Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand

Desensitization of recombinant human thrombin receptors expressed in Sf9 insect cells was compared with native thrombin receptors in megakaryoblast erythroleukaemia (HEL) cells. Addition of thrombin (2 units/ml) or agonist peptide SFLLRN (10 μM) to HEL cells, or to Sf9 cells infected with recombinant baculovirus containing the thrombin receptor cDNA, produced an increase in the free cytosolic Ca2+ concentration ([Ca2+]i) as measured by fura-2. The response in HEL cells was transient, reflecting a rapid homologous desensitization. In contrast, [Ca2+]i in Sf9 cells expressing the thrombin receptor increased rapidly to a peak value that slowly declined, but remained elevated for at least 12 min following stimulation by thrombin. The sustained [Ca2+]i response to thrombin was not reversed by washout of thrombin or by subsequent addition of hirudin. Pretreatment of Sf9 cells with either thrombin (2 units/ml) or SFLLRN (10 or 50 μM) for 5 min produced a shift in the ED50 for SFLLRN (added 10 min after washout) from 0.4 μM to 20 and 7 μM, respectively. Thus, desensitization of thrombin receptors expressed in Sf9 cells occurs slowly and reflects a decrease in receptor affinity. The sustained [Ca2+]i response in Sf9 cells stimulated by thrombin may reflect continuous activation by the tethered ligand. To test this hypothesis, the effect of protease treatment during the sustained phase of the response was examined. Addition of either aminopeptidase M or thermolysin reversed the sustained response to SFLLRN, but only thermolysin reversed the sustained response to thrombin. Thermolysin had no effect on the change in [Ca2+]i observed following carbachol stimulation of Sf9 cells expressing the M5 muscarinic receptor. Furthermore, following thermolysin treatment, the cells remained responsive to a subsequent application of SFLLRN. These results demonstrate that the tethered ligand remains active for extended periods of time after thrombin stimulation and suggests that further hydrolysis by extracellular proteases may represent an important mechanism of rapid receptor deactivation.

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Thrombin receptors modulate insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation in 1321N1 astrocytoma cells

Biochemical Journal ◽

10.1042/bj3170347 ◽

1996 ◽

Vol 317 (2) ◽

pp. 347-351 ◽

Cited By ~ 25

Author(s):

Ian H. BATTY ◽

C. Peter DOWNES

Keyword(s):

Insulin Receptors ◽

Thrombin Receptor ◽

Astrocytoma Cells ◽

Thrombin Receptors ◽

Transient Activation ◽

Ligand Type ◽

Insulin Receptor Signalling ◽

Human Thrombin ◽

Tethered Ligand ◽

1321N1 Astrocytoma

Thrombin and insulin receptor signalling via phosphoinositide (PI)-specific phospholipase C (PLC) and PI 3-kinase was studied in [3H]inositol-labelled 1321N1 cells. Thrombin stimulated a dramatic, transient activation of PLC which is probably mediated via receptors of the ‘tethered-ligand’ type, since it was both reproduced by, and abolished following, pretreatment of cells with a synthetic peptide (SFLLRN) corresponding to the ligand domain of the human thrombin receptor. However, neither thrombin nor SFLLRN stimulated PI 3-kinase. By contrast, insulin did not influence [3H]InsP3 concentrations but stimulated accumulation of [3H]PtdIns(3,4,5)P3 and [3H]PtdIns(3,4)P2, the relative steady-state concentrations of which may indicate degradation of [3H]PtdIns(3,4,5)P3 by 5- and 3-phosphatases. The independent coupling of thrombin and insulin receptors to PLC and PI 3-kinase respectively in 1321N1 cells allowed interactions between these systems to be examined. Thus insulin-stimulated [3H]PtdIns(3,4,5)P3 accumulation was attenuated on co-stimulation of the thrombin receptor, whereas concentrations of [3H]PtdIns(3,4)P2 were transiently enhanced but then reduced. These results indicate that thrombin receptors in 1321N1 cells do not activate PI 3-kinase, but can modulate signalling by this enzyme.

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Properties of single Drosophila Trpl channels expressed in Sf9 insect cells

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.1.c27 ◽

1997 ◽

Vol 272 (1) ◽

pp. C27-C34 ◽

Cited By ~ 35

Author(s):

D. L. Kunze ◽

W. G. Sinkins ◽

L. Vaca ◽

W. P. Schilling

Keyword(s):

Transient Receptor Potential ◽

Insect Cells ◽

Single Channel ◽

Recombinant Baculovirus ◽

Sf9 Cells ◽

Single Channels ◽

Channel Activity ◽

Whole Cell ◽

Bradykinin Receptor ◽

Sf9 Insect Cells

The transient receptor potential (trp)-like (trpl) gene is thought to encode an ion channel important for signal transduction in Drosophila photoreceptor cells. Consistent with this hypothesis, heterologous expression of the trpl-encoded protein (Trpl) is associated with the appearance of an outwardly rectifying, nonselective cation current. In the present study, single channels were recorded in cell-attached, inside-out, and outside-out membrane patches from Sf9 insect cells infected with recombinant baculovirus-containing trpl cDNA under control of the polyhedrin promoter. The single-channel current-voltage relationship was linear from -100 to +80 mV with a slope conductance of 89-110 pS. The probability of opening was voltage sensitive, increasing at positive potentials contributing to the outwardly rectifying properties of the whole cell currents. The single channels 1) were never observed in Sf9 cells infected with recombinant baculovirus containing the B2 bradykinin receptor cDNA or in noninfected Sf9 cells; 2) appear at the same time postinfection as the Trpl whole cell current; 3) were nonselective with respect to Na+, Ca2+, and Ba2+; 4) were blocked by 1-2 mM La3+ and Gd3+ (but not 10 microM); and 5) were blocked by 4-8 mM Mg2+. The single Trpl channel activity increased spontaneously with time after patch formation, and the activity was further increased by application of bradykinin to cells expressing both the B2 bradykinin receptor and the Trpl protein. These results suggest that this single-channel activity reflects expression of the Trpl protein and provides conclusive evidence that trpl encodes a nonselective cation channel consistent with its proposed role in Drosophila phototransduction.

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

FEBS Letters ◽

10.1016/0014-5793(92)81354-o ◽

1992 ◽

Vol 311 (1) ◽

pp. 7-11 ◽

Cited By ~ 24

Author(s):

Subhash Vasudevan ◽

Louis Premkumar ◽

Sally Stowe ◽

Peter W. Gage ◽

Helmut Reiländer ◽

...

Keyword(s):

Ion Channels ◽

G Proteins ◽

Acetylcholine Receptor ◽

Insect Cells ◽

Muscarinic Acetylcholine Receptor ◽

Recombinant Baculovirus ◽

Muscarinic Acetylcholine ◽

Sf9 Insect Cells

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Ins(1,4,5)P3 activates Drosophila cation channel Trpl in recombinant baculovirus-infected Sf9 insect cells

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.5.c1332 ◽

1995 ◽

Vol 269 (5) ◽

pp. C1332-C1339 ◽

Cited By ~ 37

Author(s):

Y. Dong ◽

D. L. Kunze ◽

L. Vaca ◽

W. P. Schilling

Keyword(s):

