Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide.
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 microg 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. 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),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 [alpha-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 gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-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.
Shimada
Masako
M
Endocrine Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA.
Chen
Xin
X
Cvrk
Tomas
T
Hilfiker
Helene
H
Parfenova
Maria
M
Segre
Gino V
GV
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry; D014162:Transfection
D010446:Peptide Fragments; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D012243:Rhodopsin