Muscarinic acetylcholine receptor produced in recombinant baculovirus infected <i>Sf9</i> insect cells couples with endogenous G‐proteins to activate ion channels

Vasudevan, Subhash G.; Premkumar, Louis S.; Stowe, Sally; Gage, Peter W.; Reiländer, Helmut; Chung, Shin-Ho

doi:10.1016/0014-5793(92)81354-o

Cited by 32 publications

(11 citation statements)

References 16 publications

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“…Indirect evidence for such a GTP-binding protein has been reported (Mumby et al, 1986;Graber et al, 1992), but up to now a detailed analysis has never been made. Although the immunoblotting experiments suggest that G,,o subunits are absent in insect cells, there has been a recent report that rat muscarinic M, receptors, produced in Sf9 cells, can couple with endogenous pertussis-toxin-sensitive GTP-binding proteins to modulate calcium-activated K' channels (Vasudevan et al, 1992). It is interesting that the fi subunit of the endogenous insect GTP-binding protein differs in apparent molecu-lar mass from mammalian and avian p subunits, although it cross-reacts with the antibody which is directed against the C-terminus of the subunit.…”

Section: Western-blot Analysis Of Endogenous Gtp-binding Proteins Fromentioning

confidence: 99%

Human β₂‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase

Kleymann

Boege²,

Hahn³

et al. 1993

European Journal of Biochemistry

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Spodoptera frugiperda insect cells (Sf9) containing the stably integrated human P,-adrenergic receptor gene under the control of the baculovirus IE1 promoter expressed up to 350000 human receptorskell. The number of receptors did not change with cell density or age of culture. The adrenergic receptors overexpressed in the insect cells were functional with respect to their ligand binding and signalling properties. Coupling of the receptors to endogenous GTP-binding proteins is demonstrated by hormone-dependent stimulation of GTPase and adenylyl cyclase activity in the transformed insect cells. Western-blot analysis revealed that the endogenous GTP-binding protein appears to be of the heterotrimeric type. Antibodies raised against the mammalian a subunit of stimulatory GTP-binding proteins cross-react with the insect a subunit of GTP-binding proteins, which also exhibits the same apparent molecular mass as its mammalian counterpart. The p subunit of GTP-binding proteins from insect cells reacts with anti-peptide serum directed against the Cterminal amino acids of the mammalian / 3 subunit of GTP-binding proteins, but is about =2 kDa larger than that of the P subunit of GTP-binding proteins from bovine brain. Exposure of the transformed insect cells to L-isoproterenol rapidly induces uncoupling and internalization of 30 % of the heterologously expressed receptors. In contrast to the situation in mammalian cells, prolonged exposure of the agonist (24 h) does not result in down regulation of the remaining 70% of the receptors.Mammalian P-adrenergic receptors CB,AR) have been introduced into several heterologous expression systems for extensive pharmacological and biochemical analysis, including Escherichia coli (Marullo et al., 1989(Marullo et al., ,1990, yeast (King et al., 1990), Xenopus oocytes (Frielle et al., 1988;Kobilka et al., 1988), several mammalian cell lines Fraser et al., 1987;Hen et al., 1989;Lohse, 1992;Rands et al., 1990;Wang et al., 1989) and insect cells, using the baculovirus expression system (George et al., 1989;Reiliinder et al., 1991). Evidence has been presented that mammalian P,AR functionally couple to insect adenylyl cyclase in cells of Spodoptera frugiperda (Sf9) . However, little is known about the signal-transduction pathway in these invertebrate cells. Despite the several advantages which have made the baculovirus expression system a popular method of engineering richer sources of recombinant protein, analysis of insect proteins that are known to functionally interact with the foreign proteins has been hampered. This is because the transient expression that occurs after the cells have been infected with the recombi- nant baculovirus is a cytotoxic event that stops the transcription and translation of insect proteins and ultimately kills the cells. To facilitate further biochemical and biophysical characterization of P,AR, taking advantage of the stability and robust nature of insect ovarian cells, we have used the promoter of the IE1 gene (an immediate early gene of the baculovirus Aut...

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Section: Western-blot Analysis Of Endogenous Gtp-binding Proteins Fromentioning

confidence: 99%

Human β₂‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase

Kleymann

Boege²,

Hahn³

et al. 1993

European Journal of Biochemistry

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“…Still, functional coupling to endogenous G proteins was depicted for receptors known to be coupled to pertussissensitive G proteins such as human serotonin 5HT 1A (46) and 5HT 1B (45). Similarly, Vasudevan et al (49) reported that the rat muscarinic acetylcholine receptor subtype m 3 activates potassium channels, and Oker-Blom et al found (50) that the human ␣ 2 C-C4 adrenergic receptor inhibits forskolin-stimulated adenylyl cyclase in Sf9 cells. These observations suggest that receptors coupled to G o and/or G i may interact with Sf9 endogenous G␣ subunits.…”

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

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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...

