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Abstract: Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29–32pmol/mg and it binds to angiontensin II with high affinity (K d =0.98–1.1nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Gαq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression. [Copyright &y& Elsevier]

Bradykinin, a vasoactive peptide, plays a crucial role in many cardiovascular processes via activation of the bradykinin subtype 2 receptor (B2R). B2R, a member of the G protein-coupled receptor (GPCR) superfamily, is a potential drug target in the treatment of cardiovascular disorders, pain and inflammation. In this study, human B2R was expressed at high levels in baby hamster kidney (BHK) cells using Semliki Forest virus-based vectors. The recombinant receptor was produced as a fusion protein with affinity tags and an expression level of 11 pmol/mg (i.e., approx. 0.2 mg of active receptor per liter of culture) was obtained. Radioligand binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity ( Kd=0.12 nM) and its pharmacological profile was similar to that of B2R in native tissues. Bradykinin-stimulated accumulation of inositol phosphate was observed in BHK cells expressing the recombinant receptor, which indicated the activation of endogenous Gαq protein by the recombinant B2R. Confocal laser scanning microscopy and immunogold staining revealed that the recombinant receptor was predominantly localized intracellularly. To the best of our knowledge, this is the first report of an affinity-tagged recombinant B2R been expressed at high levels in BHK cells and extensively characterized. [ABSTRACT FROM AUTHOR]

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]