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 C-protein coupled receptor clas s B, for example, receptors for secretin, glucagon, or parathyroid hormone, are involved in ligand binding. To obtain structural and functional inform ation on the N-terminal receptor fragment of human parathyroid hormone rece ptor 1 (PTHR1), the truncated receptor was expressed in the cytosol of Esch erichia coli in the form of inclusion bodies. Oxidative refolding of inclus ion body material resulted in stable, soluble, monomeric protein. Ligand bi nding was proved by surface plasmon resonance spectroscopy and isothermal t itration calorimetry, Refolded receptor fragment was able to bind parathyro id hormone with an apparent dissociation constant of 3-5 mu M. Far-UV circu lar dichroism spectra showed that the refolded polypeptide contained approx imately 25% alpha-helical and 23% beta-sheet secondary structures. Analysis of the disulfide bond pattern of the refolded receptor fragment revealed d isulfide 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, stabl e conformation, which shows a significant ligand binding activity.