Mapping the bimolecular interface of the parathyroid hormone (PTH)-PTH1 receptor complex: Spatial proximity between Lys²⁷ (of the hormone principal binding domain) and Leu²⁶¹ (of the first extracellular loop) of the human PTH1 receptor

In an effort to characterize the bimolecular interface between parathyroid hormone (PTH) and its human receptor PTH1-Rc (hPTH1-Rc), we previously identified two contact sites in the receptor: one for position 1 and another for position 13 (located at the ends of the principal activation domain) in PTH(1-34). The present study reports a third, novel 'contact site' between hPTH1-Rc and Lys²⁷ of PTH(1-34). Lys²⁷ is located in the principal binding domain of the hormone (residues 25-34). The photoreactive PTH(1-34) analogue K27 contains a benzophenone (BP) moiety on Lys²⁷. The analogue binds to stably transfected HEK 293/C-21 cells (which express a high level of recombinant hPTH1-Rc) and stimulates adenylyl cyclase activity with a potency similar to PTH(1-34). In addition, ¹²⁵I-K27 cross-links effectively and specifically to the hPTH1-Rc. Enzymatic (Glu-C and Lys-C) and chemical (CNBr and BNPS-skatole) digestions of the photoconjugate between ¹²⁵I-K27 and hPTH1-Rc were performed. In addition, photoconjugates involving the bioactive mutants [L261M]- and [R262K]-hPTH1-Rc, transiently expressed in COS-7 cells, were also digested. The data obtained clearly identify L²⁶¹ or R²⁶² of the first extracellular loop of hPTH1-Rc as the contact site for Lys²⁷ in the hormone. On the basis of (i) the similarity in molecular mass between the CNBr digest of the ¹²⁵I-K27- [L261M]hPTH1-Rc conjugate and free ¹²⁵I-K27 and (ii) the failure to cross- link ¹²⁵I-K27 to a bioactive mutant receptor [L261A]hPTH1-Rc, we conclude that L²⁶¹ is the cross-linking site. These results provide the first demonstration of an interaction between the principal binding domain of PTH and the first extracellular loop of hPTH1-Rc. Revealing proximity of Lys²⁷ (in PTH) to L²⁶¹ (in hPTH1-Rc) provides additional insight into the nature of the ligand-receptor bimolecular interface and clearly illustrates that the extracellular loops of the receptor contribute to the specificity of the PTH- PTH1-Rc interaction. Taken together with previous studies, the new findings add important constraints on the possible positioning of the C-terminal helix of PTH (which contains the principal binding domain) relative to the first extracellular loop and the distal C-terminal helix of the large extracellular amino terminal domain of the PTH1-Rc.