Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

Abstract

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18μg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyra23, consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (Kd = 16.5 ± 1.3 versus 11.9 ± 1.9 nM), and the purified receptors bound rat [Nle8,21,Tyr34]PTH-(1-34)-NH2 (PTH-(1-34)), and rat [Ile5,Trp23,Tyr36]PTHrP-(5-36)- NH2 with indistinguishable affinity. Maximal displacement of 125I-PTH-(1-34) binding by rat [α-aminoisobutyric acid (Aib) 1.3,Nle8,Gln10,Har11, Ala12,Trp14,Arg19,Tyr21] PTH-(1-21)-NH2 and rat [Aib1,3,Gln10,Har11,Ala12, Trp14]PTH-(l-14)-NH2 of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [35S]GTPγS incorporation into Gαs in a time- and dose-dependent manner, when recombinant hPTHiR, Gαs-, and βγ-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.