Sf9 Cell Membranes Research Articles

Recombinant scorpion insect excitatory toxin BmK IT accelerates the growth of insect Spodoptera frugiperda 9 cells

Several scorpion insect toxins are selectively active on the lepidopterous and dipterous insects. The gene encoding insect excitatory neurotoxin (BmK IT) from the scorpion Buthus martensii Karsch was expressed in Escherichia coli BL21(DE3) at a high level of 3 mg/0.5 L using the prokaryotic expression system pTWIN1. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), whole-cell patch-clamp technique and immunofluorescence assays were used to evaluate the toxicity of rBmK IT to insect Spodoptera frugiperda 9 (Sf9) cells and to analyze the potential mechanism of this toxicity. rBmK IT accelerated the growth of Sf9 cells in a dose-dependent manner. Voltage-gating sodium channel activity could not be detected in Sf9 cells using a whole-cell patch-clamp technique. However, immunofluorescence analysis clearly showed co-localization of tetrodotoxin (TTX) and rBmK IT on the Sf9 cell membrane, which demonstrated that rBmK IT could bind to and act on the voltage-gated sodium channels on the Sf9 cells by the high affinity action power. The findings presented in this study are essential for further study of this peptide.

Millimolar Mn2+ influences agonist binding to 5-HT1A receptors by inhibiting guanosine nucleotide binding to receptor-coupled G-proteins

Manganese is an essential trace element but its overexposure causes poisoning (called manganism) that shares several symptoms with Parkinson's disease, but with a mechanism that is still not well understood: in addition to involvement of the dopaminergic system, both serotonergic and peptiergic systems have been implicated. In the present report we have studied the influence of Mn2+ on 5-HT1A receptor signaling complexes in rat brain and found that Mn2+ in millimolar concentration caused an increase of high-affinity agonist binding to rat hippocampal membranes in comparison with experiments in the presence of Mg2+, but not in rat cortical membranes and in Sf9 cell membranes expressing 5-HT1A receptors and Gi1 heterotrimers. Activation of G proteins with 30μM GTPγS turned all 5-HT1A receptors in these preparations into a low-affinity state for agonist binding in the presence of 1mM Mg2+, but not in the presence of 1mM Mn2+ in rat hippocampal membranes. However, if 1μM GTPγS was used for G protein activation, a substantial amount of high affinity agonist binding was detected in the presence of Mn2+ also in cortical membranes and Sf9 cells, but not with Mg2+ or EDTA. Comparison of the abilities of GDP and GTPγS to modulate high affinity agonist binding to 5-HT1A receptors indicated that both nucleotides were almost 10-fold less potent in the presence of MnCl2 compared to MgCl2. This means that by inhibiting guanosine nucleotide binding to G proteins in complex with 5-HT1A receptors, Mn2+ acts as an enhancer for agonist binding and signal transduction. As the influence of Mn2+ resembles the hypersensitivity of dopaminergic system in Parkinsonial models, it can be proposed that at least some symptoms of manganism are connected with a change of signal transduction complex caused by manganese-nucleotide complexes.

Baculovirus surface display of E envelope glycoprotein of Japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models

Japanese encephalitis virus (JEV), an important pathogen in humans and animals, is capable of causing febrile syndrome, encephalitis and death. The E glycoprotein of JEV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. In this work, we have succeeded in construction of one recombinant baculovirus BacSC-E expressing His6-tagged E with the baculovirus envelope protein gp64 TM and CTD. After infection, E was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E glycoprotein was successfully displayed on the viral surface. Vaccination of mouse and swine with recombinant baculovirus BacSC-E successfully induced neutralizing antibody response and protective immunity toward a lethal challenge of the JEV. Taken all findings together, our results indicate that the recombinant baculovirus BacSC-E can be a potential vaccine against JEV infections. This finding provides valuable information for establishing subunit vaccines for JEV antigenic complex viruses. This is a fresh research demonstrating the potential of E-pseudotyped baculovirus as a JEV vaccine.

