Backbone-methylated Analogues of the Principle Receptor Binding Region of Human Parathyroid Hormone

Barbier, J.-R.; Gardella, Thomas J.; Dean, Thomas R.; MacLean, Susanne; Potetinova, Zhanna; Whitfield, J. F.; Willick, Gordon E.

doi:10.1074/jbc.m500817200

Cited by 16 publications

(17 citation statements)

References 54 publications

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“…This observation raised the possibility that the C-terminal binding domain of PTH can functionally interact with the receptor J domain. Consistent with this possibility, we recently showed that methylation of several backbone nitrogen atoms in region 17-31 of PTH-(1-31) impairs, albeit modestly, the capacity of the ligand to stimulate cAMP formation in cells expressing PTHR-delNt (8). Taken together, these observations point to the uncertainty that exists in our understanding of the specific mechanisms by which the C-terminal domain of PTH contributes to the PTHR-binding process.…”

Section: Parathyroid Hormone (Pth)mentioning

confidence: 53%

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Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor

Dean

Khatri

Potetinova

et al. 2006

Journal of Biological Chemistry

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Parathyroid hormone (PTH)2 plays a key role in calcium and phosphate homeostasis and has potent effects on the bone-remodeling process. PTH interacts with a Class 2 G protein-coupled receptor that is prominently expressed in bone osteoblasts and in cells located in the proximal and distal portions of the renal convoluted tubules. The PTH receptor (PTHR) is also expressed in the primordia of developing long bones, heart, mammary glands, and other tissues, where it mediates the morphogenic actions of PTH-related protein (PTHrP) (1).For both PTH and PTHrP, the bioactive portions of the molecule reside within the first 34 amino acids of the processed polypeptides. Within region 1-34, the principal determinants of receptor-binding affinity and receptor-signaling activity map to the C-and N-terminal domains, respectively (2, 3). Solutionphase NMR studies of PTH-(1-34)-based ligands typically show a well formed ␣-helix in the region of the C-terminal binding domain (4,5 , and Leu 28 , which form the hydrophobic face of this ␣-helix, are particularly important for efficient interaction with the receptor (9 -11).The mechanism by which PTH interacts with its receptor has been investigated via the approaches of ligand analog design, receptor mutagenesis, and photochemical cross-linking (reviewed in Ref. 12). The view that has emerged from these studies is that the overall mechanism consists of two principal and, to some extent, autonomous components: 1) an interaction between the C-terminal helical domain of the ligand and the N-terminal extracellular domain (N domain; spanning Tyr 23 to approximately Ile 190 ) of the mature receptor and 2) an interaction between the N-terminal portion of the ligand and the juxtamembrane domain (J domain) of the receptor containing the extracellular loops and seven transmembrane helices. The N domain component of the interaction is thought to provide the major portion of binding energy and stability to the complex, and the J domain component is thought to mediate the conformational changes involved in receptor activation (13). It now seems likely that most, if not all, of the 15 or so other Class 2 G protein-coupled receptors utilize a similar two-site binding mechanism for interacting with their cognate peptide ligands (14 -16).Consistent with such a two-site binding mechanism for PTH and the PTHR, we have shown that N-terminal PTH peptide fragments, such as PTH-(1-14), bind only extremely weakly to the receptor, but can nevertheless induce at least measurable increases in cAMP levels in PTHR-expressing cells (17). The potency of such N-terminal PTH fragments can be significantly enhanced by introducing substitutions that improve the affinity

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Section: Parathyroid Hormone (Pth)mentioning

confidence: 53%

“…The instrument was calibrated with ammonium (ϩ)-10-camphorsulfonate. Data are expressed as the number of helical residues/peptide chain as calculated from Ϫ[] 222 ϫ 30/28,000, where [] 222 is the mean residue ellipticity at 222 nm, as we described previously (8).…”

Section: Methodsmentioning

confidence: 99%

Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor

Dean

Khatri

Potetinova

et al. 2006

Journal of Biological Chemistry

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“…The presence of a methylation site can therefore be expected to be reflected in the masses of the cand z-ions that contain the methylation site, as is the case with side-chain post-translational modifications. While the methyl modification is expected to be strongly bound, its presence on the amide nitrogen might have been expected to affect the overall ETD process, at least based on one proposed mechanism for ECD/ETD [18]. N-methylation affects modestly the relative abundances of the sequence informative fragment ions but this observation may very well result from conformational differences between modified and unmodified versions of the peptide cations.…”

