The N-terminal fragment of human parathyroid hormone receptor 1 constitutes a hormone binding domain and reveals a distinct disulfide pattern.
The N-terminal extracellular parts of human G-protein coupled receptor class B, for example, receptors for secretin, glucagon, or parathyroid hormone, are involved in ligand binding. To obtain structural and functional information on the N-terminal receptor fragment of human parathyroid hormone receptor 1 (PTHR1), the truncated receptor was expressed in the cytosol of Escherichia coli in the form of inclusion bodies. Oxidative refolding of inclusion body material resulted in stable, soluble, monomeric protein. Ligand binding was proved by surface plasmon resonance spectroscopy and isothermal titration calorimetry. Refolded receptor fragment was able to bind parathyroid hormone with an apparent dissociation constant of 3-5 microM. Far-UV circular dichroism spectra showed that the refolded polypeptide contained approximately 25% alpha-helical and 23% beta-sheet secondary structures. Analysis of the disulfide bond pattern of the refolded receptor fragment revealed disulfide bonds between Cys170 and Cys131, Cys148 and Cys108, and Cys117 and Cys48. These results demonstrate that the extracellular N-terminal domain of the parathyroid hormone receptor (PTHR1) possesses a well-defined, stable conformation, which shows a significant ligand binding activity.
Institut für Biotechnologie der Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany.
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U
Grauschopf
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H
Lilie
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K
Honold
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M
Wozny
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Reusch
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Esswein
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Schäfer
K P
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Rücknagel
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R
Rudolph
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D018016:Receptors, Parathyroid Hormone / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D004220:Disulfides; D008024:Ligands; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins