High-efficiency expression of mammalian β-adrenergic receptors in baculovirus-infected insect cells

George, Shaji T.; Arbabian, Marty; Ruoho, Arnold E.; Kiely, Joan; Malbon, Craig C.

doi:10.1016/0006-291x(89)91114-5

Cited by 64 publications

(17 citation statements)

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“…Preparation of Recombinant ␤ 2 AR and Insulin Receptor-Recombinant hamster ␤ 2 -adrenergic receptor was expressed using the baculovirus-Sf9 insect cell expression system (5) and purified by affinity, HPLC, and lectin chromatography (6). Recombinant human insulin receptor (rIR) was purified by lectin chromatography (7) from Chinese hamster ovary (CHO) T cells, which stably overexpress the human insulin receptor (8), or from COS-1 cells, which were transiently transfected with the human insulin receptor cDNA (9).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of Tyrosyl Residues 350/354 of the β-Adrenergic Receptor Is Obligatory for Counterregulatory Effects of Insulin

Karoor

Baltensperger

Paul

et al. 1995

Journal of Biological Chemistry

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Insulin stimulates a loss of function and increased phosphotyrosine content of the ␤ 2 -adrenergic receptor in intact cells, raising the possibility that the ␤ 2 -receptor itself is a substrate for the insulin receptor tyrosine kinase. Phosphorylation of synthetic peptides corresponding to cytoplasmic domains of the ␤ 2 -adrenergic receptor by the insulin receptor in vitro and peptide mapping of the ␤ 2 -adrenergic receptor phosphorylated in vivo in cells stimulated by insulin reveal tyrosyl residues 350/354 and 364 in the cytoplasmic, C-terminal region of the ␤ 2 -adrenergic receptor as primary targets. Mutation of tyrosyl residues 350, 354 (double mutation) to phenylalanine abolishes the ability of insulin to counterregulate ␤-agonist stimulation of cyclic AMP accumulation. Phenylalanine substitution of tyrosyl reside 364, in contrast, abolishes ␤-adrenergic stimulation itself.The counterregulatory effects of insulin and catecholamines on carbohydrate and lipid metabolism are well known, whereas the molecular details of insulin regulation of G-protein-linked pathways remain unknown. Upon ligand binding, the insulin receptor displays tyrosine kinase activity which is critical to signal propagation (1). G-protein-linked receptors (like the ␤ 2 -adrenergic receptor, ␤ 2 AR), 1 in contrast, activate adenylyl cyclase via G s and are phosphorylated during agonist-induced desensitization (2, 3). We demonstrated recently that the well known counterregulatory actions of insulin included loss of function and increased phosphorylation of the ␤ 2 -adrenergic receptor (4). In the current study the structural basis for these counterregulatory effects of insulin exerted on the ␤ 2 -adrenergic receptor is explored. MATERIALS AND METHODSPreparation of Recombinant ␤ 2 AR and Insulin Receptor-Recombinant hamster ␤ 2 -adrenergic receptor was expressed using the baculovirus-Sf9 insect cell expression system (5) and purified by affinity, HPLC, and lectin chromatography (6). Recombinant human insulin receptor (rIR) was purified by lectin chromatography (7) from Chinese hamster ovary (CHO) T cells, which stably overexpress the human insulin receptor (8), or from COS-1 cells, which were transiently transfected with the human insulin receptor cDNA (9).Phosphorylation of ␤ 2 AR in Vivo-In vivo, DDT 1 MF-2 hamster vas deferens smooth muscle cells were cultured in Dulbecco's modified Eagle's medium (DMEM), metabolically labeled in phosphate-free DMEM containing 0.5% fetal bovine serum and [ 32 P]orthophosphate (1 mCi/ml) for 4 h at 37°C (4). At the end of the 4-h incubation, insulin or vehicle was added as indicated in the figure legends. To terminate phosphorylation, cells were washed and then lysed. The lysis buffer was composed of Triton X-100 (1%), sodium dodecyl sulfate (0.1%), dithiothreitol (6.0 M), aprotinin (5 g/ml), leupeptin (5 g/ml), bacitracin (100 g/ml), benzamidine (100 g/ml), sodium orthovanadate (2 mM), NaCl (150 mM), EDTA (5 mM), NaF (50 mM), sodium pyrophosphate (40 mM), KH 2 PO 4 (50 mM), sodium molybdate (10 mM), and ...