Section: Discussionmentioning

confidence: 99%

“…ECD [9 -12] and ETD [13] have already been shown to be effective in identifying side-chain and N-terminal methylation in histones, for example. Amide nitrogen methylation occurs in nature, such as in bacterial and fungal peptides/proteins that have possible medicinal properties [14 -17], and has been used as a tool for protein binding studies [18]. The dissociation behavior of N-methylated peptide ions subjected to collisional activation has been reported, and it was noted that there was an increase in relative abundance of the products coming from cleavage of the amide bond at the methylation site [19,20].…”

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confidence: 99%

Electron transfer dissociation of amide nitrogen methylated polypeptide cations

Crizer

McLuckey

2009

J. Am. Soc. Mass Spectrom.

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Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process. (J Am Soc

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“…The crystal structure of the PTH1R extracellular domain-PTH(15-34) complexes provides direct evidence for the model that the C-terminal domain of the ligand binds to the N terminus of the receptor, thus positioning the N terminus of the ligand at a favorable distance to allosterically activate the J-domain of the receptor (52). Therefore, it is interesting to compare the data from photoaffinity cross-linking of PTH1R (22)(23)(24)(25)(53)(54)(55)(56)(57)(58)(59)(60) with those of CRFR1. Similar to our finding that residues 16 and 17 of sauvagine cross-link to the J-domain of CRFR1, residues within the C terminus of PTH , such as 19 and 27, are reported to cross-link to the second transmembrane domain and within the first extracellular loop, respectively; both are within the J-domain of PTH1R (58).…”

Section: Discussionmentioning

confidence: 99%

Residue 17 of Sauvagine Cross-links to the First Transmembrane Domain of Corticotropin-releasing Factor Receptor 1 (CRFR1)

Assil-Kishawi

Samra

Mierke

et al. 2008

Journal of Biological Chemistry

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Corticotropin-releasing factor (CRF), 2 the main regulator of the hypothalamic pituitary adrenal axis (1), binds to specific G protein-coupled receptors CRFR1 (2, 3) and CRFR2 (4 -7) cloned from several mammalian and nonmammalian species. A third CRF receptor, CRFR3, was found only in fish (8). Several CRF-like peptides were shown to interact with the CRF receptors with high affinity: sauvagine (SVG) characterized from the frog skin (9), urotensin I from the urophysis of fish (10), and urocortin I (UCN1) from the mammalian brain (11). Later, UCN2 and UCN3 (or stresscopins) (12-14) where characterized as CRFR2-specific ligands with little or no affinity to CRFR1.The CRF receptors, which belong to group B of the G protein-coupled receptor superfamily, have six conserved cysteine residues and several N-linked glycosylations in their N-terminal extracellular domains. The extracellular cysteine residues form disulfide bridges that stabilize the structure of the N terminus (15, 16). The N-linked glycosylation moieties are important for processing through the Golgi system and cell surface expression (17). Activation of the cloned CRF receptors stimulates adenylate cyclase (4 -7), phospholipase C (18), and mitogen-activated protein kinases (MAPKs) (19,20).Chemical and/or photoaffinity cross-linking have been extensively used for characterization of ligand-receptor interaction within group B of the G protein-coupled receptor superfamily (21-25), including the CRF receptors (26,27). Recently, the photosensitive amino acid analog p-benzoylphenylalanine (Bpa) was used to substitute several residues within the UCN1 sequence, and photoaffinity cross-linking was used to map the interaction site; the data showed that the very N-terminal ligand residues (1-11) that are responsible for receptor activation are in a close proximity to the second extracellular loop within the juxtamembrane (J-) domain of CRFR1 (26). These data contradict the proposed model derived from the interaction of the free receptor N terminus with the ligand (28 -31). We have previously used an oxidation-resistant SVG analog, [Tyr 0 , Gln 1 , Leu 17 ]SVG (YQL-SVG), which binds to CRFR1 and CRFR2 with high affinity and which cross-links to the CRF receptors with a high efficiency (32), to map the disuccinimidyl suberate-cross-linked residues, and identified covalent crosslinking between Lys 16 of SVG and Lys 257 of CRFR1 in the second extracellular loop (27). In this paper we have used a Bpasubstituted SVG analog, [Tyr 0 , Gln 1 , Bpa 17 ]SVG (YQB-SVG), which has high affinity for CRFR1, and mapped the Bpa 17 crosslinking site to His 117 of CRFR1 and thus established that position 17 of the radioligand lies within 9 Å of position 117 located in the first transmembrane-spanning domain (TM1) of CRFR1.

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Backbone-methylated Analogues of the Principle Receptor Binding Region of Human Parathyroid Hormone

Cited by 16 publications

References 54 publications

Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor

Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor

Electron transfer dissociation of amide nitrogen methylated polypeptide cations

Residue 17 of Sauvagine Cross-links to the First Transmembrane Domain of Corticotropin-releasing Factor Receptor 1 (CRFR1)

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