Establishment of recombinant cannabinoid receptor assays and characterization of several natural and synthetic ligands

Cannabinoid receptors (CBR) are important drug targets for the treatment of various inflammatory, metabolic and neurological diseases. Therefore, sensitive test systems for the assessment of ligands are needed. In this study, a steady-state GTPase assay for human CBR subtypes 1 and 2 was developed to characterize the pharmacological property of ligands at a very proximal point of the signal transduction cascade. Establishing these in vitro test sytems, we studied cell or tissue membranes heterogenously or endogenously expressing CBR, such as CBR-infected Human Embryonic Kidney (HEK) 293 cells, rat cerebellum and spleen cells. The lack of effects in the GTPase assay and in [(35)S]GTPgammaS binding experiments in these expression system, directed us to use Spodoptera frugiperda (Sf9) cells. Co-expressing CBR, different Galpha-subunits, Gbetagamma heterodimer, and RGS (Regulator of G-protein signaling)-proteins in Sf9 cell membranes greatly improved the sensitivity of the assay, with highest GTPase activation in the CBR + Galpha(i2) + Gbeta(1)gamma(2) + RGS4 system. We examined exogenous and endogenous standard ligands as well as secondary metabolites as Delta(9)-tetrahydrocannabinol (Delta(9)-THC), dodeca-2E,4E-dienoic acid isobutylamide, an alkylamide from Echinacea purpurea, and an E. purpurea hexane extract according their agonistic and antagonistic properties. The suitability of the assay for screening procedures was also proven by detecting the activity of Delta(9)-THC in a matrix of other less active compounds (Delta(9)-THC-free Cannabis sativa extract). In conclusion, we have developed highly sensitive test systems for the analysis of CBR ligands.

Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors

Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC(4)) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligands, Cy3B-NDP-alpha-MSH and TAMRA-NDP-alpha-MSH, were used and compared in assays that monitored their binding to MC(4) receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC(4) receptors was saturable and with high affinity. All studied MC(4) receptor-specific nonlabeled ligands displaced fluoroligands' binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters.

Involvement of lipid rafts and cellular actin in AcMNPV GP64 distribution and virus budding

GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.

Baculovirus surface display of NS3 nonstructural protein of classical swine fever virus

Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. NS3 proteins of CSFV, which include serine protease and RNA helicase/nucleotide triphosphatase (NTPase) activities, are multifunctional proteins involved in polyprotein processing and viral replication. Previous reports showed that NS3 protein can induce apoptosis in host cells that present cytopathic effects (CPE). Baculovirus/insect cell systems are used widely for recombinant protein production. In this study, one recombinant baculovirus BacSC-NS3 expressing histidine-tagged NS3 with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 of baculovirus was constructed. After infection, NS3 was expressed and anchored to the plasma membrane of Sf-9 cells, as demonstrated by Western blot assay and confocal microscopy. Immunogold electron microscopy demonstrated that the NS3 glycoprotein successfully displayed on the baculoviral envelope. Animal vaccine tests showed that recombinant baculovirus BacSC-NS3 elicited significantly higher NS3 antibody titers in the treated mouse models than the control group. This report demonstrated the potential of NS3-pseudotyped baculovirus expression of NS3 protein successfully.

Redistribution Of The Human Mu-opioid Receptor (hMor) In HEK 293 Cell Membranes In Response To Agonists

