Chromatography studies on bio-affinity of nine ligands of α1-adrenoceptor to α1D subtypes overexpressed in cell membrane

To improve selectivity and specificity of cell membrane chromatography (CMC), the chromatography affinities of nine ligands of α₁-adrenergic receptor(AR)to α_1D-AR subtype were investigated. The human embryonic kidney (HEK) 293 cells expressed by cDNA of α_1D-AR subtypes were cultured and cell membrane stationary phase (CMSP) was prepared. Then the interactions between ligands and α_1D-AR in CMSP were investigated using CMC. The affinity rank order to α_1D-AR subtype obtained from CMC for the nine α₁-adrenoceceptor ligands is: prazosin, BMY7378, phentolamine, oxymetazoline, 5-methylurapidil, norepinephrine, phenyle-phrine, methoxamine, RS-17053. The affinity rank order is similar and correlates well with that obtained from others’ radioligand binding assays (RBA). CMSP prepared by transfected HEK293 cells with α_1D-adrenoceptor cDNA and CMC method could be used to evaluate affinities of drug-receptor and drug-receptor subtypes and to screen drugs selective to α_1D-AR.     

Chromatography studies on bio-affinity of nine ligands of a1-adrenoceptor to a1D subtypes overexpressed in cell membrane

Copyright by Science in China Press 2004 The a1-AR is present in many tissues including heart, blood vessels, liver, brain, kidney, prostate and spleen. In these tissues, the a1-AR mediates a variety of physiological effects such as neurotransmission, vasoconstriction, cardiacinotropy, chronotropy and glycogenolysis[1]. In 1986, Morrow and Greese pro-posed that a1-AR should be subclassified into two subtypes. Later this hypothesis was proved by Q.D Han in 1987. He not only verified a1-A…     

Chromatography studies on bioaffinity of nine ligands of α1 adrenoceptor to α1D subtypes overexpressed in cell membrane

To improve selectivity and specificity of cell membrane chromatography (CMC), the chromatography affinities of nine ligands of α₁-adrenergic receptor(AR)to α_1D-AR subtype were investigated. The human embryonic kidney (HEK) 293 cells expressed by cDNA of α_1D-AR subtypes were cultured and cell membrane stationary phase (CMSP) was prepared. Then the interactions between ligands and α_1D-AR in CMSP were investigated using CMC. The affinity rank order to α_1D-AR subtype obtained from CMC for the nine α₁-adrenoceceptor ligands is: prazosin, BMY7378, phentolamine, oxymetazoline, 5-methylurapidil, norepinephrine, phenylephrine, methoxamine, RS-17053. The affinity rank order is similar and correlates well with that obtained from others’ radioligand binding assays (RBA). CMSP prepared by transfected HEK293 cells with α_1D-adrenoceptor cDNA and CMC method could be used to evaluate affinities of drug-receptor and drug-receptor subtypes and to screen drugs selective to α_1D-AR.     

The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.     

Differences in the subcellular localization of alpha1-adrenoceptor subtypes can affect the subtype selectivity of drugs in a study with the fluorescent ligand BODIPY FL-prazosin

G protein-coupled receptor (GPCR) subtypes are differentially distributed in the cell; however, it remains unclear how this affects the subtype selectivity of particular drugs. In the present study, we used flow cytometry analysis with the fluorescent ligand, BODIPY FL-prazosin, to study the relationship between the subcellular distribution of subtype receptors and the subtype-selective character of ligands using alpha1a and alpha1b-adrenoceptors (ARs). Alpha1a-ARs predominantly localize inside the cell, while alpha1b-ARs on the cell surface. Flow cytometry analysis and confocal laser-scanning micrographs of living cells showed that BODIPY FL-prazosin can label not only alpha1-ARs on the cell surface, but also those localized inside the cell. Furthermore, flow cytometry analysis of alpha1A-AR-selective drug, KMD-3213, and alpha1B-AR-selective drug, CEC, revealed that the major determinant of the subtype selectivity of each drug is different. The alpha1A-AR selectivity of KMD-3213 can be explained by its much higher affinity for alpha1a-AR than alpha1b-AR (affinity-dependent selectivity), while the alpha1B-AR selectivity of the hydrophilic alkylating agent CEC is due to preferential inactivation of alpha1-ARs on the cell surface (receptor localization-dependent selectivity). This study illustrates that factors in addition to the affinity of the drug for the receptor, such as subcellular localization of the receptor, should be taken into account in assessing the subtype selectivity of a drug.     

