Expression of μ-, δ- and κ-opioid receptors in baculovirus-infected insect cells

Obermeier, Hans; Wehmeyer, Andrea; Schulz, Rüdiger

doi:10.1016/s0014-2999(96)00743-1

Cited by 25 publications

(14 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, these expression levels are highly dependent on receptor subtype: using exactly the same expression strategy, the values obtained for each of the five muscarinic receptor subtypes were comprised between 0.6 and 16 pmol/mg [81]. Less variability was observed for the three opioid receptors subtypes, which were weakly expressed by this system [75,76,[148][149][150][151]. Some parameters, such as the presence and the position of tags can also influence the expression level of a given receptor.…”

Section: Results Obtained With Insect Cellsmentioning

confidence: 93%

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

Sarramegna¹,

Talmont²,

Demange³

et al. 2003

CMLS, Cell. Mol. Life Sci.

216

201

View full text Add to dashboard Cite

G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs. However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available. This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells. After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results.

show abstract

Section: Results Obtained With Insect Cellsmentioning

confidence: 93%

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

Sarramegna¹,

Talmont²,

Demange³

et al. 2003

CMLS, Cell. Mol. Life Sci.

216

201

View full text Add to dashboard Cite

show abstract

“…While preparing this manuscript expression of rodent opioid receptors in insect cells was reported (54). Using homologous competition binding assays, agonist binding affinities in the nanomolar range were measured on High Five membrane preparations.…”

Section: Effect Of the Amino-terminal Histidine Tag On Receptor Exprementioning

confidence: 99%

Characterization of δ, κ, and μ Human Opioid Receptors Overexpressed in Baculovirus-infected Insect Cells

Massotte¹,

Baroche²,

Simonin³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

The cDNAs encoding human ␦ (hDOR), (hKOR) and (hMOR) opioid receptors were cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. Expression levels were optimized in Spodoptera frugiperda (Sf9) cells and were in the following order hMOR > hDOR > hKOR. The receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian cells. They also retained selectivity toward specific antagonists. The three receptors bound peptidic agonists with low affinity, suggesting that they might not be functionally coupled to intracellular effectors. Introduction of an amino-terminal hexahistidine tag decreased the levels of expression markedly. Only hMOR-his was expressed at a level allowing binding study, but no difference could be detected in the affinities of both agonists and antagonists compared with the nontagged protein. hMOR expression was also optimized in High Five cells leading to a further increase in protein production. The pharmacological profile was similar to the one obtained when the receptor was expressed in Sf9 cells. Our results show that the baculovirus expression system is suitable for large scale production of human opioid receptors.Opioid receptors and endogenous opioid peptides form a neuromodulatory system that plays a major role in the control of nociceptive pathways. The opioid system is not only a key element for pain perception but also modulates affective behavior as well as neuroendocrine physiology and controls autonomic functions such as respiration, blood pressure, thermoregulation, and gastrointestinal motility. It affects locomotor activity and could be involved in learning and memory. The receptors are otherwise targets for exogenous narcotic drugs, a major class of drugs of abuse.Genes coding for ␦, , and opioid receptor subtypes have now been identified and isolated from different vertebrates. Primary sequence analysis indicated that opioid receptors belong to the G-coupled receptor family whose structure shows a seven-transmembrane domain topology (for review, see Refs. 1 and 2). Engineering of protein chimeras together with generation of point mutants led to a better identification of the receptor regions interacting specifically with different ligands and/or involved in the signal transduction pathway. However, almost no structural data are available but only a few models based on rhodopsin and bacteriorhodopsin structures (2-5). Recently a new insight was brought by a model drawn without template for the ␦ receptor (6).The baculovirus expression system is extremely efficient to produce large amounts of mammalian proteins. Post-translational modifications are identical to those observed in mammalian cells with the exception of glycosylation, which is mostly of the high mannose type. Several G-coupled receptors, some of human origin, have already been expressed successfully under a functional state including adrenergic; ␥-aminobuty...

show abstract

“…In order to perform structural and functional studies of opioid receptors, heterologous expression attempts have already been made, in the yeast Pichia pastoris [2] and in insect cells [3,4]. Based on the proven ability of Escherichia coli to accommodate functional GPCRs in the periplasmic membrane [5–7], we undertook to overexpress human opioid receptors in E. coli , with the goal of determining their functional parameters in a system totally deprived of endogenous interference.…”

mentioning

confidence: 99%

Expression of δ, κ and µ human opioid receptors in Escherichia coli and reconstitution of the high‐affinity state for agonist with heterotrimeric G proteins

Stanasila

Massotte

Kieffer

et al. 1999

European Journal of Biochemistry

View full text Add to dashboard Cite

Human opioid receptors of the d, m and k subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors´cell 21 or 0.5 pmol´mg 21 membrane protein. Binding of [ 3 H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, K D , of 2.5 nm, 0.66 nm and 0.75 nm for human d, m and k opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coliexpressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for m opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.Keywords: opioid receptor; GTP-binding protein; expression in Escherichia coli; reconstitution; pharmacology.Opioid receptors belong to the wide family of G protein coupled receptors (GPCRs). Their predicted topology is that of a polytopic membrane protein with seven membrane-spanning segments, an extracellular N-terminus and an intracellular C-terminus. Opioid receptors and their endogenous ligands, opioid peptides, form a neuromodulatory system that is involved in stress-induced analgesia, affects locomotive activity and regulates neuroendocrine physiology and autonomic functions such as respiration, blood pressure and gastrointestinal motility. The opioid system has also been shown to play a role in learning and memory and possibly in the modulation of immune function. Pharmacological studies allowed the classification of opioid receptors into three subtypes, d, m and k, each possessing distinct ligand selectivities and distribution profiles, but little is known to date of their structure or functional mechanisms [1].In order to perform structural and functional studies of opioid receptors, heterologous expression attempts have already been made, in the yeast Pichia pastoris [2] and in insect cells [3,4]. Based on the proven ability of Escherichia coli to accommodate functional GPCRs in the periplasmic membrane [5±7], we undertook to overexpress human opioid receptors in E. coli, with the goal of determining their functional parameters in a system totally deprived of endogenous interference. All the components of the complex cascade involved in GPCRs signal transduction, from the heterotrimeric G proteins to the proteins responsible for receptor desensitization and internalization (ar...

show abstract

Expression of μ-, δ- and κ-opioid receptors in baculovirus-infected insect cells

Cited by 25 publications

References 33 publications

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

Characterization of δ, κ, and μ Human Opioid Receptors Overexpressed in Baculovirus-infected Insect Cells

Expression of δ, κ and µ human opioid receptors in Escherichia coli and reconstitution of the high‐affinity state for agonist with heterotrimeric G proteins

Contact Info

Product

Resources

About