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-Re and Lys(27) Of PTH(1-34). Lys(27) 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 Lys(27). 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, I-125-K27 crosslinks effectively and specifically to the hPTH1-Rc. Enzymatic (Glu-C and Lys-C) and chemical (CNBr and BNPS-skatole) digestions of the photoconjugate between I-125-K27 and hPTH1-Re 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 L-261 or R-262 of the first extracellular loop of hPTH1-Rc as the contact site for Lys(27) in the hormone. On the basis of (i) the similarity in molecular mass between the CNBr digest of the I-125-K27-[L261M]hPTP1-Rc conjugate and free I-125-K27 and (ii) the failure to cross-link I-125-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 Lys(27) (in PTH) to L-261 (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.