-2, was not influenced by the presence of either JBU or Jaburetox-2Ec indicating that the urease isoforms do not compete with RhoprCAPA-2 for binding and activation of RhoprCAPAr1. Taken together, these results suggest that at least two distinct mechanisms leading to inhibition of fluid secretion by MTs exist in R. prolixus and, unlike the urease-related effects, the eicosanoid metabolite pathway is not involved in RhoprCAPA-2 mediated anti-diuresis.
Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration
2011, Journal of Biomolecular Screening
Citation Excerpt :
With this approach, genomic loci are identified (and reused) that favor the expression of the recombinant gene. The overexpression of transmembrane-spanning proteins such as GPCRs depends on a complex maturation process within the host cell, and this is known as a rate-limiting step to obtaining appropriate cell lines.31,32 The correct folding and posttranslational modification leads to stress, which counterselects for nonexpressing cells.
The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase “cut-and-paste” engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.
Heterologous investigation of metabotropic and ionotropic odorant receptors in ab3A neurons of Drosophila melanogaster
2023, Frontiers in Molecular Biosciences

View all citing articles on Scopus

View full text

Trends in Biotechnology

FocusHeterologous expression of G-protein-coupled receptors

Eur. J. Pharmacol.

J. Biol. Chem.

Cell

J. Biol. Chem.

Mol. Cell. Endocrinol.

J. Biol. Chem.

Eur. J. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

Mol. Cell. Endocrinol.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Biotechnol.

Life Sci.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell. Endocrinol.

Protein Expression Purific.

Anal. Biochem.

Curr. Opin. Biotechnol.

Trends Neurosci.

Anal. Biochem.

Focus
Heterologous expression of G-protein-coupled receptors