Direct identification of two contact sites for parathyroid hormone (PTH) in the novel PTH-2 receptor using photoaffinity cross-linking

Direct examination of the interacting sites between PTH and the human PTH2 receptor (PTH2r) was conducted by photoaffinity cross-linking followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. Photoreactive analogs of PTH, individually substituted with an L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in cells stably expressing recombinant PTH2r. One highly bioactive analog, [Bpa¹,Nle^8,18,Arg^13,26,27,L-2-Nal²³,Tyr³⁴]PTH-(1 -34)NH₂ (Bpa¹-PTH), was chosen for cross-linking studies. In addition, a PTH analog in which the photoreacive moiety is at the mid-region position 13 (K13) was demonstrated to be bioactive, then cross-linked to PTH2r. The minimal digestion-restricted domain containing the contact site ('contact domain') for ¹²⁵i-Bpa¹-PTH is in the sixth transmembrane domain and part of the third extracellular loop, spanning residues Ser³⁶⁴-Met³⁹⁵ of the receptor. This domain was further confirmed and refined by cross-linking ¹²⁵I-Bpa¹-PTH to two receptor mutants, PTH2R[V380M]- and PTH2R[V380M,M395L]-receptors. Treatment of the cross-linked conjugates with cyanogen bromide identified a single amino acid (position 380) as the putative contact point. The contact domain for ¹²⁵I-K13 is located in the N-terminal extracellular tail of the receptor (in the C-terminal portion) and spans Gln¹³⁸-Met¹⁴⁷. Further validation of this contact domain was accomplished by photocross-linking to point-mutated PTH2R[K137R] receptor. Previous studies in which PTH analogs were cross-linked to human PTH/PTHrP receptor (PTH1R) identified Met⁴²⁵ and Phe¹⁷³-Met¹⁸⁹ as the contact sites for Bpa¹-PTH and K13, respectively. These studies demonstrate that both receptor subtypes, PTH1- and PTH2-receptors, use analogous sites for interaction with positions 1 and 13 in PTH.