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Direct mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking
- Source :
- Proceedings of the National Academy of Sciences. 94:3644-3649
- Publication Year :
- 1997
- Publisher :
- Proceedings of the National Academy of Sciences, 1997.
-
Abstract
- Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are calciotropic hormones interacting with a shared seven-transmembrane domain G protein-coupled receptor, which is located predominantly in bone and kidney. To map the interface of the bimolecular interaction between hormone and receptor, we designed and radioiodinated a bioactive, photoreactive PTH agonist, 125 I-[Nle 8,18 ,Lys 13 (ɛ- p -(3-I-Bz)Bz), l -2-Nal 23 ,Arg 26,27 ,Tyr 34 ]bPTH-(1–34)NH 2 ( 125 I-all-R-K13). This ligand contains a photoreactive benzophenone moiety attached to the side chain of Lys 13 . All other lysyl residues are substituted by argynyls. The analog photocrosslinks specifically to the recombinant hPTH/PTHrP receptor stably transfected into human embryonic kidney cells (HEK-293/C-21 cells, ≈400,000 receptors per cell), generating a diffuse ≈87-kDa band on SDS/PAGE autoradiography. To identify the “contact domain” within the hPTH/PTHrP receptor involved in binding of 125 I-all-R-K13, the radiolabeled band containing the ligand–receptor conjugate was subjected to chemical and enzymatic cleavage. Two independent pathways of sequential digestion were used: Route A, lysyl endopeptidase C, then endo-N-glycosidase F, followed by cyanogen bromide; Route B, cyanogen bromide followed by endo- N -glycosydase F. The identified domain is in contact with position 13 in 125 I-all-R-K13 and corresponds to amino acids 173–189 of the hPTH/PTHrP receptor, located at the C-terminal region of the N-terminal extracellular domain.
- Subjects :
- Binding Sites
Multidisciplinary
Parathyroid hormone-related protein
Parathyroid hormone receptor
Chemistry
Parathyroid Hormone-Related Protein
Proteins
Parathyroid hormone
Biological Sciences
Peptide Mapping
Molecular biology
Cell Line
Cross-Linking Reagents
Lysyl endopeptidase
Biochemistry
Parathyroid Hormone
Hormone receptor
Thyrotropin-releasing hormone receptor
Humans
Receptors, Parathyroid Hormone
Receptor
Binding domain
Subjects
Details
- ISSN :
- 10916490 and 00278424
- Volume :
- 94
- Database :
- OpenAIRE
- Journal :
- Proceedings of the National Academy of Sciences
- Accession number :
- edsair.doi.dedup.....bb11a6317dfbbc4cb0243b0988050c34