Studies of the N-terminal region of a parathyroid hormone-related peptide(1-36) analog:: Receptor subtype-selective agonists, antagonists, and photochemical cross-linking agents

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 [(I,W23)-W-5,Y-36]-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, [I-5,W-23]PTHrP(5-36) was a potent antagonist for both PTH receptor subtypes. We then prepared and characterized photolabile analogs of [I-5,W-23,Y-36]-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 [Bpa(2)]- and [Bpa(4)]-substituted analogs selectively antagonized and preferentially cross-linked to the PTH-1 receptor and PTH-S receptor, respectively. These results demonstrate that the 1-5 region of [I-5,W-23] -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.