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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 Gαi1β1γ2 proteins were colocalized in the cells. GTPγS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Gαi1 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/Gαi1β1γ2 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. [ABSTRACT FROM AUTHOR]

Abstract: High-level overexpression of G protein-coupled receptors GPCRs in mammalian cells remains a difficult task inspite of newly developed virus based expression systems. Here, we show that the functional expression level of the recombinant bradykinin receptor (B2R) in mammalian cells can be increased up to sixfold just by the addition of dimethylsulphoxide in the culture medium. Total expression level, cellular localization and binding affinity of the recombinant receptor for its endogenous ligand remains unaltered. The strategy presented here, with recombinant B2R as a case example, is applicable to other GPCRs and provides a generic tool to improve the functional expression level of recombinant GPCRs in mammalian cells. [Copyright &y& Elsevier]

Abstract: Neuromedin U (a neuropeptide) plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow and pain. The physiological actions of NmU are mediated via two recently identified G protein-coupled receptors namely the neuromedin U type 1 receptor (NmU1R) and the neuromedin U type 2 receptor (NmU2R). Despite their crucial roles in cell physiology, structural information on these receptors is limited, mainly due to their low expression levels in native tissues. Here, we report the overexpression of the human NmU2R in the methylotrophic yeast Pichia pastoris and baby hamster kidney (BHK) cells using the Semliki Forest virus (SFV) system. The recombinant receptor was expressed as a fusion protein with three different affinity tags namely, the Flag tag, the histidine 10 tag and the biotinylation domain of Propionobacterium shermanii. Expression level of the recombinant receptor was 6–9pmol/mg under optimized conditions, which is significantly higher than the expression level in the native tissues. The recombinant receptor binds to its endogenous ligand neuromedin U with high affinity (Kd=0.8–1.0nM) and the binding constant for the recombinant receptor is similar to that of the wild type NmU2R. Enzymatic deglycosylation suggested that the recombinant NmU2R was glycosylated in P. pastoris, but not in BHK cells. Confocal laser scanning microscopy and immunogold labelling experiment revealed that the recombinant receptor was predominantly localized in the intracellular membranes. To our knowledge, this is the first report of heterologous overexpression of an affinity tagged recombinant NmU2R and it should facilitate further characterization of this receptor. [Copyright &y& Elsevier]

Abstract: Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields. [Copyright &y& Elsevier]