Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Masako Shimada, Xin Chen, Tomas Cvrk, Helene Hilfiker, Maria Parfenova, Gino V. Segre
2002 Journal of Biological Chemistry  
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 18 g 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 Tyr 23 , consistent with removal of the 22-amino acid signal peptide.
more » ... sons 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 (K d ‫؍‬ 16.5 ؎ 1.3 versus 11.9 ؎ 1.9 nM), and the purified receptors bound rat [Nle 8,21 ,Tyr 34 ]PTH-(1-34)-NH 2 (PTH-(1-34)), and rat [Ile 5 ,Trp 23 ,Tyr 36 ]PTHrP-(5-36)-NH 2 with indistinguishable affinity. Maximal displacement of 125 I-PTH-(1-34) binding by rat [␣؊aminoisobutyric acid (Aib) 1,3 ,Nle 8 ,Gln 10 ,Har 11 ,Ala 12 ,Trp 14 ,Arg 19 ,Tyr 21 ]PTH-(1-21)-NH 2 and rat [Aib 1,3 ,Gln 10 ,Har 11 ,Ala 12 ,Trp 14 ]PTH-(1-14)-NH 2 of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [ 35 S]GTP␥S incorporation into G␣ s in a time-and dose-dependent manner, when recombinant hPTH1R, G␣ s -, and ␤␥-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.
doi:10.1074/jbc.m204166200 pmid:12080067 fatcat:lg5y7lsi5vhc5b6mardegmwlia