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J. Biol. Chem., Vol. 280, Issue 3, 1797-1807, January 21, 2005

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Novel Parathyroid Hormone (PTH) Antagonists That Bind to the Juxtamembrane Portion of the PTH/PTH-related Protein Receptor^*

Naoto Shimizu, Thomas Dean, Janet C. Tsang, Ashok Khatri, John T Potts, Jr, and Thomas J. Gardella

From the Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Current antagonists for the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR) are N-terminally truncated or N-terminally modified analogs of PTH(1–34) or PTHrP(1–34) and are thought to bind predominantly to the N-terminal extracellular (N) domain of the receptor. We hypothesized that ligands that bind only to PTHR region comprised of the extracellular loops and seven transmembrane helices (the juxtamembrane or J domain) could also antagonize the PTHR. To test this, we started with the J domain-selective agonists [Gln¹⁰,Ala¹²,Har¹¹,Trp¹⁴,Arg¹⁹ (M)]PTH(1–21), [M]PTH(1–15), and [M]PTH(1–14), and introduced substitutions at positions 1–3 that were predicted to dissociate PTHR binding and cAMP signaling activities. Strong dissociation was observed with the tri-residue sequence diethylglycine (Deg)¹-para-benzoyl-L-phenylalanine (Bpa)²-Deg³. In HKRK-B7 cells, which express the cloned human PTHR, [Deg^1,3,Bpa²,M]PTH(1–21), [Deg^1,3,Bpa²,M]PTH(1–15), and [Deg^1,3,Bpa²,M]PTH(1–14) fully inhibited (IC₅₀s = 100–700 nM) the binding of ¹²⁵I-[-aminoisobutyric acid^1,3,M]PTH(1–15) and were severely defective for stimulating cAMP accumulation. In ROS 17/2.8 cells, which express the native rat PTHR, [Deg^1,3,Bpa²,M]PTH(1–21) and [Deg^1,3,Bpa²,M]PTH(1–15) antagonized the cAMP-agonist action of PTH(1–34), as did PTHrP(5–36) (IC₅₀s = 0.7 µM, 2.6 µM, and 36 nM, respectively). In COS-7 cells expressing PTHR-delNt, which lacks the N domain of the receptor, [Deg^1,3,Bpa², M]PTH(1–21) and [Deg^1,3,Bpa²,M]PTH(1–15) inhibited the agonist actions of [-aminoisobutyric acid^1,3]PTH(1–34) and [M]PTH(1–14) (IC₅₀s 1 µM), whereas PTHrP(5–36) failed to inhibit. [Deg^1,3,Bpa²,M]PTH(1–14) inhibited the constitutive cAMP-signaling activity of PTHR-tether-PTH(1–9), in which the PTH(1–9) sequence is covalently linked to the PTHR J domain, as well as that of PTHR_camH223R. Thus, the J-domain-selective N-terminal PTH fragment analogs can function as antagonists as well as inverse agonists for the PTHR. The new ligands described should be useful for further studies of the ligand binding and activation mechanisms that operate in the critical PTHR J domain.

Received for publication, July 21, 2004 , and in revised form, November 1, 2004.

^* This work was supported by Grant DK-11794 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 617-726-3966; Fax: 617-726-7543; E-mail: gardella{at}helix.mgh.harvard.edu.

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