Direct mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking

Direct mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking

Division of Bone and Mineral Metabolism, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215

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, ¹²⁵I-[Nle^8,18,Lys¹³(ɛ-p-(3-I-Bz)Bz),l-2-Nal²³,Arg^26,27,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.

Footnotes

↵ * To whom reprint requests should be addressed at: Division of Bone and Mineral Metabolism, Beth Israel Deaconess Medical Center-Harvard Medical School, 330 Brookline Ave (HIM 944), Boston MA 02215. e-mail: mchorev{at}warren.med.harvard.edu.
Irwin C. Gunsalus, Gulf Breeze, FL
ABBREVIATIONS:

pBz2,

p-benzoyl-benzoyl;

p(3-I-Bz)Bz,

p-(3-I-benzoyl)-benzoyl;

Endo-F,

endoglycosidase F/N-glycosidase F;

Lys-C,

lysyl endopeptidase;

Nal,

naphthylalanyl;

PTH,

parathyroid hormone;

PTHrP,

PTH-related protein;

all-R-K13,

[Nle8,18,Lys13(ɛ-p-(3-I-Bz)Bz, l-2-Nal23,Arg26,27,Tyr34)]bPTH-(1–34)NH2;

125I-all-R-K13,

125I-[Nle8,18,Lys13(ɛ-p-(3-I-Bz)Bz);

l-2-Nal23,

Arg26,27,Tyr34]bPTH-(1–34)NH2;

Tricine,

N-[tris(hydroxymethyl)methyl]glycine;

rt,

room temperature