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 crosslinking followed by pr otein digestion and mapping of the radiolabeled photoconjugated receptor. P hotoreactive analogs of PTH, individually substituted with an L-p-benzoylph enylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmac ologically evaluated in cells stably expressing recombinant PTH2R. One high ly bioactive analog, [Bpa(1),Nle(8,18),Arg(13,26,27),L-2-Nal(23),Tyr(34)]PT H-(1-34)NH2 (Bpa(1)-PTH), was chosen for cross-linking studies. In addition , a PTH analog in which the photoreacive moiety is at the mid-region positi on 13 (K13) was demonstrated to be bioactive, then cross-linked to PTH2R. T he minimal digestion-restricted domain containing the contact site ("contac t domain") for I-125-Bpa(1)-PTH is in the sixth transmembrane domain and pa rt of the third extracellular loop, spanning residues Ser(364)-Met(395) of the receptor. This domain was further con firmed and refined by cross-linki ng I-125-Bpa(1)-PTH to two receptor mutants, PTH2R[V380M]- and PTH2R[V380M, M395L]-receptors. Treatment of the cross-linked conjugates with cyanogen br omide identified a single amino acid (position 380) as the putative contact point. The contact domain for I-125-K13 is located in the N-terminal extra cellular tail of the receptor (in the C-terminal portion) and spans Gln(138 )-Met(147). Further validation of this contact domain was accomplished by p hotocross-linking to point-mutated PTH2R[K137R] receptor. Previous studies in which PTH analogs were cross-linked to human PTH/PTHrP receptor (PTH1R) identified Met(425) and Phe(173)-Met(189) as the contact sites for Bpa(1)-P TH and K13, respectively. These studies demonstrate that both receptor subt ypes, PTH1- and PTH2-receptors, use analogous sites for interaction with po sitions 1 and 13 in PTH.