Direct mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking
AUTOR(ES)
Zhou, Alice T.
FONTE
The National Academy of Sciences of the USA
RESUMO
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, 125I-[Nle8,18,Lys13(ɛ-p-(3-I-Bz)Bz),l-2-Nal23,Arg26,27,Tyr34]bPTH-(1–34)NH2 (125I-all-R-K13). This ligand contains a photoreactive benzophenone moiety attached to the side chain of Lys13. 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 125I-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 125I-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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=20494Documentos Relacionados
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