Human neuropeptide Y1 receptor expressed in Escherichia coli retains its pharmacological properties.
The coding region of the gene for the human neuropeptide Y (NPY) Y1 receptor was fused to the maltose binding protein gene. Expression of this transcription unit, after derepression with isopropyl beta-D-thiogalactopyranoside, was evidenced by the appearance of a protein of higher molecular weight, as well as the native maltose binding protein, both of which were immunoreactive with anti-maltose binding protein antibodies. Specific [125I]NPY binding activity was found associated mainly with the inner bacterial membrane fraction, suggesting that the receptor is correctly folded in this membrane. Competition binding experiments using NPY Y1- and Y2-specific ligands clearly exhibited a NPY Y1 specific pharmacological profile with Kd values indistinguishable from those of the native receptor. These results suggest that the membrane environment required by the human NPY Y1 receptor for specific high-affinity ligand binding is conserved in this heterologous bacterial system and can be used for detailed analyses of ligand-receptor interaction and drug screening.
Herzog
H
H
Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, NSW Australia.
Münch
G
G
Shine
J
J
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007544:Isopropyl Thiogalactoside; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009478:Neuropeptide Y / Q000031:analogs & derivatives / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009478:Neuropeptide Y; D010455:Peptides; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D007544:Isopropyl Thiogalactoside