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J. Biol. Chem., Vol. 277, Issue 35, 31774-31780, August 30, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/35/31774    most recent M204166200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shimada, M. Articles by Segre, G. V. Search for Related Content PubMed PubMed Citation Articles by Shimada, M. Articles by Segre, G. V. Social Bookmarking                      What's this?

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, and Gino V. Segre

From the Endocrine Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

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 µg 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 Tyr²³, 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 (K_d = 16.5 ± 1.3 versus 11.9 ± 1.9 nM), and the purified receptors bound rat [Nle^8,21,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 [aminoisobutyric acid (Aib)^1,3,Nle⁸,Gln¹⁰,Har¹¹,Ala¹²,Trp¹⁴,Arg¹⁹,Tyr²¹]PTH-(1-21)-NH₂ and rat [Aib^1,3,Gln¹⁰,Har¹¹,Ala¹²,Trp¹⁴]PTH-(1-14)-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]GTPS 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.

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