Heterologous expression and comparative characterization of the human neuromedin U subtype II receptor using the methylotrophic yeast Pichia pastoris and mammalian cells.
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 (NmU(1)R) and the neuromedin U type 2 receptor (NmU(2)R). 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 NmU(2)R 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 NmU(2)R. Enzymatic deglycosylation suggested that the recombinant NmU(2)R 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 NmU(2)R and it should facilitate further characterization of this receptor.
Shukla
Arun Kumar
AK
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany. Arun.Shukla@mpibp-frankfurt.mpg.de
Haase
Winfried
W
Reinhart
Christoph
C
Michel
Hartmut
H
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D007457:Iodine Radioisotopes; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D009479:Neuropeptides / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D017981:Receptors, Neurotransmitter / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D009479:Neuropeptides; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; C082444:neuromedin U receptor; C056900:neuromedin U; D004121:Dimethyl Sulfoxide