Opioid receptors play a role in a whole battery of physiological processes, including affective behaviour, neuroendocrine physiology and pain perception. The cell membrane, which is the physical environment of the receptor, is known to consist of domains with distinct lipid/protein composition and physical characteristics. Previous work (Moffet et al. (2000) J. Biol. Chem. 275: 2191-2198.) has shown that the G-protein signalling partners of hMOR localize to the “so called” detergent resistant membrane (DRM). Our previous studies using ligand-affinity atomic force microscopy (AFM) of hMOR-expressing Sf9 cell membranes indicated that hMOR localizes to mixed lipid domains, presumably corresponding to detergent soluble membranes (DSM). These results would indicate a physical separation of the receptor and its signalling partner prior to activation. However, the relationship between microdomains characterized using biochemical techniques (DSM/DRM) and membrane rafts defined by biophysical techniques remains unclear. Here we track active hMOR localization and lipid composition in detergent and detergent-free separated membranes. For each fraction, hMOR activity was assessed using a modified binding assay, and Western blot analysis was used to determine the relative amount of lipid raft marker, flotillin-1, and G-proteins. We show a redistribution of the receptor in response to its agonists, implying that hMOR distribution at the cell membrane helps to regulate it function.

Baculovirus surface display of σC and σB proteins of avian reovirus and immunogenicity of the displayed proteins in a mouse model

Avian reovirus (ARV), an important pathogen in poultry, causes arthritis, chronic respiratory disease, and malabsorption syndrome that cause considerable economic losses to the poultry industry. In present study, we have succeeded in construction of a universal baculovirus surface display system (UBSDS) that can display different foreign proteins on the envelope of baculovirus. Sequences encoding the signal peptide (SS), transmembrane domain (TM), and cytoplasmic domain (CTD) derived from the gp64 protein of baculovirus and histidine tag, respectively were inserted into the pBacCE vector. Four restriction enzyme sites between the histidine tag and gp64 transmembrane domain were established for expression of different foreign proteins. The transmembrane domain and CTD of gp64 in the platform were designed in order to improve stability and quantity of foreign proteins on the envelope of baculovirus. The σC and σB proteins of ARV are known to elicit neutralizing antibodies against ARV. The UBSDS was therefore used to express σC and σB proteins on the envelope of baculovirus. Two recombinant baculoviruses BacSC-σC and BacSC-σB have been successfully constructed. After infection, both His 6-tagged recombinant σC (rσC) and σB (rσB) proteins were displayed on the envelope of recombinant baculoviruses and the recombinant viral proteins were anchored on the plasma membrane of Sf-9 cells, as revealed by immunofluorescence staining (IFS) and confocal microscopy. The antigenicity of rσC and rσB proteins was demonstrated by Western blotting assay. Immunogold electron microscopy demonstrated that both recombinant viruses displayed rσC and rσB proteins on the viral surface. Immunization of BALB/c mice with recombinant viruses, demonstrated that serum from the BacSC-σC and BacSC-σB treated models had significant higher levels of virus neutralization activities than the control groups. This demonstrates that the recombinant baculoviruses BacSC-σC and BacSC-σB can be a potential vaccine against ARV infections.

Baculovirus surface display of E rns envelope glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. E rns is an envelope glycoprotein of CSFV which is known to induce virus-neutralizing antibodies and protective immunity in the natural host. In this study, one recombinant baculoviruses BacSC-E rns expressing histidine-tagged E rns with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 was constructed and its immunizing efficacy was evaluated in a mouse model. After infection, E rns was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western-blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E rns glycoprotein was successfully displayed on the baculoviral envelope. Vaccine tests in animals showed that BacSC-E rns elicited significantly higher E rns antibody titers in the immunized mouse models than the control group. This demonstrates that the BacSC-E rns vaccine can be used potentially against CSFV infections. This is the first report demonstrating the potential of E rns-pseudotyped baculovirus as a CSFV vaccine.

Baculovirus surface display of E2 envelope glycoprotein of classical swine fever virus and immunogenicity of the displayed proteins in a mouse model

Classical swine fever virus (CSFV) causes significant losses in pig industry in many countries. The E2 glycoprotein of CSFV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. In this study, one recombinant baculoviruses BacSC-E2 expressing histidine-tagged E2 with the CTD and TM derived from baculovirus envelope protein gp64 was constructed and evaluated its vaccine efficacy in mice model. After infection, E2 was expressed and anchored on the plasma membrane of Sf-9 cells, as revealed by confocal microscopy. Immunogold electron microscopy demonstrated that the BacSC-E2 was displayed E2 glycoprotein on the viral surface. Animal vaccine tests showed that BacSC-E2 elicited significantly higher E2 antibody titers in the treated mouse models than the control group. Virus neutralization test showed that serum from the BacSC-E2 treated models had significant levels of virus neutralization activities. This demonstrates that the BacSC-E2 vaccine can be a potential vaccine against CSFV infections. This is the first report demonstrating that the potential of E2-pseudotyped baculovirus as a classical swine fever virus vaccine.

