Direct mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking.
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),Lys13(epsilon-p-(3-I-Bz)Bz),L-2-Nal(23),Arg(26,2 7),Tyr34] bPTH-(1-34)NH2 ((125)I-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, approximately 400,000 receptors per cell), generating a diffuse approximately 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.
Zhou
A T
AT
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
Bessalle
R
R
Bisello
A
A
Nakamoto
C
C
Rosenblatt
M
M
Suva
L J
LJ
Chorev
M
M
D001665:Binding Sites; D002460:Cell Line; D003432:Cross-Linking Reagents; D006801:Humans; D010281:Parathyroid Hormone / Q000378:metabolism*; D044162:Parathyroid Hormone-Related Protein; D010449:Peptide Mapping; D011506:Proteins / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone / Q000819:agonists*
D003432:Cross-Linking Reagents; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D018016:Receptors, Parathyroid Hormone