Sf9 cells: A versatile model system to investigate the pharmacological properties of G protein-coupled receptors

Schneider, Erich H.; Seifert, Roland

doi:10.1016/j.pharmthera.2010.07.005

Cited by 65 publications

(56 citation statements)

References 217 publications

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“…This may be due to the lack of mammalian G proteins coupling to the receptor protein thereby allosterically modulating the agonist binding site [40]. In the presence of G proteins, GPCRs may undergo conformational changes reaching a state to which agonists bind with high affinity [39]. When the rAdeR was cotransfected with mammalian G proteins, the affinity of adenine increased, not dramatically, but the difference was statistically significant (Fig.…”

Section: Discussionmentioning

confidence: 98%

“…Insect cells have previously been shown to be a suitable system for expressing AdeRs and to permit radioligand binding studies due to a very low background of [ 3 H]adenine binding (see Fig. 3) [3,38,39].…”

Section: Discussionmentioning

confidence: 99%

“…Four injections were performed before serum collection. To gain better specificity, additional antibodies were raised against the following peptides: [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] (N-term), 181-194 (extracellular loop 2 (ECL2)), 222-235 (intracellular loop 3 (ICL3)), 295-314 (C-terminal), and 313-331 (C-terminal). These antibodies were produced in two rabbits each by Thermo Scientific.…”

Section: Antibodiesmentioning

confidence: 99%

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The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

Knospe

Müller

Rosa

et al. 2013

Purinergic Signalling

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The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110 3.24 47 , were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenineinduced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Antibodiesmentioning

confidence: 99%

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The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

Knospe

Müller

Rosa

et al. 2013

Purinergic Signalling

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“…Insect cells are lacking suitable G proteins for mammalian GPCR signal transduction [32]. Therefore, the determined K D values represent adenine affinities for the nonactivated low-affinity state (for agonists) of the receptors.…”

Section: Discussionmentioning

confidence: 99%

“…The differences in affinity in both expression systems can be explained by the lack of suitable G proteins in Sf9 cells and the presence of these G proteins in CHO cells, which allosterically modulate the receptors. GPCRs have been shown to bind agonists with higher affinity when G proteins are present [32] because they can form a ternary complex with GPCRs, thus inducing an active conformation. In this state, the receptor's affinity for agonists is maximally increased [32].…”

Section: Discussionmentioning

confidence: 99%

Characterization of new G protein-coupled adenine receptors in mouse and hamster

Thimm

Knospe

Abdelrahman

et al. 2013

Purinergic Signalling

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The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation "P0-receptors" has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [ 3 H] adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure-activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC 50 9.77 nM) and the cAdeR (EC 50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC 50 6.24 nM) and was found to be additionally coupled to G q proteins.

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Enhanced early expression of membrane receptors with the Rous sarcoma virus promoter in baculovirus‐infected insect cells

Näreoja

Åkerman

Näsman

2012

Biotech and App Biochem

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The effect of the Rous sarcoma virus (RSV) long terminal repeat enhancer/promoter on expression levels of complementary DNAs (cDNAs) encoding seven transmembrane receptors was studied using the baculovirus expression vector system. Expression of the human α2B‐adrenoceptor (AR) cDNA under the control of the polyhedrin (POL) promoter produced up to 7.6 pmol/mg protein at 28 H post infection (p.i.) in Sf9 cells. The addition of the RSV promoter increased the expression to 11.6 pmol/mg protein. Dramatic increases in expression levels at early times were also obtained with the α2A‐AR, the M1 and M4 muscarinic receptors, and the orexin OX1 receptor. Analysis of the time‐dependent expression revealed that expression driven by the RSV promoter reaches almost maximum 24 H p.i. and that this promoter is superior to the often used POL promoter at early times p.i. when functional studies need to be performed. Functional enhancement of signaling as a result of early expression is demonstrated with the α2B‐AR and the OX1 receptor. Finally, enhanced green fluorescent protein fluorescence in living cells was used to monitor expression by various viral promoters. The results verified the early transcriptional activity of the RSV promoter, whereas the cytomegalovirus promoter was found to be poorly active in Sf9 cells.

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Sf9 cells: A versatile model system to investigate the pharmacological properties of G protein-coupled receptors

Cited by 65 publications

References 217 publications

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

Characterization of new G protein-coupled adenine receptors in mouse and hamster

Enhanced early expression of membrane receptors with the Rous sarcoma virus promoter in baculovirus‐infected insect cells

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