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Direct Identification of Two Contact Sites for Parathyroid Hormone (PTH) in the Novel PTH-2 Receptor using Photoaffinity Cross-Linking¹

Division of Bone and Mineral Metabolism, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Dr. Michael Chorev, Division of Bone and Mineral Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue (HIM 944), Boston, Massachusetts 02215. E-mail: mchorev{at}warren.med.harvard.edu

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.

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