Insulin induces alpha1B-adrenergic receptor phosphorylation and desensitization

The ability of insulin to induce alpha1B-adrenoceptor phosphorylation and desensitization was tested in two model systems: rat-1 cells that stably express alpha1B-adrenoceptors, through transfection, and endogenously express insulin receptors and DDT1 MF2 cells that endogenously express both receptors. Insulin induced concentration-dependent increases in the phosphorylation state of the adrenergic receptors in the two models with similar EC50 values (0.5-2 nM). The effect was rapid in the two systems but it was sustained in rat-1 cells and transient in DDT1 MF2 cells. In both cell lines, the insulin-mediated phosphorylation of alpha1B-adrenoceptors was blocked by wortmannin and LY 294002, and by staurosporine and bisindolylmaleimide I, indicating that the effect involved phosphoinositide 3-kinase and protein kinase C activities. The adrenoceptor phosphorylation induced by insulin was associated to desensitization as evidences by a diminished elevation of intracellular calcium in response to noradrenaline. Inhibitors of phosphoinositide 3-kinase and protein kinase C blocked the functional desensitization induced by insulin.     

激动剂作用下α1-肾上腺素受体亚型在HEK293A细胞中的分布变化

为了明确α1-肾上腺素受体（α1-adrenergic receptor,α1-AR）三种亚型在人胚胎肾（human embryonic kidney,HEK）293A细胞株中的分布特点,及其在激动剂作用下在细胞内的定位改变,本研究采用放射配体结合实验、实时荧光共聚焦成像和Western blot方法检测α1-AR三种亚型在细胞中的定位及蛋白质表达的变化。结果发现：（1）α1-AR三种亚型在HEK293A细胞株转染效率相同,均达90％以上。三株细胞的粗制膜上α1B-AR表达量最高,α1D-AR最低,α1A-AR居中,但三者的解离常数（配）相等;（2）在无激动剂作用时,α1A-AR均匀地分布在HEK293A细胞的胞膜和胞浆,α1B-AR主要位于胞膜,而α1D-AR则主要分布在胞浆中：（3）用α1-AR激动剂苯‘肾上腺素（phenylephrine,PE）刺激细胞1h后,α1A-和α1B-AR在胞膜上分布明显减少,而在胞浆中分布增加,其中α1B-AR变化更为显著,α1D-AR的分布在PE作用下无明显变化。以上结果提示,在激动剂作用下,α1-AR二种亚型在HEK293A细胞中的定位特点和分布变化各有不同。     

TRPC1 protein forms only one type of native store-operated channels in HEK293 cells

TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I_max channels, while the properties of I_min and I_NS channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current–voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I_max channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I_max channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I_max channels but is not an essential subunit for other store-operated channel types in HEK293 cells.     

High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line

The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.     

α1-肾上腺素受体亚型介导细胞内游离Ca2+增高的信号转导途径

本文探讨了α１ａ,α１ｂ,α１ｄ三种亚型肾上腺素受体激动时细胞内Ｃａ６２＋浓度升高的信号转导途径。在稳定表达三亚型α１－ＡＲ的ＨＥＫ２９３细胞２系中,用ｆｕｒａ－２方法细胞内Ｃａ＾２＋信号强弱的变化。结果显示,百日咳毒素对去甲肾上腺素激动三亚型α１－ＡＲ而引起的「Ｃａ＾２＋」ｉ升高无影响,Ｕ－７３１２２和ＰＭＡ明显抑制「Ｃａ＾２＋」ｉ升高．     

Subcellular localization of human neutral ceramidase expressed in HEK293 cells

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.     

