The human PTH2 receptor: binding and signal transduction properties of the stably expressed recombinant receptor.
We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.
Behar
V
V
Division of Bone and Mineral Metabolism, Harvard-Thorndike and Charles A. Dana Laboratories, Boston, Massachusetts 02215, USA.
Pines
M
M
Nakamoto
C
C
Greenberg
Z
Z
Bisello
A
A
Stueckle
S M
SM
Bessalle
R
R
Usdin
T B
TB
Chorev
M
M
Rosenblatt
M
M
Suva
L J
LJ
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D010281:Parathyroid Hormone / Q000378:metabolism / Q000494:pharmacology*; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology*; D011869:Radioligand Assay; D044183:Receptor, Parathyroid Hormone, Type 2; D018016:Receptors, Parathyroid Hormone / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D019379:Teriparatide; D014162:Transfection
D003432:Cross-Linking Reagents; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D044183:Receptor, Parathyroid Hormone, Type 2; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; D019379:Teriparatide; D004533:Egtazic Acid; D015759:Ionomycin; D000242:Cyclic AMP; D002118:Calcium