Parathyroid Hormone-Receptor Interactions Identified Directly by Photocross-linking and Molecular Modeling Studies

Bisello, Alessandro; Adams, A. Elizabeth; Mierke, Dale F.; Pellegrini, Maria; Rosenblatt, Michael; Suva, Larry J.; Chorev, Michael

doi:10.1074/jbc.273.35.22498

Cited by 178 publications

(306 citation statements)

References 48 publications

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“…Many photoaffinity crosslinking reports (including studies from us and others working on different ligand-receptor systems) which identified methionine as the crosslinked residue [1,3,15,16], may now warrant interpretation in a new light. The identified sites of interaction might have been significantly shifted by the presence of methionine.…”

Section: Discussionmentioning

confidence: 99%

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Methionine acts as a “magnet” in photoaffinity crosslinking experiments

et al. 2006

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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.

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Section: Discussionmentioning

confidence: 99%

“…Peptide synthesis was carried out as described [1]. Cyanogen bromide (CNBr) was from Aldrich (Milwaukee, WI), Na 125 I from Amersham-Pharmacia (Arlington Heights, IL), and endoglycosidase F/N-glycosidase F (Endo-F) from New England BioLabs (Beverly, MA).…”

Section: Methodsmentioning

confidence: 99%

Methionine acts as a “magnet” in photoaffinity crosslinking experiments

et al. 2006

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“…We initially focused our attention on residues in the upper region of TM6 since our previous finding, using Bpa1-PTH, showed that M425 appeared to be a contact point for position 1 of the peptide (6). While investigating this region of TM6, ongoing work in our laboratory suggested that, upon activation of the receptor, the upper regions of TM5 and TM6 move closer together (unpublished data).…”

Section: Identification Of Residues In Tm5 In Close Proximity To Posimentioning

confidence: 99%

“…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.…”

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“…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.…”

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

et al. 2008

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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...

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Structural characterization of peptide hormone/receptor interactions by NMR spectroscopy

Pellegrini

Mierke

1999

Biopolymers

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The structural characterization of peptide hormones and their interaction with G‐protein (guanine nucleotide‐binding regulatory protein) coupled receptors by high‐resolution nmr is described. The general approaches utilized can be categorized into three different classes based on their target: the ligand, the receptor, and the ligand/receptor complex. Examples of these different approaches, aimed at facilitating the rational design of peptides and peptidomimetics with improved pharmacological profiles, based on work carried out in our own laboratory, are given. In the ligand‐based approach, the high‐resolution structures of bradykinin analogues allowing for the development of a structure–activity relationship for activation of the B1 receptor are described. Studies targeting the receptor are to a large extent theoretical, based on computational molecular modeling. However, experimentally based structural features provided by high‐resolution nmr can be used to great advantage, providing insight into the mechanism of receptor function, as illustrated here with results from parathyroid hormone. A similar combination of theoretical methods, supplemented by high‐resolution structures from nmr has been utilized to probe the formation and stabilization of the ligand/receptor complex both for parathyroid hormone and cholecystokinin. In each of these three approaches, the importance of well‐designed peptide mimetics and accurate structural analysis by high‐resolution nmr, will be highlighted. © 1999 John Wiley & Sons, Inc. Biopoly 51: 208–220, 1999

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Parathyroid Hormone-Receptor Interactions Identified Directly by Photocross-linking and Molecular Modeling Studies

Cited by 178 publications

References 48 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

Structural characterization of peptide hormone/receptor interactions by NMR spectroscopy

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