Adaptor protein Ruk1 forms protein-protein complexes with endonuclease activity in HEK293 cells

The structural and functional organization of the adaptor protein Ruk₁ is characterized by the presence of three SH3-domains at the N-terminus followed by Pro- and Ser-rich sequences and a C-terminal coiled-coil region. Multiple modules in the Ruk₁ structure involved in protein–protein interactions can provide for formation of ligand clusters with varied properties and subcellular location. To study the nature and biological role of such complexes, the recombinant protein Ruk₁ with a Glu-epitope at the C-terminus (Ruk₁ Glu-tagged) was purified from transfected HEK293 cells by affinity chromatography on protein G-Sepharose with covalently conjugated anti-Glu-tag antibodies. By SDS polyacrylamide gel electrophoresis with subsequent staining with silver, a set of minor bands in addition to the 85-kD Ruk₁ Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the Ruk₁ Glu-tagged preparation by retardation of electrophoretic mobility of ³²P-labeled oligodeoxyribonucleotides in gel. The Ruk₁ Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl₂ and heparin inhibited the DNAse activity. These findings suggest the presence of DNases associated with the Ruk₁ protein in HEK293 cells. Such complexes were isolated from lysates of HEK293 cells by chromatography on heparin-Sepharose. By elution with 0.5 and 1.0 M NaCl, two fractions with DNase activity and containing proteins with molecular weights of 83, 80 and 72 kD were obtained. The reaction was inhibited by ZnCl₂ and heparin, and previous precipitation of Ruk-related proteins with anti-Ruk antibodies resulted in the exhaustion of nuclease activity. By immunoblotting with anti-Ruk antibodies, 83-kD protein immunologically related to the Ruk₁ protein was identified in the fractions. It was concluded that the adaptor protein Ruk₁ forms complexes with endonucleases in HEK293 cells.     

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process.     

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.     

Cell surface expression of alpha1D-adrenergic receptors is controlled by heterodimerization with alpha1B-adrenergic receptors

alpha(1)-Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (alpha(1A), alpha(1B), and alpha(1D)). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged alpha(1)-ARs showed that alpha(1A)- and alpha(1B)-ARs localize to the plasma membrane, whereas alpha(1D)-ARs accumulate intracellularly. We recently showed that alpha(1D)- and alpha(1B)-ARs form heterodimers, whereas alpha(1D)- and alpha(1A)-ARs do not. Here, we examined the role of heterodimerization in regulating alpha(1D)-AR localization using both confocal imaging of GFP- or CFP-tagged alpha(1)-ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with alpha(1B)-ARs caused alpha(1D)-ARs to quantitatively translocate to the cell surface, but co-expression with alpha(1A)-ARs did not. Truncation of the alpha(1B)-AR extracellular N terminus or intracellular C terminus had no effect on surface expression of alpha(1D)-ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant alpha(1B)-AR (Delta12alpha(1B)) increased both alpha(1D)-AR surface expression and coupling to norepinephrine-stimulated Ca(2+) mobilization. Finally, GFP-tagged alpha(1D)-ARs were not detected on the cell surface when expressed in rat aortic smooth muscle cells that express no endogenous ARs, but were almost exclusively localized on the surface when expressed in DDT(1)MF-2 cells, which express endogenous alpha(1B)-ARs. These studies demonstrate that alpha(1B)/alpha(1D)-AR heterodimerization controls surface expression and functional coupling of alpha(1D)-ARs, the N- and C-terminal domains are not involved in this interaction, and that alpha(1B)-AR G protein coupling is not required. These observations may be relevant to many other Class I G protein-coupled receptors, where the functional consequences of heterodimerization are still poorly understood.     

