Probing the bimolecular interactions of parathyroid hormone and the human parathyroid hormone/parathyroid hormone-related protein receptor. 2. Cloning, characterization, and photoaffinity labeling of the recombinant human receptor.
Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and cross-linking of a benzophenone-substituted PTH analog, [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of approximately 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of 125I-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Rc, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.
Adams
A E
AE
Division of Bone and Mineral Metabolism, Harvard-Thorndike Research Laboratories, Beth Israel Hospital, Boston, Massachusetts, USA.
Pines
M
M
Nakamoto
C
C
Behar
V
V
Yang
Q M
QM
Bessalle
R
R
Chorev
M
M
Rosenblatt
M
M
Levine
M A
MA
Suva
L J
LJ
D000345:Affinity Labels; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000196:embryology; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010777:Photochemistry; D010920:Placenta / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000235:genetics*; D011994:Recombinant Proteins; D014162:Transfection
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; C095415:parathyroid hormone (1-34)amide, Nle(8,18)-Lys(13)(epsilon-pBz2)-2-Nal(23)-Tyr(34)-