Parathyroid hormone-receptor interactions identified directly by photocross-linking and molecular modeling studies

Direct mapping of the interface between parathyroid hormone (PTH) and its receptor (hPTH1-Rc) was carried out by photoaffinity scanning studies. Photoreactive analogs of PTH singularly substituted with a p-benzoyl-phenylalanine (Bpa) at each of the first six N-terminal positions have been prepared. Among these, the analog [Bpa(1),Nle(8,18),Arg(13,26,27),L-2-Nal(23),Tyr(34)] bPTH-(1-34)NH2 (Bpa(1)-PTH-(1-34)) displayed in vitro activity with potency similar to that of PTH-(1-34). The radioiodinated analog I-125-Bpa(1)-PTH-(1-34) cross-linked specifically to the hPTH1-Rc stably expressed in human embryonic kidney cells. A series of chemical and enzymatic digestions of the hPTH1-Rc-I-125-Bpa(1)-PTH-(1-34) conjugate suggested that a methionine residue (either Met(414) or Met(425)) within the contact domain hPTH1-Rc-(409-437), which includes the transmembrane helix 6 and part of the third extracellular loop, as the putative contact point. Site-directed mutagenesis (M414L or M425L) identified Met(425) as the putative contact point. Molecular modeling of the hPTH1-Rc together with the NMR-derived high resolution structure of hPTH-(1-34), guided by the cross-linking data, strongly supports Met(425), at the extracellular end of transmembrane helix 6, as the residue interacting with the N-terminal residue of the hPTH-(1-34). The photocross-linking and molecular modeling studies provide insight into the topologic arrangement of the receptor-ligand complex.