C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity

We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(i1) protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Galpha(i1)beta(1)gamma(2) complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.

Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter

The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.

Avian Influenza Virus Hemagglutinin Display on Baculovirus Envelope: Cytoplasmic Domain Affects Virus Properties and Vaccine Potential

Hemagglutinin (HA) is the major immunogen on the envelope of avian influenza virus (AIV). Therefore we constructed two recombinant baculoviruses: Bac-HA, expressing histidine-tagged HA with the cytoplasmic domain (CTD) derived from HA, and Bac-HA64, expressing histidine-tagged HA with the CTD derived from baculovirus envelope protein gp64. After infection, HA with either CTD was expressed and anchored on the plasma membrane of Sf-9 cells, as revealed by confocal microscopy. Immunogold electron microscopy demonstrated that both Bac-HA and Bac-HA64 displayed HA on the viral surface. However, analyses of purified viruses revealed that significantly more HA was incorporated into Bac-HA64 than into Bac-HA. In comparison with Bac-HA, Bac-HA64 significantly improved the gene delivery and transgene expression in mammalian cells, as determined by quantitative real-time polymerase chain reaction and flow cytometry. Bac-HA64 elicited significantly higher hemagglutination inhibition titers in mouse models than Bac-HA and the negative controls. These data collectively confirmed that the gp64 CTD, in comparison with HA CTD, resulted in more efficient HA incorporation into baculovirus, more efficient transgene delivery and expression, and elevated immunogenicity. This is the first report demonstrating the potential of HA-pseudotyped baculovirus as an avian influenza vaccine and that the choice of CTD tremendously affects baculovirus properties and vaccine efficacy.

CONDITIONAL STIMULATION OF TYPE V AND VI ADENYLYL CYCLASES BY G PROTEIN βγ SUBUNITS

In a yeast two hybrid screen of mouse brain cDNA library, using the N-terminus of human type V adenylyl cyclase (hACV) as bait, we identified G protein β2 subunit as an interacting partner. Additional yeast two hybrid assays showed that the Gβ1 subunit also interacts with the N-terminus of hACV and human type VI adenylyl cyclase (hACVI). In-vitro adenylyl cyclase activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gβγ subunits enhance the activity of these enzymes in presence of Gαs or forskolin. Deletion of residues 77–151, but not 1–76, in the N-terminus of hACVI obliterated the ability of Gβγ subunits to stimulate the enzyme. Likewise, activities of the recombinant soluble forms of ACV and ACVI, which lack the N-termini, were not enhanced by Gβγ subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gβγ subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when Gi was inactivated by pertussis toxin. Therefore, we conclude that the N-termini of human hACV and hACVI are necessary for their interactions with, and regulation by Gβγ subunits both in-vitro and in intact cells. Moreover, Gβγ subunits derived from source(s) other than Gi are necessary for the full activation of hACVI by isoproterenol in intact cells. Supported by NIH grants HL59679 and GM073181 to TBP and GM060419 to CWD.

Cholesterol Potentiates ABCG2 Activity in a Heterologous Expression System: Improved in Vitro Model to Study Function of Human ABCG2

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2.

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein beta2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the Gbeta(1) subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gbetagamma subunits enhance the activity of these enzymes provided either Galpha(s) or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of Gbetagamma subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by Gbetagamma subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gbetagamma subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G(i) was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, Gbetagamma subunits both in vitro and in intact cells. Moreover, Gbetagamma subunits derived from a source(s) other than G(i) are necessary for the full activation of hACVI by isoproterenol in intact cells.

Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles

The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants K(m) and V(max) of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry.

Human Multidrug Resistance Protein 2 Transports the Therapeutic Bile Salt Tauroursodeoxycholate

The multidrug resistance protein 2 (MRP2/ABCC2) mediates the biliary excretion of glucuronide and glutathione conjugates of endogenous and exogenous compounds. We examined the activation of human MRP2-mediated ATP-dependent transport by the choleretic bile salt ursodeoxycholic acid (UDC) and its taurine and glycine amidates in Sf9 cell membranes expressing MRP2 using beta-estradiol 17-(beta-D-glucuronide) (E(2)17G) and beta-estradiol 3-(beta-D-glucuronide) (E(2)3G) as substrates. MRP2 transported E(2)3G via classic Michaelis-Menten kinetics (K(m) = 122 microM; V(max) = 3.0 nmol/mg/min), whereas E(2)17G transport showed positive cooperativity (Hill slope, 2.15; K(m) = 75 microM; V(max) = 3.8 nmol/mg/min). UDC, tauroursodeoxycholate, and glycoursodeoxycholate (80-100 microM) maximally stimulated E(2)3G transport 9-, 7.9-, and 3.6-fold, respectively, whereas higher concentrations (1-2 mM) inhibited transport. At low (0.3 microM) concentrations, tauroursodeoxycholate was transported only in the presence of E(2)17G or E(2)3G, but not other MRP2 substrates such as methotrexate, leukotriene C(4), or S-methylglutathione. Kinetic analysis of higher concentrations of tauroursodeoxycholate transport by MRP2 showed positive cooperativity (Hill slope, 1.84; K(m) = 127 microM; V(max) = 779 pmol/mg/min). Taurocholate (2-100 microM) was not detectably transported by MRP2 either alone or in the presence of E(2)17G but was transported in the presence of E(2)3G. Thus, UDC, tauroursodeoxycholate, and glycoursodeoxycholate activated MRP2 transport. Tauroursodeoxycholate was transported by MRP2 and demonstrated positive cooperativity, identifying it as the second MRP2 substrate able to stimulate its own transport. The data suggest MRP2 binding sites that can require specific complementarities between substrates and modulators of MRP2-mediated transport.

Modulation of dopamine D 1 receptor signaling by adenosine A 1 receptors in Sf9 cells requires expression of G i proteins

There are several evidences that some functions of D 1 dopamine receptors can be modulated by colocalized adenosine A 1 receptors. To elucidate the role of particular components of the receptor complex in the ligand binding and second messenger activation level we have used Sf9 cell expression system. The expression of D 1 and A 1 receptors was confirmed by proper binding of specific radioligands [ 3 H]SCH23390 ( K d = 1.1 ± 0.1 nM, B max = 2.2 ± 0.1 pmol/mg protein) and [ 3 H]DPCPX ( K d = 2.1 ± 0.8 nM, B max = 2.9 ± 0.4 pmol/mg protein), respectively. The kinetics of [ 3 H]SCH23390 binding corresponded to the simplest reversible bimolecular binding reaction of complex formation, with k on = 0.20 ± 0.02 min −1 nM −1 and k off = 0.13 ± 0.01 min −1. Dopaminergic agonists increased the accumulation of cAMP in the transfected cells in concentration-dependent manner, indicating a correct coupling of receptor to second messenger system. The coupling of the A 1 receptor to G i proteins was confirmed by both GTPγS dependent agonist binding and inhibition of cAMP accumulation by N-cyclopentyladenosine (NCPA). Activation of the A 1 receptor by NCPA had no significant influence on neither affinities of dopaminergic ligands nor the radioligand binding kinetics to the co-exprssed D 1 receptors in Sf9 cell membranes. On the other hand, the activation of the A 1 receptors inhibited the D 1 receptor-specific accumulation of cAMP, but only in cells where G i proteins were expressed with the receptors.