The Mid-Region of Parathyroid Hormone (1−34) Serves as a Functional Docking Domain in Receptor Activation

Wittelsberger, Angela; Corich, Martina; Thomas, Beena; Lee, Byoung Kwon; Barazza, Alessandra; Czodrowski, Paul; Mierke, Dale F.; Chorev, Michael; Rosenblatt, Michael

doi:10.1021/bi051833a

Cited by 54 publications

(56 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Our recent results suggest that this helix might re-arrange and fold back towards the helical bundle during ligand-binding [8]. This conformational flexibility might explain how methionines on different sides of the helix are captured by Bpa.…”

Section: Discussionmentioning

confidence: 92%

“…Abbreviations: Bpa, p-benzoylphenylalanine; Bpa n -PTH, [Bpa n , Nle 8,18 ,Arg 13,26,27 ,L-2-Nal 23 ,Tyr 34 ]bPTH(1-34)NH 2 ; CNBr, cyanogen bromide; Endo-F, endoglycosidase F/N-glycosidase F; N-ECD, N-terminal extracellular domain; Nle, norleucine; PTH, parathyroid hormone; PTHR1, PTH receptor-1…”

Section: Radioiodination and Photoaffinity Crosslinkingmentioning

confidence: 99%

“…Binding affinities of the PTH analogs for mutant receptors were determined using 125 I-[Nle 8,18 ,Tyr 34 ]-bPTH-(1-34)NH 2 as tracer [9].…”

Section: Binding Assaymentioning

confidence: 99%

See 2 more Smart Citations

Methionine acts as a “magnet” in photoaffinity crosslinking experiments

et al. 2006

Self Cite

View full text Add to dashboard Cite

Photoaffinity crosslinking has been utilized to probe the nature of the ligand-receptor interface for a number of G protein-coupled receptor systems. Often the photoreactive benzophenone moiety incorporated in the ligand is found to react with a methionine in the receptor. We introduced methionines one-ata-time into the region 163-176 of the parathyroid hormone receptor, and find that crosslinking occurs to the side-chain of methionine over a range of 11 amino acids. We call this the ''Magnet Effect'' of methionine. Hence, crosslinking contact points can be significantly shifted by the presence of methionine in a receptor domain.

show abstract

Section: Discussionmentioning

confidence: 92%

Section: Radioiodination and Photoaffinity Crosslinkingmentioning

confidence: 99%

See 1 more Smart Citation

Methionine acts as a “magnet” in photoaffinity crosslinking experiments

et al. 2006

Self Cite

View full text Add to dashboard Cite

show abstract

“…The Ser1 residue used in the simulations adopts a position equidistant between the extracellular portion of TM5 and TM6, consistent with the general mode of GPCR activation by ligand. (4)(5)(6)8,9,12). By combining data collected from extensive photoaffinity cross-linking studies with molecular modeling, a model of the PTH-PTHR1 bimolecular complex has been generated that identifies regions of contact at the interface between the hormone and the receptor.…”

Section: Molecular Modeling Of the Site Of Interaction Of Position 1 mentioning

confidence: 99%

“…Posttransfection (18-24 h), the media were replaced with DMEM containing 5% FBS. The luciferase assay for measuring hormone-stimulated adenylyl cyclase activity was carried out as described (12). The EC 50 was calculated by nonlinear regression analysis using the GraphPad Prism software.…”

Section: Adenylyl Cyclase Activitymentioning

confidence: 99%

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

et al. 2008

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Backbone dynamics of human parathyroid hormone (1–34): Flexibility of the central region under different environmental conditions

et al. 2005

Self Cite

View full text Add to dashboard Cite

The presence of a stable tertiary structure in the bioactive N-terminal portion of parathyroid hormone (PTH), a major hormone in the maintenance of extracellular calcium homeostasis, is still debated. In this work, 15N relaxation parameters of the 33 backbone amides of human PTH(1-34) were determined in phosphate-buffered saline solution (PBS) and in the presence of dodecylphosphocholine (DPC) micelles. The relaxation parameters were analyzed using both the model-free formalism (G. Lipari and A. Szabo, Journal of the American Chemical Society, 1982, Vol. 104, pp. 4546-4549) and the reduced spectral density functions approach (J.-F. Lefevre, K. T. Dayie, J. W. Peng, and G. Wagner, Biochemistry, 1996, Vol. 35, pp. 2674-2686). In PBS, the region around Gly12 possesses a high degree of flexibility and the C-terminal helix is less flexible than the N-terminal one. In the presence of DPC micelles, the mobility of the entire molecule is reduced, but the stability of the N-terminal helix increases relative to the C-terminal one. A point of relatively higher mobility at residue Gly12 is still present and a new site of local mobility at residues 16-17 is generated. These results justify the lack of experimental nuclear Overhauser effect (NOE) restraints with lack of tertiary structure and support the hypothesis that, in the absence of the receptor, the relative spatial orientation of the two N- and C-terminal helices is undefined. The flexibility in the midregion of PTH(1-34), maintained in the presence of the membrane-mimetic environment, may enable the correct relative disposition of the two helices, favoring a productive interaction with the receptor.

show abstract

The Mid-Region of Parathyroid Hormone (1−34) Serves as a Functional Docking Domain in Receptor Activation

Cited by 54 publications

References 33 publications

Methionine acts as a “magnet” in photoaffinity crosslinking experiments

Methionine acts as a “magnet” in photoaffinity crosslinking experiments

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

Backbone dynamics of human parathyroid hormone (1–34): Flexibility of the central region under different environmental conditions

Contact Info

Product

Resources

About