Mapping peptide hormone-receptor interactions using a disulfide-trapping approach.

Class II G protein-coupled receptors (GPCRs)1 interact with physiologically important peptides. There is intense study of how these peptides associate with their cognate receptors since elucidation of these interactions should provide important insights for the rational design of ligands with enhanced pharmacological properties for use in treating an array of diseases (1, 2). Over the past decade, photoaffinity cross-linking has been employed to study the interaction of a number of peptide–GPCR interactions (3). This technique involves systematically probing the receptor for regions of interaction using a peptide that incorporates a photoreactive moiety that irreversibly cross-links to the receptor. We and others have used this approach extensively to study the interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1) (4-12). This hormone–receptor system plays an integral role in calcium metabolism and bone biology. Our research is focused on studying the bimolecular interface of the PTH–PTHR1 complex in order to gain insights that will aid the design of ligands of PTHR1 for the treatment of osteoporosis and other disorders. The extreme N-terminus of PTH is essential for its activity (13). Therefore, elucidating the interaction between this region of the hormone with the receptor should provide valuable information for the future design of novel PTHR1 agonists. Using benzoylphenylalanine- (Bpa-) mediated photoaffinity cross-linking, we previously reported that an analogue of PTH incorporating a Bpa moiety at position 1 (Bpa1-PTH) cross-links to M425 of the receptor, close to the top of transmembrane (TM) 6 (6). However, based on recent findings that Bpa exhibits a strong cross-linking preference for methionine, the precision of this contact site is not well-defined (14-19). Given the methionine preference, coupled with the rotational freedom of the Bpa moiety (encompassing a large radius for cross-linking), further refinement of this PTH/PTHR1 region of interaction using a Bpa photoaffinity cross-linking approach is precluded. In order to obtain a more detailed and accurate molecular model of the PTH–PTHR1 complex, we used a recently developed approach for cross-linking peptides to receptor in a novel manner. The disulfide bond-mediated approach for cross-linking small molecules and peptides to GPCRs has been used successfully (20, 21), primarily as a means for screening thiol-containing compound libraries in order to identify novel ligands. We now utilize this technique for studying the interactions of PTH and PTHR1 and mapping the interface of their biomolecular complex. After the original submission of this paper, a report of a closely similar approach applied to the C5a receptor appeared (22). This technique monitors disulfide bond formation between a peptide analogue containing a cysteine at a defined position and mutant receptors that have individual cysteines introduced at sites predicted to be close to the natural binding site for the region of the peptide being studied. The rationale is that if the cysteine of the ligand comes into close proximity to an introduced cysteine in the receptor during ligand binding, a disulfide bond will form. Using the disulfide-trapping approach, we probed a total of 24 positions within TM5 and TM6 of PTHR1 for interaction with Cys1. The positions selected for study were based on the previous finding that M425 in TM6 is the contact point for position 1 in PTH (6). Also, the TM5 domain was included in the study based on recent related research from our laboratory (unpublished data) indicating interactions between TM5 and TM6 upon ligand binding. A portion of extracellular loop 3 (EC3), namely, positions 427–429, was included. But the lack of cross-linking confirmed our interest in focusing on TM5 and TM6. Although EC3 is involved in ligand binding, it likely interacts with positions of PTH other than position 1. Application of the disulfide approach enabled us to identify four residues of PTHR1, situated toward the extracellular portions of TM5 and TM6, which form part of the binding pocket for the extreme N-terminus of PTH. Importantly, although 24 positions were probed, only 4 cross-linked. This contrasts to the observation of many cross-linking sites detected due to the “methionine magnet effect” (14 -19). The novel sites of interaction identified by this study generated a molecular model that exhibits important differences from our previous model based on Bpa photoaffinity cross-linking data. In this report, we demonstrate that the disulfide-trapping approach, when guided by a model based on the widely employed Bpa-mediated photoaffinity cross-linking methodology, can provide higher resolving power than Bpa photo-cross-linking alone. This method should have important applications in mapping ligand–receptor interfaces and in the further refining of existing models, not just for the PTH–PTHR1 system but also for class II GPCRs, in general.