Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

Abstract

Efforts to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1), have relied heavily on benzoylphenylalanine-(Bpa-) based photoaffinity cross-linking. However, given the flexibility, size, and shape of Bpa, the resolution at the PTH-PTHR1 interface appears to be reaching the limit of this technique. Here we employ a disulfide-trapping approach developed by others primarily for use in screening compound libraries to identify novel ligands. In this method, cysteine substitutions are introduced into a specific site within the ligand and a region in the receptor predicted to interact with each other. Upon ligand binding, if these cysteines are in close proximity, they form a disulfide bond. Since the geometry governing disulfide bond formation is more constrained than Bpa cross-linking, this novel approach can be employed to generate a more refined molecular model of the PTH-PTHR1 complex. Using a PTH analogue containing a cysteine at position 1, we probed 24 sites and identified 4 in PTHR1 to which cross-linking occurred. Importantly, previous photoaffinity cross-linking studies using a PTH analogue with Bpa at position 1 only identified a single interaction site. The new sites identified by the disulfide-trapping procedure were used as constraints in molecular dynamics simulations to generate an updated model of the PTH-PTHR1 complex. Mapping by disulfide trapping extends and complements photoaffinity cross-linking. It is applicable to other peptide-receptor interfaces and should yield insights about yet unknown sites of ligand-receptor interactions, allowing for generation of more refined models.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. † This work was supported in part by Grants DK47490 (to M.R.) and GM54082 (to D.F.M.) from the National Institutes of Health.* Address correspondence to this author. Phone: (617) 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 o...

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

“…E364 within TM6 is in a region critical for peptide binding in other family B GPCRs including the secretin (48) and PTH (49,50) receptors. Our model suggests a direct interaction of this residue with R310 (TM5) and through this hydrogenbonding network onto Q234 (TM3), providing key structural constraints between transmembrane helices.…”

“…Similarly, potency estimates and E max values for cAMP generation derived from experiments on all receptor constructs are given in Table 3. 50 and E max values. Data are mean Ϯ SEM with n ϭ 7 for the WT receptor and n ϭ 3 for each of the mutants.…”

Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor

“…This is the analogous region of PTH 1 (18,58) and PTH 2 (59) receptors labeled by the amino-terminal PTH probes. Recently, the amino terminus of PTH has also been shown to additionally interact with a residue within TM5 of its receptor using a disulfide trapping approach (60). In addition, mid-region position 19 and carboxyl-terminal position 27 residues of PTH have also been shown to interact with the body of its receptor, with distinct residues in TM2 and ECL1, respectively (55,56).…”

International Union of Basic and Clinical Pharmacology. XCIII. The Parathyroid Hormone Receptors—Family B G Protein–Coupled Receptors

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