G418抗性HEK293细胞的培育

目的　培育具有G418抗性的HEK2 93细胞 ,用于建立猪内源性反转录病毒感染人HEK2 93细胞的模型。方法　通过脂质体转染的方法 ,将含有neo基因的质粒pIRESneo导入HEK2 93细胞中 ,利用G418的选择特性 ,对转染细胞进行压力筛选 ,并对其进行了PCR鉴定。结果　经 6 0 0 μg ml的G418压力筛选后 ,获得了抗性细胞克隆。抗性细胞的形态和生长速度与筛选前细胞没有差异 ,特异性核苷酸引物检测抗性细胞基因组DNA ,可以扩增出对应的核苷酸片段。结论　成功地培育了G418抗性HEK2 93细胞 ,为建立猪内源性反转录病毒感染人HEK2 93细胞的模型奠定了基础。     

Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.     

Epac- and Rap- independent ERK1/2 phosphorylation induced by Gs-coupled receptor stimulation in HEK293 cells

Serotonin activates Ras and Ras-dependent ERK1/2 phosphorylation in HEK293 cells expressing G(s)-coupled 5-HT(4) or 5-HT(7) serotonin receptors through unknown mechanisms. Both Epac/Rap-dependent and -independent pathways for Ras-dependent ERK1/2 activation have been suggested. Epac overexpression or Epac-specific 8-CPT-2'-O-Me-cAMP did not cause ERK1/2 phosphorylation, despite Rap activation. The data did not support a role for PLCepsilon or DAG-dependent Ras GEFs of the Ras-GRP family in Ras-dependent ERK1/2 phosphorylation. However, serotonin stimulated phosphorylation of endogenous and recombinant Ras-GRF1, increased [Ca(2+)](i) and caused Ca(2+)- and calmodulin-dependent ERK1/2 phosphorylation. Different signalling pathways seem to be utilised by G(s)-coupled receptors in various isolates of HEK293 cells.     

Subtype-selective noncompetitive or competitive inhibition of human alpha1-adrenergic receptors by rho-TIA

The 19-amino acid conopeptide (rho-TIA) was shown previously to antagonize noncompetitively alpha(1B)-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)-over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B(max)) for human alpha(1B)-ARs without changing affinity (K(D)), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing K(D) without changing B(max), suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated [(3)H]inositol phosphate formation in HEK293 cells expressing human alpha(1B)-ARs but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino-terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA noncompetitively inhibits alpha(1B)-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.     

A novel method to study insect olfactory receptor function using HEK293 cells

The development of rapid and reliable assays to characterize insect odorant receptors (ORs) and pheromone receptors (PRs) remains a challenge for the field. Typically ORs and PRs are functionally characterized either in vivo in transgenic Drosophila or in vitro through expression in Xenopus oocytes. While these approaches have succeeded, they are not well suited for high-throughput screening campaigns, primarily due to inherent characteristics that limit their ability to screen large quantities of compounds in a short period of time. The development of a practical, robust and consistent in vitro assay for functional studies on ORs and PRs would allow for high-throughput screening for ligands, as well as for compounds that could be used as novel olfactory-based pest management tools. Here we describe a novel method of utilizing human embryonic kidney cells (HEK293) transfected with inducible receptor constructs for the functional characterization of ORs in 96-well plates using a fluorescent spectrophotometer. Using EposOrco and EposOR3 from the pest moth, Epiphyas postvittana as an example, we generated HEK293 cell lines with robust and consistent responses to ligands in functional assays. Single-cell sorting of cell lines by FACS facilitated the selection of isogenic cell lines with maximal responses, and the addition of epitope tags on the N-termini allowed the detection of recombinant proteins in homogenates by western blot and in cells by immunocytochemistry. We thoroughly describe the methods used to generate these OR-expressing cell lines, demonstrating that they have all the necessary features required for use in high-throughput screening platforms.