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“…Recently, several groups have reported the purification of G-protein-coupled receptors from insect cells infected by a recombinant baculovirus (George et al, 1989;Parker et al, 1991 ;Reilander et al, 1991 ;Vasudevan et al, 1991). They also showed that the purified receptors were able to couple with Gproteins in reconstituted systems (Parker et al, 1991 ;Reilander et al, 1991) as well as in in vivo (Kleyman et al, 1993;Vasudevan et al, 1992).…”

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Characterization of the Rat m3 Muscarinic Acetylcholine Receptor Produced in Insect Cells Infected with Recombinant Baculovirus

Vasudevan¹,

Hulme²,

Bach³

et al. 1995

European Journal of Biochemistry

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ThLe m3 muscarinic acetylcholine receptor from rat heterologously produced in insect cells after infection with a recombinant baculovirus has an apparent molecular mass of approximately 75 kDa. Polyclonal antibodies raised against a carboxy-terminal nonapeptide that is unique to the m3 subtype can detect the receptors produced in the insect cells by Western blot and can also immunoprecipitate solubilized receptor. Immunofluorescence microscopy as well as electron microscopy revealed that the receptor was located intracellularly, visualized as a ring around the nucleus of the infected insect cells. Solubilization of the receptor was accomplished with digitonin which was added in increments (over 10 min) to a final concentration of 0.8%) (masshol.). The solubilized receptor is unstable when the ligand-binding site is not protected by a ligand. Here the low-affinity ligand propylbenzilylcholine (= 10 nM) has demonstrable protective ability during solubilization, but the usefulness of this ligand is limited by a very slow off rate. From the behaviour of the solubilized receptor during DEAE-Sephacel chromatography and lectin-affinity chromatography it can be deduced that the receptor produced in insect cells is heterogenously glycosylated in the producing insect cells.Keywords. Muscarinic receptor; baculovirus, expression; solubilization; glycosylation; purification.The muscarinic acetylcholine receptors (mAChR) are singlechain polypeptides predicted to traverse the membrane seven times in an a-helical configuration (for review see Hulme et al., 1989Hulme et al., , 1990 Hose:y, 1992). The receptor family modulates a variety of intracellular events (e.g. opening of K' channels, activation or inhibition of enzymes that control levels of intracellular second messengers such as cyclic AMP and inositol trisphosphate) upon agonist activation via the appropriate guanosinenucleotide-binding regulatory proteins (G-protein ; Ashkenazi et al., 1987;Fukuda et al., 1988Fukuda et al., , 1989Pfaffinger et al., 1985;Neher et al., 1988;Yatani et al., 1990). Cloning and nucleotide sequencing have idlentified five distinct subtypes of muscarinic receptors (ml -m5) from mammalian sources Fukuda at al., 1987;Bonner et al., 1987Bonner et al., , 1988Braun et al., 1987; Kubo et ,aL, 1986a,b; Liao et al., 1989; Peralta et al., 1987b). The receptors are not abundant in mammalian organs and their biochemical studies are complicated by the occurence of mixed subtypes in tissues and also cell lines. This makes the accurate identification of subtype-specific agents virtually impossible . To circumvent these prob- Abbreviations. AbhO, 3-(2'-aminobenzhydryloxy)tropane; BCIP, 5-bromo-4-chloro-3-indolyl phosphate p-toluidinium salt ; Con A, concanavalin A ; mAChR, muscarinic acetylcholine receptor; m3, muscarinic acetylcholine recepto:r subtype 3 ; m.o.i., multiplicity of infection; pfu, plaque-forming unit; PhMeSO,F, phenylmethylsulfonyl fluoride; Sf9 cells, Spodopteru frugiperdu insect cells ; WGA, wheatgerm agglutinin; G-protein, guanosine-nucle...

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Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G‐proteins to activate ion channels

Cited by 32 publications

References 16 publications

Human β₂‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase

Human β₂‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase

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

Characterization of the Rat m3 Muscarinic Acetylcholine Receptor Produced in Insect Cells Infected with Recombinant Baculovirus

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Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G‐proteins to activate ion channels

Cited by 32 publications

References 16 publications

Human β2‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase

Human β2‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase

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

Characterization of the Rat m3 Muscarinic Acetylcholine Receptor Produced in Insect Cells Infected with Recombinant Baculovirus

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Human β₂‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase

Human β₂‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase