Photoaffinity cross-linking identifies differences in the interactions of an agonist and an antagonist with the parathyroid hormone/parathyroid hormone-related protein receptor

Analogs of parathyroid hormone (PTH)-related protein (PTHrP), singularly substituted with a photoreactive L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in human embryonic kidney cells stably expressing the recombinant human PTH/PTHrP receptor. Two of these analogs, in which the photoreactive residue is either in position 1 or 2 (Bpa¹- and Bpa²-PTHrP, respectively) displayed high affinity binding. Bpa¹-PTHrP also displayed high efficacy for the stimulation of increased cAMP levels. Surprisingly, Bpa²-PTHrP was found to be a potent antagonist, despite the presence of the principal activation domain (sequence 1-6). Analysis of the digestion profiles of the ligand- receptor photoconjugates revealed that both the agonist and the antagonist cross-link to the S-CH₃ group of Met⁴²⁵ in transmembrane domain 6 of the human PTH/PTHrP receptor. However, the antagonist Bpa²-PTHrP also cross- links to a proximal site within the receptor domain Pro⁴¹⁵-Met⁴²⁵. Unlike the antagonist Bpa²-PTHrP, the potent agonist Bpa²-PTH, also bearing the Bpa residue in position 2, cross-links only to the S-CH₃ group of Met⁴²⁵ (similar to Bpa₁-PTHrP and Bpa¹-PTH). Taken together, these results suggest that the antagonist Bpa²-PTHrP is able to distinguish between two distinct conformations of the receptor. The comparison between PTHrP analogs substituted by Bpa at two consecutive positions and across PTH and PTHrP reveals insights into the PTH/PTHrP ligand-receptor bimolecular interaction at the level of a single amino acid.