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Section: Methodsmentioning

confidence: 99%

Phosphorylation of Tyrosyl Residues 350/354 of the β-Adrenergic Receptor Is Obligatory for Counterregulatory Effects of Insulin

Karoor

Baltensperger

Paul

et al. 1995

Journal of Biological Chemistry

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“…Recombinant hamster ␤ 2 -adrenergic receptor (r␤AR) was expressed using the baculovirus-Sf9 insect cell expression system (7) and purified by affinity, HPLC, and lectin chromatography (8). Recombinant human insulin receptor (rIR) was purified by lectin chromatography (9) from Chinese hamster ovary (CHO)-T cells, which stably overexpress the human insulin receptor (10) or from COS-1 cells, which were transiently transfected with the human insulin receptor cDNA (11).…”

Section: Recombinant ␤ 2 Ar Insulin Receptor and Purified Igf-i Recmentioning

confidence: 99%

The β-Adrenergic Receptor Is a Substrate for the Insulin Receptor Tyrosine Kinase

Baltensperger

Karoor

Paul

et al. 1996

Journal of Biological Chemistry

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101

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Protein phosphorylation plays a prominent role in the regulation of cell signaling. A populous family of G-protein-linked receptors mediate activation of a diverse class of effectors, such as adenylyl cyclase, phospholipase C, and various ion channels, via a less populous class of G-proteins (1). These G-proteinlinked receptors share many features, including regulation via protein phosphorylation (2, 3). Insulin counter-regulates the action of ␤-adrenergic catecholamine stimulation, at a point proximal to the ␤-adrenergic receptor (4). Cells stimulated by insulin show increased phosphotyrosine content and loss of function of ␤ 2 -adrenergic receptors (4). The insulin receptor, upon ligand binding, expresses intrinsic tyrosine kinase activation (5), raising the intriguing hypothesis that G-proteinlinked receptors and intrinsic tyrosine kinase growth receptors may interact directly, the former a substrate for the latter. Recently, we have deduced structural information on sites of phosphotyrosine labeling in vivo (6). In the current work, we directly test the hypothesis that the ␤ 2 -adrenergic receptor is a substrate for growth factor receptor tyrosine kinase, using recombinant receptors and a defined reconstitution assay in vitro. The results demonstrate that growth factor tyrosine kinase receptors (e.g. insulin receptor and the IGF-I 1 receptor) can directly phosphorylate a G-protein-linked receptor. MATERIALS AND METHODSRecombinant ␤ 2 AR, Insulin Receptor, and Purified IGF-I ReceptorRecombinant hamster ␤ 2 -adrenergic receptor (r␤AR) was expressed using the baculovirus-Sf9 insect cell expression system (7) and purified by affinity, HPLC, and lectin chromatography (8). Recombinant human insulin receptor (rIR) was purified by lectin chromatography (9) from Chinese hamster ovary (CHO)-T cells, which stably overexpress the human insulin receptor (10) or from COS-1 cells, which were transiently transfected with the human insulin receptor cDNA (11). The IGF-I receptor (IGF-IR) was prepared from lectin chromatography of cell extracts of human osteogenic sarcoma, a cell line replete in IGF-IR (11).Phosphorylation of ␤ 2 AR in Vitro-In vitro, phosphorylation of the r␤AR was achieved in a reconstitution assay, whereby 5 l (100 -200 fmol) of rIR and 20 l (10 -20 pmol) of ␤ 2 AR (or 20 l of buffer) were incubated at 22°C in a final volume of 45 l in (final concentrations) 25 mM Tris/HCl (pH 7.4), 50 mM NaCl, 10 mM MgCl 2 , 3 mM MnCl 2 , 100 M Na 3 VO 4 , 1 mM dithiothreitol, 0.1% (w/v) Triton X-100. 5 l of [␥-32 P]ATP (40 Ci/mmol) were then added to give a final concentration of 5 M ATP. The phosphorylation reaction was terminated at 30 min by the addition of 50 l of 2ϫ concentrated Laemmli sample buffer containing 100 mM dithiothreitol. Proteins were denatured for 5 min at 95°C and then separated by SDS-PAGE. Phosphorylated proteins were made visible by exposing the dried gel to X-Omat AR film (Kodak). The amount of label incorporated into r␤ 2 AR by insulin-stimulated rIR in this detergent-dispersed reconstitution syst...

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“…The baculovirus expression system has been employed to produce higher levels of several G-protein-coupled receptors. 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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confidence: 99%

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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High-efficiency expression of mammalian β-adrenergic receptors in baculovirus-infected insect cells

Cited by 64 publications

References 10 publications

Phosphorylation of Tyrosyl Residues 350/354 of the β-Adrenergic Receptor Is Obligatory for Counterregulatory Effects of Insulin

Phosphorylation of Tyrosyl Residues 350/354 of the β-Adrenergic Receptor Is Obligatory for Counterregulatory Effects of Insulin

The β-Adrenergic Receptor Is a Substrate for the Insulin Receptor Tyrosine Kinase

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

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