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Studies of the N-Terminal Region of a Parathyroid Hormone-Related Peptide(1–36) Analog: Receptor Subtype-Selective Agonists, Antagonists, and Photochemical Cross-Linking Agents¹

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

Address all correspondence and requests for reprints to: Thomas J. Gardella, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: gardella{at}helix.mgh.harvard.edu

The N-terminal regions of PTH and PTH-related peptide (PTHrP) are involved in receptor-mediated signaling and subtype selectivity. To better understand the molecular basis for these processes, we first prepared a series of [I⁵,W²³,Y³⁶]-PTHrP(1–36)NH₂ analogs having stepwise deletions of residues 1–4 and characterized them with the human (h)PTH-1 and hPTH-2 receptor subtypes stably transfected in LLC-PK₁ cells. Deletions beyond residue 2 caused progressive and severe losses in cAMP-signaling efficacy without dramatically diminishing receptor-binding affinity; consistent with this, [I⁵,W²³]-PTHrP(5–36) was a potent antagonist for both PTH receptor subtypes. We then prepared and characterized photolabile analogs of [I⁵,W²³,Y³⁶]-PTHrP(1–36)NH₂ that were singly modified with para-benzoyl-L-phenylalanine (Bpa) along the first six residues. These full-length analogs exhibited receptor subtype-selective agonism, antagonism, and photochemical cross-linking profiles. In particular, the [Bpa²]- and [Bpa⁴]-substituted analogs selectively antagonized and preferentially cross-linked to the PTH-1 receptor and PTH-2 receptor, respectively. These results demonstrate that the 1–5 region of [I⁵,W²³]-PTHrP(1–36) is critical for activating the PTH-1 and PTH-2 receptors and suggest that the individual residues in this region play distinct roles in modulating the activation states of the two receptors. The cross-linking of both agonist and antagonist ligands to these PTH receptors lays the groundwork for identifying critical signaling determinants in the ligand binding pocket of the receptor.

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