Signal Transduction ◽

Insect Cells ◽

Recombinant Baculovirus ◽

Cation Channel ◽

Physical Interaction ◽

Release Channel ◽

Pipette Solution ◽

Whole Cell ◽

Sf9 Insect Cells ◽

Inositol 1,4,5 Trisphosphate

The trp-like (trpl) gene product (Trpl) is thought to form a nonselective cation channel important for signal transduction in Drosophila photoreceptor cells. This channel may be the insect homologue of mammalian channels involved in Ca2+ signal transduction. To determine the mechanism of receptor-mediated activation of Trpl, whole cell membrane currents were examined in Sf9 insect cells after infection with recombinant baculovirus. Stimulation by bradykinin increased whole cell Trpl currents three- to fivefold. Similar activation of Trpl was observed by inclusion of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the pipette solution during whole cell recordings. These currents were 1) not seen in noninfected cells or in cells expressing only the B2 receptor, 2) mimicked by D-myo-inositol 2,4,5-trisphosphate, and 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate, 3) not seen with D-myo-inositol 1,4-bisphosphate or D-myo-inositol 1,3,4,5-tetrakisphosphate, and 4) blocked by heparin, but not by de-N-sulfated heparin. In contrast, Trpl currents were unaffected by thapsigargin. These results demonstrate that the Trpl cation channel is activated by Ins(1,4,5)P3 in a heparin-sensitive fashion. Regulation of channel activity by Ins(1,4,5)P3 may occur by a number of mechanisms, including direct binding of Ins(1,4,5)P3 to the Trpl channel or direct physical interaction between the Ins(1,4,5)P3 receptor/Ca(2+)-release channel of the endoplasmic reticulum and the Trpl protein.

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Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain

Biochemical Journal ◽

10.1042/bj3050277 ◽

1995 ◽

Vol 305 (1) ◽

pp. 277-283 ◽

Cited By ~ 22

Author(s):

H Boudin ◽

A Grauz-Guyon ◽

M P Faure ◽

P Forgez ◽

A M Lhiaubet ◽

...

Keyword(s):

Cerebral Cortex ◽

Molecular Mass ◽

Cho Cells ◽

Insect Cells ◽

Rat Cerebral Cortex ◽

Molecular Forms ◽

Sf9 Cells ◽

Neurotensin Receptor ◽

Sf9 Insect Cells ◽

Peptide Antibody

In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied in the visualization of the receptor.

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Effects of thrombin receptor activating peptide on phosphoinositide hydrolysis and protein kinase C activation in cultured rat aortic smooth muscle cells: evidence for “tethered-ligand” activation of smooth muscle cell thrombin receptors

Biochemical Pharmacology ◽

10.1016/0006-2952(93)90297-a ◽

1993 ◽

Vol 45 (8) ◽

pp. 1577-1582 ◽

Cited By ~ 10

Author(s):

Maria L. Webb ◽

David S. Taylor ◽

Christopher J. Molloy

Keyword(s):

Smooth Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Thrombin Receptor ◽

Ligand Activation ◽

Aortic Smooth Muscle ◽

Kinase C ◽

Thrombin Receptors ◽

Tethered Ligand ◽

Protein Kinase C Activation

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Relationship between oxygen uptake rate and time of infection of Sf9 insect cells infected with a recombinant baculovirus

Cytotechnology ◽

10.1007/bf00762390 ◽

1994 ◽

Vol 15 (1-3) ◽

pp. 157-167 ◽

Cited By ~ 40

Author(s):

T. K. Kathy Wong ◽

Lars K. Nielsen ◽

Paul F. Greenfield ◽

Steven Reid

Keyword(s):

Oxygen Uptake ◽

Uptake Rate ◽

Insect Cells ◽

Oxygen Uptake Rate ◽

Recombinant Baculovirus ◽

Sf9 Insect Cells ◽

Time Of Infection

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High-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells

Biochemical Journal ◽

10.1042/bj3300959 ◽

1998 ◽

Vol 330 (2) ◽

pp. 959-966 ◽

Cited By ~ 13

Author(s):

K. Michael SIEVERT ◽

S. David THIRIOT ◽

H. Robert EDWARDS ◽

E. Arnold RUOHO

Keyword(s):

Synaptic Vesicle ◽

High Efficiency ◽

Insect Cells ◽

Expression System ◽

Broad Band ◽

Recombinant Baculovirus ◽

Baculovirus Expression System ◽

Sf9 Cells ◽

Monoamine Transporter ◽

Infected Cells

The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8×106 transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1 l culture of infected cells produced approx. 15 nmol (900 μg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 μM tetrabenazine and 10 μM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63 kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.

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