Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a well characterized phenomenon believed to contribute to receptor desensitization. The 5-hydroxytryptamine (5-HT) 2C subtype of serotonin receptor is a GPCR that we have shown to internalize upon agonist incubation. In this study, we have examined the effects of 5-HT 2C on the internalization of a C-terminal green fluorescent protein (GFP)-tagged 5-HT 2C receptor (VSV isoform) expressed in transiently transfected human embryonic kidney cells. We detected internalization with an automated, cell-based fluorescence-imaging system (Arrayscan) and monitored function with intracellular Ca 2ϩ measurements (flourometric imaging plate reader). The 5-HT 2C -GFP construct exhibited appropriate pharmacology, and we observed that although all three agonists resulted in similar magnitudes of dose-dependent internalization, the partial agonists resulted in ϳ50% less internalization, and the inverse agonists and neutral antagonists failed to induce internalization. These results were confirmed by confocal microscopy. They demonstrate that the 5-HT 2C receptor is internalized by incubation with agonists and partial agonists but not with inverse agonists or neutral antagonists.Agonist induced internalization of G protein-coupled receptors (GPCRs) is a well characterized phenomenon believed to contribute to receptor desensitization. Upon agonist binding, GPCRs undergo conformational changes that result in activation of heterotrimeric G protein subunits, which activate or inhibit downstream targets such as nucleotide cyclases, phospholipases, and kinases. Subsequent to receptor-effector coupling, second messenger-dependent protein kinases and G protein receptor kinases phosphorylate the receptor, which promotes recruitment of ␤-arrestins, uncoupling of heterotrimeric G proteins, and internalization in clathrin-coated vesicles (for review, see Ferguson, 2001). Although such internalization is typically associated with agonist induction, antagonists have been shown to induce internalization of several GPCRs, including the 5-hydroxytryptamine (5-HT) 2A receptor (Willins et al., 1998), the cholecystokinin receptor (Roettger et al., 1997), the vasopressin V 2 receptor (Pfeiffer et al., 1998), and the A 1 adenosine receptor (Navarro et al., 1999), indicating that the ligand dependence of GPCR internalization is a receptor-specific property.Although agonist-and antagonist-induced internalization has been studied for the 5-HT 2A serotonin receptor, it has not been examined for the closely related 5-HT 2C receptor subtype. The 5-HT 2C receptor functions through the same mechanism as the other members of the 5-HT 2 family, activating phospholipase C, promoting the hydrolysis of membrane phospholipids, leading to increases in the intracellular levels of inositol phosphates and intracellular calcium (for review, see Boess and Martin, 1994). Both the 5-HT 2A and 5-HT 2C receptor subtypes have received considerable attention in pharmaceutical drug discovery. In...

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1 In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2 HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. 3 Na + -dependent L-glutamate uptake activity (3.2 pmol min 71 mg 71 ) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/ EAAT2 cells to the ecdysone analogue Ponasterone A (10 mM for 24 h) resulted in a 510 fold increase in the Na + -dependent activity. 4 L-glutamate uptake into induced HEK/EAAT2 cells followed ®rst-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+7.5 mM, V max 3.8+0.9 nmol min 71 mg 71 protein.5 The pharmacological pro®le of the EAAT2 subtype was characterized using a series of Lglutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6 Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+5% control in the presence of 30 mM) of the L-glutamate transport capacity in the induced HEK/EAAT2 cells. 7 This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone-inducible mammalian expression system.

A recombinant baculovirus was constructed containing an expression cassette with a reporter gene, green fluorescent protein, directed by a constitutive mammalian promoter: a human cytomegalovirus immediate early promoter/enhancer (CMV-IE). High titer virus was prepared with ultracentrifugation. Efficient gene delivery and expression were observed in the virus-treated chicken primary culture, myoblast cells, and whole embryonic fibroblast cells. It was noticed that an addition of sodium butyrate (a selective histone deacetylase inhibitor) to viral transduction medium extremely enhanced the reporter-gene expression. However, there is no effect of presence of trichostatin A observed. To maximize the reporter-gene expression, the baculoviral infection condition was optimized with both cell types. Our approaches demonstrated that recombinant baculovirus could efficiently deliver its genome DNA into chicken primary cells and that CMV-IE, a mammalian-cell-active promoter, was functional in chicken primary cells and could direct a high level of gene expression. Clearly, the recombinant baculovirus provides an alternative means for foreign gene delivery into avian cells.